effect of elevated carbon dioxide and high temperature on
TRANSCRIPT
Effect of Elevated Carbon Dioxide and High
Temperature on Major Micronutrients in
Strawberry
Himali Balasooriya
A thesis submitted in total fulfillment of the requirements
for the degree of
Doctor of Philosophy
December 2019
School of Agriculture and Food
Faculty of Veterinary and Agricultural Sciences
The University of Melbourne
AUSTRALIA
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ABSTRACT
Climate changes have become a main challenge in the modern agriculture due to their
significant effects on both quantity and quality of the crops. This study investigated the effect
of increased temperature, carbon dioxide concentration [CO2] and their interactions on plant
growth and development, physical quality of fruit (yield, size, and colour), major antioxidant
compounds (polyphenols, vitamin C, and folate) and their in vitro bioaccessibility in
strawberries. Using strawberry as an example, it demonstrated that the physical and chemical
composition, metabolism and bioaccessibility of micronutrients of this fruit were significantly
(p<0.05) affected by the increase in atmospheric [CO2] and air temperature.
This investigation involved two different strawberry cultivars; ‘Albion’ and ‘San Andreas’
grown at six different [CO2] and temperature combinations in controlled environmental plant
growth facility of the glasshouse complex at the Parkville Campus of the University of
Melbourne, Australia. The CO2 levels were 400 (ambient), 650, and 950 ppm, and the
temperature treatments were 25 °C (ambient) and 30 °C. Physiological performances,
vegetative growth, and reproductive growth of plants were studied to determine the
physiological adjustment and estimate yield characteristics of strawberry in response to climate
stress. Fresh and freeze-dried (FD) strawberry samples were then analysed for polyphenols,
folates and vitamin C to determine the effect of elevated [CO2] and temperature on nutritional
quality of strawberry. Finally, strawberry samples grown under ambient (400 ppm and 25 °C)
and elevated (950 ppm and 30 °C) growth conditions were subjected to simulated in vitro
gastrointestinal digestion to determine the bioaccessibility of polyphenols, folates and vitamin
C in fruits of strawberries. The in vitro colonic fermentation study was carried out using human
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faecal in a basal media to measure bioactive compounds and microbial metabolites in the
soluble fraction of fermented strawberry digests.
The results of the study showed that elevated [CO2] and increased temperature individually and
interactively influenced the growth and yield of both strawberry cultivars. Elevated [CO2] from
400 to 650 ppm significantly (P≤0.05) increased the plant height by 25% and 30% at 25 °C and
30 °C, respectively. The elevated growth conditions ([CO2] and temperature) significantly
(p<0.05) influenced the reproductive development of strawberry. Increasing the temperature
by 5 °C remarkably reduced the fruit setting by 43% at 400 ppm and supressed the fruit
development by reducing size, weight and total yield of strawberry fruits. Cultivar ‘Albion’
had maximum fruit yield of 45.09±4.90 g per plant at 650 ppm and 25 °C. Cultivar ‘San
Andreas’ had higher yields at both 400 ppm (60.05±10.4 g per plant) and 650 ppm (59.48±7.92
g per plant) at 25 °C. Both cultivars had the lowest yields of 9.60±2.41 g and 7.00±1.70 g per
plant in ‘Albion’ and ‘San Andreas’, respectively at 400 ppm and 30 °C. In conclusion, plant
growth associated with 650 ppm and 25 °C environmental conditions seemed to be more
favourable for vegetative growth and fruit development of strawberry. However, elevated
[CO2] was not fully able to compensate the negative effects of increased temperature. In
comparison to ambient growth conditions (25 °C and 400 ppm), the combination of both
elevated temperature and [CO2] did not noticeably influence on plant growth or development.
Different colorimetric methods revealed that polyphenol contents of strawberry were
significantly (P≤0.05) influenced by [CO2] and temperature (individually and interactively)
and the responses were cultivar dependent. The greatest amounts of flavonoid (48±7 mg per
100 g FW) and total antioxidant compounds (11.5±1.5 µmol per g of FW) were detected in
‘Albion’ grown under 30 °C and 950 ppm, and total polyphenols (335±12 mg GAE per 100 g
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of FW) and anthocyanin (33±2 mg per 100 g of FW) in ‘San Andreas’ grown at 25 °C and 950
ppm. Both elevated temperature (30 °C) and [CO2] (950 ppm) increased TPC by 182% in
‘Albion’ and 206% in ‘San Andreas’, and TAC by 179% and 110% in ‘Albion’ and ‘San
Andreas’, respectively. Results from HPLC-UV analyses revealed that individual phenolic
compounds of strawberries were also increased by elevated [CO2] and/or temperature and their
interactions. In this study, twelve major polyphenolic compounds in strawberry were identified
and quantified. Pelargonidin glucoside (Pel-3-Glu), pelargonidin rutinoside (Pel-3-Rut) and a
cyanidin derivative were detected at 510 nm. Quercetin-glucoside and -glucuronide,
kaempferol glucoside and glucuronide, resveratrol, catechin, p-coumaric acid, ferulic acid and
p-coumaroyl acid were also identified as phenolic compounds at different wavelengths (280,
320 and 360 nm). These individual phenolic compounds in strawberries were significantly
(P≤0.05) increased by elevated [CO2] and higher temperature. The Pel-3-Glu content ranged
from 11.5±2.3 to 27.3±3.0 mg per 100 g fresh weight (FW) in cultivar ‘Albion’ and from
11.4±2.4 to 34.8±3.3 mg per 100 g FW in cultivar ‘San Andreas’. The interaction of elevated
[CO2] (950 ppm) and increased temperature (30 °C) enhanced Pel-3-Glu contents by 90% and
103% in ‘Albion’ and ‘San Andreas’, respectively in comparison with plants grown under
ambient conditions (400 ppm [CO2] and 25 °C). Highest elevated [CO2] and higher temperature
interactively enhanced the Pel-3-Rut contents of fruits from 1.8±0.3 to 4.1±0.4 mg per 100 g
of FW for cultivar ‘Albion’ and from 2.4±0.4 to 5.8±0.6 mg per 100 g of FW for cultivar ‘San
Andreas’, respectively. Elevated [CO2] from 400 ppm to 950 ppm at 30 °C increased the
cyanidin content by 600% and 435% in ‘Albion’ and ‘San Andreas’, respectively.
In this study, four different folate derivatives (tetrahydrofolic acid – THFA, 10-formylfolic
acid – 10FFA, 5-formyltetrahydrofolic acid – 5FTHFA, 5-methytetrahydrofolic acid
(5MTHFA)) were identified in fresh and freeze-dried strawberry samples. The individual and
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interaction effects of increased [CO2] and temperature on total folates content were significant
(P≤0.05), and the responses were cultivar dependant. Total folate content in strawberries varied
from 52.6 ± 5.1 µg to 364.8 ± 16.0 µg/100 g FW in cultivar ‘Albion’ and from 48.6 ± 7.0 µg
to 237.4 ± 23.8 µg/100 g FW in cultivar ‘San Andreas’. Although, increased temperature
positively affected the total folates content under lower [CO2] levels, the effects turned negative
at the highest [CO2] concentration (950 pm). Higher temperature reduced the content of total
folates in strawberries by 26% and 13% in cultivar ‘Albion’ and ‘San Andreas’, respectively.
Impacts of elevated [CO2], higher temperature and their interactions on total vitamin C content
in strawberries were statistically significant (P≤0.05) and the responses were cultivar
dependent. Vitamin C contents in cultivar ‘Albion’ and ‘SA’ fresh strawberries were in a range
of 59 ± 7 mg to 133 ± 15 mg/100 g FW and 56 ± 9 mg to 132 ± 9 mg/100 g FW, respectively.
Increased growth temperature to 30 °C at 650 ppm [CO2] enhanced the amounts of vitamin C
significantly (P≤0.05) to a maximum by 123% and 132% in cultivars ‘Albion’ and ‘San
Andreas’, respectively. However, that effect wasn’t detected when the CO2 concentration was
increased further to 950 ppm, and vitamin C concentrations drastically decreased by 36% and
31% in Albion’ and ‘San Andreas’, respectively. In general, folates and vitamin C contents
were significantly (P≤0.05) higher in FD strawberry than fresh fruits.
The next step of the study was to study the accessibility of increased polyphenols, vitamin C
and folates in the fruits of fresh and frozen strawberries using simulated in vitro gastrointestinal
digestion and colonic fermentation. Elevated [CO2] (ambient to 950 ppm) and higher
temperature (ambient to 30 °C) enhanced the accessibility of polyphenols, folate and vitamin
C in strawberries. Bioaccessibility of Pel-3-Glu increased from 67% to 88% in fresh
strawberries when exposed to elevated growth. The exact amounts of individual polyphenols
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in accessible fraction were significantly (P≤0.05) higher in fresh fruits of strawberries grown
under elevated growth conditions. For example, the highest amounts of Pel-3-Glu (19.89±0.4
mg/100 g FW), Pel-3-Rut (2.55±0.5 mg/100 g FW), p-coumaric (0.23±0.02 mg/100 g FW),
ferulic (1.33±0.05 mg / 100 g FW), quercetin (1.97±0.2 mg/100 g FW) and p- coumaroyl
(0.65±0.05 mg/100 g FW) were detected in fed state simulated gastrointestinal digesta of fresh
strawberry grown under elevated growth conditions. Fresh strawberries grown under ambient
growth contained 93.09±6.2 µg/100g folates and 18.55±0.5 mg/100g vitamin C as
bioaccessible fractions under fed state while, elevated growth enhanced soluble folates and
vitamin C up to 188.63±7.5 µg/100g and 30.48±0.3 mg/100g, respectively. Fresh strawberries
contained higher amounts of accessible micronutrients than frozen strawberries, while
increased bile contents in intestinal fluid (fed state) facilitated the release of bioactive
compounds to gastrointestinal fluid.
The insoluble fraction of strawberry digests after gastrointestinal digestion was then subjected
to in vitro colonic fermentation using human faecal cultures and basal media. The soluble
fraction of fermented strawberry digests was extracted to analyse polyphenols, folates and
vitamin C. Higher contents of folate (7.90±0.05 µg/100 g FW), vitamin C (33.6±1.0 ng/100 g
FW), Pel-3-Glu (2.00±0.14 mg/100 g FW), and p-coumaric (39±5 µg/100 g FW) were observed
in soluble fraction of fermented precipitate after simulated gastrointestinal digestion at fasted
state in frozen strawberries. These bioactive compounds and their metabolites would play an
important role in the human colon by maintaining a healthy environment via scavenging the
free radicals. According to the current study, the amount of bioaccessible bioactive compounds
in strawberry could vary quantitatively and qualitatively based on growth and storage
conditions as well as the status of digestion (fed or fasted state). Increased carbon dioxide and
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temperature in the growth environment enhanced the bioaccessibility of polyphenols, folates
and vitamin C in strawberries.
It can be concluded that strawberry fruits grown under elevated [CO2] and temperature may
not be visually attractive comparing to normal strawberries. However, considering their
nutritional value, those fruits can be promoted as freeze-dried strawberry in value added foods
such as dairy products. Additionally, these research outcomes would help the commercial
growers to focus on the nutritional aspects of fruits and vegetables grown under such elevated
and extreme environmental conditions in the future. However, as a very little information is
available concerning the interactive effects of elevated [CO2] and high temperature on fruits
and vegetables in the field, more researches are needed to confirm the results from glasshouse
studies.
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DECLARATION
This is to certify that:
1.The thesis comprises only the original work of the author and none of this work has
been submitted for any other degree;
2.Due acknowledgment has been made in the relevant part of the text to all other
methodologies used and adapted;
3.The thesis is fewer than 100 000 words in length, exclusive of words in figures, tables,
lists of references and appendices.
Himali Balasooriya
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PREFACE
I was the lead investigator in all chapters included in this thesis, responsible for planning, data
collection and analysis, and manuscript composition. In Chapter 4, I planned and performed all
the controlled environment experiments for growing strawberry plants in the glasshouse of
Parkville campus of the University of Melbourne. In Chapters 5, 6, and 7, I developed and
performed all HPLC experiments including HPLC spiking, identifications and quantifications.
I was the primary author of published articles which comprises Chapter 2.2, 2.3, and 4, and
also in the accepted manuscript in Chapter 5 and 6. I have planned another two journal papers
from this project which will be presented in chapters 7, and 8.
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LIST OF PUBLICATIONS
Published papers:
1. Balasooriya HN, Dassanayake KB, Seneweera S, and Ajlouni S. (2019) Impact of
Elevated Carbon Dioxide and Temperature on Strawberry Polyphenols. The Journal of
the Science of Food and Agriculture (https://doi.org/10.1002/jsfa.9706).
2. Balasooriya HN, Dassanayake KB, and Ajlouni S. (2019) The Impact of elevated CO2
and High Temperature on the Nutritional Quality of Fruits – A Short Review. American
Journal of Agricultural Research, 4;26. (https://doi.org/10.28933/ajar-2018-12-1608).
3. Balasooriya HN, Dassanayake KB, Seneweera S, and Ajlouni S. (2018). Interaction of
elevated carbon dioxide and temperature on strawberry (Fragaria × ananassa) growth
and fruit yield. International Journal of Agricultural and Biosystems Engineering. 12:9.
(https://waset.org/Publication/10009464).
4. Balasooriya HN, Dassanayake KB, Tomkins B, Seneweera S, and Ajlouni S. (2017)
Impacts of Elevated Carbon Dioxide and Temperature on Physicochemical and Nutrient
Properties in Strawberries. Journal of Horticultural Science and Research (1):19-29.
(https://www.scholarlypages.org/Articles/horticulture/jhsr-1-004.php?jid=horticulture).
Accepted papers:
5. Balasooriya HN, Dassanayake KB, and Ajlouni S. (2019). High temperature effects on
strawberry fruit quality and antioxidant contents.
Accepted by Acta Horticulturae
Accepted date: 9th December 2018
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Manuscripts Submitted and under review:
6. Balasooriya HN, Dassanayake KB, Seneweera S, and Ajlouni S. (2019). Folates and
vitamin C contents in strawberries grown under elevated carbon dioxide and temperature.
Submitted to: Journal of Food Chemistry
Submitted on: 13th May 2019
7. Balasooriya HN, Dassanayake KB, and Ajlouni S. (2019). Bioaccessibility of
micronutrients in fresh and frozen strawberry fruits grown under elevated carbon dioxide
and temperature.
Submitted to: Journal of Food Chemistry
Submitted on: 7th March 2019
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ACKNOWLEDGMENTS
I would like to thank my supervisors A/Prof Said Ajlouni, Dr Kithsiri Dassanayake, Dr Saman
Seneweera and Mr Bruce Tomkins for their invaluable support in completing this PhD project.
I specially thank my principal supervisor, A/Prof Ajlouni who provided me an opportunity to
be his PhD student and for providing me a comfortable environment in the past three years to
complete this research study. I appreciate his patience and motivation with me even I appeared
suddenly in his office during my hard times in laboratory works. I am also indebted to Dr
Dassanayake for introducing me to A/Prof Said and for his continuous and constructive support
on both academic and personal matters. He helped me in planning, establishing and
successfully completing the glasshouse experiments. I was fortunate to have these supervisors
who are experts in their fields. Their guidance and insightful comments bring the fields of crop
science and food science into this interdisciplinary thesis. They always encouraged me in
improving my writing with patience and committed their time and scholarly in preparing and
publishing manuscripts. I have always been able to communicate effectively with them and
they often gave me constructive feedback and helped me to hone my skills. I would like to
thank the rest of my supervisors for their invaluable guidance and suggestions during the
research study and also, their time, expertise and scholarly in preparing manuscripts. I am also
grateful to my committee chair; Dr Graham Ian Brodie, and all academic, technical staff and
my colleagues that helped me in numerous ways during my candidature at the School of
Agriculture and Food. I greatly acknowledge the 2017 Innovation Seed Fund for Horticulture
Development of The University of Melbourne and Department of Economic Development,
Jobs, Transport and Resources (DEDJTR) for the financial assistance in this project. I would
like to express my sincere gratitude to The University of Melbourne for offering me
scholarships which financially support my living in Melbourne. Finally, I thank my husband
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for his continuous support to my project works and sharing my ups and downs; and also, for
my family for supporting me spiritually throughout this challenging journey.
xiii
CONTENTS
ABSTRACT…………………………………………………………………………………....i
DECLARATION …………………………………………………………………………....vii
PREFACE……………………………………………………………………………….......viii
LIST OF PUBLICATIONS…………………………………………………………………..ix
ACKNOWLEDGEMENTS…………………………………………………………………..xi
LIST OF TABLES…………………………………………………………………………..xvi
LIST OF FIGURES…………………………………………………………………….........xix
Chapter 1: INTRODUCTION.................................................................................................... 1
1.1. Background ..................................................................................................................... 1
1.2. Research Questions ......................................................................................................... 4
1.3. Thesis Outline ................................................................................................................. 6
Chapter 2: LITERATURE REVIEW......................................................................................... 7
2.1. Introduction ..................................................................................................................... 7
2.2. Paper ............................................................................................................................... 8
2.3. Paper ............................................................................................................................. 17
Chapter 3: GENERAL MATERIALS AND METHODS ....................................................... 28
3.1. Introduction ................................................................................................................... 28
3.2. Experimental Design ..................................................................................................... 28
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3.2.1. Planting materials .................................................................................................. 29
3.2.2. Physiological performances, plant growth, flowering and fruiting of strawberry
plants ................................................................................................................................ 31
3.2.3. Physical quality of strawberry fruit ........................................................................ 32
3.2.4. Strawberry sampling............................................................................................... 33
3.2.5. Polyphenol extraction ............................................................................................. 34
3.2.6. Total Polyphenol Content ....................................................................................... 35
3.2.7. Total Antioxidant Capacity ..................................................................................... 37
3.2.8. Total Flavonoid Content ......................................................................................... 38
3.2.9. Total Monomeric Anthocyanin Content ................................................................. 38
3.2.10. HPLC-UV Analysis for Identification and Quantification of Strawberry Nutrients
.......................................................................................................................................... 39
3.2.11. Simulated Gastrointestinal Digestions and Colonic Fermentation for
Bioaccessibility Assessment .............................................................................................. 44
Chapter 4: INTERACTION OF ELEVATED CARBON DIOXIDE AND TEMPERATURE
ON STRAWBERRY (Fragaria × ananassa) GROWTH AND FRUIT YIELD………........ 46
4.1. Introduction ................................................................................................................... 46
4.2. Paper ............................................................................................................................. 47
Chapter 5: HIGH TEMPERATURE EFFECTS ON STRAWBERRY FRUIT QUALITY AND
ANTIOXIDANT CONTENTS ................................................................................................ 56
5.1. Introduction ................................................................................................................... 56
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5.2. Manuscript .................................................................................................................... 57
Chapter 6: IMPACT OF ELEVATED CARBON DIOXIDE AND TEMPERATURE ON
STRAWBERRY POLYPHENOLS ......................................................................................... 79
6.1. Introduction ................................................................................................................... 79
6.2. Accepted Paper ............................................................................................................. 80
Chapter 7: FOLATES AND VITAMIN C CONTENTS IN STRAWBERRIES GROWN
UNDER ELEVATED CARBON DIOXIDE AND TEMPERATURE ................................... 91
7.1. Introduction ................................................................................................................... 91
7.2. Manuscript .................................................................................................................... 92
Chapter 8: BIOACCESSIBILITY OF MICRONUTRIENTS IN FRESH AND FROZEN
STRAWBERRY FRUITS GROWN UNDER ELEVATED CARBON DIOXIDE AND
TEMPERATURE .................................................................................................................. 126
8.1. Introduction ................................................................................................................. 126
8.2. Manuscript .................................................................................................................. 127
Chapter 9: GENERAL CONCLUSIONS AND RECOMMENDATIONS ........................... 173
9.1. General Conclusion ..................................................................................................... 173
9.2. Recommendations ....................................................................................................... 178
xvi
LIST OF TABLES
Chapter 3 (in a published paper)
Table 3.1 Composition of fresh strawberry (per 100 g of edible fruit) based on country of
origin. Values depend on cultivar, growth conditions, and the geographical
location (pp. 18)
Table 3.2 Phenolic compounds found in Strawberry (pp. 19)
Table 3.3 Effect of increased CO2 concentrations on physical and chemical properties of
strawberry fruit (pp. 22)
Table 3.4 Effect of increasing temperature on physical and chemical properties of
strawberry fruit (pp.24)
Chapter 4 (in a published paper)
Table 4.1 Leaf gas exchange, Ci/Ca ratio, photosynthetic water use efficiency and leaf
photosynthesis efficiency of strawberry plants as affected by elevated
temperature and CO2 concentrations (pp. 50)
Table 4.2 Vegetative growth of strawberry plants as affected by different combinations of
temperature and CO2 concentrations (pp. 53)
Table 4.3 Effect of various growth temperatures and CO2 concentrations on fruit yield of
strawberry plants (pp. 53)
Chapter 5 (in a manuscript)
Table 5.1 Fruit yield and size of strawberry grown at different temperatures (pp. 64)
Table 5.2 Colour of strawberry fruits grown at different temperatures (pp. 65)
Table 5.3 Antioxidant properties of strawberry grown at different temperatures (pp. 67)
Table 5.4 Total AA content in strawberry grown at different temperatures (pp. 70)
xvii
Chapter 6 (in a published paper)
Table 6.1 Pearson’s correlation matrix between TPC, TFC and TMAC in strawberry
cultivars (pp.114)
Table 6.2 Identification of strawberry polyphenols (pp. 114)
Table 6.3 Effect of different temperature and [CO2] on different anthocyanins and
flavonols compounds in strawberry cultivars (pp. 115)
Table 6.4 Effect of different temperature and [CO2] on other polyphenol compounds in
strawberry varieties (pp. 116)
Chapter 7 (in a manuscript)
Table 7.1 Effect of different temperature and [CO2] on other polyphenol compounds in
strawberry varieties (pp. 151)
Table 7.2 Total folate and vitamin C contents in fresh and FD strawberry grown under
different [CO2] and temperature combinations (pp. 151)
Chapter 8 (in a manuscript)
Table 8.1 Quantification of free and bound polyphenols from fresh and frozen strawberry
fruits grown at ambient (400 pm and 25 ºC) and elevated (950 ppm and 30 ºC)
growth conditions (pp. 194)
Table 8.2 Quantification of free and bound folates and vitamin C from fresh and frozen
strawberry fruits grown at ambient (400 pm and 25 ºC) and elevated (950 ppm
and 30 ºC) growth conditions (pp. 195)
xviii
Table 8.3 Quantification of micronutrients after colonic fermentation of the precipitate of
gastric digests (bound nutrients) from fresh and frozen strawberry fruits grown
at ambient (400 pm and 25 ºC) and elevated (950 ppm and 30 ºC) conditions
(pp.196)
Table 8.4 Changes in the pH of the sediment of gastric digests of fresh and frozen
strawberry fruits grown at ambient (400 pm and 25 ºC) and elevated (650 ppm
and 30 ºC) conditions after in vitro colonic fermentation (pp.197)
Table 8.5 Anaerobic and aerobic bacterial counts in gastric digests of fresh and frozen
strawberry fruits grown at ambient (400 pm and 25 ºC) and elevated (650 ppm
and 30 ºC) conditions after being subjected to in vitro colonic fermentation
(pp.198)
xix
LIST OF FIGURES
Chapter 3
Figure 3.1 The major stages of the project (pp. 29)
Figure 3.2 Different stages of strawberry plants during the glasshouse experiments (pp. 30)
Figure 3.3 Automated digital image analysis for estimating strawberry canopy area (pp.
32)
Figure 3.4 Images of strawberry cultivars grown in the study (pp. 33)
Figure 3.5 Schematic diagram illustrating the process of extraction and analysis of
strawberry polyphenols (pp. 36)
Figure 3.6 Reactions involved in ABTS radicals and antioxidant compounds (pp. 37)
Figure 3.7 The schematic diagram for using HPLC-UV analysis in the study (pp. 40)
Figure 3.8 HPLC chromatographs for an individual polyphenol analysis at different
wavelengths (pp. 42)
Figure 3.9 Schematic diagram for determination of bioaccessibility of polyphenols, AA
and folates of strawberry fruits (pp.45)
Chapter 4 (in a published paper)
Figure 4.1 (a) The influence of different combinations of temperature and CO2 concentrations
on strawberry leaf photosynthesis (A) photosynthesis rate, (B) transpiration rate
in different strawberry cultivars (pp. 50)
Figure 4.1 (b) The influence of different combinations of temperature and CO2 concentrations
on strawberry leaf photosynthesis. (A) photosynthesis rate, (B) stomatal
conductance, (C) intercellular CO2 concentration (Ci), (D) transpiration rate, (E)
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WUE and (F) photosynthesis efficiency of leaf commonly in different
strawberry cultivars (pp. 51)
Figure 4.2 Reproductive characteristics of strawberry plants as affected by different
combinations of temperature and CO2 concentrations (pp. 52)
Chapter 5 (in a manuscript)
Figure 5.1 HPLC chromatogram of Pel-3-Glu analysis in strawberries (pp. 67)
Figure 5.2 HPLC chromatogram of AA in strawberries obtained at 245 nm (pp. 69)
Chapter 6 (in a published paper)
Figure 6.1 Total polyphenolic contents (TPC) of two different strawberry cultivars
(‘Albion’ and ‘San Andreas’) at different combinations of temperature and
[CO2] (pp. 110)
Figure 6.2 Total flavonoid contents (TFC) of two different strawberry cultivars (‘Albion’
and ‘San Andreas’) at different combinations of temperature and [CO2] (pp.
