THE ROLE OF STARCH PHYSICOCHEMICAL

PROPERTIES IN DETERMINING THE GLYCAEMIC

INDEX OF NOVEL POTATO VARIETIES

by

Tracy Sousa Moreira

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Nutritional Sciences

University of Toronto

© Copyright by Tracy Sousa Moreira 2012

ii

THE ROLE OF STARCH PHYSICOCHEMICAL PROPERTIES IN DETERMINING

THE GLYCAEMICINDEX OF NOVEL POTATO VARIETIES

Master of Science, 2012

Tracy Sousa Moreira

Department of Nutritional Sciences

University of Toronto

ABSTRACT

Potatoes are often thought of as high GI. It is known that cooking and cooling affect GI

and that these effects may be mediated through the physicochemical properties of their starch.

As part of a Canadian initiative to develop low GI potatoes, novel potato varieties which differed

in starch composition were tested in 2 separate studies. In study 1, we determined the GI of 8

varieties and found that cooling produced a wide range of effects (0-50% reduction in GI). In

study 2, four previously tested varieties were re-examined. A significant variety-x-treatment

interaction (p<0.01) was observed with cooling reducing GI in some potatoes. Examination of

the starch properties and their role in determining GI showed that RDS was positively associated

with GI (r2= 0.85, p= 0.001) and SDS (r

2=-0.60, p= 0.02) and amylose (r

2=-0.99, p=0.007)

negatively associated with GI. No relationship between RS and phosphorous content and GI was

observed.

iii

ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr. Thomas Wolever for giving me the opportunity

to pursue graduate studies at the University of Toronto and for always being available to answer

my many questions.

I would also like to thank my committee members Dr. David Jenkins and Dr. Richard Bazinet for

providing me with great insight and constructive feedback for my studies. I would also like to

thank Dr. Mary Keith for acting as my external examiner and to Dr. Mary L‟Abbe for

chairing my defense.

Thank-you to all the members of the Wolever laboratory, especially Tara Kinnear for engaging

me in exciting conversations about potatoes!!

A special thank-you to everyone at Glycemic Index Laboratories, for training me and aiding me

in the organization and day-to-day operations of my studies.

Thank-you to my parents Jack and Dionisia Moreira for always supporting me and encouraging

my pursuit for higher knowledge. I would like to especially thank my dad for encouraging me

and instilling in me the idea that I could truly be whatever I wanted to be as long as I worked

hard at it.

Last, but not least, I want to thank my fiancé, James Lucas for his constant support and

encouragement and for allowing me to ramble on about my research, when often he had no idea

what I was talking about.

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TABLE OF CONTENTS

Page

ABSTRACT ii

ACKNOWLEDGMENTS iii

LIST OF ABBREVIATIONS vii

LIST OF FIGURES viii-ix

LIST OF TABLES x

CHAPTER 1. INTRODUCTION 1-4

CHAPTER 2. REVIEW OF LITERATURE 5-21

2.1 Nutritional Composition of Potato 6-7

2.2 Potato Starch Structure and Composition 7

2.3 Effects of cooking and cooling on starch structure and 7-8

digestibility

2.4 Measurement and Classification of Dietary Carbohydrates 9-12

2.4.1 Glycaemic Index 9-10

2.4.2 In vitro Starch Digestibility 10-12

2.5 Potato Digestibility and the Glycaemic Index 12-15

2.6 The Role of Rapidly Digestible Starch, Slowly Digestible 15-17

Starch and Resistant Starch in Health

2.7 Physicochemical Properties that Affect Starch 17-19

Digestibility of Potato

2.7.1 Amylose:Amylopectin 17-18

2.7.2 Fine Structure of Amylopectin 18-19

2.7.3 Phosphorous Content 19

v

2.7.4 Maturity 19

2.8 Summary and Research Objectives 20-21

2.8.1 Summary 20

2.8.2 Research Objectives 21

2.8.3 Hypotheses 21

CHAPTER 3. STUDY 1- THE EFFECT OF COOLING

ON THE GLYCAEMIC INDEX OF NOVEL

POTATO VARIETIES 22-44

3.1 Introduction 23-24

3.2 Study Design and Methods 23-32

3.2.1 Subjects 25

3.2.2 Test Meals 27-28

3.2.3 Protocol 29

3.2.4 Calculation of Glycaemic Index 30-31

3.2.5 Statistical Analysis 31-32

3.3 Results and Discussion 33-43

3.4 Conclusion 44

CHAPTER 4. STUDY 2- THE ROLE OF STARCH

PHYSICOCHEMICAL PROPERTIES IN

DETERMINING THE GLYCAEMIC INDEX OF

POTATO 45-76

4.1 Introduction 46-47

4.2 Study Design and Methods 48-50

4.2.1 Subjects 48

4.2.2 Test Meals 48

4.2.3 Protocol 49

4.2.4 Calculation of Glycaemic Index 50

4.2.5 Statistical Analysis 50

vi

4.3 Materials and Methods- In vitro starch digestibility 51

4.3.1 Preparation of dry Matter 51

4.3.2 Chemical Composition 51

4.3.3 In vitro digestibility of cooked potatoes 54

4.3.4 Statistical Analysis 54

4.4 Results and Discussion 55-74

CHAPTER 5. GENERAL DISCUSSION, CONCLUSIONS AND 75-79

FUTURE DIRECTIONS

5.1.1 Study 1 76

5.1.2 Study 2 76-77

5.2 Conclusions 78

5.3 Future Directions 79

REFERENCES 80-90

vii

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

AUC Area under the curve

avCHO Available carbohydrate

BG Blood glucose

BMI Body mass index

BV Biological value

CHO Carbohydrate

CVD Cardiovascular disease

FBG Fasting Blood Glucose

FC Freshly cooked

GI Glycaemic Index

GR Glycaemic Response

II Insulinaemic Index

SAG Slowly available glucose

SDS Slowly digestible starch

RAG Rapidly available glucose

RDS Rapidly digestible starch

RS Resistant starch

SCFA Short chain fatty acids

SD Standard deviation

SEM Standard error of mean

T2D Type 2 diabetes

viii

LIST OF FIGURES

Page

3.1a Blood glucose response elicited by freshly cooked 37

and cold potatoes for varieties 1, 2, 3 and 4.