111)
Figure 6.3 Total monomeric anthocyanin contents (TMAC) of two different strawberry
cultivars (‘Albion’ and ‘San Andreas’) at different combinations of temperature
and [CO2] (pp. 111)
Figure 6.4 Total antioxidant contents (TAC) (µmoL Trolox equivalents per g of FW) of
two different strawberry cultivars (‘Albion’ and ‘San Andreas’) at different
combinations of temperature and [CO2] (pp. 112)
xxi
Figure 6.5 HPLC chromatographs of polyphenols of strawberries obtained at 280, 320, 360
and 510 nm (pp. 113)
Chapter 7 (in a manuscript)
Figure 7.1 Chemical structures of different folate derivatives and their chemical names (pp.
148)
Figure 7.2 HPLC chromatograph for folates obtained from fresh strawberry fruits at 290
nm (pp. 148)
Figure 7.3 HPLC-UV chromatograph for vitamin C of strawberry fruits obtained at 245
nm wavelength (pp. 149)
Figure 7.4 Total folates contents in strawberry cultivars under different growth
combinations of [CO2] and Temperature. (a) ‘Albion’ and (b) ‘San Andreas’
(pp. 149)
Figure 7.5 All the four-different mono-glutamate compounds (THFA, 10FFA, 5FTHFA
and 5MTHFA) were detected in all the strawberry samples (pp. 150)
Figure 7.6 Total vitamin C contents in strawberry cultivars at different growth
combinations of [CO2] and Temperature. (a) ‘Albion’ and (b) ‘San Andreas’
(pp. 150)
Chapter 8 (in a manuscript)
Figure 8.1 Schematic diagram for determination of bioaccessibility of polyphenols, AA
and folates of strawberry fruits (pp. 189)
xxii
Figure 8.2 HPLC chromatographs for (A) soluble fraction (extracted from supernatant) and
(B) insoluble fraction (extracted from precipitate) of simulated gastrointestinal
digesta (pp. 190)
Figure 8.3 HPLC chromatographs for vitamin identification in soluble fraction (extracted
from supernatant) of simulated gastrointestinal digesta of strawberry subjected
to in vitro gastric digestion (pp. 191)
Figure 8.4 Soluble fraction (extracted from supernatant) of folates (A) and vitamin C (B)
in simulated gastrointestinal (SGI) digesta of strawberry subjected to in vitro
gastric digestion (pp. 192)
Figure 8.5 HPLC chromatographs for colonic fermentation of fresh strawberry samples.
(A). HPLC chromatograph for individual phenolic compounds and their
metabolites at 280 nm in fresh strawberry samples at fed state (pp. 193)
1
Strawberry is a rich source of antioxidants which are mostly polyphenolic compounds, ascorbic
acid and folates (Giampieri et al., 2012). Particularly, strawberry processes a wide range of
phenolic compounds including anthocyanins, ellagitannins and phenolic acids. These
phytochemicals contribute to a higher antioxidants capacity of strawberry fruit and provide
immense health benefits in disease prevention and promotion of human health (Giampieri et al.,
2013). As a result of its healthfulness, strawberry consumption was increased not only as a fresh
fruit but also as frozen fruits, juice, and value added products (Giampieri et al., 2015). Further, in
the past few years, strawberry recorded a higher fresh fruits production around the world
(FAOSTAT, 2016). According to the Food and Agriculture Organization in 2016, strawberries
were produced in 77 countries on 401863 hectares in the globe. Interestingly, the quality and
quantity of antioxidants and individual antioxidant compounds were significantly varied with
strawberry genotype (Capocasa et al., 2008, Diamanti et al., 2008, Kosar et al., 2004, Tulipani et
al., 2008a, Tulipani et al., 2008b). However, in the last decade, few studies have reported that the
strawberry cultivation areas have been affected by climate change (Esitken et al., 2008, Husaini et
al., 2012, Krüger, 2009, Leposavić and Cerović, 2008, Neri et al., 2012, Palencia et al., 2009).
Atmospheric temperature and carbon dioxide concentration ([CO2]) are key factors of the climate
change. Intergovernmental Panel on Climate Change (IPCC) stated that atmospheric carbon
dioxide concentration would increase more than 1000 ppm and global average temperature by 5.8
°C at the end of this century (IPCC, 2014). The influence of growth temperature was thoroughly
2
investigated on strawberry in terms of vegetative plant growth (Gulen, 2003, Gulen and Eris, 2004,
Kadir, 2006, Ledesma et al., 2004, Wang and Lin, 2006), flowering and fruiting (Ledesma and
Sugiyama, 2005, Ledesma et al., 2008, Nishiyama and Kanahama, 2002, Sonsteby and Heide,
2008, Verheul et al., 2007), yield (Palencia et al., 2013), and quality of fruits (Diamanti et al.,
2008, Matsushita, 2016, Wang and Camp, 2000, Wang and Zheng, 2001). These investigations
revealed that strawberry showed significant reductions in plant net photosynthesis, growth, yield
and fruit physical quality at increased than the ambient temperature. Similarly, strawberry was
found to be sensitive to the rising carbon dioxide concentration in the growth environment,
however in a positive way. Elevated carbon dioxide levels more than the ambient during
strawberry growth enhanced plant photosynthesis (Bunce, 2001, Bushway, 2002, Chen et al.,
1997a, Chen and Lenz, 1997b, Keutgen et al., 1997, Peixi, 2002), plant growth (Chen et al., 1997b,
c, Chen et al., 1997d, Chen and Lenz, 1997a, b), fruit yield and fruit quality (Chen et al., 1997e,
Deng and Woodward, 1998, Wang and Bunce, 2004) of strawberries. Particularly, increased
temperature, as well as carbon dioxide enhanced the antioxidant content and nutritional quality of
strawberries. Therefore, the effects of these individual climatic factors on strawberry have been
clearly examined.
However, carbon dioxide and temperature increase concomitantly in the atmosphere and climate
change would often enforce more than one abiotic stress on plants. Though these factors were
individually studied, according to the reviewed literature, the information on the interactive effects
of elevated carbon dioxide and temperature on strawberry is lacking. Therefore, it is important to
investigate the combined effects of elevated carbon dioxide and high temperature on strawberries
to examine whether elevated carbon dioxide will compensate for the negative effects of high
temperature. Similarly, no published data are available to demonstrate the combined effects of
3
carbon dioxide and temperature on the contents of individual phenolic compounds, ascorbic acid
and folates. Further, the bioaccessibility and bioavailability of strawberry nutrients were also
unresearched under these elevated growth conditions. Therefore, this project was planned to
address these research gaps and to alert the commercial strawberry growers about the anticipated
effect of the future climates. Additionally, in this project, two different strawberry genotypes were
examined under the elevated growth conditions in order to evaluate the response of different
strawberry cultivars to the future climatic conditions.
4
1. What would be the impact of elevated [CO2] and high temperature combinations on plant
photosynthesis, growth and yield of different strawberry cultivars?
Considering the predicted atmospheric [CO2] and global warming in future, six different
[CO2] and temperature combinations were tested on two popular strawberry cultivars in
Australia. The physiological performances, vegetative growth, reproductive development
and fresh fruit yields of strawberry plants were analysed under different growth
combinations.
2. Does high temperature significantly affect strawberry fruit quality and antioxidant levels?
Effect of high temperature (30 °C) in growth environment on physical and nutritional
quality of strawberry fruit were studied. The harvested fruits of strawberry cultivars
‘Albion’ and ‘San Andreas’ were analysed for their weight, size, shape and colour. Total
polyphenol, flavonoid, anthocyanin, and antioxidant contents in fruits were also measured
to analyse the impact of high temperature on nutritional attributes of strawberries. High
Performance Liquid Chromatography (HPLC) were used to identify and quantify the
pelargonidin-3-glucoside content in strawberries.
3. How do the elevated [CO2] and high temperature combinations in growth environment
influence the antioxidant compounds and individual polyphenols of strawberry fruit?
Effect of elevated carbon dioxide and/or high temperature on polyphenol and antioxidant
properties in strawberry cultivars ‘Albion’ and ‘San Andreas’ were studied. Total
polyphenol, flavonoid, anthocyanin, and antioxidant contents in strawberry fruits were
5
detected using different colorimetric methods. Individual polyphenol compounds of
strawberry fruits were identified and quantified by using HPLC-UV analysis.
4. What are the contents of vitamin C and folates in strawberry fruits that were grown under
different [CO2] and temperature conditions?
Elevated carbon dioxide and /or high temperature effect on vitamin C and folate contents
of strawberry fruits were studied. Different chemical methods were used to extract vitamin
C and folates from fresh strawberries and freeze-dried strawberry powder. HPLC-UV
analysis was performed to identify and quantify vitamin C and different derivatives of
folate.
5. What are the bioaccessibilities of the polyphenols and vitamins in strawberry fruits grown
under elevated [CO2] and temperature?
Strawberry fruits of cultivar ‘San Andreas’ were obtained from ambient growth conditions
(400 ppm and 25 °C) and elevated [CO2] 950 ppm and high temperature (30 °C). Fresh and
frozen strawberry samples were subjected to simulated in vitro gastro-intestinal digestion.
Individual polyphenol contents were analysed following the method outlined previously in
research question 3 and determined the bioaccessibility of selected polyphenol compounds,
vitamin C and folates. The residue of gastric-intestinal digestion was then subjected to in
vitro colonic fermentation and polyphenols, vitamin C and folates of the fermented product
were determined by HPLC.
6
This thesis consists eight chapters, including the current chapter; chapter 1– Introduction.
Chapter 2 is the literature review on the impact of elevated carbon dioxide and high temperature
on physicochemical and nutrient properties of strawberries. This chapter outlines the nutrient
properties and health benefits of strawberry based on the recent literature. It summarizes also
the effects of high temperature and elevated carbon dioxide in the growth environment on
growth and development of strawberry plant and physical and nutritional attributes of
strawberry fruits. Chapter 3 details the experimental design of the study and general materials
and methods used. Chapter 4 explains the strawberry production under six different
combinations of carbon dioxide and temperature. It further discusses the combined effect of
elevated carbon dioxide and high temperature on photosynthesis, growth, flowering and fruit
yield of strawberry plants. Chapter 5 illustrates the high temperature effect on physical and
nutritional attributes of strawberry fruits. The effects of elevated carbon dioxide and/or high
temperature on total polyphenols, antioxidants and individual phenolic contents are explained
in Chapter 6. Chapter 7 discusses the impact of elevated carbon dioxide and high temperature
on vitamin C and folates in strawberry fruits. Chapter 8 involves the assessment of
bioaccessibility and colonic fermentation of strawberry polyphenols, vitamin C and folates.
Finally, Chapter 9 summarises the general conclusions and recommendations from the study.
7
Chapter 2 consists of two separate literature reviews (2.2 and 2.3). Chapter 2.2 is a short review
on “the impact of elevated [CO2] and high temperature on the nutritional quality of fruits”. This
short review discusses the individual and interaction effects of the investigated environmental
factors on fruit phytochemicals and physical fruit quality. The short review was published to
the American Journal of Agricultural Research as “The Impact of Elevated CO2 and High
Temperature on the Nutritional Quality of Fruits - A Short Review” by Balasooriya et al. (2019)
(https://doi.org/10.28933/ajar-2018-12-1608).
Chapter 2.3 is an overview of previous studies of elevated [CO2] and high temperature and
their interactions on strawberry. This literature review discusses the profile of micronutrients
of strawberry fruit and its health benefits and responds of strawberry to different [CO2] and
temperature experiments. This review was published in the Journal of Horticultural Science
and Research as ‘Impact of elevated carbon dioxide and temperature on physicochemical and
nutrient properties in Strawberries’ by Balasooriya et al. (2017)
(http://scholarlypages.org/Articles/horticulture/jhsr-1-004.php?jid=horticulture).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
The experimental design, materials and methods performed in this project to address the
research questions in Chapter 1. Most of the methods used in this project were based on
previously published literature with slight modifications. Lists of materials and most methods
are included in published papers and prepared manuscripts attached in Chapters 4, 5, 6, 7 and
8.
The major stages of the experimental design in this project is shown in figure 3.1. Strawberry
plants were grown under different [CO2] and temperature combinations in two identical
controlled growth chambers. Two popular strawberry cultivars were selected and there were
three plants per cultivar and four replicates per each experiment.
Each laboratory experiment and in vitro digestions and colonic fermentation were performed
at least twice, and individual measurement was done in triplicate within each experiment. Data
were statistically analysed in Minitab® 17 Statistical Software using analysis of variance
utilising General Linear Model (Proc GLM). The mean separation was performed using
Tukey’s multiple comparison method at 95% confidence level. The results were presented as
mean ± standard deviation (SD).
29
Planting materials
The experiments were carried out under controlled growth chambers in the glasshouse complex
at Parkville, the University of Melbourne, Australia. The main variables were; three [CO2]
concentrations (400 ppm, 650 ppm, and 950 ppm), two different temperatures (25 and 30 C),
and two different day neutral strawberry cultivars (Albion and San Andreas). Each experiment
combination included four replicates from each cultivar and three plants per each replicate.
Altogether twelve strawberry plants were randomized inside the growth chamber. All the six
treatment combinations were performed as six experiments in three different steps from May
2016 to October 2017. Fresh strawberry runners were purchased from Toolangi Certified
Strawberry Runner Growers' Co-Op Ltd, Toolangi, Victoria, Australia. For each experiment a
new set of six weeks old strawberry plants were used inside the controlled growth chambers.
Ripen fruits with 90% red colour, free from defects and decay were harvested separately. The
detailed method is included in a published paper attached in chapter 4. Figure 3.2 shows the
different stages of the glasshouse experiment.
Growing strawberries
under different [CO2] and
temperatures
Analysing the physiological
performances, growth and
development of plants
Analysing colour,
weight, size, and total weight of
fruits
Quantification of total
polyphenol, flavonoid,
anthocyanin and antioxidant
contents of fruits
Identification and
quantification of individial polyphenols, vitamin C and
folates of fruits by HPLC-UV
analysis
Determination of bio-
accessibility of polyphenols, vitamin C and
folates
Figure 3.1 The major stages of the project
30
Strawberry runners
Strawberry plant at the start Strawberry runners in pots
Strawberry plants after 6 weeks
Strawberry plant after 8 weeks
inside the controlled growth
chamber
Strawberry plants in controlled growth chambers
Figure 3.2 Different stages of strawberry plants during the glasshouse experiments
31
Physiological performances, plant growth, flowering and fruiting of strawberry
plants
During the phonological development, strawberry plants were assessed for plant physiological
performances, growth, reproductive development and fruit yield as detailed in chapter 4.
Leaf gas exchange parameters including, net photosynthesis rate, stomatal conductance,
intercellular [CO2] and transpiration rate were measured using a portable photosynthesis
system (LI-COR 6400, Lincoln, NE, USA). The LI-COR 6400 gas exchange instrument was
operated as instructed by Evans and Santiago (2014). Photosynthesis water use efficiency
(WUE), leaf photosynthesis efficiency and the ratio of intercellular to atmospheric [CO2]
(Ci/Ca) were calculated as follows;
𝑃ℎ𝑜𝑡𝑜𝑠𝑦𝑛𝑡ℎ𝑒𝑠𝑖𝑠 𝑤𝑎𝑡𝑒𝑟 𝑢𝑠𝑒 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 𝑁𝑒𝑡 𝑝ℎ𝑜𝑡𝑜𝑠𝑦𝑛𝑡ℎ𝑒𝑠𝑖𝑠 𝑟𝑎𝑡𝑒
𝑇𝑟𝑎𝑛𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒
𝐿𝑒𝑎𝑓 𝑝ℎ𝑜𝑡𝑜𝑠𝑦𝑛𝑡ℎ𝑒𝑠𝑖𝑠 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =𝑁𝑒𝑡 𝑃ℎ𝑜𝑡𝑜𝑠𝑦𝑛𝑡ℎ𝑒𝑠𝑖𝑠 𝑟𝑎𝑡𝑒
𝐼𝑛𝑡𝑒𝑟𝑐𝑒𝑙𝑙𝑢𝑙𝑎𝑟 [𝐶𝑂2]
𝐶𝑖
𝐶𝑎𝑟𝑎𝑡𝑖𝑜 =
𝐼𝑛𝑡𝑒𝑟𝑐𝑒𝑙𝑙𝑢𝑙𝑎𝑟 [𝐶𝑂2]
𝐴𝑡𝑚𝑜𝑠𝑝ℎ𝑒𝑟𝑖𝑐 [𝐶𝑂2]
Vegetative growth of strawberry plants was measured as the means of plant height, number of
crowns, number of leaves and canopy area. Automated digital image analysis (Easlon and
Bloom, 2014) was performed to estimate the canopy cover of strawberry plants. In this method,
Easy Leaf Area software was used to estimate the canopy area as shown in figure 3.3. The
software calibrates a known red area in each image to estimate the unknown canopy area of an
image. This software accurately monitors the canopy cover regardless of source of image,
distance of the camera and focal length. Accordingly, canopy area was estimated as follows;
32
𝐶𝑎𝑛𝑜𝑝𝑦 𝑎𝑟𝑒𝑎 = 𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑔𝑟𝑒𝑒𝑛 𝑐𝑎𝑛𝑜𝑝𝑦 𝑝𝑖𝑥𝑒𝑙𝑠 ×𝐶𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑖𝑜𝑛 𝑟𝑒𝑑 𝑎𝑟𝑒𝑎
𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑖𝑜𝑛 𝑟𝑒𝑑 𝑝𝑖𝑥𝑒𝑙𝑠
The reproductive development of strawberry plants was determined by monitoring total
number of inflorescences, flowers, and fruits. Fruit setting percentage was calculated using
total number of flowers and total number of fruits, according to:
𝐹𝑟𝑢𝑖𝑡 𝑠𝑒𝑡𝑡𝑖𝑛𝑔% = 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑓𝑟𝑢𝑖𝑡𝑠
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑓𝑙𝑜𝑤𝑒𝑟𝑠 × 100
Physical quality of strawberry fruit
Physical quality of strawberry fruits was monitored via the means fruit surface colour, size,
shape, and weight. A Minolta Chroma Meter CR-300 (Minolta Co., Ltd., Japan) was used to
measure the L*, a* and b* values of whole strawberry fruits. The L*, a* and b* colour system
consists of a luminance or lightness component (L*) and two chromatic components for green
(-a*) to red (+a*) and the b* component for blue (-b*) to yellow (+b*) colours. The hue angle
(h° = arctan b*/a*) and chroma value (C* = (a*2 + b*2)1/2) which indicates the colour intensity
Digital image of strawberry canopy Measured canopy area in Easy Leaf
Area Software
Red calibration area
Figure 3.3 Automated digital image analysis for estimating strawberry canopy area
33
was calculated using the correspondence L*, a*, and b* values. A standard white plate was
used to calibrate the colorimeter. Figure 3.4 shows the fresh fruits of strawberry cultivars
‘Albion’ and ‘San Andreas’. Maximum length and width of strawberry fruits were measured
using a measuring ruler to determine the size and shape of the fruits. Finally, fresh weight of
strawberry fruits was measured to determine the average individual fruit weight and the final
fruit yield.
Strawberry sampling
The nutritional properties of strawberry fruits were determined in both fresh fruits and freeze-
dried powder (FDP). Fruits were stored in a cold room at 4 ºC until use for fresh analysis.
Strawberry samples for freeze draying were stored at -80 ºC freezer for at least overnight before
transferred to the freeze drier (Dynavac Engineering FD3 freeze dryer, NSW, Australia). The
freeze-dried strawberry fruits were immediately ground into fine powder. The FDP was sealed
in polyethylene bags and stored in a laboratory desiccator at room temperature until used.
Figure 3.4 Images of strawberry cultivars grown in the study
(Received from Toolangi Certified Strawberry Runner Growers' Co-Op Ltd (Section 3.2.1))
Cultivar ‘Albion’ Cultivar ‘San Andreas’
34
Polyphenol extraction
Fresh fruits and FDP of Strawberry were extracted using 70% methanol and 0.18 N HCl
following the method of Tow et al. (2011). Polyphenols have strong hydrogen bonds in pure
organic solvent and low solubility. Different proportions of water and solvent (methanol)
mixtures modulate the polarity of polyphenols, weakness the hydrogen bonds and increase the
ionization of polyphenols there by increase the solubility. To obtain the best yield of
polyphenols, low concentrations of strong acids are used (Dai and Mumper, 2010). Fresh
strawberry (5 g) or FDP (2 g) was thoroughly mixed with 70% methanol (15 mL) and 0.18 N
HCl (5 mL) in a 50 mL polyethylene tube using Ultra Turrax homogenizer (Janke and Kunnel,
IKA-Labortechnik Ultra-Turrax T25). The supernatant was collected quantitatively after
centrifugation (Centrifuge, Thermoline, Scientific Equipment Pvt Ltd) of the homogenate at
14,000 rpm for 15 min at room temperature. The residue was extracted again with additional
5 mL methanol in the same way and supernatants were combined. The supernatant was
concentrated by evaporating the methanol using a rotary evaporator at 60 C with 8 rpm.
Temperature less than 70 °C facilitates the release of polyphenols from plant tissues, however,
extraction times longer than 30 minutes can degrade the polyphenols (Dai and Mumper, 2010).
The concentrated extract was dissolved in Milli-Q water in a volumetric flask to adjust the
final volume to 25 mL. This final polyphenol extract contained the major polyphenolic
compounds including flavonoids and phenolic acids. This extract was then used for analysing
the total antioxidants, polyphenols, flavonoid and anthocyanin contents in strawberries using
different colorimetric methods describe in later sections in this chapter. An aliquot (2 mL) of
the extract was filtered through 0.45 µm membrane when used for HPLC-UV analysis which
is detailed in section 3.1.9. Further, the final extraction method and polyphenol quantification
35
methods are described briefly in the accepted manuscripts attached in Chapter 5 and 6. Figure
3.5 illustrates process of the extraction and analysis of strawberry polyphenols in this study.
Total Polyphenol Content
Folin–Ciocalteu (FC) method is the commonest in determination of total polyphenols in plant
materials. This colorimetric assay is based on electron transfer under alkaline condition from
polyphenol to phosphomolybdic/phosphotungstic acid complex to generate a blue colour
complex than can be measured spectroscopically at approximately 760 nm (Dai and Mumper,
2010).
Phenolic protons are dissociated to phenolate anions by the sodium carbonate in the solution.
Then these phenolate anions can reduce the FC regent via Mo+6 to Mo+5 and form the
phosphomolybdic/phosphotungstic acid complex. The Total Polyphenol Content of strawberry
fruits was determined following the method of Tow et al. (2011), and adjusted to measure OD
using a microplate reader (Multiskan GO, Thermo Scientific, Australia). In this method, a
mixture of 20 µl of polyphenol sample extract, solvent blank or standard gallic acid and 100 µl
of 0.2 N Folin–Ciocalteu reagent (FCR) were added in a 96 well microplate. Within 5 minutes,
200 µl of Milli-Q water and 80 µl of sodium carbonate (7.5% w/v) were added and incubated
at room temperature for one hour in a 96 well microplate. The absorption was measured at 756
nm using a micro plate reader and a total polyphenol content was expressed as milligrams of
Gallic acid equivalent (GAE) per 100 g of fresh or dry weight of strawberry.
36
Fresh strawberry
fruits Stored in -80 °C freezer for
overnight
Laboratory Freeze-drier
Freeze-dried strawberry fruits
Freeze-dried strawberry
powder (FDP)
Solvent extraction with 70%
methanol and 0.18 N HCl
Supernatant (Polyphenols)
Total Polyphenol Content
Total Flavonoid Content
Total Monomeric Anthocyanin
Content
Total Antioxidant Content
Filtering through
0.45 µm nylon filter
Individual
Polyphenol analysis
Colorimetric methods
using Microplate reader
HPLC-UV system
Figure 3.5 Schematic diagram illustrating the process of extraction and analysis of
strawberry polyphenols
37
Total Antioxidant Contents
The 2,2′-Azinobis-3-ethylbenzotiazoline-6-sulphonic acid (ABTS) assay by Re et al. (1999)
with slight modifications (as described here) was performed to determine the antioxidant
content of strawberry fruits. The ABTS solution was prepared by mixing equal amounts of 2.45
mM potassium persulfate and 7 mM ABTS. The mixture was incubated overnight in the dark
to generate the green ABTS• organic free radicals (Fig 3.6). Then it was diluted with methanol
to an absorbance value between 0 to 1 at λ=734 nm. Antioxidants scavenge and stabilize the
ABTS• free radical by electron transfer or hydrogen donation as shown in Fig 3.6. The Trolox
equivalent antioxidant capacity (TEAC) is an electron transfer based assay and the colour from
the TEAC assay solution becomes light blue when the reaction is completed. The degree of
colour change is proportional to the concentration of antioxidants. Sample extract or standard
Trolox solution (25 μl) was mixed with ABTS solution (250 μl) directly in a 96 wells
microplate and the absorbance was read at 734 nm after 6 mins of incubation at room
temperature. TAC was reported as μmol Trolox equivalents per grams of strawberry fresh or
dry weight.
Figure 3.6 Reactions involved in ABTS radicals and antioxidant compounds
38
Total Flavonoid Content
Total flavonoid content can be determined by a colorimetric method using aluminium chloride
reagent. In this method, the flavonoid molecules react with aluminium ions (Al+3) in a basic
medium. The coloured product of red aluminium flavonoid chelates (flavonoid-Al+3) are read
under 415 nm wavelength. A slightly modified aluminium chloride method (Meyers et al.,
2003) was used in determination of TFC of strawberry samples. Adapted to a microscale, 125
μl of strawberry extract or solvent blank or quercetin standard was mixed with 25 μl of 1 M
sodium acetate, 25 μl of 10% aluminium chloride, and 175 μl of Milli-Q water. The mixtures
in microplate were then incubated at room temperature for 1 h. The absorbance was measured
at 415 nm against a blank solution using a micro plate reader. The result for total flavonoids
content were expressed as mg of quercetin equivalents per 100 grams of strawberry fresh or
dry weight.
Total Monomeric Anthocyanin Content
The total monomeric anthocyanin content (TMAC) of strawberries was determined with pH
differential method as described by Giusti and Wrolstad (2001). This method is based on the
significant reversible changes in absorption of the anthocyanin pigments when the pH changes
between 1.0 (highest absorption = coloured oxonium form) and 4.5 (lowest = colourless
hemiketal). This method accurately measures total monomeric anthocyanins when there are
interfering compounds in the sample. Initially, the samples were diluted with 0.025 M
potassium chloride (pH 1.0) and 0.4 M sodium acetate (pH 4.5) buffer to make the absorbance
within linear range (1-0) of the microplate reader. The final volume of the sample was divided
by the initial volume of the sample to calculate the dilution factor (DF) for the final calculation.
The dilutions were equilibrated for 15 mins at room temperature and then the absorbance of
39
the mixtures was measured at 496 and 700 nm against a Milli-Q water blank using the
microplate reader. The final absorbance of diluted sample was obtained as follows;
Absorbance (A) = (A496 nm – A700 nm) pH 1.0 – (A496 nm – A700 nm) pH 4.5
TMAC was calculated using the equation below and expressed as mg per 100 grams of
strawberry fresh or dry weight. The terms in equation stand for molar absorptivity (ε) of 27300
L cm-1 mol-1
and molecular weight (MW) of 433.2 g mol-1 for the anthocyanin Pel-3-glu.
𝑀𝑜𝑛𝑜𝑚𝑒𝑟𝑖𝑐 𝑎𝑛𝑡ℎ𝑜𝑐𝑦𝑎𝑛𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 =(𝐴 × 𝑀𝑊 × 𝐷𝐹 × 1000)
(𝜀 × 1)
HPLC-UV Analysis for Identification and Quantification of Strawberry
Nutrients
HPLC-UV analysis was performed in several stages of this study. Different HPLC analyses
were used in identification and quantification of individual polyphenols, vitamin C and folate
derivatives in fresh and FDP samples of strawberry and samples from in vitro bioaccessibility.
The methods are included in different Chapters, specifically, in Chapters 5 and 6 (individual
polyphenol analysis of fresh strawberry), Chapter 7 (vitamin C and folate analysis) and Chapter
8 (selected polyphenols and vitamin analyses of in vitro bioaccessibility samples). The
schematic outline of HPLC-UV methods is demonstrated in Figure 3.7.
40
Supernatant
filtered
through 0.45
µm
Fresh Strawberry Freeze-dried
Strawberry Powder Frozen Strawberry
Polyphenol
extraction with
70% methanol
and 0.18N HCl
Vitamin C
extraction with
4.5%
metaphosphoric
acid
Folate extraction
with 10%
phosphoric buffer
In vitro Bioaccessibility
Concentration of
supernatant and
re-suspension in
Milli-Q water
HPLC-UV
analysis
Individual polyphenol
characterization with a
gradient of 0.2% (v/v)
formic acid in Milli Q
water, (A) and acetonitrile
(B) as mobile phases
Vitamin C
quantification
with an isocratic
elution of 0.01%
H2SO4 at pH 2.6
as mobile phase
Folate identification and
qualification with a
gradient of 40 mM
sodium hypophosphate
buffer (A) and 8%
acetonitrile (pH 5.5) (B)
as mobile phases
Figure 3.7 The schematic diagram for using HPLC-UV analysis in the study
41
3.2.10.1. Individual Polyphenol Analysis
Polyphenols were separated using a Gemini C18 silica 250 × 4.6 mm, 5 µm column
(Phenominex Inc., Lane Cove West, NSW, Australia) connected to a HPLC-UV system. The
HPLC-UV system was equipped with a Water 2690 Alliance separation module (Waters,
Rydalmere NSW, Australia) coupled with a Waters 2998 Photodiode Array (PDA) detector.
The mobile phases included Milli-Q water acidified with 0.2% (v/v) formic acid (A) and
acetonitrile (B). The gradient used was 5% B in A for 1 min, up to 100% B over 35 min, and
returned to 5% B at 40 min. The injection volume was 20 µl and the flow rate was 1 mL/min.
Polyphenol separation and quantification were performed at room temperature (25 °C).
Identification of individual polyphenol peaks were based on both internal and external
standards. Identified peaks in strawberry extracts were quantified based on peak area and mass
of its corresponding HPLC calibration curve of the external standard. Results were expressed
as milligram or microgram per 100 grams of strawberry fresh weight. During the quantification,
mean area of a targeted peak was averaged from duplicated HPLC injections. Different
polyphenol compounds were detected and quantified at optimum and different wavelengths as
required.
The resulted HPLC chromatographs are presented in Figure 3.8. These included, Pel-3-Glu,
Pel-3-Rut and Cyanidin at 510 nm; Kaempferol-3-Glucoside and Kaempferol-3-Glucurnoride
at 360 nm; Resveratrol at 320 nm; and Catechin, Ferulic acid, Quercertin-3,4-di Glucoside,
Quercertin-3-O- Glucuronide, p-coumaric and p-Coumaroyl at 280 nm. The detailed
chromatographs are included in a manuscript attached in Chapter 6.
42
3.2.10.2. Vitamin C Analysis
Vitamin C consists of two active forms of ascorbic acid (AA) and dehydroascorbic acid
(DHAA). The HPLC method is an easy, sensitive, very accurate and the cheapest method to
determine the individual types of vitamin C (Sanchez Mata et al., 2000, Seeram et al., 2006).
Fresh strawberry sample (5 g), or FDP (2 g) was thoroughly mixed with 15 mL of 4.5%
metaphosphoric acid for 2 min at room temperature using the laboratory homogenizer. The
mixture was centrifuged at 4000 rpm for 20 min at 4 °C. The supernatant was collected and
filtered again through Whatman No. 4 filter paper and 0.45 µm membrane prior injecting to
the HPLC. To analyse the total amount of vitamin C, 5 mL of the filtrate was mixed with 1
mL of dithiothreitol (DTT) (20 mg\mL). DTT was added to convert all the vitamin C into AA
form (Zuo, 2014). This mixture was kept in dark for another 2 hrs at room temperature prior
Figure 3.8 HPLC chromatographs for an individual polyphenol analysis at different wavelengths
(Refer to Chapter 6 for the detailed HPLC chromatographs)
280 nm
320 nm
360 nm
510 nm
43
injecting 20µL of each sample into HPLC. Among the three different mobile phases of HPLC
assayed; (1) methanol/0.5% KH2PO4-buffer/H2O (28:60:12 and 20:60:0); (2) 0.05 M KH2PO4,
pH 2.6; and (3) H2SO4, pH 2.6 and 2.2, Sanchez Mata et al. (2000) suggested 0.01% H2SO4 at
pH 2.6 was better to obtain accurate vitamin C contents. The flow rate was 0.9 mL\min. The
AA peaks were then quantified at 245 nm wavelength. The results were reported as µg vitamin
C/100 g.
3.2.10.3. Folate Analysis
The folate derivatives of strawberry samples were identified and quantified following the
method of Lebiedzinska et al. (2008) with some modifications as described here. Fresh
strawberry (5 g) or FDP of strawberry (2 g) was thoroughly homogenised with 20 ml of 10%
phosphoric buffer (pH 6.8). The homogenate was kept in a water bath at 100 C for 10 min.
Then, it was cooled down in an ice bath and the pH was adjusted to 4.9. The mixture was then
subjected to enzyme treatment to release the folates bound to proteins or sugars. Hog kidney
folate conjugase (3 mL of 5 mg/mL solution) was added to the mixture and incubated at 37 C
for 3 hrs. The samples were then heated at 100 C for 5 mins to inactivate the enzyme and
rapidly cooled in ice. The samples were centrifuged at 10000 rpm for 20 min at 4 °C. The
supernatant was collected separately, and the residue was extracted again following the same
procedure to collect any left folates in the samples. The combined supernatant was adjusted to
25 mL in a volumetric flask, centrifuged again, filtered through a Whatman 4 filter paper and
stored in dark at -20 C freezer until used. An aliquant (2 mL) of that sample extract was filtered
through 0.45 µm cartridges before injection into the HPLC.
Sample extract (10 µL) was injected into the HPLC to separate and quantitate the folate forms.
A gradient elution of 40 mM sodium hypophosphate buffer (A) and 8% acetonitrile (pH 5.5)
44
(B) was used as mobile phases in characterization of folate derivatives. A flow rate was
maintained at 0.45 mL/min, and detection and quantification were performed against the folate
standards at 270-300 nm. The mobile phases were used as a gradient started with 5% B in A
and linearly increased to 50% B in A over 5 min, to 70% B in A over 7 min, followed by
recycling to initial conditions over 8 min before the following injection (Lebiedzinska et al.,
2008). The results were recorded as µg folic acid/100g.
Simulated Gastrointestinal Digestions and Colonic Fermentation for
Bioaccessibility Assessment
Simulated in vitro digestions were carried out to study the bioaccessibility of the identified
polyphenols and vitamins in human. It also provides the information on the stability and
structural transformation of different nutrient compounds. Although these in vitro systems may
be associated with some limitations in simulating the human and animal gastrointestinal tract,
these analyses provide important information on food digestion and bioaccessibility of food
nutrients. This study followed Fu et al. (2015) to assess the bioaccessibility of selected food
nutrients using simulated in vitro gastrointestinal digestion models combined with HPLC
methods. The resulted residue from in vitro digestions was next subjected to in vitro colonic
fermentation. The detailed methods used in this study were described in Chapter 8. A schematic
diagram of the main stages of the bioaccessibility study is also illustrated in Figure 3.9.
45
Figure 3.9 Schematic diagram for determination of bioaccessibility of
polyphenols, AA and folates of strawberry fruits
Fresh Strawberry Frozen Strawberry
In vitro
Gastrointestinal
digestion
Centrifugation
In vitro intestinal
digestion
(37 °C, 2 hrs)
In vitro gastric
digestion
(37 °C, 3 hrs)
Residue/
insoluble
fraction
In vitro colonic
fermentation
Centrifugation
Supernatant
Solvent extractions for polyphenol, AA and
Folate analysis
HPLC-UV
analysis
Supernatant/
Soluble/
bioaccessible
fraction
46
Chapter 4 includes the detailed methodology of growing two strawberry cultivars under
different [CO2] and temperature conditions. The chapter also discussed the individual and
interactive effects of elevated [CO2] and high temperature on plant growth, development and
yield responses of two different strawberry cultivars. The strawberry fruits produced under this
simulated climate stress were then used Chapter 5, 6, 7, and 8 to analysis of polyphenols,
vitamin C, folates and finally their bioaccessibility. The results were published in International
Journal of Agricultural and Biosystems Engineering as “Interaction of elevated carbon dioxide
and temperature on strawberry (Fragaria ananassa) growth and fruit yield” by Balasooriya et
al. (2018) (https://waset.org/publications/10009464/interaction-of-elevated-carbon-dioxide-
and-temperature-on-strawberry-fragaria-ananassa-growth-and-fruit-yield).
47
48
49
50
51
52
53
54
55
56
This chapter investigated the impact of high temperature in the growth environment on the
contents of polyphenols, flavonoids, anthocyanins and antioxidants in strawberry fruits.
Additionally, it determined the effects of high temperature on the pelargonidin 3 glucoside
content of strawberry fruits by using HPLC-UV methods. It also analysed the impact of high
temperature on physical quality of strawberry fruits. The results of this chapter are included in
a manuscript which has been accepted to publish in Acta Horticulturae.
57
“High temperature effects on strawberry fruit quality and antioxidant contents”
B.L.H.N. Balasooriya1, K. Dassanayake1, 2, and S. Ajlouni1
1Faculty of Veterinary and Agriculture Sciences, The University of Melbourne, Australia;
2Faculty of Engineering, The University of Melbourne, Australia.
Abstract
Strawberry (Fragaria × ananassa Duch.) is a demanded edible fruit with exceptional
nutritional qualities. Nowadays, strawberries are commercially cultivated across a wide range
of climatic and geographical regions in the world. However, it is anticipated that quality and
quantity of strawberry might be affected by the recent trend in climate changes. This research
evaluates the effects of anticipated increase in temperature of the growth environment on fruit
development, yield, and antioxidant contents and bioavailability of strawberries. Strawberry
cultivars, ‘Albion’ and ‘San Andreas’ were grown at 25/20 and 30/20 C day/night
temperatures under ambient CO2 levels in controlled environment chambers. Ripen fruits were
harvested and analysed for their physical and nutritional qualities. Increase in temperature by
5 C significantly (p<0.05) reduced fruit growth and yield of both cultivars. Average individual
fruit weight and total fruit weight per plant were declined by 70% and 80%, respectively at
30/20 C compared to plants grown at 25/20 C. Fruits produced by plants grown at higher
temperature were irregular in shape, smaller in size and lower intensity in red colour; however,
those fruits had significantly (P <0.05) higher total polyphenols (171.1±29 mg 100 g-1) content
and antioxidant capacity (10.32±1.9 µmol g-1), compared to the fruits grown at 25/20 °C
(93.9±10 mg 100 g-1 and 6.42±1.22 µmol g-1, respectively). Additionally, at 30/20 C, fruit
surface colour became darker (decreased lightness). HPLC results revealed remarkably higher
58
amounts (15.2±5.5 mg 100g-1) of Pelargonidin-3-glucoside, the main phenolic compound in
strawberry in comparison with 11.3±3.4 mg 100 g-1at 25 °C.
climate, cultivar, plant growth, polyphenols, Pelargonidin-3-glucoside
Introduction
Strawberry (Fragaria × ananassa Duch.) is a demanded edible fruit, cultivated and consumed
worldwide. It is highly appreciated for its nutritional and medicinal properties. Among all the
other fruits, strawberry has a universally recognized identity for its bright red colour, sweet
taste and distinct aroma. Ripen strawberries evoke all the senses in consumers including, taste,
vision, tactile sensation and olfaction (Schwieterman et al., 2014) which are related to quality
in general (Zhao, 2007). For quality fruit production, strawberry requires cool and moist
climates (Sinha, 2008) with 25/12 °C (day/night) optimum temperature (Wang and Camp,
2000). However, recent studies have reported significant changes in strawberry production
areas due to climate change (Calleja, 2011; Esitken et al., 2008; Neri et al., 2012; Palencia et
al., 2009; Palencia et al., 2013). Nevertheless, the predicted increase in temperature and CO2
concentrations will affect strawberry nutritional quality, especially its antioxidant contents. It
has been reported that plants tend to synthesise greater amounts of antioxidants as a defensive
response to the external environmental stress (Atkinson et al., 2005). However, most of the
recent studies extensively examined the strawberry plant responses to high temperature in
terms of vegetative growth (Kadir, 2006; Serce and Hancock, 2005), reproductive growth
(Kumakura, 1995; Ledesma and Sugiyama, 2005; Ledesma et al., 2008; Nishiyama and
Kanahama, 2002; Sonsteby and Heide, 2008; Verheul et al., 2007), plant chemical composition
(Gulen and Eris, 2004; Wang and Lin, 2006), and fruit nutritional properties (Diamanti et al.,
2008; Wang and Camp, 2000; Wang and Zheng, 2001). However, very little is known about
59
the influence of high temperature on strawberry antioxidants. Therefore, the current study
focused mainly on investigating the effect of high temperature on the fruit antioxidant
properties of two common day neutral strawberry cultivars in Australia ‘Albion’ and ‘San
Andreas’.
Materials and methods
5.2.2.1. Experimental Location, Plant Materials and Treatments
The experiments were carried out in controlled environment (CE) chambers at Parkville
Campus of the University of Melbourne, Australia. Fresh strawberry runners purchased from
Toolangi Certified Strawberry Runner Growers' Co-Op Ltd, Victoria, Australia, were planted
in plastic pots (1.65 L) filled with well drained commercial potting media and kept under
ambient environmental conditions in a glasshouse for a six-week period for initial growth.
Uniform sized plants were then transferred to the experimental CE chambers. Temperature of
the two CE chambers were set to 25/20 and 30/20 °C day/night. However, other environmental
conditions in each CE chamber were maintained at same levels. For example, relative humidity
(RH), 70%; ambient CO2 level (400 ppm); and photosynthetically active radiation, nearly 500
µmol m-2 s-1 for 14 hours (8:00-22:00 h) day length. All the plants were automatically irrigated
to maintain moisture content of the growth medium at field capacity during the whole
experimental period. All the other crop management practices were applied equally to all the
plants. There were four replicates per each treatment and three plants per each strawberry
cultivar. The plants were randomised within the chamber to eliminate the position effect and
swop between the chambers to avoid any chamber effect during the experimental period. The
temperature treatments were continued for around 10 weeks until sufficient quantity of ripen
fruits were harvested for analyses. Ripen fruits with 90% red colour, free from defects and
60
decay were harvested separately from each chamber for each cultivar and for each replicate.
Fresh fruits were stored in a fridge at 4 C until used. The harvested fruits were then analysed
for their physical properties (weight, size, shape, and colour) on the same day of harvested, and
antioxidants properties within maximum two days. Each laboratory analytical test (described
below) was performed at least twice, and individual measurements were repeated three times.
5.2.2.2. Chemicals and standards
Hydrochloric acid, acetonitrile, methanol, formic acid, sulfuric acid, metaphosphoric acid,
Folin–Ciocalteu’s regent (FC), ABTS (2,2′-Azinobis-3-ethylbenzotiazoline-6-sulphonic acid),
Trolox solution (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), potassium
persulfate, potassium chloride, sodium acetate, aluminium chloride, anhydrous sodium
carbonate, dithiothreitol (DTT), Pelargonidin-3-glucoside (Pel-3-Glu), ascorbic acid (AA),
gallic acid (GA), and quercertin were purchased through the Central Chemical Store at the
University of Melbourne, Chem supply, Gillman, SA, Australia and from Sigma-Aldrich,
NSW, Australia.
5.2.2.3. Colour assessment
A Minolta Chroma Meter CR-300 (Minolta Co. Ltd., Japan) was used to measure the L*
(lightness), a* (greenness (-) to redness (+)), and b* (blueness (-) to yellowness (+)) values.
The hue angle (h° = arctan b*/a*) and chroma value (C* = (a*2 + b*2)1/2) which indicates the
colour intensity was calculated using the correspondence L*, a*, and b* values. At least twenty
fruits were assessed per each treatment and three readings were taken at different sides of fruit
surface (Wang and Camp, 2000) for colour assessments.
61
5.2.2.4. Chemical analysis
5.2.3.4.a. Individual polyphenol quantification
Strawberry polyphenols were extracted using 70% methanol as described by Tow et al. (2011)
with some modifications. Waters 2996 High Performance liquid Chromatography (HPLC)
equipped with a workstation computer (Class VP) and a photodiode array (PDA) detector
(Waters 2998) was used to perform the individual polyphenol quantification. LC18 stainless
steel column of 25 cm × 4.6 mm packed with C18 reserved-phase material with a particle size
of 5µm (SUPELCO) was used. The extract was filtered through 0.45 µm filter before injecting
20 µL sample into the column. The column was eluted at a flow rate of 1.0 mL/min with 0.2%
(v/v) formic acid in Milli-Q water (A)/ 0.2% (v/v) formic acid in acetonitrile (B) as the mobile
phase; the gradient was changed as follows: 95% A/5% B for 1 min, 90% A/10% B in 10 min,
87% A/13% B in 5 min, 80% A/20% B in 5 min, 70% A/30% B in 5 min, 0% A/100% B in 5
min, 100% B for 5 min, and 95% A/5% B in 5 min. Pel-3-Glu, the main phenolic compound
peak was identified at its maximum absorbance of 500 nm using both internal and external
standard curves. The amounts of Pel-3-Glu was calculated using a linear regression equation.
5.2.3.4.b. Determination of total polyphenol content (TPC)
Modified FC reagent method (Tow et al., 2011) was used to measure TPC. A mixture of 20 µL
of polyphenol sample extract or solvent blank and 100 µL of FC reagent (1:10) were directly
mixed in a microplate well. Then after 2 min, 80 µL of Na2CO3 (7.5% w/v) was added and the
absorption was measured at 760 nm using a microplate reader (Thermo, Multiskan Spectrum)
after incubating 1 hour at room temperature (RT). A standard curve was generated using
different concentrations of Gallic acid. Results were expressed as milligrams of Gallic acid
equivalent (GAE) per 100 g of strawberry FW.
62
5.2.3.4.c. Determination of total antioxidant capacity (TAC)
The ABTS assay was performed to determine TAC of phenolic extract (Re et al., 1999). The
ABTS radical was prepared by mixing 10 mL of 2.45 mM potassium persulfate with 10 ml of
7 mM ABTS•. The mixture was incubated overnight and then diluted with methanol to an
absorbance of 0.7 (±0.02) at λ=734 nm. Directly in microplate well, 25 μL of sample extract
was mixed with 250 μL of ABTS• reagent and the absorbance was read at 734 nm after 6 min.
Trolox solutions were used for calibrating standard curve. The results were expressed as μmol
Trolox equivalents antioxidant capacity (TEAC) per g of strawberry sample.
5.2.3.4.d. Determination of total monomeric anthocyanin content (TMAC)
The TMAC of strawberries was determined with pH differential method as described by Giusti
and Wrolstad (2001). Briefly, the samples were appropriately diluted in 0.025 M potassium
chloride (pH 1.0) and 0.4 M sodium acetate (pH 4.5). The mixtures were kept at RT before the
absorptions were measured at 496 and 700 nm using the microplate reader. TMAC was
calculated using molar absorptivity of 27300 L cm-1 mol-1 and molecular weight of 433.2 g
mol-1 for Pel-3-glu and expressed as mg TMAC 100 g-1 strawberry FW.
5.2.3.4.e. Determination of total flavonoid content (TFC)
The modified colorimetric aluminium chloride method (Meyers et al., 2003) was used to
determine the TFC of samples. Briefly, 125 µL of strawberry extract was separately mixed
with 25 µL of 1 M sodium acetate, 25 µL of 10% aluminium chloride, and 175 µL of Milli-Q
water in microplate wells. The mixtures were then left at RT for 1 h and the absorbance was
measured at 415 nm in the microplate reader. TFCs were calculated as quercetin from a
calibration curve and expressed as mg of quercetin equivalents per 100 g strawberry FW.
63
5.2.3.4.f. Determination of total vitamin C
Vitamin C consists of two active forms of ascorbic acid (AA) and dehydroascorbic acid
(DHAA). Strawberry vitamin C was extracted in 4.5% metaphosphoric acid and quantified in
HPLC following the method of Sanchez Mata et al. (2000). One millilitre of the sample
extract was mixed with 0.2 mL of 20 mg mL-1 DTT and kept in the dark at RT for 2 h to convert
all the vitamin C into AA form (Zuo, 2014). Then the mixture was filtered through 0.45 µm
filter prior injecting 10 µL into HPLC. Isocratic mobile phase of 0.01% H2SO4 at pH 2.6 was
used in a flow rate of 0.9 mL min-1. The AA peak was determined around 245 nm. The results
were reported as µg 100 g-1 strawberry FW.
Statistical Analysis
Data were statistically analysed using Minitab® 17 Statistical Software and differences of
means were determined using one-way analysis of variance (ANOVA) and Tukey’s multiple
comparison method at 95% confidence level. All data were reported as means ± standard
deviation.
Results and discussion
5.2.4.1. Impact of high temperature on physical characteristics
Changes in fruit growth and development in strawberries can be exhibited by variations in size,
shape, yield, colour, texture, flavour, chemical composition and nutrient contents. Fruit yield
is the economically important part in commercial strawberry production. In the present study,
high temperature markedly reduced the fresh fruit yields of both strawberry cultivars. Average
fruit weight was reduced from 9.4±0.7 g to 2.6±0.3 g, and total fruit yields from 49.86±7.34 g
to 8.47±1.5 g per plant (Table 1). The size and the unique shape of strawberry fruit are
64
important quality characters which attract the consumers’ visual attention. As a result of high
temperature, plants produced smaller and irregular shaped fruits due to restricted fruit growth.
The maximum fruit length ranged from 1.72±0.6 to 3.05±0.4 cm, and width from 1.67±0.2 to
2.69±0.2 cm at 30/20 and 25/20 °C, respectively (Table 1). These results are in agreement with
those reported by Kadir (2006); Miura et al. (1994); Wang and Camp (2000). It has been further
reported that heat stress under high temperatures can cause pollen sterility, lower fruit set and
yields, and may cause even a complete crop failure under extreme high temperature conditions
(Hancock, 1999; Ledesma et al., 2008; Palencia et al., 2013). Therefore, it is anticipated that
high temperatures will have a detrimental effect on reproductive development of strawberry
and will produce lower quality fruits.
Table 1. Fruit yield and size of strawberry grown at different temperatures
Categories Parameter
Temperature main effect
Average
fruit weight
*
Total
fruit yield**
Max. fruit
length ***
Max. fruit
width***
25/20 °C 9.4±0.72 a 49.9±7.34 a 3.05±0.43 a 2.69±0.23 a
30/20 °C 2.6±0.30 b 8.5±1.55 b 1.72±0.57 b 1.67±0.24 b
Temperature × cultivar interaction effect
25/20 °C × Albion 8.2±1.34 a 33.8±6.44 a,b 2.98±0.40 b 2.59±0.25 b
25/20 °C × San Andreas 10.3±0.73 a 60.1±10.4 a 3.10±0.47 a 2.76±0.20 a
30/20 °C × Albion 3.2±0.36 b 9.6±2.41 b 1.94±0.52 b 1.82±0.15 b
30/20°C × San Andreas 1.9±0.34 b 7.0±1.70 b 1.48±0.55 b 1.51±0.21 b
*, **, and *** indicates the units g fruit-1, g plant-1and cm respectively, Max. = maximum.