Values are means ± SEM (n= 10).

3.1b Blood glucose response elicited by freshly cooked 38

and cold potatoes for varieties 5, 6, 7 and 8.

Values are means ± SEM (n= 10).

3.2 GI of potato chips for group A (n=10) and group B (n=10). 39

3.3 GI of potato varieties 1, 2, 3, 4, 5, 6, 7, and 8 served to subjects 40

(n= 10) freshly cooked and cold.

3.5 Percent reduction in GI upon cooling for potato varieties 1, 2, 3, 41

4, 5, 6, 7 and 8

4.1 Blood glucose response elicited by freshly cooked and 62

cold potatoes for varieties 2, 3, 4 and 5.

Values are means ± SEM (n= 10)

4.2 Insulin response elicited by freshly cooked and 63

cold potatoes for varieties 2, 3, 4 and 5

4.3 GI of potato varieties 2, 3, 4, and 5 served to subjects 64

(n= 10) freshly cooked and cold

4.4 GI of potato varieties 2, 3, 4 and 5 served to ten subjects in two 65

ways, freshly cooked and cold and harvested in 2009 and 2010

4.5 Relationship between glycaemic index (GI) and insulinaemic 66

index (II) values of 50g available carbohydrate portions of

potato varieties 2, 3, 4 and 5 freshly cooked and cold

4.6 Relationship between the amount of rapidly digestible starch (RDS) 67

in a portion of potato containing 50g of available carbohydrate and

the GI freshly cooked and cold potatoes

4.7 Relationship between the amount of slowly digestible starch (SDS) 68

in a portion of potato containing 50g of available carbohydrate and

the GI freshly cooked and cold potatoes

ix

4.8 Relationship between the amount of resistant starch (RS) in a portion 69

of potato containing 50g of available carbohydrate and the GI freshly

cooked and cold potatoes

4.9 Relationship between percent amylose (%) for each potato variety and GI 70

4.10 Relationship between the GI of potato and phosphorous content 71

x

LIST OF TABLES

Page

3.1. Subject Characteristics 26

3.2 Energy and Macronutrient Composition of Test Meals 29

3.3 Blood glucose AUC for potato varieties 1-8 35

served to subjects (n= 10) freshly cooked and Cold

3.4 GI values for potato varieties 1-8 served to subjects 36

(n= 10) freshly cooked and Cold

4.1 Subject Characteristics 56

4.2 Energy and Macronutrient Composition of Test Meals 57

4.3 Blood glucose and Insulin AUC values for potato 58

varieties 2, 3, 4 and 5

4.4 GI values for potato varieties 2, 3, 4 and 5 59

4.5 GI values for potato varieties 2, 3, 4 and 5 harvested in 2009 60

and 2010 and served to subjects (n= 10) freshly cooked and cold

4.6 In vitro digestibility of cooked and cooled potatoes 61

1

CHAPTER 1

INTRODUCTION

2

1. INTRODUCTION

The potato (solanum tuberosum) was first identified over 8,000 years ago in the Andean

mountains of Peru (Lutaladio, 2009). In the late 16th

century they were brought to Europe by the

Spaniards and from there were spread globally (Lutaladio, 2009). Canada has become an

important contributor to potato production worldwide and is currently the 12th

largest producer of

potatoes with over 160 potato varieties and 5 million metric tonnes grown annually (AAFC,

2007). Potatoes are ranked the fourth most important food crop world-wide, after maize, wheat

and rice, making them a staple in the human diet on a global scale (CIP, 1996).

Potatoes are a nutrient dense, low calorie food. Potatoes are a good source of

carbohydrates (CHO), high quality protein and when eaten with the skin contain appreciable

amounts of several micronutrients including vitamin C, phosphorous and potassium (Camire,

Kubow & Donnelly, 2009). Potatoes also contain phenolic compounds such as caffeic acid and

chlorogenic acid, however the composition and amount varies between potato cultivars (Brown

et al., 2003; Brown et al., 2005; Camire, Kubow & Donnelly, 2009). Despite their favourable

nutrient profile, potato consumption has steadily declined in several countries including Canada

(AAFC, 2007). Potatoes represent 44% of all fresh vegetables consumed in Canada, however

consumption has declined by 29% from 1996 to 2005 (AAFC, 2007). Processed potato

consumption including that of frozen potatoes, chips, and other processed potato products have

increased slightly, however not enough to account for the decline in fresh potato consumption

observed in the Canadian population (AAFC, 2007). In 1996 Canadians were consuming 75kg

per person; this declined to 65kg per person in 2005 (AAFC, 2007). So why has potato

consumption declined? There are several potential explanations for this decline, one potential

3

variable is the market saturation of other CHO foods that are often well-marketed to the public

and present with specific health claims such as low-fat, fat-free or reduces cholesterol. In

addition, information found in popular diet books (Brand-Miller, 1996), magazines (Golden,

2002) and scientific literature (Halton et al., 2006; Liu & Willett, 2002) may be to blame for the

paradigm shift from advocating a high CHO diet to the ever popular low CHO diet (Halton et al.,

2006). Moreover, results from some research studies reporting high glycaemic index (GI) values

for potatoes have been extrapolated to all potatoes thus reinforcing the erroneous notion that

potatoes are an unhealthy food choice and should be avoided (Crapo et al., 1977; Crapo et al.,

1980; Soh & Brand-Miller, 1999). Interestingly enough some of the studies showing high GI

values for potatoes were conducted before the GI term was coined and thus the appropriate GI

testing protocol may not have been utilized.

The GI is a ranking system that allows CHO foods to be classified on a scale of 0-100

based on their effects on postprandial blood glucose (BG) levels (Wolever, 2006). Using this

system foods can be classified as either low (≤ 55), medium (≥ 56-69) or high (≥ 70) GI

(Wolever, 2006). High GI foods are disconcerting due to their rapid and exaggerated effects on

postprandial BG and insulin levels (Wolever, 2006). This has significant health implications

considering the association between high GI diets and an increased risk for developing chronic

diseases such as, type 2 diabetes (T2D) and coronary heart disease (CHD) (Salmeron et al., 1997;

Liu et al., 2000). Although potatoes tend to be thought of as high GI there is still no definitive

evidence to support this notion. In fact the GI values reported in the literature vary significantly

from as low as 23 for an unidentified variety to 111 for a baked russet potato (Soh & Brand-

Miller, 1999; Foster-Powell, Holt & Brand-Miller, 2002). The possibility exists that these

4

differences in GI values reported in the literature may be due to random experimental error or

flaws in the GI methodology used by individual laboratories. However, a more plausible

explanation for these variations in reported GI values is that there are inherent differences in the

potatoes‟ starch structure and morphology that affect the way they respond to cooking and

cooling processes.

The following literature review will examine the several factors that are thought to affect

the digestibility of potatoes and hence their GI. Focus will be on the effect of cooking, cooling

as well as starch physicochemical properties in determining the GI of potatoes.

5

CHAPTER 2

REVIEW OF LITERATURE

6

2.1 Nutritional Composition of Potato

Potatoes are comprised of ~80% water and ~20% dry matter; 66-80% of dry matter is

represented as CHO, mainly in the form of starch (Camire, Kubow & Donnelly, 2009). Despite

their reputation as an unhealthy food choice, potatoes are a good source of CHO, high quality

protein and contains appreciable amounts of several micronutrients (Golden et al., 2002; Camire,

Kubow & Donnelly, 2009). One medium size potato (150g, boiled with skin intact) contains 100

kcals and is virtually free of fat, sodium and cholesterol (Monro & Mishra, 2008). Due to their

high water content potatoes are less energy dense than other common staple CHO foods, such as

bread, rice and pasta (Monro and Mishra, 2008; Lynch et al., 2007). This is important to note

considering these foods are often touted as healthy alternatives to potatoes (Brand-Miller, 1996).

Potatoes also contain a small amount protein (2.5g/ 150g serving) compared to other raw

vegetables. Furthermore, the protein it does contain has a high biological value (90-100) which

is comparable to that of whole egg (100) and soybean (84) (Camire, Kubow & Donnelly, 2009).

In addition, potatoes are high in several vitamins and minerals including vitamin C, potassium

and phosphorous (Camire, Kubow & Donnelly, 2009; Monro and Mishra, 2008). One 150g

serving of potato provides 126mg to 218mg of vitamin C (45% of the recommended dietary

allowance), 93mg of phosphorous and 610mg of potassium (Camire, Kubow & Donnelly, 2009;

Monro and Mishra, 2008). The amounts of potassium found in potato are comparable to that

found in banana (118g serving contains 442mg), a food that is often touted for its high potassium

content (Canadian Nutrient File, 2010; Monro and Mishra, 2008). Potatoes are also known to

contain several phytochemicals including anthocyanins and polyphenols, particularly

chlorogenic acid which is the most abundant polyphenol found in potatoes (Camire, Kubow &

7

Donnelly, 2009). An assessment of over 100 diverse potato varieties identified more than 60

different phytochemicals. (Science Daily, 2007).

2.2 Potato Starch Structure and Composition

Potato starch is made up of two α-glucans, amylopectin, a highly branched, high

molecular weight fraction and amylose, a smaller, un-branched linear fraction (Hasjim et al.,

2010). Amylopectin is made up of a series of glucose molecules bound by α1-4 glycosidic bonds

and α1-6 glycosidic bonds at its branch points (Englyst, Liu & Englyst, 2007). Amylose is a

linear fraction comprised of a series of glucose molecules bound by α1-4 glycosidic bonds

(Englyst, Liu & Englyst, 2007). In potato, amylopectin and amylose are contained in large

spherical structures referred to as starch granules (Singh, Kaur, McCarthy, 2008). In its native

form, potato starch exhibits a large granular size and a B-type crystalline structure that is highly

resistant to enzymatic hydrolysis by digestive enzymes, however with cooking, cooling and

processing this structure is altered irreversibly (Englyst & Cummings, 1986; Englyst, Liu &

Englyst, 2007; Lynch et al., 2007). Most potato starch is comprised of 70-80% amylopectin and

20-30% amylose, although the exact amounts vary depending on the potato variety (Hoover,

2001). The composition of potato varies with the cultivar, growing area and fertilization regime,

while the granular structure is determined by genetic factors that govern starch biosynthesis (Liu

et al., 2007; Guilbot & Mercier, 1985).