Means in each category and within each column followed by different superscript letters are
significantly different (P<0.05).
Strawberry fruit colour is also unique and important as a physical quality parameter. It is
perhaps considered a key fruit-intrinsic cue that consumers decide visually before buy or taste.
The anthocyanin compounds mainly, Pel-3-Glu and Cyanidin-3-Glucoside are highly
65
responsible to create the attractive red colour in strawberry fruits (Wang and Camp, 2000).
Fruit surface colour can be varied as affected by many factors such as, external environmental
conditions, cultural practices, cultivar, maturity, harvesting and postharvest handling
(Capocasa et al., 2008; Crecente-Campo et al., 2012; Yoshida et al., 2002). The hue angle
describes the overall colour where 0 = red-purple, 90 = yellow, 180 = bluish-green and 270° =
blue. The overall reported hue angle values were 34.2±0.4 and 33.9±0.5 at 25 °C and 30/20 °C,
respectively. These values were between 0 and 90 degree, and not significantly different
(P>0.05), which indicated a similar level of red colour on these fruits. On the contrary,
examining a* (redness) values showed significantly larger value (37.7±0.5) at 25/20 °C than at
30/20 °C (34.8±0.8) (Table 2). Additionally, the fruit surface appearance was significantly
(P<0.05) darker (lower L*) and exhibited a lower surface pigment intensity (lower C*) at high
temperature (Table 2). All these colour readings confirmed the negative impact of high
temperature on strawberry red colour. These results were contradictory to those presented by
Wang and Camp (2000) who reported redder fruit surface (decreased hue angle) at high
temperature. As darker and redder fruit surface are considered better sensory colour
characteristics for strawberries (Wang and Camp, 2000), our results showed negative effect of
high temperature on strawberry red colour. Data in Table 2 also revealed the same trend and
negative effect of high temperature on fruit colour of individual cultivar.
Table 2. Colour of strawberry fruits grown at different temperatures
Categories Measured colour indicators
Temperature main effect L* a* b* C* h°
25/20 °C 39.9±0.4a 37.7±0.5 a 25.7±0.5 a 45.6±0.6 a 34.2±0.4a
30/20 °C 37.1±0.7b 34.8±0.8 b 23.5±0.4 b 42.0±0.8 b 33.9±0.5a
Cultivar × temperature combination effect
25/20 °C × Albion 38.3±0.77 b 35.4±0.2 b 23.6±1.77 b 42.6±1.22 b 33.7±1.85a
25/20 °C × San Andreas 41.0±1.22 a 39.2±0.26 a 27.0±1.62 a 47.6±0.98 a 34.5±1.86 a
30/20 °C × Albion 36.8±2.13 b 35.1±0.97 b 23.4±1.75 b 42.3±3.0 b 33.7±2.12 a
30/20 °C × San Andreas 37.5±3.28 b 34.2±1.41 b 23.7±1.68 b 41.6±3.5 b 34.3±0.87 a
66
Means in each category and within each column followed by different superscript letters are
significantly different (P<0.05).
5.2.4.2. Effect of high temperature on polyphenol content and antioxidant capacity
Phenolic compounds and vitamins are the most deliberated antioxidants in fruits and
vegetables, which are comparatively abundant in strawberry phytochemical profile. These
plant antioxidants are naturally synthesized protective compounds which react against diverse
types of free radicals generated under stressful external environmental conditions. The group
of phenolic compounds in strawberry includes; flavonoids represented as anthocyanins,
phenolic acids, lignans, stilbens, tannins and coumarins (Skinner and Hunter, 2013). Among
few selected popular fruits, strawberry gives the second highest polyphenol after blueberries
and over orange and apple (Williamson and Holst, 2008). Zhao (2007) investigated the levels
of total phenols content in strawberry fruit and reported the values to range between 43 to 273
mg 100 g-1 FW. In the present study, strawberry grown at 30/20 °C had significantly (P<0.05)
greater amounts of total polyphenols (171±29 mg/100 g FW) than those grown at 25/20 °C
(93.9±10 mg 100 g-1 FW) (Table 3). In the strawberry polyphenol profile, the flavonoid group
holds a significant position as it contains anthocyanins, flavanols and flavonols. TFC increased
from 18.4±9.6 to 30.2±8.7 mg 100 g-1 FW when the temperature was raised by 5 °C.
Anthocyanin is the best-known polyphenol compound classified under flavonoids and it
increased remarkably with rising temperature. The TMAC increased significantly (P<0.05) and
reached 12.76±3.2 mg/100 g FW when strawberry was grown at 30/20 °C (Table 3). Diverse
67
polyphenolic classes in strawberries have been identified to fulfil different important functions
during fruit development (Halbwirth et al., 2006).
Pel-3-Glu is the major and predominant anthocyanin compound identified in strawberries
(Aaby et al., 2012; Buendia et al., 2010; da Silva Pinto et al., 2008; Giampieri et al., 2013;
Oszmianski and Wojdylo, 2008) and accounted between 75% to 91% of total anthocyanins
Table 3. Antioxidant properties of strawberry grown at different temperatures
Categories Antioxidant Property
Temperature main
effect TPC * TFC * TMAC * Pel-3-Glu * TAC **
25/20 °C 93.9±10 b 18.4±9.6 b 6.89±1.4 b 11.3±3.4 b 6.42±1.2 b
30/20 °C 171±29 a 30.2±8.7 a 12.76±3.2 a 15.2±5.5 a 10.32±1.9 a
Temperature × cultivar combination effect
25/20 °C × Albion 97.1±7.5 b 26.6±10 a 6.98±1.4 b 11.2±3.9 b 6.79±1.5 b
25/20 °C × San Andreas 90.4±11.5 b 12.6±2.5 b 6.78±3.2 b 11.4±3.0 b 6.01±0.6 b
30/20 °C × Albion 163.2±27.3 a 27.4±9 a 13.2±2.9 a 13.2±4.2 a,b 9.94±1.5 a
30/20 °C × San Andreas 178.0±27.5 a 31.7±6 a 12.4±3.5 a 17.2±6.0 a 10.76±2.4 a
* and ** indicates the units mg per 100 g FW and µmol g-1 FW respectively, ns stands for not
significant. Means in each category and within each column followed by different superscript are
significantly different (P<0.05).
Figure 1. HPLC chromatogram of Pel-3-Glu analysis in strawberries
68
(Buendia et al., 2010). This compound was clearly detected at 500 nm in the HPLC
chromatograph as it was found in greater amounts in strawberries (Figure 1). Data in Table 3
showed Significant (P<0.05) increase in Pel-3-Glu content (from 11.3±3.4 to 15.2±5.5 mg/100
g FW) at higher temperature (30/20 °C). Anthocyanins contribute to fruits colour development,
and also responsible for the generation of distinctive flavour in strawberries. However, the
general phenolic profile presented in strawberry can further rely on pre and post cultivation
conditions other than the genotype (Ariza et al., 2015; de Jesus Ornelas-Paz et al., 2013;
Pincemail et al., 2012).
Antioxidant capacity derived from the accumulative and synergistic power of the individual
antioxidants creating total antioxidant capacity (TAC). Such antioxidant capacity of plant
phytochemicals is an important biological property in humans’ diet. Naturally, strawberry has
the third highest TAC value among most of the commercial fruits after pomegranate and guava,
respectively (Skinner and Hunter, 2013). In both cultivars, strawberries harvested from high
growth temperature recorded greater antioxidant capacity (10.32 µmol TAC g-1) in comparison
with lower temperature (6.42 µmol g-1) (Table 3).
5.2.4.3. Effect of high temperature on total AA content
Polyphenols are the principal group of compounds responsible for the TAC (Wang and Zheng,
2001). Among the antioxidants in strawberries, vitamin C contributes about 15 to 39% of the
TAC (Szeto et al., 2002; Tulipani et al., 2008; Wang et al., 1996). Strawberry had the highest
AA and total vitamin C over 17 commercial fruits including lemon, orange, kiwifruit,
grapefruit, green and red apple, mandarin, mango, green and red grape, banana, pineapple and
pear (Wang et al., 1996). AA was detected by HPLC at 245 nm (Figure 2).
69
Figure 2. HPLC chromatogram of AA in strawberries obtained at 245 nm
Data of Vitamin C analysis using HPLC techniques exhibited slight increment in AA content
at high temperature (from 58.17±8.5 to 60.54±8.2 mg 100g-1 FW at 25/20 and 30/20 °C,
respectively), however, such increase was not statistically significant (P>0.05)(Table 4). These
results were different from those reported by Wang and Camp (2000), who observed decreased
AA contents at higher growth temperature. However, it should be noted that Wang and Camp
(2000) used a titration method based on the interaction of sample extract with 2, 6-
dichloroindophenol, which is a less sensitive method compared to HPLC analysis. It has been
suggested that, ascorbate degradation when fruits are grown at high temperature (30/20 °C),
could be attributed to increased oxidation (Gautier et al., 2008) and/or down regulated of AA
biosynthesis (Richardson et al., 2004) at high temperature.
70
Conclusion
Our results showed that higher temperatures (30/20 °C) had negative effects on fruit yield and
physical characteristics (weight, size, colour, and shape) of strawberries. However, high
temperature increased health promoting polyphenols contents. Therefore, further studies
should be carried out using different strawberry cultivars for screening heat resistant cultivars.
Better heat tolerant cultivars will be beneficial in future commercial strawberry production to
promote quality fruits, both physically and nutritionally. However, a little is known about the
combined effects of climate parameters (CO2 and temperature) on physical, chemical and
biological properties of strawberry. Consequently, scientific evidence is still required to
elucidate the climate change effect on strawberry yield and actual nutritional values.
Acknowledgement
The authors would like to thank Steven Elefteriadis, the glasshouse manager for his assistance
during the glasshouse experiments and our colleague, Ruchika Perera for his assistance
throughout the study and writing this paper.
Table 4. Total AA content in strawberry grown at different temperatures
Categories Total AA Content*
Temperature main effect
25/20 °C 58.17±8.5 a
30/20 °C 60.54±8.2 a
Temperature × cultivar combination effect
25/20 °C × Albion 59.85±7.5 a
25/20 °C × San Andreas 56.41±9.4 a
30/20 °C × Albion 59.57±9.3 a
30/20 °C × San Andreas 61.50±7.1 a
* indicates the units mg per 100 g FW. Means in each category and within each column followed
by different superscript are significantly different (P<0.05).
71
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Chapter 6 investigated the combined effect of elevated carbon dioxide and high temperature
on strawberry polyphenols. In this chapter, the effect of different carbon dioxide and
temperature combinations on total polyphenol, flavonoid, anthocyanin and antioxidant
contents was determined using different colorimetric methods. Additionally, the individual
polyphenols including Pel-3-Glu, Pel-3-Rut, cyanidin, kaempferol glucoside, kaempferol
glucuronide, resveratrol, catechin, ferulic acid, quercetin glucoside, quercetin glucuronide, p-
coumaric and p-coumaroyl (6-O-p-coumaroyl-1,2-digalloylglucose) were identified and
quantified using HPLC analysis. The results were published in The Journal of the Science of
Food and Agriculture.
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Chapter 7 investigated the individual and combination effects of elevated carbon dioxide and
high temperature on folates and vitamin C contents in two strawberry cultivars ‘Albion’ and
‘San Andreas’. In this study, different folate derivatives and ascorbic acid were identified and
quantified in fresh and freeze-dried strawberry samples using HPLC-UV system. The
manuscript has been submitted to The Journal of the Science of Food and Agriculture.
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“Folates and vitamin C contents in strawberries grown under elevated carbon dioxide and
temperature”
Himali N. Balasooriya1*, Kithsiri B. Dassanayake1,2, Saman Seneweera1,3, Said Ajlouni1*
1. School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The
University of Melbourne, Parkville, VIC 3010 Australia.
2. Department of Infrastructure Engineering, Faculty of Engineering, The University of
Melbourne, Parkville, VIC 3010 Australia.
3. National Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka.
*Corresponding author; Himali N. Balasooriya – phone: +61420620730;
e-mail: [email protected]
Abstract
Background: Composition of micronutrients in plants is influenced by various factors, but
importantly the environmental factors including [CO2] and temperature of growth
environment. This study evaluated folates and vitamin C contents of strawberries produced
from cultivars ‘Albion’ and ‘San Andreas (SA)’ under different combinations of [CO2] levels
(400, 650, 950 ppm) and day temperatures (25 and 30 °C). HPLC-UV system was used in
identification and quantification of the above vitamins in fresh and freeze-dried strawberry
fruits.
Results: The study identified four different folate derivatives in strawberries using the HPLC-
UV analyses. Total folate contents in strawberries varied from 52.6 ± 5.1 µg to 364.8 ± 16.0
µg/100 g FW in cultivar ‘Albion’ and from 48.6 ± 7.0 µg to 237.4 ± 23.8 µg/100 g FW in
cultivar ‘SA’. In this study, four-different mon-glutamate compounds of THFA, 10FFA,
5FTHFA and 5MTHFA were found in strawberries. Effects of elevated [CO2], higher
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temperature and their interactions on vitamin C content of strawberries were statistically
significant (P<0.05) and the responses were cultivar dependent. The highest total vitamin C
contents in ‘Albion’ and in ‘SA’ were 132.9 ± 14.9 mg/100g FW and 132.4 ± 9.7 mg/100g
FW, respectively, under 650 ppm and 30 °C growth combination.
Conclusion: Rising [CO2] levels up to 950 ppm during plant growth can result in enhanced
total folates and vitamin C contents in strawberries. However, increases in temperature would
be significantly negative at 950 ppm [CO2]. Freeze drying found to be an effective method to
preserve vitamins in strawberries.
Keywords: Carbon dioxide, climate change, folates, strawberry, temperature, vitamin C
Introduction
Folate (vitamin B9) and vitamin C are vital nutrients which provide many health benefits to
humans. These vitamins play an important role in protecting humans from a range of chronic
diseases and cancers1. More specifically; folate is the naturally occurring vitamin B9 which is
a functional nutrient in a range of metabolic processes. It reduces the risk of neural tube defects,
risks of heart deceases and promotes cell division and growth. Thus, folate is essentially
required during pregnancy, lactation and infancy2. Vitamin C is mainly required for
biosynthesis of collagen, carnitine and neurotransmitters and preventing scurvy disorder and
cold. Nonetheless, it is also a powerful important antioxidant, anti-atherogenic, anti-
carcinogenic, and immunomodulator3.
The basic chemical structure of folate consists of a p-aminobenzoate molecule, pteridine ring
and a glutamic acid. Different mono-glutamate folate compounds are available, which differ in
one carbon unit linked to the pteridine ring, such as methyl (5-CH3), methylene (5,10-CH2),
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formimino (5-CHNH), formyl (5- or 10-HCO), and methenyl (5,10-CH)1 (Figure I).
Strawberries, like many other fruits and also vegetables contain 5MTHFA as the main mono-
glutamate compound 4 and THFA is found in small amounts5. These folate compounds can be
interconverted chemically and enzymatically1. All these derivatives are commonly called
folates, which act as cofactors for enzymes in one-carbon metabolism. Vitamin C is chemically
termed as 2-oxo-L-threo-hexono-1,4-lactone2,3-enediol. L-ascorbic acid (AA) and
dehydroascorbic acid (DHAA) are the main dietary forms of ascorbic acid and make up the
total vitamin C content in human foods.
Both folates and vitamin C are water soluble and chemically liable. Moreover, they are highly
sensitive to light, heat, oxygen and pH3,6 where they undergo oxidative degradation.
Altogether, they are naturally vulnerable to degradation during food handling, processing,
cooking and storage.
Fruits and vegetables are main natural sources of the above two micronutrients and there is
serious global concern on potential adverse effects of synthetic forms of those vitamins. In
addition, the socioeconomic value of those two critical vitamins in terms alleviating human
health risks is highly recognised, especially in an era where the aging population continuously
grow. Among the fruits, strawberries contain relatively very high levels of both vitamins, folate
and C, therefore promoted as a key source of dietary those vitamins.
The above vitamins are metabolic products of plant processes. Earlier reviews by Balasooriya
et al.7,8 reported possible effects of environmental factors such as atmospheric [CO2] and
temperature on the nutritional quality of strawberries in terms of micronutrients. According to
the Intergovernmental Panel on Climate Change (IPCC), atmospheric [CO2] may increase up
to around 1000 ppm from the current level of 400 ppm and surface air temperature by 2.5 to
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7.8 °C by 21009. These increases in [CO2] and temperature would significantly influence the
vitamin contents in fruits and vegetables. However, information about the effect of changes in
climatic factors of plant growth environment on fruit phytonutrients, especially on vitamins
such as folates and vitamin C are scarce. Therefore, this study investigated the impact of
individual and interaction effects of elevated [CO2] and temperature on folates and vitamin C
contents in strawberry.
Materials and Methods
7.2.3.1. Chemicals
HPLC gradient grade acetonitrile and dithiothreitol (DTT) were obtained from Thermo Fisher
Scientific, Melbourne, Victoria, Australia. HPLC gradient grade sulphuric acid (84733),
dipotassium phosphate (17835), hydrochloric acid, sodium hydroxide, phosphoric acid,
metaphosphoric acid, kidney acetone powder porcine type II (K7250), folinic calcium salt
(F7878), formylfolic acid (F0380000), 5-methyltetrahydrofolic acid disodium salt (M0132),
tetrahydrofolic acid (T3125) and ascorbic acid (95210) were purchased from Sigma-Aldrich
Co, NSW, Australia. Ultrapure Milli-Q® water (Purification System (ZMQP60001),
Massachusetts, United States) was used in samples preparation, extractions of vitamins and
running the HPLC mobile phase.
7.2.3.2. Strawberry Samples
Strawberry plants were grown in controlled environment (CE) chambers (Model: TPG-2400-
TH-CO2, Thermoline Scientific Equipment Pty. Ltd., Wetherill Park, NSW, Australia) in
Parkville Campus at The University of Melbourne, Australia as described by Balasooriya et
al.10 to obtain the fruits for this study. Briefly, strawberry cultivars, ‘Albion’ and ‘SA’ were
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grown under six different [CO2] and temperature combinations of 400 ppm×25 ºC, 400
ppm×30 ºC, 650 ppm×25 ºC, 650 ppm×30 ºC, 950 ppm×25 ºC and 950 ppm×30 ºC inside
automated CE chambers. In this experiment, there were four replicates and each replicate had
three strawberry plants. The treatments were applied throughout the plant growth, development
until harvesting. Healthy fruits were harvested separately from each treatment within each
replicate and for each cultivar when they are at approximately 90% red colour.
The folate and ascorbic acid contents of strawberries were analysed in both fresh fruits and
freeze-dried powder (FDP). Fresh strawberry Fruits were stored in a cold room at 4 ºC until
used. Strawberry fruits for preparing FD samples were cleaned from calyx and peduncle and
then stored at -80 ºC freezer for at least overnight. Frozen fruits were then transferred to the
freeze-dryer (Dynavac Engineering FD3 freeze dryer, NSW, Australia). Freeze-dried
strawberry fruits were immediately ground into fine powder using a molar and pestle before
storage in sealed polyethylene bags. These samples were stored in a lab desiccator at room
temperature. The initial fruits weight and weight soon after freeze-drying were recorded to
calculate the moisture content in strawberries.
7.2.3.3. Extraction of vitamins
7.2.3.3.a. Extraction of folate
The folate derivatives of strawberry samples were identified and quantified following the
method of Lebiedzinska et al.11 with some modifications. Fresh strawberry sample (5 g) or FDP
strawberry (1 g) was thoroughly homogenised in 20 ml of 10% phosphoric buffer (pH 6.8)
using and Ultra-Turrax T25 (Janke and Kunnel, IKA-Labortechnik. Staufen/Germany). The
homogenate was kept in a water bath at 100 C for 10 mins. Then it was cooled down in an ice
bath and the pH was adjusted to 4.9. Kidney acetone powder (3 ml of 5 mg/ml solution) was
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added to the mixture and incubated at 37 C for 3 hours. The Samples were then heated at 100
C for 5 mins to terminate the enzyme activity and rapidly cooled in ice. The samples were
centrifuged at 10000 rpm for 20 min at 4 °C (Centrifuge, Thermoline, Scientific Equipment
Pvt Ltd). The supernatant was collected from each sample separately, and the residue was
extracted two more times following the same procedures, in order to extract all the folate. The
combined supernatants were filtered through a Whatman 4 filter paper and adjusted to 25 ml
final volume in a volumetric flask. An aliquant (2 ml) of the sample extract was then filtered
through 0.45 µm cartridges. This procedure was performed under dark and the filtrate was kept
in dark in the -20C freezer until used.
7.2.3.3.b. Extraction of ascorbic acid
Fresh strawberry sample (5 g), or FDP (1 g) was thoroughly mixed with 15 ml of 4.5%
metaphosphoric acid for 2 minutes at room temperature using the laboratory homogenizer
(Ultra-Turrax T25). The mixture was centrifuged at 4000 rpm for 20 minutes at 4 °C. The
supernatant was collected, and the residue was extracted two more times following the same
procedures. The combined supernatants were filtered through Whatman No. 4 filter paper and
brought to a final volume of 25 ml in a volumetric flask12. To analyse the total amount of
vitamin C, 5 ml of the filtrate was mixed with 1 ml of dithiothreitol (DTT) (20 mg\ml). DTT
was added as the reducing agent to convert all the DHAA into AA form and to calculate the
total vitamin C in strawberry samples12. This mixture was kept in dark for another 2 hours at
room temperature prior used in HPLC analysis. All vitamin C extracts were filtered through
0.45 µm membrane before injecting into the HPLC.
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7.2.3.4. Chromatographic conditions in identification and quantification of vitamins
A Gemini C18 silica 250 × 4.6 mm, 5 µm column (Phenominex Inc., Lane Cove West, NSW,
Australia) connected to a HPLC system equipped with Water 2690 Alliance Separation Module
(Waters, Rydalmere NSW, Australia) and coupled with a Waters 2998 Photodiode Array
(PDA) Detector was used to identify and quantify the vitamins. Both internal and external
standards were used in identification of folates and AA peaks. Quantification of vitamin
contents in strawberry samples was based on peak area and mass of the external calibration
standards that generated the corresponding curve to the peak. Finally, the average peak area
was calculated as the average of two targeted peaks from duplicated HPLC injections.
7.2.3.4.a. Analysis of folates
A validated HPLC technique by Lebiedzinska et al.11 was performed to analyse the folate
derivatives in strawberries. Sample extract (20µl) was injected into the HPLC to separate and
quantitate the folate forms. A gradient elution of 8% acetonitrile (pH 5.5) (A) and 40 mM
dipotassium phosphate buffer was used as mobile phases in characterization of folate
derivatives. A flow rate was maintained at 0.9 ml/min, and detection and quantification were
performed against the folate standards at 290 nm. The gradient was started at 8% acetonitrile
and linearly increased from 5-50% over 5 min, 50-70% over 7 min, followed by recycling to
initial conditions over 8 min. The column was washed out with acetonitrile (8%) for 10 mins
between injections. Four different reference folate standards were used for external peak
identification including tetrahydrofolic acid (THFA), 10-formyltetrahydrofolic or 10-
formylfolic acid (10FFA), 5-formyltetrahydrofolic acid (5FTHFA), and 5-methyl
formyltetrahydrofolic acid (5MTHFA). The quantities were calculated and recorded as µg of
folate derivative or total folates/100g fresh weight (FW) as well as based on dry weight (DW).
99
7.2.3.4.b. Analysis of vitamin C
Among the three different HPLC mobile phases; (1) methanol/0.5% KH2PO4-buffer/H2O
(28:60:12 and 20:60:0); (2) 0.05 M KH2PO4, pH 2.6; and (3) H2SO4, pH 2.6 and 2.2, Sanchez
Mata et al.13 suggested that isocratic 0.01% H2SO4 at pH 2.6 (mobile phase 3) was the best to
obtain accurate ascorbic acid contents. Using mobile phase 3, the flow rate was maintained at
0.9 ml\min and 20 µl of sample was injected. L-AA reference standard was used in
identification of AA peaks in strawberry samples at 245 nm wavelength. The total vitamin C
content of samples was reported as mg/100 g FW or DW.
Statistical analysis
Each extraction procedure was performed at least in triplicate (3 trials) and each sample was
duplicated in HPLC analysis. Minitab® 17 Statistical Software was used in data analysing
following the Analysis of Variance procedure with a General Linear Model (Minitab 17
Statistical Software (2010). Tukey’s multiple comparison method at 95% confidence level was
performed to separate the means. Finally, the results were reported as mean ± standard
deviation (SD).
Results
7.2.5.1. Identification of vitamins in HPLC-UV analysis
7.2.5.1.a. Folates
In this study, four different folate derivatives were identified in fresh and freeze-dried
strawberry samples using HPLC-UV system at 290 nm wavelength. These four folate
100
compounds appeared at retention times between 8 and 20 mins (Figure II) and the exact
retention times of these compounds are shown in Table I.
7.2.5.1.b. Ascorbic acid
AA was detected at 245 nm wavelength within the first 5 mins of the sample chromatograph
and the average retention time of AA peak was 3.1±0.02. HPLC-UV chromatograph obtained
for vitamin C in strawberry is presented in Figure III. The solvent buffer used in vitamin C
extraction always appeared along with AA peak in the chromatograph.
7.2.5.2. Effect of elevated [CO2] and higher temperature on vitamins in fresh
strawberry fruits
7.2.5.2.a. Folates
Total folate contents in strawberries varied from 52.6 ± 5.1 µg to 364.8 ± 16.0 µg/100 g FW in
cultivar ‘Albion’ and from 48.6 ± 7.0 µg to 237.4 ± 23.8 µg/100 g FW in cultivar ‘SA’
depending on growth conditions. The highest total folate content was found in strawberry fruits
grown under the combination of 950 ppm [CO2] and 25 °C for both cultivars. In general
increases in [CO2] and temperature significantly favoured total folate contents of strawberry
fruits however the responses were cultivar dependent and [CO2] x temperature interactions
were significant (P<0.05). Main effects of [CO2], temperature and their interactions on total
folates contents for the cultivar ‘Albion’ and ‘SA’ are shown in Figure IV.