2.3 Effects of Cooking and Cooling on Starch Structure

Native potato starch is highly resistant to digestive enzymes because it is encapsulated

within starch granules (Ring et al., 1988; Gallant et al., 1992). In order to make the starch in

8

granules become available to α-amylase, they must undergo gelatinization (Englyst and

Cummings, 1987). In the presence of water and heat at temperatures between 60-70°C water

molecules form bonds with the hydroxyl groups of amylose and amylopectin causing potato

starch granules to take in water and swell resulting in irreversible structural changes, this process

is termed gelatinization (Singh, Kaur & McCarthy, 2008). The collapse in crystalline structure

within the starch granule causes irreversible changes to starch properties such as swelling power,

starch solubility and crystalline order (Hoover, 2001). Potato starch granules swell up to 100

times their original size which is significant when compared to other starches such as maize and

wheat which swell up to 30 times their original size (de Willigen, 1976). Gelatinization occurs

more readily in the amylopectin fraction due to its highly branched structure while amylose

restricts granule swelling and hence is resistant to gelatinization (Fredriksson et al., 1998).

Gelatinized starch is rapidly hydrolyzed by digestive enzymes which causes a rapid rise in

postprandial BG and insulin levels (Lehman & Robin, 2007). Conversely, when cooled, starch

granules re-associate forming an irregular structure that is more resistant to digestion; this

process is termed starch retrogradation (Englyst, Liu & Englyst, 2007). When cooled, amylose

re-associates rapidly to form a single or double helical structure that is highly resistant to α-

amylase (Liu et al., 2007; Zhang, Ao & Hamaker, 2008). In contrast, amylopectin is more

resistant to retrogradation due to its highly branched structure and upon cooling re-associates to

form bonds that are less resilient than those formed by amylose (Zhang, Ao & Hamaker, 2008).

9

2.4 Classification and Measurement of Dietary Carbohydrate

Both in vivo and in vitro methods have been employed to measure starch digestibility, a

review of two such methods, the in vivo GI test (Jenkins et al., 1981; FAO, 1998) and the in vitro

starch digestibility assay (Englyst et al. in 1992; Englyst et al., 1999) follows.

2.4.1 The Glycaemic Index

The GI is considered to be a more relevant and practical method of assessing starch

digestibility because it takes into account the physiological changes that occur with food

consumption (Wolever, 2006; Cummings & Englyst, 1995). The GI is a classification system

that allows CHO foods to be classified on a scale of 0-100 based on their effects on postprandial

BG levels (Wolever, 2006). The GI is defined as the incremental area under the BG response

curve (iAUC) elicited by a portion of a given test food which contains 50g of available CHO

(avCHO) (total CHO minus fibre) expressed as a fraction of the incremental area under the BG

response curve (iAUC) elicited by 50g of glucose (reference food) taken by the same subject

(Wolever et al. 1991). The GI classifies foods as either low (≤ 55), medium, (56-69) or high (≥

70) GI. The GI is an assessment of the quality of CHO present in a given food and is

independent of the amount consumed (Wolever, 2006). To correct for the day to day variation

that occurs within subjects, the reference food (glucose) is tested at least two, preferably three

times for each subject (Wolever et al., 1991, 2002). The GI adjusts for each individuals

glycaemic response to a given test food by using their individual response to the reference food

(Wolever, 2006). The GI value for a test food is taken as the mean GI value obtained from at

least 10 subjects, however more subjects are required if greater precision is desired (Brouns et

al., 2005)

10

Prior to 1998 the GI testing protocol was not clearly defined resulting in different

laboratories using their own version of an existing protocol. This often resulted in a wide

variation in GI values reported for seemingly identical foods (Atkinson et al., 2008; Lynch et al.,

2007). In order to prevent this, in 1998 a standard GI testing protocol was developed and

published by the Food and Agriculture Organization (FAO, 1998).

2.4.2 In vitro Starch Digestibility

In vitro methods have been used to estimate starch digestibility in vivo. Using these in

vitro methods starch can be classified into the nutritionally important starch fractions rapidly

digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) (Englyst et al.,

1992). RDS is identified as the amount of starch hydrolyzed in the first 20mins after incubation

with α-amylase, SDS as the amount hydrolyzed between 20-120mins; RS is calculated by

subtracting RDS and SDS from the total starch measured and represents the amount that is not

hydrolyzed at 120 min post incubation (Englyst et al., 1992). This analytical method allows us

to predict the likelihood of a given CHO food to be rapidly digested (RDS), slowly digested

(SDS) or escape digestion (RS) in the human small intestine (Englyst et al., 1992).

In 1999, Englyst et al. modified the existing in vitro digestibility assay for measuring

RDS, SDS and RS in order to measure rapidly available glucose (RAG) and slowly available

glucose (SAG). This modification is designed to reflect the rate at which glucose (from both

starch and sugar) becomes available for absorption in the human small intestine (Englyst et al.,

1999). RAG is the amount of free glucose measured after a given food is incubated with

digestive enzymes for 20 minutes (G20= RAG). SAG is the amount of free glucose measured

11

after 120 minutes of incubation minus that measured at 20 minutes (G120 - G20= SAG). RDS can

be determined by subtracting free sugar glucose (FSG) from RAG and multiplying by 0.9 (G20-

FSG x 0.9= RDS). SDS is determined by subtracting the free glucose measured after 120

minutes incubation from glucose measured after 20 minutes and multiplied by 0.9 [(G120 - G20) x

0.9= SDS]. Finally, RS is calculated by subtracting the glucose measured at G120 from the total

glucose measured and then multiplied by 0.9 (total glucose- G120= resistant starch) (Englyst et

al., 1999). Values for starch fractions are expressed as polysaccharides by using a conversion

factor of 0.9 (Englyst et al., 1999). The modification of the original in-vitro method has allowed

foods which are not predominantly made up of starch to be assessed as well.

In vitro starch digestibility assays have been shown to be a good predictor of the in vivo

glycaemic response of starchy foods (Englyst et al., 1999, Englyst et al., 2003). Foods which

contain high amounts of RDS are rapidly hydrolyzed by α-amylase in the intestinal lumen

causing an rapid influx of RAG which becomes readily available for absorption in the small

intestine; the result is a rapid rise in postprandial BG and insulin levels. Foods containing SDS

are metabolized at a slower rate allowing for a slower more sustained glucose release into the

circulation followed by a slower and lower insulin response (Wolever, 2006; Englyst et al.,

2003). RS is not digested and instead travels to the colon where it can be acted upon by colonic

bacteria to produce short chain fatty acids (SCFA) (Wolever, 2006). There is evidence to

support the efficacy of increasing SDS and RS content in the diet because of its ability to prevent

the exaggerated BG and insulin responses that occur with consumption of high GI foods

(Lehman & Robin, 2007). This very important fact has lead researchers to explore the potential

12

for modifying starch composition with the hope of increasing SDS and RS content in foods

commonly consumed like potatoes.

In vitro starch digestibility assays have been shown to be good predictor of the glycaemic

response of several different CHO foods (Garcia-Alonso and Goni, 2000; Kingston & Englyst,

1994; Mishra et al., 2008) In vitro methods have also been successful in measuring the

proportions of SDS and RS which is advantageous when looking to assess of the effect of

cooling on starch retrogradation (Monro & Mishra, 2008). A study examining the relationship

between the in vitro digestibility of 23 cereal products and their GI showed a close positive

relationship; they also determined that the difference in the relative amounts of RAG and SAG

could explain 68% of the variability in GI between foods (Englyst et al., 2003).

2.5 Potato Digestibility and the Glycaemic Index of Potatoes

It is well accepted that foods which are rapidly digested cause hyperglycaemia and

hyperinsulinaemia which are problematic because of their contribution to the development of the

metabolic syndrome, T2D and secondary conditions affecting the kidney‟s, heart and eyes

(Brownlee, 2001; Ricciardi et al., 2008). The CHO in potatoes is generally thought to be rapidly

digested and thus classified as high GI, however the assumption that all potatoes are high GI is

unwarranted, in fact the GI values reported in the literature show a wide variation from a very

low 23 to a very high 111 (Foster-Powell, 2002; Lynch et al., 2007). According to the 2002

international GI tables, only ~50% of potatoes tested are classified as high GI with 28%

classified as medium and 22% classified as low GI (Brand-Miller, 2002). These variations in

glycaemic responses and resultant GI values may be due to the several factors thought to affect

13

the GI of potatoes, including variety, method of preparation (baked, boiled, mashed etc.), state

consumed (freshly cooked; cooked, cooled and reheated; cooked, cooled), time of harvest (early

vs. mature) and the physicochemical properties of the starch (RDS, SDS, RS, phosphorous and

amylose content) within them (Svegmark et al., 2002; Lynch et al., 2007).

In a study conducted on three Australian potato varieties that were prepared in different

ways (boiled, baked, microwaved) all potato varieties had high GI values that ranged from 87 to

101 (Soh & Brand-Miller, 1999). They also determined there was no effect of variety, cooking

method or maturity on GI despite observing a 36% lower GI in canned new potatoes when

compared to boiled potatoes that presumably were harvested at maturity (Soh and Brand-Miller,

1999). In contrast, a group in the UK found that eight potato varieties had GI values ranging

from 56 to 94 (Henry et al., 2005). So there are definite differences between varieties and within

varieties as well. For example one group in the UK found that the “Nicola” variety had a mean

GI of 56 ± 7 (mean ± SEM) while a group in Finland determined that the same „Nicola‟ variety

had a GI of 104 ± 39 (mean ± SD) (Henry et al., 2005; Tahvonen et al., 2006). These

noteworthy differences in GI could be due to differences in methodology or experimental

inconsistencies, however, it is more likely that their different growing environments may have

resulted in different starch structure and composition, particularly the amylose: amylopectin

which has been thought to play a significant role in determining how potatoes respond to

cooking and cooling processes (Zhang, Ao & Hamaker, 2008).

14

Cooling cooked potatoes results in the formation of retrograded starch, a form of starch

that is resistant to digestion (Lynch et al., 2007). It is thought that increasing the amount of SDS

and RS could lead to a decrease in the glycaemic potency of a potato meal, which from a health

perspective may be seen as beneficial. Monro et al. (2008) looked at the effects of cooking and

cooling on RDS, SDS and RS content of the „Frisia‟ potato variety and found that cooled

potatoes reduced their RDS content by ~20% while increasing the proportion of SDS and RS.