An increase in [CO2] from 400 ppm to 650 ppm did not affect the content of total folates;
however, further increases [CO2] up to 950 ppm significantly (P<0.05) increased the total
folates content in fresh strawberry by several folds, i.e. from 52.6 ± 5.1 µg to 364.8 ± 16.0
µg/100 g FW and from 48.6 ± 7.0 µg to 237.4 ± 23.8 µg/100 g FW, respectively for cultivars
101
‘Albion’ and ‘SA’. Similarly, 5 °C increase in growth temperature significantly (P<0.05)
increased the total folate content in fresh strawberries and there was a significant cultivar and
temperature interaction. As illustrated in Figure IV, Albion grown at 30 °C under 950 ppm
[CO2] produced fruits with a significantly (P<0.05) greater folate content. Although, increases
in growth temperature positively influenced total folates contents of strawberry at lower [CO2]
levels, the effects turned negative at the highest [CO2] (950 pm). Total folate content was
reduced by 26% and 13% in cultivar ‘Albion’ and ‘SA’, respectively (Figure IV) by higher
temperature under 950 ppm.
Concentrations of individual folate derivatives found in strawberry are presented in Figure V.
All the four-different mon-glutamate compounds of THFA, 10FFA, 5FTHFA and 5MTHFA
were present in all the strawberry samples of this experiment. Compared to the other folate
derivatives, THFA levels were low in both strawberry cultivars and values ranged from 2% to
8% of the total folates. Concentrations of the folate derivative 10FFA varied from 4% to 47%
and 10% to 48% respectively in cultivars ‘Albion’ and ‘SA’. In general, relative levels of
5FTHFA were always greater in Albion than in SA and the values ranged from 24% – 91%
and 4% – 82%, respectively for those two strawberry cultivars. Additionally, fruits from both
strawberry cultivars grown at the highest [CO2] concentration, 950ppm, constantly maintained
greater concentrations of 5FTHFA, irrespective of the temperature level and the values ranged
from 77% – 91%. However, the relative concentrations of foliate derivative, 5MTHFA were
always found to be lower (1%-7%) at 950 ppm in both cultivars compared to the other [CO2]
levels.
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7.2.5.2.a. Vitamin C
Effects of elevated [CO2], higher temperature and their interactions on vitamin C content of
strawberries were statistically significant (<0.05) and the responses were cultivar dependent.
Vitamin C contents of fresh strawberries in cultivar ‘Albion’ and ‘SA’ varied from 59 ± 7 mg
to 133 ± 15 mg/100 g FW and 56 ± 9 mg to 132 ± 9 mg/100 g FW, respectively. The individual
and interaction effects of elevated [CO2] and temperature on vitamin C contents in fresh
strawberry are shown in Figure VI.
Vitamin C content in fresh strawberry fruits was not influenced by elevated [CO2] from 400
ppm to 650 ppm at 25 °C. However, 950 ppm [CO2] at 25 °C significantly (P<0.05) enhanced
the vitamin C contents in fresh strawberries by 116% and 48%, in cultivar ‘Albion’ and ‘SA’,
respectively. Even though the effect of higher temperature (30 °C) on vitamin C content under
400 ppm [CO2] was insignificant (P<0.05), increasing the growth temperature to 30 °C at 650
ppm [CO2] enhanced the amounts of vitamin C significantly (P<0.05) to a maximum of 123%
and 132% in cultivars ‘Albion’ and ‘SA’, respectively. However, that effect wasn’t detected
when the [CO2] concentration was increased further to 950 ppm, and the vitamin C
concentrations drastically decreased by 36% and 31% in Albion’ and ‘SA’, respectively at 30
°C and 950 ppm [CO2].
7.2.5.3. Effect of freeze drying on vitamin contents in strawberry fruits
Moisture content in strawberries ranged from 87% to 90% when calculated using freeze drying
techniques. The calculated total folate and vitamin C contents in fresh and freeze-dried
strawberry samples under different growth combinations are presented in Table II.
103
The total folate content in FD strawberry ranged from 106.8 ± 11.5 µg/100g FW to 322.0 ±
29.1 µg/100g FW in cultivar ‘Albion’ and from 99.5 ± 17.1 µg/100g FW to 395.6 ± 25.1
µg/100g FW in cultivar ‘SA’. Vitamin C content of FD strawberry in cultivar ‘Albion’ and
‘SA’ were varied in a range of 74.8 ± 6.7 mg/100g FW to 165.4 ± 12.6 mg/100g FW and 77.4
± 6.7 mg/100g FW to 162.7 ± 10.9 mg/100g FW, respectively. In general, folates and vitamin
C contents were significantly (P<0.05) higher in FD strawberry than fresh samples.
Discussion
7.2.6.1. Analysis of folates and ascorbic acid by HPLC-UV system
7.2.6.1.a. Folates
Microbiological assay (MA) is the traditional method of analysing the total folates content in
food materials, however that method involves extensive procedures and thus is very time
consuming. Consequently, HPLC methods have recently become an increasingly popular
technique in folates analysis to quantify the mono-glutamate forms in foods with the capability
of separation and detection of individual folate compounds in a comparatively shorter period
time14,15. Stralsjo et al.4 compared different analysis methods of folates and found that HPLC
methods resulted in higher amounts of total folates than MA in the same material, but lower
than radioprotein-binding assay (RPBA). In earlier studies, Vahteristo et al.16 were able to
detect only two folates, namely THFA and 5MTHFA, while, Stralsjo et al.4 detected only
5MTHFA in strawberries. However, this study clearly identified the four main forms of
naturally occurring folate derivatives; THFA, 10FFA, FTHFA and 5MTHFA (Figure II) and
quantified those using a HPLC technique. According to the best of our knowledge, this is the
first time that a HPLC-UV study detected all these four different mono-glutamate forms in
104
strawberry. The analysis of main folates compounds in have a greater value in studying the
absorption, metabolization and bioavailability of folates15.
Apart from the specific quantification technique, sample preparation and folate extraction also
significantly influence the folate determination17. It is well established that addition of
antioxidants like AA and 2-mercaptoethanol into the extraction solvent, stabilizes the folates
during sample extraction11, however, it may interfere the detection of folates in HPLC-UV
system14. Since, strawberries are naturally rich with antioxidants, for examples, polyphenols
and vitamin C, the folates would be naturally protected from degradation during the process18-
21. Therefore, in this study, strawberry samples were extracted with no added AA and it resulted
in a good separation of poly-glutamates and successful detection of all individual folate
derivatives (Figure II). In addition, study used a single-enzyme extraction, instead of using the
tri-enzyme treatment (protease, α-amylase and folate conjugase) process. The key step in
enzymatic extraction of folates is the deconjugation of poly-glutamate chain into mono-
glutamate forms to overcome the difficulty of detecting longer poly-glutamate chains22.
Considering the availability and lower cost, the porcine kidney powder was used as the folate
conjugase. There are some controversies on addition of protease and α-amylase in folate
extraction. Earlier studies by Lim et al.23, Shrestha et al.24 revealed increased amounts of folates
in food samples extracted with protease and α-amylase but, Ndaw et al.25 found no significant
difference in their results using tri- and single (chicken pancreas conjugase) enzyme treatment.
Nonetheless, Iwatani et al.17 reported decreased folates contents in vegetable samples due to
the applied tri-enzyme treatment in comparison with single-enzyme extraction. Additionally,
folate degradation during heating at boiling temperature to deactivate the enzymes could be
eliminated or reduced by adjusting the pH of the extraction buffer and the presence of reducing
105
agents such as ascorbic acid 26. Finally, no compound from the extraction solvent appeared to
interfere in the detection of folates derivatives in strawberry samples in this recent study.
Methyl and formyl derivatives have been reported to largely dominate the plants folates27. Most
fruits and vegetable predominantly contain 5MTHFA, while 5- or 10FFA is abundant in whole
grains15, 28, 29. When it comes to mushrooms, enoki and oyster have been documented to contain
exclusively 5MTHFA, and morel and chanterelle to contain mainly FFA30.
The 5MTHFA is functionally important in human DNA methylation and brain chemistry,
reducing mental disorders and enhancing antioxidant properties30. Few studies have reported
that 5MTHFA was the most dominant folate form found in strawberries4,16,18. In this recent
study, the strawberry samples contained all four different folate derivatives in different
proportions. This could be possibly due to the interconversion of the main mono-glutamyl
5MTHFA into other folate species. A similar suggestion was presented by Wang et al.31. The
same authors (Wang et al.31) observed interconversion of 5MTHFA and 10FFA compounds
into THFA during juicing of the vegetables. Such interconversion of 5MTHFA into other folate
forms was detected in eight different vegetable juices but most importantly it had no significant
impact on the total folate contents. Interconversion of these compounds could be a result of the
stability of folate derivatives. In the folate structure (Figure I), N5 and N10 positions are more
stable, thus the other forms are readily transferred to 5 or 10 folates forms. In general,
strawberry samples in the current study showed greater proportion of 5FTHFA which is the
most stable folate form from all other than folic acid32. Additionally, 5MTHFA and 5FTHFA
have been reported to show higher bioefficacy than THFA33. Generally, folic acid is rarely
detected naturally in foodstuffs. The presence of folic acid in samples might be an indication
of folate degradation during extraction process or in samples before the analysis26.
106
In previous studies, total folate content of strawberries varied from 36 µg to 69 µg/100 g FW
and from 476 µg to 640 µg/100g DW34, and from 20 µg to 99 µg/100 g FW35, and the moisture
content of strawberries ranged from 83% to 93% under vacuum oven drying at 70 °C. The
results of strawberry fruits grown under ambient growth conditions (400 ppm and 25 °C) in the
current study values (52.6 µg/100 g FW in ‘Albion’ and 48.6 µg/100 g FW ‘SA’) were in the
agreement with data reported elsewhere. Variations between some values of total and
individual folates from the literature as compared to findings of this current study (52.6 µg/100
g FW in ‘Albion’ and 48.6 µg/100 g FW ‘SA’) could be due to several reasons. These could
include, genotypical differences, post-harvest losses, storage, growth environment, the method
of extraction and analysing, purity of reference standards used, processing techniques,
degradation and interconversion of folates in strawberry samples from harvest to analyses.
Aside from the analytical method point of view, strawberries appeared to contain considerably
higher amount of folates (113 µg/100g) compared to those in some of the other most known
high foliate fruits, such as white grapes (32 μg/100g), pineapple, mango, orange (44 μg/100g),
raspberry (31 μg/100 g), banana (29 µg/g) and kiwifruit (36 μg/100g)5, 36. Spinach, rocket and
watercress also contained high total folates respectively 140.9 µg, 139.2 µg and 127.9 µg/100
g FW.
7.2.6.1.b. Vitamin C
The current study used a previously validated method by Odriozola-Serrano et al.37 for the
analysis of vitamin C. The authors of the above study confirmed that a maximum recovery of
vitamin C was achieved when employed a C18 column connected to a HPLC-UV system with
sulphuric acid (0.01%, pH 2.6) as the mobile phase. Additionally, the same above study
demonstrated that DTT was a better reducing agent in transforming DHAA to AA where the
107
recovery of AA was almost 100%. Sanchez Mata et al.13 who used similar conditions, but
connected to different column, observed a solitary peak from AA for green beans samples,
where AA maximum absorbance was between 245 nm to 254 nm wavelength. Similarly, this
study too detected and identified AA at 245 nm wavelength. Additionally, we observed a stable
baseline in the chromatograph and apparently neither DTT nor other compounds interfered the
HPLC analysis. However, the extraction solvent always appeared with the AA peak in the
chromatograph (Figure III). The extraction solvent peak and retention time were confirmed by
injecting the solvent separately.
In the current study, vitamin C concentration in strawberry varied from 56.4 ± 9.4 mg to 59.9
± 7.5 mg/100g FW under normal growth conditions (400 ppm [CO2] and 25 ºC) using HPLC
technique. These results agree with those published in the literature. For example, vitamin C
contents in strawberry were reported to be 59.1 ± 0.9 mg/100g FW37 and 20 mg to 70 mg/100g
FW38 using HPLC-UV, 77 mg/100g FW39 using ferric reducing antioxidant power (FRAP)
assay and 26.46 mg to 37.77 mg/100g FW36 using spectrophotometry. The differences
observed in vitamin C content in strawberry could be due to cultivar, cultural practices, growth
environment and quantification techniques.
7.2.6.2. Effect of elevated carbon dioxide and higher temperature on vitamin contents
7.2.6.2.a. Folates
In plants, THFA involves in photorespiratory pathway and the conversion process of glycine
to serine in mitochondria32. This reaction is catalysed by serine hydroxymethyltransferase
(SHMT) while THFA is converted to 5,10-methylenetetrahydrofolate (5,10-MTHFA)32. In the
formation of folate derivatives, 5,10-MTHFA is irreversibly hydrolysed into 5FTHFA by
SHMT and 5FTHFA, and considered the most stable form of natural folates27. Since high
108
temperature favours photorespiration in plants, it could indirectly involve in stimulating the
synthesis of higher folates contents. It has been also previously reported that higher growth
temperature may induce production of novel plant proteins in strawberry40,41. Increased
production of more stable forms of folates like 5FTHFA could be a result of overexpression of
the committing proteins (folate bound proteins) and enzymes involved in the folate production
pathway32,42. On the other hand, the synthesis and accumulation of folates in fruits is also
enhanced in the presence of antioxidants and ascorbic acid by favouring the stability of
folates42. Strawberries produced a greater quantity of polyphenolic compounds43 and as vitamin
C (Figure VI) in response to the elevated [CO2] and higher temperature levels during plant
growth. Therefore, it can also be deduced that enhanced levels of folates34 in strawberry as
observed above would be result of a counter balancing process. The folate synthesis pathway
in plants is the same as in bacteria42, where more total folate formation has been reported at
higher temperature (28 °C) which was more favourable for folate synthesis, and that was
bacterial type dependent 44. Increased photosynthesis in strawberry plants at elevated [CO2]10
may also favour the folate synthesis since 30% of total folates in leaves involve in leaf
photorespiration and converted to 5,10-MTHFA 45. However, the decline in plant folates due
to increased temperature under 950 ppm would be a result of enzymatic or biochemical
breakdown32, where more reactive oxygen species (ROS) are produced. The increment in ROS
is attributed to environmental stress, which can promote the chemical oxidation of plant
folates45. Additionally, light in the growth environment enhances the degradation of plant
folates45. However, other than the current study, information on the influence of elevated [CO2]
and temperature on folate contents in fruits is lacking.
109
7.2.6.2.b. Ascorbic acid
Increases in vitamin C contents in plants are important in scavenging free radicals which arose
due to the external stress factors such as heat. Vitamin C protects the cells and cell components
from oxidative damages46. The role of vitamin C as an antioxidant is different from phenolic
antioxidants. vitamin C has the ability to accept the free radicals from reactive oxygen species
(ROS) but polyphenols only react with ROS to restrict the free radical amount47. Therefore,
ascorbic acid is one of the key components in enzymatic antioxidant pathways like ascorbate
peroxidase cycle46.
The increased levels of total vitamin C in strawberry fruits under 950 ppm [CO2] and 25 °C
detected in this study were in agreement with those reported by Wang et al.46. The same author
indicated also that growing strawberry plants at 650 ppm [CO2] and 25 °C did not affect vitamin
C contents significantly, which again was similar to our findings (Figure VI). However, results
from this current study showed that increasing the growth temperature by 5 °C (from 25 °C to
30 °C) at [CO2] 650 ppm significantly (P<0.05) increased vitamin C content in strawberry fruits
which did not agree with Wang and Camp48 and Richardson et al.49. In those studies48,49, the
temperature was elevated only during specific growth stage of the plant however, in the current
study, temperature was increased by 5 °C during the whole crop cycle. Wang and Camp48
reported 40% reduction in ascorbic acid content in strawberries at 30/22 °C in comparison with
25/12 °C. However, in that study the high temperature was imposed after plant blooming48.
Similarly, Richardson et al.49 found lower amounts of ascorbic acid in kiwi fruits under
elevated temperature during the phases of cell division and starch accumulation in the growth
cycle but not at the maturation stage. However, the increased vitamin C content is also
important for the stability of folate compounds in strawberry34. The interaction and positive
effect of elevated [CO2] and temperature on vitamin C content was significant (p<0.05) up to
110
temperature 25 °C. However, increasing the temperature to 30 °C at 950 ppm [CO2] negatively
affected the vitamin C content. In conclude, increased temperature by 5 °C influenced
differently on vitamin C content in strawberry under different [CO2] levels.
7.2.6.3. Maintaining strawberry vitamins by freeze drying
The contents of vitamins in processed foods vary depending on the methods of storage,
preparation and processing. Most of fruits and vegetables are commonly processed and
subjected to a long-term storage before consumption, which contribute to vitamins loss. The
effective level of nutrients in foods including vitamins depends on the amounts lost during
processing and storage. For examples, cooking by stir-frying, frying, boiling and pressuring
have been reported to degrade folates in vegetables by around 0-40%50, 1-36%, 10-68%51,52
and 10-94%52, respectively. However, folate contents was not influenced by steaming and
cooking in microwave53 hence recommended to process folate rich vegetables52. Nevertheless,
the ready to consume fresh drinks lose about 46% of its folates during the long term (one year)
storage6. Similarly, boiling in water degraded vitamin C in frozen vegetables by 51%52.
Blanching caused 51% loss in frozen vegetables vitamin C content52. Though steaming did
not cause reductions in folates, but decreased vitamin C content by approximately 70% in leafy
vegetables.
Preserved and processed fruits and vegetables market have been growing throughout the years
because such products provide convenience to consume, become available during the offseason
and preserve the excess to feed more. Strawberries are most often consumed fresh, hence
provide considerable amounts of total folates and vitamin C. However, as a short shelf-life
fruit, strawberry processing and preservation are important and critical postharvest practices
but may affect its nutrients contents. In order to protect and maintain strawberry vitamins
111
content, freeze drying (FD) could be considered the best technique for strawberry processing.
FD strawberry is appreciated as a natural source of antioxidants with low glycemic index in
controlling complications of type 2 diabetes54. As a result, consumption of FD strawberry
products with breakfasts, desserts and snacks recently became more popular among people55,56.
Meanwhile, due to the promising results, freeze dried strawberry powder has been promoted
as better health-enhancing food.
Freeze-dried strawberry processing has been reported to preserve higher contents of total
polyphenols57, folates58,59 and antioxidants including vitamin C55 than most of other
conventional drying methods60. In the current study, freeze drying increased the folates and
vitamin C in strawberry fruits in compared to fresh fruits (Table II). The apparent increase in
vitamins in strawberry fruits was mainly due to the water removal during freeze drying rather
than to actual increase of folates and vitamin C 4. Clifford et al.59 observed similar amounts of
folates in freeze dried vegetables to fresh vegetables. Lane et al.58 reported increased folates
amounts in shuttle foods such as freeze-dried corn, broccoli, and pasta with creamed spinach.
However, significant reductions were found in freeze dried asparagus, cauliflower with cheese,
green beans with broccoli and mixed Italian vegetables. Such reduction in these food products
could be attributed to oxidative degradation of folates58. Lin et al.53 found no significant losses
of vitamin C in freeze dried carrot slices. However, significant degradation of vitamin C
amounts with an average of 16% to maximum of 80% were reported by Marques et al.61 in
pineapple, cherry, guava, papaya and mango.
No folate losses found in strawberries even after stored in -20 °C for 6 months16. This could be
supported by the good stability of 5MTHFA (the main compound in strawberry) under cold
storage26. In freeze-drying of strawberry fruits, freezing is important to develop ice crystals
within the fruit matrix when temperature is dropped below its freezing point. Such low
112
temperature will stop microbial activities and slow down or stop the enzymatic and oxidative
reactions. Removal of water from the fruit matrix promotes more solutes in the rest of non-
frozen water. The formed porous structures are the results of sublimation of ice followed by
water removal. Thus, it results in a greater cell rupture to access more extraction solvent.
Finally, freeze-drying may lead to an efficient nutrient extraction and more preserved
nutrients57. In this study, vacuum freeze drying was performed which is one of the best methods
in removing water60 specially for heat sensitive food materials62. This method demands longer
time and energy to maintain high vacuum and low temperature, however it protects fruit colour,
flavour and appearance and secures more nutrients63. Even if freeze drying caused any
reductions in food folates, it has been reported that there was adequate amounts of folates
available in foods to meet the required daily folates amount58.
In conclusion, a very little information is available concerning the separate and interactive
effects of elevated [CO2] and high temperature on vitamin contents in fruits and vegetables.
Therefore, more researches are needed to confirm the results of the current study in the field.
These research outcomes would also help the commercial growers to increase the nutritional
aspects of fruits and vegetables.
Acknowledgments
The Authors greatly acknowledge and appreciate the contribution and effort provided by Bruce
Tomkins throughout this investigation. The 2017 Innovation Seed Fund for Horticulture
Development of The University of Melbourne and Department of Economic Development,
Jobs, Transport and Resources (DEDJTR) provided the financial assistance for this project.
113
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Figures and Tables
Figure I. Chemical structures of different folate derivatives and their chemical names.
Figure II. HPLC chromatograph for folates obtained from fresh strawberry fruits at 290 nm.
Peaks with their retention times (RT±SD) are as follows; 1 – THFA (10.3±0.6), 2 – 10FFA
(12.2±0.5), 3 – 5FTHFA (13.6±0.4), and 4 – 5MTHFA (16.9±0.5).
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Figure III. HPLC-UV chromatograph for vitamin C of strawberry fruits obtained at 245 nm
wavelength. Peaks are; 1 – Extraction solvent (K2HPO4), 2 – AA (RT±SD – 3.1±0.1), 3 –
unknown.
Figure IV. Total folates contents in strawberry cultivars under different growth combinations
of [CO2] and Temperature. (a) ‘Albion’ and (b) ‘San Andreas’. Error bars refer to standard
deviation of data (n = 12).
f f
a
e
de
b
0
100
200
300
400
400 650 950
Tota
l F
ola
te c
on
ten
t
(ug/1
00
g F
W)
[CO2] ppm
(a) Albion
25 °C
30 °Cf f
c
de
dec
0
100
200
300
400
400 650 950
Tota
l F
ola
te c
on
ten
t
(ug/1
00
g F
W)
[CO2] ppm
(b) SA
25 °C
30 °C
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Figure V.All the four-different mono-glutamate compounds (THFA, 10FFA, 5FTHFA and
5MTHFA) were detected in all the strawberry samples.
Figure III. Total vitamin C contents in strawberry cultivars at different growth combinations
of [CO2] and Temperature. (a) ‘Albion’ and (b) ‘San Andreas’. Error bars refer to standard
deviation of data (n = 12).
0
50
100
150
200
400 650 950
Tota
l vit
amin
C c
on
ten
t
(mg/1
00
g F
W)
[CO2] ppm
(a) Albion
25 °C 30 °C
0
50
100
150
200
400 650 950
Tota
l vit
amin
C c
on
ten
t
(mg/1
00
g F
W)
[CO2] ppm
(b) SA
25 °C 30 °C
c
a a
b
c
c c
c
a
c
b
c
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Table I Effect of different temperature and [CO2] on other polyphenol compounds in strawberry
varieties Different folate derivatives identified and quantified in strawberry samples and their
retention times (RT).
Chemical Name Abbreviation RT (min)
(Mean ± SD)
Tetrahydrofolic acid THFA 10.3±0.6
10-formylfolic acid 10FFA 12.0±0.5
5-formyltetrahydrofolic acid 5FTHFA 13.6±0.4
5-methytetrahydrofolic acid 5MTHFA 16.9±0.5
Table II Total folate and vitamin C contents in fresh and FD strawberry grown under different [CO2]
and temperature combinations
Cultivar Temperature
(°C)
[CO2]
(ppm)
Total folates content
(µg/100g)
Total Vitamin C content
(mg/100g)
Fresh FD Fresh FD
Albion 25 400 52.6 ± 5.1 f 106.8 ± 11.5 e 59.9 ± 7.5 c 74.8 ± 6.7 e
650 53.4 ± 6.9 f 112.0 ± 20.7 e 59.6 ± 7.1 c 79.1 ± 6.6 e
950 364.8 ± 16.0 a 395.6 ± 25.1 a 129.6 ± 15.1 a 149.8 ± 9.9 b
30 400 122.5 ± 11.7 e 159.5 ± 9.9 d 59.6 ± 9.3 c 81.3 ± 6.0 e
650 158.6 ± 34.5 de 179.5 ± 31.3 d 132.9 ± 14.9 a 165.4 ± 12.6 a
950 271.0 ± 7.7 b 372.33 ± 10.4 ab 83.5 ± 5.9 b 131.8 ± 1.2 c
SA 25 400 48.6 ± 7.0 f 99.5 ± 17.1 e 56.4 ± 9.4 c 83.1 ± 6.7 e
650 59.2 ± 7.9 f 93.8 ± 3.7 e 61.8 ± 6.3 c 84.4 ± 5.5 e
950 237.4 ± 23.8 c 322.0 ± 29.1 bc 83.4 ± 0.1 b 120.7 ± 9.2 c
30 400 125.3 ± 12.6 de 163.8 ± 8.7 d 61.5 ± 7.1 c 77.4 ± 6.7 e
650 156.2 ± 24.2 d 190.1 ± 18.9 d 132.4 ± 9.7 a 162.7 ± 10.9 ab
950 206.5 ± 24.8 c 302.0 ± 3.5 b 57.2 ± 5.4 c 99.8± 8.9 d
[CO2] * * * *
Temperature * * * *
Cultivar * * * *
[CO2]*Temperature * * * *
[CO2]*Cultivar * * * *
Temperature*Cultivar * NS * *
[CO2]*Temperature*Cultivar * NS * NS
Results are expressed as µg/100g FW for total folates content and mg/100g FW for total vitamin C content.