Moreover, a preliminary study conducted in our laboratory found that boiled red potatoes

consumed cold had a lower GI than their freshly cooked counterparts (56 vs. 89) (Fernandes et

al., 2005). Although we did not measure the RDS, SDS or RS content we hypothesize that the

amount of RDS would decrease and the amount of SDS and RS increase with cooling. We

suggest that upon cooling, not only is RS formed but that there is also a conversion of RDS to

SDS. Similarly, another study conducted in our laboratory found that cooling significantly

reduced the GI in some, but not all potato varieties suggesting that the effect of cooling is

dependent on variety (Kinnear et al. 2011). However, what remains unclear is whether or not

this effect of cooling on GI can be seen in other potato varieties. Because people often cook,

cool and re-heat potatoes it is also important to examine whether or not this temperature cycling

affects the GI of potatoes. Previous studies have shown that when a boiled potato was allowed to

cool at 6°C and then re-heated at 70°C the amount of RS increased from 4.5% to 9.8% (P <0.05)

(Leeman et al., 2005). Our laboratory also investigated the effect of cooling and re-heating

cycles on the GI and found no significant differences between freshly cooked and re-heated

potatoes, however we did observe a trend towards re-heated potatoes having a lower GI when

compared to those that were freshly cooked (Kinnear et al., 2011). Thus, there seems to be

15

significant variability in the way different potato varieties respond to various methods of

preparation. Furthermore, when comparing potato varieties it is not only important to know the

structure of the starch composition but it is also important to know how they are prepared and

consumed as this no doubt has an impact on GI. Additional work is needed in order to

understand the reasons for the varied effects of cooking, cooling and re-heating on the GI of

potatoes.

Potato texture may also be used as potential indicator of a potatoes GI. For example,

Henry et al. (2005) found that potatoes exhibiting a waxy like texture often have a lower GI than

those that have a floury texture. They also observed a strong positive relationship between GI

and potato texture rating (scale from 1-9, 1 being most waxy and 9 being most floury) indicating

that potatoes with a floury texture tend to have a higher GI than those that are more firm such as

„new‟ potatoes (Henry et al., 2005).

2.6 Rapidly Digestible Starch, Slowly Digestible Starch and Resistant Starch-

Health Implications

The importance of characterizing the starch fractions RDS, SDS and RS are further

justified due to their potential effects, both negative and positive, on human health. As stated

previously, RDS is rapidly digested and absorbed into the small intestine resulting in a rapid rise

in BG into the circulation and a super compensatory insulin response. These exaggerated insulin

and glucose responses are worrisome because of their potential role in developing the metabolic

syndrome and T2D (Ells et al., 2005; McKeown et al., 2004). Thus, the importance of decreasing

the consumption of foods with significant amounts of RDS is warranted.

16

Conversely, SDS is slowly hydrolyzed by digestive enzymes and is absorbed slowly into

the small intestine allowing for a slow and steady release into the circulation (Lehman & Robin,

2007). The result is a sustained glucose release with a concomitant insulin response that is not

exaggerated like that seen with foods which contain high amounts of RDS. Foods which contain

high amounts of SDS or SAG are often classified as low or medium GI which is important

considering the data suggesting that a low-GI diet may help reduce the risk of developing T2D,

CVD and some cancers (Salmeron et al., 1997; Wong & Jenkins, 2007). A meta-analysis

examining the efficacy of a low-GI diet versus high GI diet on glycaemic control (glycated

haemoglobin A1c (HbA1c) and fructosamine) and found that a low-GI diet reduced HbA1c and

fructosamine by 7.4% (Brand-Miller, 2003). In contrast, Wolever et al. (2008) found that a low-

GI diet had no effect on HbA1c,but significantly reduced C-reactive protein by 30% (2.75mg/ vs.

1.95 mg/ L, P=0.0078) in individuals with T2D.

About 3-5% of starch found in cooked potatoes is RS (Wolever, 2006). RS represents the

portion of starch that cannot be hydrolyzed and thus escapes digestion and ends up in the colon

where it can be acted upon by colonic bacteria (Wolever, 2006). Once in the colon fermentation

by bacteria can occur leading to the formation of the short chain fatty acids (SCFA) acetate,

propionate and butyrate (Cummings & MacFarlene, 1991). SCFA are thought to play a role in

health both locally and systemically. Locally, butyrate acts as a primary fuel source for

colonocytes and aids in regulating cell proliferation and differentiation (Wong & Jenkins, 2007;

Wolever & Vogt, 2003). Acetate and propionate have also been shown to effect lipid

metabolism. Acetate plays a role in cholesterol synthesis while propionate inhibits cholesterol

synthesis. It has been suggested the decreasing the acetate: propionate may reduce blood lipid

17

content and reduce CVD risk (Wong et al., 2006). RS also acts as a bulking agent that helps to

reduce the transit time of faeces resulting in less exposure of coloncytes to potential toxins

(Wolever, 2006; Hill & Fernandez, 1986).

2.7 Physicochemical Properties that Affect Starch Digestibility

As described previously, the relative amounts of RDS, SDS and RS have an impact on

potato digestibility and GI. There is also evidence to suggest that the effects of cooling on GI

may be mediated through the physicochemical properties of the starch present in potatoes. The

following will look at some of the factors thought to play an important role in starch digestibility

and GI.

2.7.1 Amylose: amylopectin

The ratio between the two starch fractions, amylose and amylopectin plays an important

role in the way potatoes respond to the processes of cooking and cooling. As previously stated

potato starch composition is mainly determined by genetic factors, however it is widely accepted

that potato starch is predominantly amylopectin (70-80%) with the remaining 20-30% comprised

of amylose.(Hoover, 2001; Bertoft & Blennow, 2008) The relative composition is important

considering amylose is more resistant to gelatinization and upon cooling forms retrograde starch

more readily. Amylose is mainly responsible for the structural changes occurring within hours

of cooling while amylopectin is responsible for the long-term (several weeks) rheological and

structural changes.(Zhang et al., 2008) In studies examining BG responses to biscuits and rice

containing different amounts of amylose and amylopectin, those containing a greater proportion

of amylose produce a lower glycaemic response than those which contain a greater proportion of

18

amylepectin (Behall et al. 1988 and Goddard et al. 1984). In the case of potatoes, genetic

modification of starch to either increase or decrease the degree of starch branching results in

either high amylose (64%) potatoes or low amylose (1%) potatoes. These modified potatoes

were then cooked and the rate of starch digestion assessed using in vitro methods, the result was

a high RS content in the high amylose potatoes when compared to the low amylose potatoes and

a lower predicted GI, 94 vs. 68, for the low and high amylose potatoes, respectively (Leeman et

al., 2006). Taking this into account we can deduce that potatoes with a greater amylose content

will become less gelatinized upon cooking thus resulting in a lower postprandial BG response

and lower GI. Conversely, when cooled, potatoes containing a greater amount of amylose will

form more retrograde starch and at a faster rate when compared to those with greater amounts of

amylopectin. It is important to note that amylose plays an important role in short-term

retrogradation, occurring within hours of cooling which is more applicable to a real-life situation

since people tend to store potatoes in the refrigerator for a few days at most. On the other hand,

amylopectin is thought to play a role in the long-term (several weeks) retrogradation properties

of starch which may be more applicable to commercial potato production.

2.7.2 Fine Structure of Amylopectin

The structure of amylopectin, particularly its branching pattern and its chain length

distribution has been shown to play a role in starch digestibility.(Zhang et al., 2008) Unlike its

starch counterpart amylose, amylopectin, due to its highly branched structure requires a lower

gelatinization temperature which from a starch digestibility stand-point is important. Zhang et

al. (2008) examined starch samples with various degrees of branching patterns (high amount of

short chains and high amount of long chains) and found whether starch samples had high

19

amounts of long or short chains did not matter, both were associated with a having a greater

proportion of RDS.

2.7.3 Phosphorous Content

Potatoes contain appreciable amounts of phosphorous which may be advantageous for the

GI of potatoes. Phosphorous is present in potato starch as monoesters and phospholipids and is

thought to significantly affect the functional properties of starch (Singh, Kaur & McCarthy,

2008). For example, phospholipids tend to form complexes with amylose and amylopectin

which when heated in water limits its ability to gelatinize. If so, potatoes which contain high

amounts of phosphorous would result in a decrease in gelatinized starch upon cooking (and a

lower GI). Phosphorous content has been shown to be positively associated with retrogradation

suggesting that the more phosphorous a potato contains the greater amount of retrograde starch

that can be formed upon cooling (Liu et al., 2007). The result is a lower glycaemic response and

lower GI.

2.7.4 Maturity

Potatoes which are harvested early (≤90 days) in the season are referred to as “new”

potatoes and tend to have a lower GI than those harvested at maturity (≥120 days). Mature

potatoes are thought to have a higher GI due to a greater degree of amylopectin branching they

contain which, as discussed previously, increases their digestibility (Soh & Brand-Miller, 1999;

Zhang et al., 2008). Indeed one study found that new potatoes had a GI that was 36% lower than

mature potatoes (Soh & Brand-Miller, 1999). The same study also observed a positive

relationship between GI and the average potato tuber size (r= 0.83, P <0.05), an indicator of

maturity (Soh & Brand-Miller, 1999).

20

2.8 SUMMARY, OBJECTIVES AND HYPOTHESES

2.8.1 Summary

Generally speaking potatoes are often thought of as a high GI food. Both, in-vitro and in-

vivo studies have shown that the starch in freshly cooked potatoes is rapidly digested and

absorbed in the small intestine causing a rapid rise in postprandial blood glucose levels. A diet

that is high in foods which are classified as high GI has been associated with an increased risk of

developing chronic diseases such as diabetes, cardiovascular disease and even some cancers.

Data collected by the AAFC (2007) shows that potato consumption has significantly declined in

Canada and this may be due the high GI classification that they have received. There are several

factors which are known to affect starch digestibility that are often not accounted for in research

studies, these include: method of preparation, variety, harvest year and the physicochemical

properties of their starch (i.e. amylopectin:amylose, phosphorous content and RDS:SDS:RS). A

network of scientists and plant breeders (Bio-Potato Network) have been working in concert to

develop low GI potato cultivars by modifying starch composition with the goal of increasing

slowly digestible starch, fibre content as well as some other properties thought to contribute to a

low GI potato. A previous study conducted in our laboratory using novel potato varieties

provided to us by the Bio-Potato network found that the effect of cooking a cooling on GI is

dependent on potato variety (Kinnear et al., 2011). However, only four potato varieties were

tested and thus we were unsure if this effect could be seen in other novel potato varieties. Also,

since the physicochemical properties of starch are also thought to play a significant role in starch

digestibility we were interested in examining some of these properties in relation to GI.