Values are Mean ± SD (n=12), *significant (p ≤ 0.05) and different letters in each column are significantly (p ≤
0.05) different.
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Chapter 8 investigated the bioaccessibility of polyphenols, folates and vitamin C in fresh and
frozen strawberries grown under elevated carbon dioxide and temperature, using simulated in
vitro gastrointestinal digestion models and HPLC-UV system. This chapter also studied the
colonic fermentation metabolites in in vitro digested strawberry by using simulated in vitro
colonic fermentation and HPLC-UV methods. The amounts of selected individual polyphenols,
folates and ascorbic acid in bioaccessible fraction, as well as in colonic fermented strawberry
were reported. This approach was based on the previous results (chapters 6 &7) which revealed
that growth combination (950 ppm and 30 °C) significantly influenced strawberry plants to
produce maximum contents of phytonutrients (polyphenols, folate and ascorbic acid). The
manuscript has been submitted to the Journal of Food Chemistry.
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“Bioaccessibility of micronutrients in fresh and frozen strawberry fruits grown under elevated
carbon dioxide and temperature”
Himali N. Balasooriya1*, Kithsiri B. Dassanayake1,2, Said Ajlouni1
1. School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The
University of Melbourne, Parkville, VIC 3010 Australia.
2. Department of Infrastructure Engineering, Faculty of Engineering, The University of
Melbourne, Parkville, VIC 3010 Australia.
*Corresponding author; Himali N. Balasooriya – phone: +61420620730;
e-mail: [email protected]
Abstract
Strawberry is a rich source of polyphenols, vitamin C and folates. These compounds are highly
appreciated in reducing of some chronic diseases. The contents of micronutrients present in
strawberry fruits have been reported to vary depending on different factors including growth
conditions. Strawberry cultivar “San Andreas” grown under normal [CO2] (400 ppm)
temperature (25 °C) and elevated [CO2] (950 ppm) and temperature (30 °C) growth conditions
were chosen for this study. The strawberries were subjected to in vitro gastrointestinal digestion
and colonic fermentation to examine the accessibility of polyphenols, vitamin C and folates in
fresh and frozen fruits using HPLC-UV analysis.
In this study, six selected major polyphenol compounds; pelargonidin-3-glucoside,
pelargonidin-3-rutinoside, p-coumaric, ferulic acid, quercertin-3-O-glucuronide and p-
coumaroyl and total folates and vitamin C were identified and the bioaccessibility was
128
quantified in different strawberry digests. Results revealed that elevated carbon dioxide and
higher temperature enhanced the amounts of accessible bioactive compounds in strawberries.
Bioaccessibility of pelargonidin-3-glucoside increased from 67% to 88% in strawberries grown
under elevated [CO2] and temperature. Fresh strawberries grown under ambient atmospheric
conditions (400 ppm [CO2] and 25 °C) contained 93.09±6.2 µg/100 g FW total folates and
18.55±0.5 mg/100 g FW vitamin C as bioaccessible fractions under the fed state condition. In
comparison, elevated growth conditions enhanced soluble folates and vitamin C up to
188.63±7.5 µg/100 g FW and 30.48±0.3 mg/100 g FW, respectively in fresh strawberries. The
results showed also that fresh strawberries contained higher amounts of accessible
micronutrients than frozen strawberries, while increased bile contents in intestinal fluid (fed
state) facilitated the release of bioactive compounds to gastrointestinal fluid.
Key words; Bioaccessibility, climate change, folate, polyphenol, strawberry, vitamin C
Introduction
Polyphenols and vitamins are major antioxidant compounds widely found in many fruits
including strawberries (Fragaria ananassa Duch.) (Forbes et al., 2016; Giampieri, Alvarez
Suarez, and Battino, 2014) and currently have gained more attraction in preventative nutrition
(Ariza et al., 2016). Strawberry contains more than 100 different individual polyphenol
compounds (La Barbera et al., 2017) and provides substantial amounts of folates and vitamin
C (Asami, Hong, Barrett, and Mitchell, 2003; Johannesson, WittHöft, and Jägerstad, 2002).
Strawberry polyphenols are associated with preventing diseases and promoting human health
by their antioxidant, anti-inflammatory, antimicrobial, antiallergy, and antihypertensive
properties (Giampieri et al., 2014; Giampieri et al., 2013; Giampieri et al., 2015; Giampieri et
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al., 2012; Hannum, 2004). In addition, vitamin compounds also have variety of health benefits
including anti-cancer and anti-chronic properties.
However, the beneficial effects of these micronutrients are strongly dependent on their
bioaccessibility from food matrixes in the human digestive tract and the bioavailability
thereafter. Bioaccessibility is the amount of a bioactive compound that is released from a solid
food matrix to the digestive tract and potentially able to cross the intestinal barrier (Mullen,
Edwards, Serafini, and Crozier, 2008). Food micronutrients are released from the food matrixes
as a result of the enzymatic digestions and the additional break down of the remaining solids
by the gut microflora in the colon. Bioavailability cab be defined as the reachable or available
fraction of a bioactive compound or its metabolite to the systemic circulation in human
(McGhie and Walton, 2007). The bioaccessibility and bioavailability of different food
constituents can be varied qualitatively and quantitatively depending on several factors
including the food type (Ariza et al., 2018; Saura Calixto, Serrano, and Goni, 2007), food
processing (Liu, 2017; Ribas, Martín Belloso, Soliva Fortuny, and Elez Martínez, 2017) and
genetic make-up (Ariza et al., 2016; Fazzari et al., 2008). McGhie et al. (2007) reported that
anthocyanins showed low bioavailability due to rapid degradation or excretion during
digestion. Fazzari et al. (2008) found that the bioaccessibility of polyphenols in sweet cherries
was significantly cultivar dependant and lower in immature fruits. Mosele, Mosele, Macià,
Romero, and Motilva (2016) demonstrated that plant-based polyphenols and fat-soluble
vitamins were less absorbable during the gastric digestion due to the presence of pectin in cell
structure. However, mechanical and thermal and/or pH modifications during food processing
can increase bioaccessibility of bioactive compounds in plant products (Liu, 2017). In addition,
food components or foodstuffs mixed in diets can also alter the bioaccessibility of bioactive
compounds in fruits (Sengul, Surek, and Nilufer Erdil, 2014). A study by da Costa and
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Mercadante (2018) found that adding sugar enhanced the bioaccessibility of carotenoids in caja
fruit (Spondias mombin L.). According to the same study, milk protein improved the
bioaccessibility carotenoids by four times in milk based caja beverages. Additionally, release
and absorbance of phenolic compounds varied significantly with different conditions in the
digestive tract. For example; Tagliazucchi, Tagliazucchi, Verzelloni, Bertolini, and Conte
(2010) indicated that gastric digestion did not affect the accessibility of polyphenols in grapes
however, phenolic acids and resveratrol were degraded during intestinal digestion. Several
other studies mentioned that bioactive compounds are more readily and highly absorbed in the
intestine than in the stomach (Ariza et al., 2018; Wu, 2002). However, gastric digestion is not
negligible (Azzini, E et al., 2010).
In this context, the increased amounts of micronutrients contents in foods would not indicate
that they are more biologically accessible and available in the same amounts in human (Ariza
et al., 2018). The releasable amounts of polyphenols or vitamins from the food matrixes into
the digestive tract are the most important, since they are potentially bioavailable. Consequently,
simulated in vitro gastric and intestinal digestions with colonic fermentation is a widely
applicable research tool in investigating the bioaccessibility of food chemical constituents.
However, the in-vivo bioavailability of bioactive compounds is the best tool in evaluating their
actual bio-efficacy in human body.
The amounts of polyphenols and vitamins in strawberry have been reported to vary depending
on different factors mainly; genetics and growth conditions. Previous work associated with
current studies (Balasooriya, Dassanayake, Seneweera, and Ajlouni, 2019a, 2019b;
Balasooriya, Dassanayake, and Ajlouni, 2017), showed that strawberry contained significantly
higher contents of polyphenols, vitamin C and folates when they were grown under elevated
carbon dioxide and increased temperature. However, chemical analysis only is not enough to
131
conclude the beneficial effects of increased bioactive compounds with respect to human
subjects. Therefore, to complete the process of evaluating the impact of growth conditions on
strawberry chemical composition and nutrients contents, it is important to investigate the
bioaccessibility of these nutrients. Additionally, the authors of this recent investigation are not
aware of any previous study so far that has been reported on the impact of the climate stresses
(elevated CO2 and temperature) on the bioaccessibility of micronutrients in strawberries.
In this study simulated in vitro gastrointestinal digestions followed by in vitro colonic
fermentation were performed to investigate the bioaccessibility of selected micronutrients
(polyphenols, vitamin C and folates) in strawberries grown under elevated carbon dioxide and
temperature using HPLC-UV.
Materials and Methods
8.2.3.1. Materials
Sodium chloride, hydrochloric acid (HCl), sodium hydroxide, calcium chloride hydrate,
sodium hydrogen carbonate, magnesium sulphate heptahydrate, potassium dihydrogen
phosphate, potassium chloride and dipotassium hydrogen phosphate meeting the American
Chemical Society (ACS) grade were purchased from Bio21 stores, The University of
Melbourne, Australia. HPLC gradient grade methanol, acetonitrile, formic acid and
dithiothreitol (DTT) were obtained from Thermo Fisher Scientific, Melbourne, Victoria,
Australia.
Phosphate buffered saline (P3813), pancreatin from porcine pancreas (P6976), pectin from
citrus peel (P9135), mucin from porcine stomach (M1778), pepsin from porcine gastric mucosa
(P6887), L-cysteine HCl (C7880), bile extract from porcine (B8631), soluble potato starch
132
(S2004), yeast extract (09182), casein (C3400), guar (G4129), Tween 80, De Man, Rogosa and
Sharpe agar (MRS), plate count agar (PCA) and Anaerocult® A were purchased from Merck
Millipore, Australia. Anaerobic indicator strips (BR55, Oxoid®), AnaeroGen® sachets (AN
0010W, Oxoid ®), peptone (LP0034B) and tryptone (LP0042R) were purchased from Thermo
Fisher Scientific, Victoria, Australia. BD Gas pak™ anaerobic jars (3.5 L) were purchased
from BD Australia, NSW, Australia. Nitrogen gas (purity 4.0, O2 free N2) was purchased from
Coregas pty Ltd, Victoria, Australia. HPLC gradient grade sulfuric acid (84733) and
dipotassium phosphate (17835), phosphoric acid, metaphosphoric acid, kidney acetone powder
porcine type II (K7250), folinic calcium salt (F7878), formylfolic acid (F0380000), 5-
methyltetrahydrofolic acid disodium salt (M0132), and ascorbic acid (95210) were purchased
from Sigma-Aldrich Co, NSW, Australia.
For laboratory analysis, polyphenol extractions and HPLC mobile phase, ultrapure water
(Milli-Q® water) was used. Milli-Q® water was generated from a Millipore Milli-Q Ultrapure
Water Purification System (ZMQP60001), Massachusetts, United States. HPLC grade
(≥99.0%) reference standards; callistephin chloride (pelargonidin-3-glucoside (Pel-3-Glu)), p-
coumaric, 6-O-p-Coumaroyl-1,2-digalloylglucose, ferulic acid, and tyrosol were also
purchased from Sigma-Aldrich Co, NSW, Australia. HPLC grade reference standards;
pelargonidin-3-rutinoside chloride (Pel-3-Rut) and quercertin-3-O-glucuronide (quercetin)
were purchased from Extrasynthase, Genay Cedex, France.
8.2.3.2. Strawberries
Strawberries used in the current study were the produce of strawberry plants grown in
controlled environment (CE) chambers (Model: TPG-2400-TH-CO2, Thermoline Scientific
Equipment Pty. Ltd., Wetherill Park, NSW, Australia) at Parkville Campus of The University
133
of Melbourne, Australia as described by Balasooriya, Dassanayake, Seneweera, and Ajlouni
(2018). Briefly, strawberry cultivar ‘San Andreas’ was grown under ambient (400 ppm [CO2]
and 25 ºC) and elevated (950 ppm [CO2] and 30 ºC) growth conditions inside automated CE
chambers. Good quality fruits with 90% red colour were harvested separately and divided into
two equal quantities. One part was used as fresh fruits, and the second was frozen and stored
at -20 ºC for at least a week before testing.
8.2.3.3. Preparation of Simulated Gastric Fluid and Simulated Intestinal Fluid
The simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) solutions were prepared
according to the U.S. Pharmacopeia Convention (United States Pharmacopeia Convention,
2009). The SGF solution consisted of 2 g of sodium chloride (NaCl) and 7 mL of hydrochloric
acid (HCl) (37%, w/v) dissolved in 800 mL of Milli-Q water. The pH of the solution was
adjusted to1.23 using 1 M NaOH. Freshly prepared pepsin (6.4 mg/mL) was added to the SGF
solution at the beginning of each experiment and stirred for 15 min before use.
The SIF was prepared using 5 mM phosphate buffered saline (PBS), 0.4 M NaCl and 15 mM
CaCl2. The pH of SIF was adjusted to 6.8 by using 1 M NaOH and 1 M HCl. The SIF solution
was stirred immediately before usage in order to keep CaCl2 in solution. The fasted (the period
before a meal) and fed (the period following a meal) state SIF were prepared with 2.5 mg/mL
and 40 mg/mL bile concentrations, respectively (Fu et al., 2015). The pancreatin was freshly
prepared in 5 mM phosphate buffer and mixed in the SIF solution to a concentration of 10
mg/mL before use. Prepared solutions were incubated with constant shaking at 37 °C prior to
experiment. The general experimental procedure for the study is illustrated in Fig 1.
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8.2.3.4. In vitro gastrointestinal digestion
Strawberry samples (5 g of fresh) or equivalent weight of frozen (-20 °C) strawberries were
treated following the same procedures. The weighed sample was mixed with 15 mL of
simulated gastric fluid (SGF, 2 g NaCl and 7 mL 37% HCl per litre, pH 1.23) containing 3.2
mg/mL pepsin, and incubated in a water bath with 100 rpm at 37 °C for 2 hrs (Fu et al., 2015).
The mixture was then adjusted to pH 6.5 with 1 M NaOH and combined with 9.6 mL of
simulated intestinal fluid (SIF) containing 3 mL of 2 M NaCl, 0.3 mL of 0.075 M CaCl2 and
6.3 mL of 2.5 or 40 mg/mL bile extract in 5 mM phosphate buffer (Fu et al., 2015). The pH
was adjusted to 6.8, and then 5.4 mL of 10 mg/mL pancreatin in 5mM phosphate buffer was
added. Samples were incubated at 37 °C, 100 rpm for 3 hrs and then placed in an ice bath to
stop the enzyme activity (Fu, 2015).
The digested sample was centrifuged (Allegra® X-126 Centrifuge, Beckman Coulter) at 10976
g for 10 mins to separate soluble and insoluble fractions for HPLC-UV analysis. The amounts
of individual polyphenols, total folate and vitamin C in the supernatants (bioaccessible
fraction/soluble) were quantitated. The residual pellet (sediments/insoluble) was separated
from digested samples and kept at -20 °C. One part of residual pellet was used for in vitro
colonic fermentation and the rest was used in quantifying the micronutrients (folate and vitamin
C) using HPLC-UV.
8.2.3.5. Colonic Fermentation
8.2.3.5.a. Faecal slurry preparation
Faecal slurries (FS); FS 1 and FS 2) were prepared using freshly void faeces from two healthy
females aged 30 yr, who did not have history if any intestinal disease, maintained a diet free of
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polyphenol and folate supplements and have not consumed any probiotics or antibiotics for at
least 3 months. Ethical approval for the study was obtained from the Ethics Advisory Group in
the Faculty of Veterinary and Agricultural Sciences, The University of Melbourne (Ethics
Approval ID; 1750292). Faecal sample (20 g) was weighed into a stomacher bag and mixed
with 80 g sterilized, anaerobic (pre-N2 flushed), 0.1 M phosphate buffer (pH 7.0) to make 20%
w/w faecal slurry. The mixture was then homogenized for 5 min in a stomacher mixer (Bag
Mixer® 400P) and filtered through sterile muslin cloth to remove particulate matter. Faecal
slurry was then transferred to 50 mL sterile, pre-N2 flushed tubes with 5 mL aliquots using
sterile pipette. The faecal slurry in tubes were used for experiment on the same day or stored
in -80 °C for analysis within a week. All work involving faecal samples were carried out
aseptically under bio-safety chamber following the method by (Fu et al., 2015).
8.2.3.5.b. Bacterial enumeration
Aerobic and anaerobic bacteria were enumerated in triplicate samples before and after the
fermentation. The enumeration was started by mixing 5 mL faecal slurry with 5 mL of sterile
basal medium. The mixture was serially diluted to 10-10 in sterile buffered peptone water (0.1%)
containing 0.5 g/L L-cysteine HCl. The procedure was also performed after the 24 hrs
fermentation.
From each selected dilution 100 µL was spread plated onto each of three plates of De Man,
Rogosa and Sharpe (MRS) agar and four plates of plate count agar (PCA) following the method
of Fu et al. (2015). PCA plates were incubated aerobically at 37 °C for 48 hrs. MRS plates were
incubated anaerobically using anaerobic chamber containing anaerobic gas generator and
indicator, at 37 °C for 48 hrs. Selected dilutions with plate counts between 30 and 300 colonies
were used for bacterial enumeration.
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8.2.3.5.c. In vitro colonic fermentation
The fermentation medium (1 L) was prepared following the method of (Fu et al., 2015) and
contained 5 g of soluble starch, 5 g of peptone, 5 g of tryptone, 4.5 of yeast extract, 4.5 g of
NaCl, 4.5 g of KCl, 2 g of pectin, 4 g of mucin, 3 g of casein, 1.5 g of NaHCO3, 0.8 g of L-
cysteine HCl, 1.23 g of MgSO4• 7H2O, 1.0 g of guar, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.4
g of bile salts, 0.11 g of CaCl2 and 1mL of Tween 80. The fermentation medium was sterilized
at 121 °C for 20 min before use. The pH of the sterilized fermentation medium was adjusted to
7.0 at 25 °C.
The fermentation samples were prepared by mixing the residual sediment (0.5 ± 0.1 g) obtained
from digested strawberry samples (section 2.4) with either 5 mL of sterilized fermentation
medium and faecal slurry (20%, w/w) already prepared and mixed at ratio of 1:1. Blank was
prepared mixing 5 mL faecal slurry in buffered saline with 5 mL of sterilized fermentation
medium without sample. All fermentation samples were placed in 15 mL tubes, flushed with
O2 free N2 and closed with caps prior to anaerobic incubation using a 3.5 L incubation jar
containing Anaerocult® A gas generator and anaerobic indicating paper. The fermentation
samples were anaerobically incubated at 37 ºC for 24 hrs. After 24 hrs fermentation, pH,
individual polyphenols, total folates and vitamin C presented in fermented samples were
analysed. The numbers of bacteria introduced into fermentation samples and remained after 24
hrs of fermentation were also enumerated. All experiments were triplicated with duplicate
measurements within each trial. Finally, soluble (supernatant) fraction after centrifuge (10976
g) was separately extracted for HPLC determination of individual polyphenol, total folate and
vitamin C as described in Balasooriya et al. (2019b) and Balasooriya et al. (2019a). The
amounts of individual polyphenols, total folates and vitamins in the soluble fraction of
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fermented strawberry digesta were analysed separately for two faecal slurries (FS 1 and FS 2).
The results were presented as the averages of both analyses.
8.2.3.6. HPLC methods for analyzing of individual polyphenols and vitamins
8.2.3.6.a. Extraction and quantification of individual polyphenols
Polyphenol compounds in simulated gastrointestinal (SGI) digests of strawberry samples were
extracted using 70% methanol and 0.18 N HCl as described by Tow, Premier, Jing, and Ajlouni
(2011) with some modifications. Supernatant (5 mL) or residual sediment (1 g) of SGI digests
or supernatant (5 mL) of fermented SGI digests was homogenised (Ultra Turrax homogenizer,
Janke and Kunnel, IKA-Labortechnik Ultra-Turrax T25) with 7.5 mL of 70% methanol and
2.5 mL of 0.18 N HCl. The supernatants of the mixtures were collected quantitatively after
centrifuged at 10976 g for 15 min at room temperature. The collected supernatant was
concentrated under vacuum in a rotary evaporator at 60 C with 8 rpm and finally, toped up to
exactly 5 mL in a volumetric flask using Milli-Q water and. An aliquot (2 mL) of the extract
was filtered through 0.45 µm membrane when used for the analysis of individual polyphenol
compounds using HPLC-UV. Briefly, a Gemini C18 silica 250×4.6 mm, 5 µm column
(Phenominex Inc., Lane Cove West, NSW, Australia) connected to a HPLC system equipped
with Water 2690 Alliance Separation Module (Waters, Rydalmere NSW, Australia) and
coupled with a Waters 2998 Photodiode Array (PDA) Detector was used in separation of
polyphenols. Milli Q water, (A) and acetonitrile (B) acidified with 0.2% (v/v) formic acid were
used as the mobile phases. The gradient was 5% B for 1 min, 10% B at 10 min, 13% B at 15
min, 20% B at 20 min, 30% B at 25 min, 100% B at 35 min, and returned to 5% B at 40 min.
The injection volume was 20 µL and the flow rate was maintained at 1 mL/min at room
temperature. Identification of individual polyphenol peaks was based on both internal and
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external standards (Balasooriya et al., 2019b). Different polyphenol compounds were detected
and quantified at 280 nm wavelength.
8.2.3.6.b. Extraction and quantification of folates
Briefly, 5 mL of supernatant or 1 g of residual sediment of strawberry digests or supernatant
(5 mL) of fermented SGI digests was thoroughly homogenised with 5 mL of 10% phosphoric
buffer (pH 6.8). The homogenate was kept in a water bath at 100 C for 10 mins and rapidly
cooled down in an ice bath before adjusted the pH to 4.9. Then the mixture was incubated at
37 C for 3 hrs with kidney acetone powder (1.5 mL of 5 mg/mL solution). The samples were
then heated at 100C for 5 mins to terminate the enzyme activity and rapidly cooled in ice
(Lebiedzinska, Dbrowska, Szefer, and Marszall, 2008). The supernatant was collected
separately after the samples were centrifuged at 10976 g for 20 min at 4 °C. Finally, the
supernatant was adjusted to 15 mL in a volumetric flask using Milli-Q water. An aliquant (2
mL) of the extraction was then filtered through 0.45 µm cartridges. Extracted folate from SGI
digests (20 µL) was injected into the HPLC-UV to separate and quantitate different folate
compounds. The mobile phases of 40 mM dipotassium phosphate buffer (A) and (B) 8%
acetonitrile (pH 5.5) were used as a gradient started with 5% B and linearly increased to 50%
B over 5 min, to 70% B over 7 min, followed by recycling to initial conditions over 8 min
(Lebiedzinska et al., 2008). The flow rate was maintained at 0.9 mL/min and folates were
quantified at 290 nm. Three different reference folate standards were used in peak identification
including 10-formyltetrahydrofolic or 10-formylfolic acid (10FFA), 5- formyltetrahydrofolic
acid (5FTHFA), and 5-methyl formyltetrahydrofolic acid (5MTHFA) (Balasooriya et al.,
2019a). The results were recorded as µg of total folate /100 g fresh weight (FW).
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8.2.3.6.c. Extraction and quantification of vitamin C
The supernatant (5 mL) or residual sediment (1 g) of SGI digests or supernatant (5 mL) of
fermented SGI digests was homogenised with 5 mL of 4.5% metaphosphoric acid. The mixture
was centrifuged at 1756 g for 20 mins at 4 °C. The supernatant was collected and brought to a
final volume of 10 mL in a volumetric flask using Milli-Q water and before filtration through
Whatman No. 4 filter paper and 0.45 µm membrane. The filtrate (5 mL) was mixed with 1 mL
of dithiothreitol (DTT) (20 mg\mL) to convert all the dihydro ascorbic acid (DHAA) into
ascorbic acid (AA) form to calculate the total vitamin C. This mixture was kept in dark for
another 2 hrs at room temperature prior used in HPLC analysis. A isocratic 0.01% H2SO4 at
pH 2.6 at 0.9 mL\min was used to quantify the ascorbic acid contents at 245 nm wavelength
(Sanchez Mata, Camara Hurtado, Diez Marques, and Torija Isasa, 2000). The total vitamin C
content of samples was reported as mg or ng/100 g FW.
8.2.3.7. Determination of Bioaccessibility of different bioactive compounds
Bioaccessibility of each micronutrient was determined based on the percentage of soluble
bioactive compounds after the strawberries were subjected SGI. It was calculated as the
percentage of solubilized polyphenol or total folates or vitamin C recovered from the
supernatant after SGI digestion of strawberry relative to total SGI digested strawberry (soluble
+ insoluble) (Sirisena, Ajlouni, and Ng, 2018). The equation as follows;
%Bioaccessibility = Mass of solubilized polyphenol / folates / vitamin C in strawberry digests
Mass of polyphenol / folates / vitamin C in total digested strawberry sample
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Statistical analysis
Statistical analyses were performed using Minitab® 17 statistical software. One-way analysis
of variance (ANOVA) was performed to analyse the collected data and the means were
separated using Tukey’s multiple comparison method at 95% confidence level. Results were
reported as mean ± standard deviation (SD) (n = 6) with 2 significant Figs according to
EURACHEM guidelines (Ellison and Williams, 2012).
Results and Discussion
8.2.5.1. Bioaccessibility of polyphenols
Strawberry is rich with more than 100 different individual polyphenol compounds (La Barbera
et al., 2017) which are different from each other with the degree of digestion and absorption in
the gastrointestinal tract (Ariza et al., 2018). The presence of these compounds in strawberry
fruits could be varied qualitatively and quantitatively due to different factors including cultivar
and growth conditions. Results from a previous study (Balasooriya et al., 2019b) identified and
quantified twelve different polyphenols in strawberry fruits of the cultivar ‘San Andreas’ and
demonstrated the influence of elevated [CO2] and higher temperature on these individual
polyphenols. In this investigation, six selected major polyphenol compounds; Pel-3-Glu, Pel-
3-Rut, p-coumaric, ferulic acid, quercetin and p-coumaroyl were identified (Fig 2) and
quantified in different strawberry digests to determine their bioaccessibility. Pel-3-Glu and Pel-
3-Rut are major anthocyanin compounds released from strawberry fruit matrix and highly
accessible in human stomach (Ariza et al., 2018).
The effects of growth condition (ambient: 400 ppm * 25 ºC and elevated: 950 ppm * 30 ºC),
storage (fresh and frozen) and bile concentration (fed and fasted) on bioaccessibility of soluble
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(free) and insoluble (bound to matrix) polyphenols in strawberry fruits were significant
(P≤0.05). Soluble polyphenols which are therefore accessible in strawberry were analysed in
the supernatant of SGI digesta after centrifugation of simulated gastrointestinal digestion
(SGID) mixture. Insoluble polyphenols were associated with precipitate of centrifuged SGID
mixture which were not accessible during gastric digestion. The amounts of soluble and
insoluble polyphenols and bioaccessibility of individual polyphenol compounds in different
strawberry digests were presented in Table 1.
Polyphenols which were released from food matrix into the SGI fluid due to the action of
digestive enzymes were considered bioaccessible and absorbable. More than 65% of detected
polyphenolic compounds were released to the digestion fluid, recording the highest of 98% of
ferulic acid in fed digesta of fresh strawberries grown at ambient conditions (Table 1).
Generally, strawberry matrix released higher amounts of polyphenols during the fed state than
fasted (Table 1). Increasing the concentration of bile salts during the fed state in the
gastrointestinal fluid was reported to facilitate the solubility of bioactive compounds (Van de
Wiele et al., 2007). The exact amounts of individual polyphenols in the bioaccessible fraction
were significantly (P≤0.05) higher in fresh fruits of strawberries grown under elevated growth
conditions (Table 1). For example; the highest amounts of Pel-3-Glu (19.89±0.4 mg/100 g
FW), Pel-3-Rut (2.55±0.5 mg/100 g FW), p-coumaric (0.23±0.02 mg/100 g Fw), ferulic
(1.33±0.05 mg/100 g FW), quercetin (1.97±0.2 mg/100 g FW) and p-coumaroyl (0.65±0.05
mg/100 g FW) were found in fed state SGI digesta of fresh strawberry grown under 950 ppm
[CO2] and 30 °C conditions (Table 1).
These results were in agreement with those reported by (Kosińska, Diering, Prim, and
Andlauer, 2015) who indicated that among all the strawberry polyphenols, Pel-3-Glu was the
most bioaccessible phenolic compound and it could be absorbed by the gastrointestinal tract in
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its original form. Pel-3-Glu showed significant (P≤0.05) increment in accessibility in fed SGI
digesta of fresh strawberries grown at elevated growth conditions (88%) with comparison to
fresh strawberries grown at ambient growth conditions (67%). The exact amounts of Pel-3-Glu
released to the SGI fluid were 13.86±0.8 mg/100 g FW and 19.89±0.4 mg/100 g FW in fed
state digests of fresh strawberry at grown under ambient and elevated growth conditions,
respectively. Similarly, Kosińska et al. (2015) detected 12 mg of accessible Pel-3-Glu in 100 g
of digested strawberries. Pel-3-Glu was more bioaccessible in fed digesta (19.89±0.4 mg/100
g FW) than in the fasted (8.46±0.5 mg/100 g FW) and fresh strawberries released significantly
higher amount of Pel-3-Glu in compared to frozen strawberries (Table 1). Fresh strawberries
grown under ambient conditions (400 ppm [CO2] and 25 °C) had significantly higher amount
of insoluble Pel-3-Glu at fed state (6.72±0.8 mg/100 g FW) than under elevated conditions
(950 ppm [CO2] and 30 °C) (2.84±0.06 mg/100 g FW).
The amounts of soluble Pel-3-Rut in fed digesta were 1.29±0.3 mg/100 g FW and 2.55±0.5
mg/100 g FW in fresh strawberries grown under ambient and elevated growth conditions,
respectively. These variations represented an increase in the bioaccessibility of Pel-3-Rut by
96% under elevated growth conditions.
The bioaccessibility of p-coumaric was the lowest (89%) at fasted state in digesta of fresh
strawberry grown at normal growth conditions when compared with all other treatments.
However, the percentages bioaccessibility of p-coumaric were greater than all other
polyphenols irrespective to the growth condition, type of fruit or bile salts level in digestive
fluid (Table 1).
According to the results, Pel-3-Glu showed the least % bioaccessibility in all treatments when
compared with other phenolic compounds. However, the same data revealed also that the
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amounts of readily available Pel-3-Glu was the highest in comparison with other polyphenols
(Table 1). Pel-3-Glu has been documented as being the most abundant phenolic form in
strawberry. Mullen et al. (2008) indicated that Pel-3-Glu was the most readily absorbed
anthocyanin in strawberries. Another study by Henning et al. (2010) mentioned that the
absorbed Pel-3-Glu was detected in the plasma and urine in its original form or as derivatives
of pelargonidin such as methylates, glucuronidates or sulfates. However, the pelargonidin-O-
glucuronide form was the most dominant in plasma after consuming strawberries. Similarly,
bioaccessible fractions of quercetin, ferulic acid, p-coumaric also undergo sulfation during the
absorption in the small intestine (Kern et al., 2003; Mullen et al., 2008). It has been revealed
also that p-coumaroyl may first be converted into p-coumaric and then found as
dihydrocoumaric acid sulphate (Kosińska et al., 2015).
In a previous study (Balasooriya et al., 2019b), the authors reported that elevated [CO2] (950
ppm) and increased temperature (30 °C) significantly increased the content of polyphenols in
strawberries. That increased amounts of individual polyphenols could be the primary source
associated with increased bioaccessibility of polyphenols in strawberries grown under elevated
growth conditions. Similar effect was demonstrated by Carkeet, Clevidence, and Novotny
(2008) that increased bioavailability of anthocyanins including Pel-3-Glu in strawberry with
increased dose of pureed strawberries. Ferruzzi and colleagues (2009) also reported higher
bioavailability of polyphenols such as gallic acid and catechins from higher doses of
polyphenol extract of grape seed with repeated dosing than taking a single dose. Carkeet et al.
(2008) reported a positive relationship between the intake dose and the absorption and
metabolism of bioactive compounds.
Results from this in vitro bioaccessibility study revealed that the soluble fractions of different
phenolic compounds varied in a broad range from 0.19±0.01 mg/100 g FW (p-coumaric) to
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13.86±0.8 mg/100 g FW (Pel-3-Glu) in fed state of fresh strawberries grown under normal
conditions. The differences of bioaccessibility of different phenolic compounds would be
influenced by dietary fiber presence in strawberries (Cassani, Gerbino, Moreira, and Gómez,
2018). Dietary fibre is well known to delay the absorption and negatively affect the
bioaccessibility of bioactive compounds (Palafox, Ayala, and González-Aguilar, 2011)
depending on individual compounds. For example, bioavailability of ferulic acid could be
significantly low due to cross-linkages of fibre fraction with arabinoxylans and lignin (Adam
et al., 2002) but, bioavailability of quercetin was enhanced significantly by pectin associate to
food matrix (Tamura et al., 2007). However, a recent study by Velderrain et al. (2016) reported
that dietary fibre rich fruits including mango, papaya and pineapple did not show any limitation
in polyphenol bioaccessibility.
In this study, bioaccessibility of individual polyphenols in strawberries was varied in between
67% (Pel-3-Glu) and 98% (ferulic acid). The different phases of human digestion including
gastric and intestinal digestion separately would also alter the bioaccessibility of polyphenols
in strawberry. Bouayed, Bouayed, Hoffmann, and Bohn (2011) found that 65% of polyphenols
absorbed by stomach and only 10% by the intestine. Both anthocyanins; Pel-3-Glu and Pel-3-
Rut were readily available during the gastric digestion however, intestinal digestion could
reduce their accessibility due to the instability of the anthocyanins under alkaline conditions
(Ariza et al., 2018). Similarly, Tagliazucchi et al. (2010) observed higher stability of
anthocyanins during acidic condition in stomach in compared to intestine. The pH changes in
the digestive fluid in stomach and intestine can degrade about 97% of anthocyanins by
transferring them into other forms like chalcone pseudobase (visibly non-red compound). As a
result, anthocyanins are not fully bioavailable in human (Hollands, Brett, Dainty, Teucher, and
Kroon, 2008). Further, these variations in pH values can change the structure of polyphenols
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and therefore influence the antioxidant activity (Tagliazucchi et al., 2010). Because of the low
bioaccessibility and absorption of anthocyanins due to alkalinity in small intestine, most
polyphenols remained bind to the insoluble fraction or precipitate and reached to the colon.
Finally, these phenolic compounds may be subjected to degradation by colonic microflora and
absorbed in the colon.
8.2.5.2. Bioaccessibility of vitamins
In addition to polyphenols, bioaccessibility of folates and vitamin C in fruits of strawberries
grown under ambient and elevated growth conditions was studied. The HPLC chromatographs
representing folates and vitamin C are shown in Fig 3. The main identified folate compounds
in the digested strawberry samples were 10FFA, 5FTHFA, and 5MTHFA, and total content of
folates were calculated as the summation of individual compounds. However, 5FTHFA was
the predominant folate compound found in digested strawberry (Fig 3A). Stability of folate
molecules are considerably different, amongst 5FTHFA has the highest stability (Witthoft,
1999) which would be the reason behind detecting folates more in the form of 5FTHFA.
Bioaccessibility of folates which ranged from 90% to 95% was comparatively higher than the
bioaccessibility of vitamin C which varied from 67% to 87% in strawberries (Table 2). Vitamin
C is highly instable during in vitro SGID and 65% decrease in vitamin C in strawberries was
reported by Cassani et al. (2018). Presence of competing substances like D-glucose and
reactions with components like nitrites, iron or copper could be the reasons behind lower
bioaccessibility of vitamin C (Davey et al., 2000). Fresh strawberries grown under elevated
growth conditions released significantly higher amounts of vitamins (both vitamin C and
folate) in comparison with the fruits grown at ambient growth conditions (Fig 4). Fresh
strawberries grown under ambient conditions contained 93.09±6.2 µg/100 g FW total folates
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(Fig 4A) and 18.55±0.5 mg/100 FW vitamin C (Fig 4B) in the bioaccessible fraction in fed
state. However, elevated growth conditions enhanced soluble folates up to 188.63±7.5 µg/100
g FW and vitamin C up to 30.48±0.3 mg/100 g FW in fed state of fresh strawberries (Fig 4A
and 4B). Similar to polyphenols, enhanced bioaccessibility in vitamins in strawberries was
associated with increased folates and vitamin contents due to increase [CO2] and temperature
(Balasooriya et al., 2019a). Davey et al. (2000) mentioned that increasing the amount of
vitamin per serve can increase the bioaccessibility and lower doses decreased the bioaccessible
fraction. Moreover, higher bioavailability of folates was demonstrated by Wei, Bailey, Toth,
and Gregory (1996) because of the higher concentration of folates in orange. However there
was no advantage of megadoses (≥200 mg per dose) depending on individuals (Davey et al.,
2000). Therefore, manipulating plant growth is one strategy to increase folate and vitamin C
content in fruits hence to increase the bioavailability (Scott, Rbeill, and Fletcher, 2000). Similar
to the polyphenols, soluble folates and vitamin C in SGI digests were significantly (P≤0.05)
higher during fed state than fasted state (Fig 4A and 4B). Strawberries released the highest
amounts of folate (188.63±7.5 µg/100 g FW) and vitamin C (30.5±0.3 mg/100 g FW) during
the fed state in gastrointestinal digestion of fresh strawberries grown under elevated growth
condition.
Bioavailability of vitamin C in oranges, one of the best source of vitamin C, varied from 80%
to 100% in normally ingested doses (Aschoff et al., 2015). In comparison, results from this
study showed that the bioaccessibility of vitamin C ranged between 78% in fasted state to 85%
in fed state in fresh strawberries grown under ambient conditions. However, Pérez, Gil
Izquierdo, and García Viguera (2002) observed more than 95% losses of vitamin C in
pomegranate juice. These losses were mainly associated with pH changes in the gastrointestinal
fluid and presence of oxygen. In the presence of oxygen, DHAA can be irreversibly broken
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down to diketogulonic acid and in the absence of oxygen, lower pH conditions would convert
DHAA into 2-hydroxyfurfural and carbon dioxide predominate (Huelin, Coggiola, Sidhu, and
Kennett, 1971). However, Aschoff et al. (2015) confirmed that both AA and DHAA in orange
juice were stable at lower pH condition. Moreover, individual differences like gastric emptying
rate and age and habits can influence the bioaccessibility of vitamin C (Brubacher, Moser, and
Jordan, 2000). Additionally, it had been suggested that vitamin C absorption can be improved
by taking it in divided and small doses rather than a single dose (Yung, Mayersohn, and
Robinson (1981).
8.2.5.3. Effect of freezing on the bioaccessibility of polyphenols and vitamins in
strawberry
The soluble amounts of individual polyphenols (Pel-3-Glu, Pel-3-Rut and ferulic acid) and
vitamins were significantly (P≤0.05) lower in frozen strawberries (at -20 °C for seven days)
compared to fresh (Table 1 and 2). However, the patterns in bioactive compounds
bioaccessibility was dependent on the insoluble fraction. For instance; bioaccessibility of Pel-
3-Glu and quercetin in frozen fed strawberries were increased up to respectively 75% (from
67% in fresh fed) and 79% (from 76% in fresh fed) while no differences were found for Pel-3-
Rut and folates (Table 1 and 2). Azzini, E et al. (2010) attributed the increases in quercetin
and kaempferol compounds in stored strawberries (+4 °C for 4 days) than fresh strawberries to
the degradation of cell structures hence their higher extractability. However, Azzini, E et al.
(2010) also observed increases in bioavailability of Pel-3-Glu due to higher amount of Pel-3-
Glu presented in fresh strawberries in compared to stored (+4 °C for 4 days) strawberries.
However, bioaccessibility of p-coumaric, ferulic acid, p-coumaroyl and vitamin C compounds
were reduced under frozen storage (Table 1 and 2). However, Sahari, Mohsen Boostani, and
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Zohreh Hamidi (2004) observed 65%, 11% and 9% vitamin C reductions during first 15 days
of storage at -12, -18 and -24 °C, respectively. Additionally, Cassani et al. (2018) observed
further decrease in vitamin C by 30% during SGI digestion. However, Azzini, E et al. (2010)
reported that there was no significant difference in bioavailability of vitamin C in fresh and
stored strawberries. The decline in vitamins bioaccessibility in frozen strawberries detected in
this investigation could be attributed with lower amounts of vitamins found in frozen fruits to
be the result of oxidative degradation of vitamins as reported by Rickman, Barrett, and Bruhn
(2007). Similarly, Oliveira et al. (2016) reported that the formation of ice crystals during
freezing can damage the food matrix and rupture the cell walls leading to increases in the
cytoplasm leaching out, oxidation and degradation of some components. Additionally, such
phenomenon, and the significant loss of nutrients from frozen fruits have been reported to be
more obvious in vulnerable and soft fruits like strawberries (Davey et al., 2000).
Many others studies have demonstrated the decline in bioactive compounds bioaccessibility
during the refrigeration and freezing storage. Cassani et al. (2018) observed significant
decrease in bioaccessibility of polyphenolic compounds and vitamin C in stored strawberries
(at 5 °C) due to enzymatic and chemical oxidations. Similarly, Dalmau et al. (2017) reported
decrease in polyphenolic amounts after SGID in frozen apples (-196 °C in liquid nitrogen).
Moreover, polyphenols favored to precipitate in strawberry fiber during the storage hence
caused less bioaccessible vitamin C (Cilla et al., 2012). However, Johannesson et al. (2002)
mentioned that freezing strawberries at -20 °C showed insignificant difference in
bioaccessibility when compared with the fresh strawberries. On the contrary, Azzini, E et al.
(2010) concluded that storage conditions or conservation strategies can alter the quantity,
quality and bioaccessibility of bioactive compounds present in fruits in general. However, the
changes would not only be dependent on individual compound, but also strongly dependent on
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the type of fruit (Rickman et al., 2007). For example; anthocyanins in fruits were easily
degradable during frozen storage, but, the impact of freezing was comparatively lower in their
amounts in red fruits (González, de Ancos, and Cano, 2003).
8.2.5.4. Colonic Fermentation
Digestive enzymes present in the gastrointestinal fluid are not able to release all the
micronutrients from the food matrixes (Palafox et al., 2011). Therefore, non-digested fraction
of strawberries (precipitate) may contain unabsorbed micronutrients and compounds bound to
dietary fiber and macro molecules like protein and carbohydrates. These matrixes bound
micronutrients are not bioavailable in the upper part of the digestive tract, but can reach to the
colon and undergo microbial degradation (Ariza et al., 2018). Human colon has approximately
1.5 kg of bacteria in 1012 cells/g density. These colonic microflora play an important role
comparable to liver and can metabolize the polyphenols into other bioactive forms and the
resulting metabolites like aglycones may be reabsorbed or further metabolites into simple
aromatic compounds (Ariza et al., 2018). These unabsorbed bioactive compounds and their
metabolites play an important role in the human colon and help maintaining a healthy
environment by scavenging the free radicals (Palafox et al., 2011). This current study analyzed
the supernatant (soluble compounds) of fermented precipitate of strawberry following the
SGID digestion, which has not been reported elsewhere. The results revealed that folate,
vitamin C, Pel-3-Glu, p-coumaric, ferulic acid and 2-(4-Hydroxyphenyl) ethanol (Tyrosol)
were identified in the soluble fraction (after centrifugation) of fermented precipitate of fresh
and frozen strawberry samples subjected to colonic fermentation (Fig 5). Higher contents of
folate (7.90±0.05 µg/100 g FW), vitamin C (33.6±1.0 ng/100 g FW), Pel-3-Glu (2.00±0.14
mg/100 g FW), and p-coumaric (39±5 µg/100 g FW) were detected in the soluble fraction of
150
fermented precipitate at fasted state of frozen strawberries grown under ambient conditions
(Table 3). In colonic fermented strawberries, Pel-3-Glu and ferulic acid were higher than other
phenolic compounds reported in this study. The detected amounts of phenolic compounds and
their metabolites were significantly (P≤0.05) greater in the fasted state than the fed (Table 3).
Tyrosol which has been reported to be the main microbial metabolite derived from Pel-3-Glu
in strawberry after colonic fermentation (López, Mosele, Macià, Ludwig, and Motilva, 2017),
was significantly higher in the soluble fraction of fermented precipitate that has been already
subjected to the SGID of fresh and frozen strawberries grown under elevated growth
conditions. The tyrosol content in frozen strawberry grown under elevated [CO2] and
temperature conditions varied from 30.65±1.37 (fed) to 87.88±4.22 µg /100 g FW (fasted) in
comparison with 59.84±1.75 (fed) and 75.88±3.46 µg/100 g FW (fasted) in fresh strawberry.
Other metabolites such as hydroxyphenylpropionic acid, hydroxyphenylacetic acid and p-
hydroxybenzoic acid have been reported in smaller amounts after the colonic fermentation of
other polyphenolic compounds (López et al., 2017). Moreover, the same authors reported that
those compounds contributed significantly to the death of the colon cancer cells.
No significant difference was found in vitamin C contents in colonic fermented strawberry
precipitates that have subjected to SGID and the amounts were smaller and varied from
31.1±0.5 to 33.6±1.0 ng/100 g FW. Different amounts of folates were recovered in the soluble
fraction of fermented precipitate of strawberry and varied from 4.76±0.08 to 7.92±0.05 µg/100
g FW depending on level of bile salts, storage and growth conditions (Table 3). Higher amounts
of folates were found in fermented strawberry digests in fasted states than fed states except in
fresh strawberries grown under elevated growth (Table 3). This was totally opposite from the
observed folates amounts in fed and fasted states after SGID. The optimum pH for intestinal
folate deconjugation is around 6-7 and pH changes could result incomplete release of folates
151
in intestine (McNulty and Pentieva, 2004). However, decrease in colonic pH might encourage
more active fermentation in colon (Louis, Hold, and Flint, 2014). According to the previous
studies, folates is mainly absorbed by the small intestine but can also be absorbed in the colon
(Kumar, Moyer, Dudeja, and Said, 1997; Zimmerman, 1990). Therefore, the results showed
that higher amounts of total folates in strawberry were bio-accessible during gastrointestinal
digestion and the rest in colonic fermentation. However, a little information was found on the
fate of matrix bound folates and vitamin C in fruits subjected to in vitro colonic fermentation.
8.2.5.5. pH changes and bacterial counts
In addition to the analyses of bioactive compounds and their fermentation products, pH changes
and bacterial counts were also examined. The initial pH values in faecal slurry 1 and 2
(described in section 2.5.1) were 6.8 and 6.9, respectively (Supplementary Table 1). However,
those values decreased in all samples to about 6.6 – 6.7 after 24 hours fermentation, which was
due to the activity of colonic microflora, mainly, lactic acid bacteria. Similar observations and
decline in pH were reported by Fu et al. (2015); Osuka et al. (2012); Sirisena et al. (2018).
Aerobic bacterial counts in the original faecal slurry 1 and 2 were 4.36±0.19 and 5.64±0.71
log10 CFU/mL and did not show considerable changes after 24 hours (Supplementary Table 2).
However, anaerobic bacterial counts increased after 24 hours of colonic fermentation from
8.07±0.19 to 8.85±0.06 and 8.72±0.03 to 9.42±0.20 log10 CFU/mL in initial faecal slurry 1 and
2, respectively. Increased anerobic bacterial counts by around 1 log10 CFU/mL in faecal slurry
indicates that simulated fermentation was favoured more by anaerobic bacteria, such as lactic
acid bacteria.
152
Conclusion
Results from this current in vitro bioaccessibility study showed that the amount of bioaccessible
bioactive compounds in strawberry may differ quantitatively and qualitatively. Additionally,
in vitro bioaccessibility of different micronutrients were affected by growth condition, storage
and different bile salt concentrations in intestinal fluid. Increased carbon dioxide and
temperature in the growth environment enhanced the bioaccessibility of polyphenols, folates
and vitamin C in strawberries. Fresh strawberries can release higher amount of these
compounds than frozen fruits (-20 °C) while higher bile salt contents in fed state encouraged
more release of micronutrients to SGI fluid.
It should be noted also the bioaccessibility is only a pre-requisite of bioavailability but, not
necessary the actual amounts of bioavailable bioactive compounds. Consequently, further
investigations to find out the efficiency of absorption of these bioaccessible compounds from
strawberries grown under elevated [CO2] and high temperature using an in vivo model is
recommended. It is also recommended that similar investigation should be repeated to examine
the response of other fruits to the future anticipated changes in climate conditions.
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163
Figures and Tables
164
Figure 2. HPLC chromatographs for (A) soluble fraction (extracted from supernatant) and (B) insoluble fraction
(extracted from precipitate) of simulated gastrointestinal digesta. Polyphenol compounds and their retention
times (RT) are as follows; (1) Pelargonidin-3-Glucoside (RT- 20.0±0.16), (2) Pelargonidin-3-Rutinoside (RT -
20.8±0.12), (3) p-Coumaric (RT - 24.2±0.80), (4) Ferulic acid (RT - 25.4±0.21), (5) Quercetin-3-O-Glucuronide
(RT - 26.8±0.21), and (6) p-Coumaroyl (RT - 27.4±0.22) at 280 nm wavelength.
165
B A
Figure 3. HPLC chromatographs for vitamin identification in soluble fraction (extracted from supernatant)
of simulated gastrointestinal digesta of strawberry subjected to in vitro gastric digestion.
(A)Folate derivatives and their retention times (RT±SD) are as follows; 1 – 10FFA (12.2±0.5), 2 –
5FTHFA (13.6±0.4), and 3 – 5MTHFA (16.9±0.5) at 290 nm wavelength.
(B) Peaks are; 1 – Extraction solvent (K2HPO4), 2 – AA (RT±SD – 3.1±0.1), 3,4 – unknown.
166
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250
Fed Fasted Fed Fasted
Fresh Frozen
Solu
ble
Fo
late
co
nte
nt
(µg
/ 1
00
g F
W)
Strawberry SGI digests
(A) FolateAmbient
Elevated
Figure 4. Soluble fraction (extracted from supernatant) of folates (A) and vitamin C (B) in simulated
gastrointestinal (SGI) digesta of strawberry subjected to in vitro gastric digestion. Error bars refer to standard
deviation of data (n = 6).
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Figure 5. HPLC chromatographs for colonic fermentation of fresh strawberry samples. (A). HPLC
chromatograph for individual phenolic compounds and their metabolites at 280 nm in fresh strawberry
samples at fed state. Peaks are; 1. Tyrosol; 2. Pel-3-Glu; 3. p-Coumaric; 4. Ferulic acid. (B). HPLC
chromatograph for folates at 290 nm in fresh strawberry samples at fed state. Peak 1. MTHFA (C)
HPLC chromatograph for vitamin C at 245 nm in fresh strawberry samples at fed state. Peaks are; 1.
K2HPO4; 2. Ascorbic acid; 3. Unknown. (Note: Frozen strawberry samples showed very similar
HPLC chromatograms to above after their colonic fermentation therefore not shown here).
A B
C
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Table 1. Quantification of free and bound polyphenols from fresh and frozen strawberry fruits grown
at ambient (400 pm and 25 ºC) and elevated (950 ppm and 30 ºC) growth conditions.
Polyphenol compound Pel-3-Glu Pel-3-Rut p-Coumaric Ferulic Acid Quercetin p-Coumaroyl
Strawberries grown at 400 ppm* 25 ºC
Fresh
Fed
FPP 13.86±0.8c 1.29±0.3bcd 0.19±0.01b 1.02±0.05b 1.38±0.1b 0.28±0.04e
BPP 6.72±0.8A 0.08±0.00CD 0.011±0.001AB 0.016±0.002C 0.44±0.03 0.019±0.004D
TPP 20.58 1.37 0.201 1.036 1.82 0.47
Accessibility 67% 94% 95% 98% 76% 93%
Fasted
FPP 5.84±0.1g 0.67±0.1d 0.20±0.02ab 0.73±0.04e 1.12±0.1bc 0.37±0.08de
BPP 2.89±0.3BC 0.08±0.00D 0.013±0.002A 0.152±0.002B 0.20±0.00 0.026±0.004CD
TPP 8.73 0.75 0.213 0.882 1.32 0.396
Accessibility 67% 89% 89% 83% 85% 93%
Frozen
Fed
FPP 11.63±0.3d 2.00±0.3ab 0.18±0.03b 0.88±0.06d 1.40±0.1b 0.29±0.05e
BPP 3.83±0.5B 0.12±0.01B 0.013±0.001A 0.127±0.007B 0.37±0.08 0.04±0.007B
TPP 15.46 2.12 0.193 1.007 1.77 0.33
Accessibility 75% 94% 93% 87% 79% 88%
Fasted
FPP 5.46±0.2g 1.12±0.2cd 0.18±0.02b 0.70±0.04e 1.35±0.3b 0.32±0.07e
BPP 2.87±0.6BC 0.10±0.00C 0.010±0.005AB 0.29±0.03A 0.47±0.01 0.047±0.004B
TPP 8.33 1.32 0.19 1.02 1.82 0.367
Accessibility 66% 85% 95% 72% 74% 87%
Strawberries grown at 950 ppm* 30 ºC
Fresh
Fed
FPP 19.89±0.4a 2.55±0.5a 0.23±0.02a 1.33±0.05a 1.97±0.2a 0.65±0.05a
BPP 2.84±0.06BC 0.12±0.01B 0.014±0.005A 0.29±0.04A 0.84±0.01 0.091±0.004A
TPP 22.73 2.67 0.244 1.62 2.81 0.741
Accessibility 88% 96% 94% 82% 70% 88%
Fasted
FPP 8.46±0.5e 1.50±0.8bc 0.12±0.02c 0.83±0.06d 0.90±0.06c 0.45±0.09cd
BPP 2.51±0.2C 0.22±0.00A 0.012±0.001AB 0.05±0.00C 0.64±0.001 0.044±0.007B
TPP 10.97 1.72 0.232 0.92 1.54 0.494
Accessibility 77% 87% 95% 95% 58% 91%
Frozen
Fed
FPP 17.14±0.8b 2.53±0.9a 0.21±0.01ab 0.97±0.01bc 1.28±0.2bc 0.61±0.03ab
BPP 2.05±0.05C 0.08±0.00CD 0.005±0.000AB ND ND ND
TPP 19.19 2.61 0.215 - - -
Accessibility 89% 97% 98% - - -
Fasted
FPP 7.00±0.6f 1.82±0.3abc 0.25±0.02a 0.90±0.04cd 0.94±0.1c 0.52±0.04bc
BPP 2.54±0.4C 0.09±0.00CD 0.006±0.000B 0.053±0.003C ND 0.035±0.005BC
TPP 9.54 1.91 0.26 0.95 - 0.56
Accessibility 73% 95% 98% 94% - 94%
FPP – Free polyphenols (soluble polyphenols) associate with the supernatant of SGID
BPP – Bound polyphenols (insoluble polyphenols) associate with the precipitate of SGID
Accessibility% = FPP / (FPP+BPP) * 100
All the amounts of polyphenolic compounds are reported in mg / 100 g of FW
Values are mean ± SD (n=12), different letters a,b,c,.. and A,B,C in each column are significantly (p ≤ 0.05) different.
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Table 2. Quantification of free and bound folates and vitamin C from fresh and frozen strawberry fruits
grown at ambient (400 pm and 25 ºC) and elevated (950 ppm and 30 ºC) growth conditions
Folate Vitamin C
µg/100g FW mg/100g FW
Strawberry grown at 400 ppm* 25 ºC (ambient)
Fresh
Fed
Free vitamins 93.09±6.2d 18.55±0.5f
Bound vitamins 8.32±0.06C 3.29±0.07F
Total vitamins 101.41 21.84
Accessibility% 92 85
Fasted
Free vitamins 75.40±5.1e 15.51±0.7g
Bound vitamins 7.20±0.14D 4.46±0.09E
Total vitamins 83.71 19.97
Accessibility% 91 78
Frozen
Fed
Free vitamins 92.58±5.6d 21.01±0.6e
Bound vitamins 8.31±0.2C 7.24±0.07D
Total vitamins 100.89 28.25
Accessibility% 92 74
Fasted
Free vitamins 66.02±2.8e 20.90±0.7e
Bound vitamins 7.30±0.08D 8.28±0.2C
Total vitamins 73.32 29.18
Accessibility% 90 72
Strawberry grown at 950 ppm* 30 ºC (elevated)
Fresh
Fed
Free vitamins 188.63±7.5a 30.48±0.3a
Bound vitamins 10.60±0.3B 4.65±0.7E
Total vitamins 199.23 35.13
Accessibility% 95 87
Fasted
Free vitamins 161.38±11.7b 27.90±0.1b
Bound vitamins 14.69±0.8A 4.60±0.1E
Total vitamins 176.07 32.5
Accessibility% 92 86
Frozen
Fed
Free vitamins 150.85±7.8bc 26.20±0.4c
Bound vitamins 11.20±0.8B 12.90±0.9A
Total vitamins 162.05 39.1
Accessibility% 93 67
Fasted
Free vitamins 128.71±3.8c 24.40±0.3d
Bound vitamins 11.36±0.3B 11.65±0.7B
Total vitamins 140.07 36.05
Accessibility% 92 68
Free vitamins – Free vitamins (soluble) associate with the supernatant of SGID
Bound vitamins – Bound vitamins (insoluble) associate with the precipitate of SGID
Accessibility% = Free vitamin / (Free vitamin + Bound vitamin) * 100
Values are mean ± SD (n=12), different letters a,b,c,.. and A,B,C in each column are significantly (p ≤ 0.05) different.
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Table 3. Quantification of micronutrients after colonic fermentation of the precipitate of gastric digests (bound nutrients) from fresh and frozen
strawberry fruits grown at ambient (400 pm and 25 ºC) and elevated (950 ppm and 30 ºC) conditions.
Values are mean ± SD (n=6), different letters a,b,c,.. in each raw are significantly (p ≤ 0.05) different.
Fresh Strawberry Frozen Strawberry
Chemical compounds Ambient growth Elevated growth Ambient growth Elevated growth
Fed Fasted Fed Fasted Fed Fasted Fed Fasted
Vitamins:
Folate (µg/100 g FW) 5.15±0.09f 5.58±0.02
e 7.92±0.05
a 5.88±0.07
d 6.33±0.08
c 7.90±0.05
a 4.76±0.08
g 6.82±0.07
b
Vitamin C (ng/100 g FW) 31.5±1.1a 31.8±2.0
a 33.4±0.5
a 33.2±0.9
a 33.3±0.6
a 33.6±1.0
a 32.3±0.7
a 31.1±0.5
a
Phenolic compounds:
Pel-3-Glu (mg/100 g FW) 1.46±0.06b 2.10±0.17
a 1.24±0.11
c 1.58±1.77
b 0.65±0.09
d 2.00±0.14
a 0.58±0.03
d 0.72±0.03
d
p-Coumaric (µg/100 g FW) 11±2de
25±3b 13±4
d 20±5
bc 15±2
cd 39±5
a 6.3±0.6
e 17±2
cd
Ferulic (µg/100 g FW) 140±8b 181±8
a 105±9
c 146±20
b 87±6
c 157±21
b 49±6
d 98±4
c
2-(4-Hydroxyphenyl) ethanol
(Tyrosol) (µg/100 g FW) 21.90±0.57
f 43.44±1.18
d 59.84±1.75
c 75.88±3.46
b 29.74±1.58
ef 58.73±5.19
c 30.65±1.37
e 87.88±4.22
a
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Table 4. Changes in the pH of the sediment of gastric digests of fresh and frozen strawberry fruits
grown at ambient (400 pm and 25 ºC) and elevated (650 ppm and 30 ºC) conditions after in vitro
colonic fermentation.
Faecal slurry 1 and 2 were prepared separately from faeces collected from two different donors.
Sample sources
pH of fermented samples
In vitro faecal slurry fermentation
t = 0 t = 24
Faecal slurry
1
Faecal slurry
2
Faecal slurry
1
Faecal slurry
2
Faecal slurry in buffer without sample 6.80±0.00 6.90±0.00 6.70±0.00 6.82±0.00
Faecal slurry in basal media without sample 6.73±0.03 6.77±0.05 6.48±0.01 6.54±0.01
Under Ambient Growth Conditions (400 ppm and 25 ºC)
Fresh Strawberry Fasted 6.65±0.00 6.70±0.00 5.49±0.02 5.45±0.03
Fresh Strawberry Fed 6.63±0.01 6.69±0.00 5.62±0.02 5.68±0.04
Frozen Strawberry Fasted 6.61±0.00 6.69±0.01 5.38±0.05 5.42±0.04
Frozen Strawberry Fed 6.61±0.00 6.69±0.00 5.54±0.04 5.58±0.00
Under Elevated CO2 and Temperature (650 ppm and 30
ºC)
Fresh Strawberry Fasted 6.67±0.00 6.73±0.02 5.20±0.01 5.25±0.03
Fresh Strawberry Fed 6.66±0.00 6.70±0.02 5.24±0.03 5.53±0.04
Frozen Strawberry Fasted 6.65±0.00 6.66±0.01 5.29±0.04 5.39±0.04
Frozen Strawberry Fed 6.65±0.00 6.67±0.01 5.50±0.05 5.56±0.05
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Table 5. Anaerobic and aerobic bacterial counts in gastric digests of fresh and frozen strawberry fruits grown at ambient (400 pm and 25 ºC) and
elevated (650 ppm and 30 ºC) conditions after being subjected to in vitro colonic fermentation.
Faecal slurry 1 and 2 were prepared separately from faeces collected from two different donors as described in section 2.5.3.
Sample sources
Faecal slurry 1 Faecal slurry 2
Anaerobic bacterial counts
(log10 CFU/ml)
Aerobic bacterial counts
(log10 CFU/ml)
Anaerobic bacterial counts
(log10 CFU/ml)
Aerobic bacterial counts
(log10 CFU/ml)
Before
fermentation
After
fermentation
Before
fermentation
After
fermentation
Before
fermentation
After
fermentation
Before
fermentation
After
fermentation
Faecal slurry in buffer without sample 8.07±0.19 8.85±0.06 4.36±0.19 4.46±0.15 8.72±0.03 9.42±0.20 5.64±0.71 5.68±0.09
Faecal slurry in basal media without
sample 7.00±0.30 8.56±0.05 4.36±0.19 4.49±0.26 7.33±0.60 8.40±0.30 5.64±0.71 5.74±0.15
Under Ambient Growth Conditions (400
ppm and 25 ºC)
Fresh Strawberry Fasted 7.00±0.30 8.56±0.05 4.36±0.19 4.56±0.11 7.33±0.60 8.47±0.11 5.64±0.71 5.67±0.12
Fresh Strawberry Fed 7.00±0.30 8.39±0.14 4.36±0.19 4.60±0.19 7.33±0.60 8.44±0.19 5.64±0.71 5.74±0.19
Frozen Strawberry Fasted 7.00±0.30 8.38±0.12 4.36±0.19 4.56±0.17 7.33±0.60 8.36±0.17 5.64±0.71 5.70±0.13
Frozen Strawberry Fed 7.00±0.30 8.41±0.10 4.36±0.19 4.50±0.18 7.33±0.60 8.50±0.18 5.64±0.71 5.72±0.09
Under Elevated CO2 and Temperature
(650 ppm and 30 ºC)
Fresh Strawberry Fasted 7.00±0.30 8.42±0.03 4.36±0.19 4.60±0.12 7.33±0.60 8.45±0.12 5.64±0.71 5.69±0.10
Fresh Strawberry Fed 7.00±0.30 8.43±0.12 4.36±0.19 4.79±0.03 7.33±0.60 8.38±0.03 5.64±0.71 5.79±0.07
Frozen Strawberry Fasted 7.00±0.30 8.42±0.05 4.36±0.19 4.60±0.15 7.33±0.60 8.37±0.15 5.64±0.71 5.75±0.11
Frozen Strawberry Fed 7.00±0.30 8.41±0.04 4.36±0.19 4.45±0.06 7.33±0.60 8.44±0.06 5.64±0.71 5.74±0.04
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Strawberry is a rich source of antioxidants and thus plays an important role in preventative nutrition.
The antioxidant profile of strawberries is strongly dependant on the genetic make-up, and significantly
affected by growth conditions. Climate change has become a main challenge in agriculture and
predicted to cause varied degrees of impact on quality and quantity of agricultural produce. Elevated
carbon dioxide concentration [CO2] and temperature are the main drivers of climatic changes and
understanding how these critical parameters influence crop growth, productivity and product quality
is critical for the industry to develop strategies for adaptation and mitigation of adverse impacts. This
study evaluated effects of [CO2], temperature and their interactions on strawberry growth,
development and micronutrients profile of two strawberry cultivars (Albion and San Andreas), which
are commonly grown in Victoria and other Australian States. It is anticipated that results from this
study can be used as indicators to predict the response of other fruits to the future predicted climate
changes. Strawberry fruits grown under elevated [CO2] and temperature were not visually attractive
comparing to normal strawberries. However, considering their nutritional value, those fruits can be
promoted as freeze-dried strawberry in value added products like incorporating in dairy.
In general, [CO2] positively affected plant growth and development of both strawberry genotypes due
to greater availability of photosynthetic substrate with increased [CO2], i.e. (650 and 950 ppm).
However, despite the synergistic relation between elevated [CO2] and plant growth and development,
increasing growth temperature reduced strawberry fruit yield. Higher temperature altered the positive
effects of elevated [CO2] while increase in [CO2] failed to compensate for the growth and yield losses
due to the increase in temperature. The results revealed also that strawberry responses to the above
growth conditions were cultivar dependant. Cultivar ‘San Andreas’ yielded more fruits than the
cultivar ‘Albion’ at 25 °C and under ambient CO2 level. However, both cultivars produced very lower
174
yields when they were grown under 30 °C temperature. Strawberry fruits grown at higher temperature
were smaller and uneven in shape while the fruit skin colour became redder and darker. Therefore, this
study suggests that significant increases in average day temperatures and also very high [CO2] (950
ppm) levels significantly affect fruit quality and yield and thereby commercial strawberry cultivations.
Elevated [CO2] and higher temperature individually and interactively increased total polyphenol,
flavonoid, anthocyanin and antioxidants in both strawberry cultivars. HPLC-UV analysis revealed that
individual phenolic compounds of strawberries were also increased by elevated [CO2] and temperature.
This study was able to identify twelve different individual phenolic compounds namely; pelargonidin-
3-glucoside, pelargonidin-3-rututinoside, cyanidin, kaempferol-3-glucoside, kaempferol-3-
glucurnoride, resveratrol, catechin, ferulic acid, quercertin-3,4-di glucoside, quercertin-3-O-
qlucuronide, p-coumaric and p-coumaroyl. However, the responses were significantly altered by the
interaction between elevated [CO2] and higher temperature. Additionally, the observed effects of
[CO2] and temperature were significantly (p<0.05) cultivar dependent. Cultivar ‘Albion’ grown under
30 °C and 950 ppm contained greatest contents of flavonoid and antioxidant and ‘San Andreas’ grown
at 25 °C and 950 ppm had higher contents of total polyphenol and anthocyanin. Since polyphenols,
flavonoids, anthocyanins, and antioxidants are considered as important fruit bioactive compounds,
increasing the contents of such fruit nutrients with increased [CO2], higher temperature and their
interactions would improve strawberry functional properties. Consequently, strawberry grown under
higher temperature (30 °C) and increased [CO2] (650 ppm and 950 ppm), could be of a better
nutritional value.
Folates and vitamin C are important bioactive compounds found in strawberries. However, the studies
addressing the impact of elevated [CO2], higher temperature and their interactions on vitamins in
strawberries have been scarce in recent literature. In this investigation, four different folate derivatives
175
namely; tetrahydrofolic acid (THFA), 10-formyltetrahydrofolic or 10-formylfolic acid (10FFA), 5-
formyltetrahydrofolic acid (5FTHFA), and 5-methyl formyltetrahydrofolic acid (5MTHFA) and
ascorbic acid were identified and quantified in fresh and freeze-dried strawberry samples using HPLC-
UV system. Increases in [CO2], temperature, and their interactions favoured total folate contents of
strawberry fruits however, the responses were cultivar dependent. Although, increases in growth
temperature positively influenced total folates contents of strawberry at lower [CO2] levels, the effects
turned negative at the highest CO2 (950 pm). Additionally, fruits from both strawberry cultivars grown
at the highest [CO2] (950ppm) constantly maintained greater concentrations of 5FTHFA, irrespective
of the temperature level. The relative concentrations of the 5MTHFA folate derivative were always
found to be lower at 950 ppm in both cultivars compared to the other [CO2] levels. These finding will
provide important information on the changes of different folate derivatives under the predicted future
changes in [CO2] and temperature. The analysis of main folates compounds would have a greater value
in studying the absorption, metabolization and bioavailability of folates.
Elevated [CO2], higher temperature and their interactions altered the vitamin C content of strawberries
and the responses were cultivar dependent. Vitamin C content in fresh strawberry fruits was not
influenced by lower levels of [CO2] up to 650 ppm at 25 °C, however, 950 ppm [CO2] at 25 °C
enhanced the vitamin C contents in fresh strawberries. Additionally, increasing the temperature to 30
°C at 950 ppm [CO2] negatively affected the vitamin C content.
Vitamins contents in processed fruits and vegetables vary depending on the methods of storage,
preparation and processing. Most fruits and vegetables are commonly processed and subjected to a
long-term storage before consumption, which contribute to vitamins loss. Strawberries are most often
consumed fresh, hence provide considerable amounts of total folates and vitamin C. However, as a
short shelf-life fruit, strawberry processing and preservation are important and critical postharvest
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practices but may affect its nutrients contents. In order to protect and maintain strawberry vitamins
content, freeze drying (FD) could be considered a good technique for strawberry processing.
Meanwhile, due to the promising results reported from previous studies, freeze dried strawberry
powder has been promoted as better health-enhancing food.
This study demonstrated that elevated [CO2] and higher temperature enhanced polyphenols, folates
and vitamin C levels in strawberries. However, the beneficial effects of those micronutrients are
strongly dependent on their bioaccessibility from food matrixes during digestion. Therefore, the final
stage of this study involved bioaccessibility assessment of selected bioactive compounds in fresh and
frozen strawberries grown under elevated [CO2] and higher temperature. Simulated in vitro gastric and
intestinal digestions with colonic fermentation were used in investigating the bioaccessibility of
polyphenols, folates and vitamin C. Results revealed that six selected major polyphenol compounds
namely; pelargonodin-3-glucoside, pelargonidin-3-rutinoside, p-coumaric, ferulic acid, quercetin and
p-coumaroyl were identified and quantified in different strawberry digests using HPLC-UV. The
soluble and accessible fraction was always greater than insoluble fraction in strawberry. The amount
of bio-accessible bioactive compounds in strawberry differed quantitatively and qualitatively based on
growth condition, storage and physiological variations in individual treatments. Increased [CO2] and
temperature in growth environment enhanced the bioaccessibility of polyphenols, folates and vitamin
C in strawberries. Fresh strawberries released higher amount of these compounds than frozen fruits
while higher bile salts content in fed state encouraged more release of micronutrients to gastrointestinal
fluid. Digestive enzymes present in the gastrointestinal fluid are not able to release all the
micronutrients from the food matrixes. Therefore, non-digested fraction of strawberries (precipitate)
may contain unabsorbed micronutrients and compounds bound to dietary fiber and macro molecules
like protein and carbohydrates. These matrixes bound micronutrients are not bioavailable in the upper
part of the digestive tract but, reaches to the colon for microbial degradation. Colonic microflora can
177
metabolize the polyphenols into other bioactive forms and the resulting metabolites like aglycones
may be reabsorbed or further metabolites into simple aromatic compounds. In this study, folate,
vitamin C, pelargonidin-3-glucoside, p-coumaric, ferulic acid and 2-(4-Hydroxyphenyl) ethanol
(Tyrosol) were identified in soluble fraction (the supernatant) of fermented precipitate of fresh and
frozen strawberry samples after colonic fermentation. In colonic fermented strawberries, pelargonidin-
3-glucoside, ferulic acid were higher than the other detected compounds. These unabsorbed bioactive
compounds by stomach or intestine and their metabolites play an important role in the human colon
by maintaining a healthy environment via scavenging the free radicals. Furthermore, the
bioaccessibility is only a pre-requisite of bioavailability but, it doesn’t necessary represent the actual
amount of bioavailable bioactive compounds all the times. However, the knowledge of bioaccessibility
of bioactive compounds is an important and valuable tool in evaluating their bio-efficacy in the human
body.
The results of the current study contribute to the knowledge and understanding the best management
practices and planning by the strawberry industry in order to prepare and to adapt to the anticipated
future changes in climate and weather patterns.
178
Climate change has become a major challenge in modern agriculture and it is expected to cause
significant reductions in food production in future (IPCC, 2014). In order to identify the potential
impacts of climate change and implement any adaptation techniques, it requires a better understanding
of the behaviour of different crop species under the anticipated climate conditions. Even in the same
crop species, there could be varied responses similar to the observations experienced in this current
research. However, only two strawberry cultivars were involved in this study. Thus, it is recommended
that future investigations should try to cover larger number of cultivars. Information generated would
be more beneficial in breeding and screening programmes to develop cultivars which can succeed
under higher [CO2] and temperature. Introducing new cultivars with improved ability to produce
quality fruits under future climate would protect the commercial fruit growers and future fruit industry
from any unexpected negative effect caused by climate changes. It is also suggested that repeating the
study using other fruits will provide the fruit industry and other parts of horticultural industry and the
scientific community with better understanding of fruits responses to climate changes.
Due to limited availability of resources, the current study covered a limited range of temperature
variation from 25 °C to 30 °C and maintained the night temperature at 20 °C. Therefore, it is
recommended that future studies could be extended further by using a wider range of temperature and
different day and night temperatures. These proposed future research conditions would provide a more
accurate and specific assessment on potential impact of future increases in temperature.
Results from the interaction between [CO2] and temperature and their effects on fruit quality and
quantities were of a great value and provided some new information. However, it also recommended
to include several other factors such as water availability, soil type, soil nutrition, fertilizer
management and other management practices and abiotic stresses in future studies. Since both,
elevated [CO2] and high temperature have been shown to cause positive impacts on strawberry fruits
179
nutritional value, [CO2] enrichment has become a popular technique in some commercial glasshouse
crop production. Similarly, it can be suggested that sudden heat stresses can be provided to plants by
exposing them to short term temperature increase. These kinds of treatments will produce high
nutritional quality fruits without reducing the fruit physical quality.
As this current study was conducted under controlled conditions (growth cabinets), it is foremost
important to retest the findings under field conditions since controlled environmental or glass house
conditions do not reflect the natural conditions. Additionally, under filed conditions it will make an
opportunity to test fruits against natural pest and diseases arising with climate change. Such
understanding will guarantee better preparation to protect our fruit industry and meet the consumers’
demands for nutritious and high-quality fruits.
Apart from the fruit yield and quality, the proper preservation of fruits surplus to maintain the best
nutritional value should also be investigated. Additionally, the knowledge and understanding of fruit
preservation techniques will help to reduce the wastage and overcome the threat to food security. In
this study, the analyses of micronutrients contents and their bioaccessibility were only examined in
fresh and frozen strawberries. Including the other types of consumed strawberries such as, freeze-dried,
canned and jam strawberries in the future studies would provide additional and beneficial information.
Finally, even though the strawberries grown under elevated [CO2] and temperature were of poor
quality with less attractive colour, size and shape, the micronutrient contents and nutritional value were
much better. Considering the increased and better nutritional quality of fruits grown under such climate
conditions, may suggest the use of such fruits in value added products such as jam, juices, purees, dried
berries and powder. Freeze dried fruits and powder can be incorporated into breakfast cereals, dairy
products, snacks, bakery products, sweets, drinks, etc. Additionally, future assessment of the
180
bioaccessibility and bioavailability of micronutrients in fruits and their value-added products using in-
vivo technique will be of a better approach but requires more resources and financial support.
181
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Minerva Access is the Institutional Repository of The University of Melbourne
Author/s:
Balasooriya, Himali
Title:
Effect of elevated carbon dioxide and high temperature on major micronutrients in strawberry
Date:
2019
Persistent Link:
http://hdl.handle.net/11343/225119
File Description:
PhD Thesis - Himali Balasooriya - 667663
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