21

2.8.2 Research Objectives

Study 1

- To screen additional novel potato varieties and determine their GI

- To confirm our previous findings that the effect of cooling on the GI is dependent on variety

Study 2

- Re-test the GI of four varieties of interest from 2009 in 2010 and examine the role of the

physicochemical properties (RDS, SDS, RS, amylose and phosphorous content) of their starch

in determining GI

- Determine the mechanism for the low glycaemic responses observed for low GI potatoes

2.8.3 Hypotheses

Study 1

- The effect of cooling is dependent on variety

- Cold potatoes elicit a lower glycaemic response and have a lower GI than freshly

cooked potatoes

Study 2

- The GI of each potato variety does not significantly differ between harvest years- 2009 vs.

2010

- The low GR produced by low GI potatoes is not due to a concomitant increase in postprandial

insulin

- There is a positive relationship between GI and RDS and negative relationship between GI and

SDS, RS, amylose and phosphorous content

22

CHAPTER 3

STUDY 1- THE EFFECT OF COOLING ON THE GLYCAEMIC

INDEX OF NOVEL POTATO VARIETIES

23

3.1 Introduction

The notion that all potatoes have an inherently high GI is unwarranted considering the

wide variation of potato GI values reported in the literature. Generally speaking, both in vitro

and in vivo studies conducted using freshly cooked potatoes have ascertained that they are

rapidly hydrolyzed by digestive enzymes. In vivo, freshly cooked potatoes result in high rises in

BG and insulin levels that are greater in magnitude and more rapid in rise when compared to

cold potatoes. This has significant health implications considering CHO foods that are rapidly

digested (high GI foods) are thought to play a role in developing T2D and CVD. However, not

all potatoes are created equal. There are several factors that are known to affect the GI of potato,

these include, variety, method of preparation (hot vs. cold), year of harvest, time of harvest (new

vs. mature) and the physicochemical properties of starch (amylose content, phosphorous content,

rate of starch digestion, etc.).

The following study is part of a collaborative effort between starch chemists, plant

breeders and nutritional scientists whose goal was to develop low-GI potatoes by modifying

starch composition through cross-breeding existing varieties to develop potatoes with high

amylose content, highly branched amylopectin and more fibre than commercially available

potatoes.

Previously, our laboratory conducted a pilot study to examine the effect of cooking,

cooling and re-heating on the GI of 4 novel potato varieties with varying starch profiles.

(Kinnear et al., 2011) In this study we observed that cooling significantly reduced the GI in

some, but not all potato varieties. Two of four potato varieties tested were classified as medium

24

to high GI when consumed freshly cooked, upon cooling all potatoes were reduced to a low GI

classification (Kinnear et al., 2011).

The objective of study 1 was to screen novel potato varieties, determine their GI and to

confirm our previous findings that the effect of cooling on the GI is dependent on variety. We

conducted these tests using novel potato varieties which were harvested at maturity in October

2009. The varieties tested were not commercially available at the time of the study. In keeping

with what was observed in previous studies conducted in our laboratory, we hypothesized that

the effect of cooling would be dependent on variety. We also hypothesized that cold potatoes

would elicit a lower glycaemic response and would have a lower GI than freshly cooked

potatoes.

25

3.2 STUDY DESIGN AND METHODS

3.2.1 Subjects

Two groups of ten healthy subjects participated in the following study. Group A

consisted of 7 males and 3 females, with a mean age of 24.2 ± 1.0 (range, 20-29), a mean weight

of 74.4 ± 4.5 (60.5- 96.7 kg), and a mean BMI= 25.5 ± 7.7 (21.6-29.4). Group B consisted of 7

males and 3 females with a mean age of 30 ± 2.7y (22-45y), a mean weight of 83.8 ± 7.7 kg

(51.5-131.9) and a mean BMI= 27.1 ± 2.4 (17.9-44.3) (Table 3.1). Two subjects, one female

from group A and one male from group B were unable to continue with the study after 6 visits;

they were replaced by two new, same sex subjects.

Subjects were screened to ensure they met the inclusion criteria for participation in the

study. Subjects were excluded from the study if they had diabetes mellitus or glucose

intolerance or any condition known to adversely affect nutrient absorption, metabolism,

excretion or gastric motility. The study was approved by the University of Toronto Research

Ethics Board and informed consent was obtained from all subjects prior to taking part in the

study.

26

Table 3.1 Subject Characteristics

Variable Group A

(mean ± SEM)

Group A

range

Group B

(mean ± SEM)

Group B

range

n (male: female) 7:3 - 7:3 -

Age (y) 24.2 ± 3.2 21-29 30 ± 8.6 22-45

Weight (kg) 74.5 ± 15.7 55.5-96.7 83.8 ± 24.3 51.5-131.9

Height (m) 1.7 ± 0.1 1.5-1.85 1.8 ± 0.1 1.5-1.9

BMI (kg/ m2) 25.5 ± 3.5 20.1-30.2 27.1 ± 7.5 17.9-44.3

27

3.2.2 Test meals

Eight novel potato varieties with varying starch profiles were tested. The potato varieties

were not commercially available at the time of the study and thus will be identified as potato

varieties 1, 2, 3, 4, 5, 6, 7 and 8. All potato varieties were grown in Lethbridge, Alberta and

were harvested at maturity. Prior to being shipped potatoes were sprout inhibited with

Chlorpropham to prevent premature spoiling. Upon arriving in Guelph, Ontario potatoes were

stored in a temperature controlled chamber at 11°C until required. While at the testing facility in

Toronto, potatoes were stored in a dark cupboard at ambient temperature.

Proximate analysis was performed by Agri-Food Laboratories (Guelph, ON) to determine

the following macronutrient content: protein: AOAC 990.03, fat: AOAC 920.39 and

carbohydrate: by difference. Analysis for total dietary fibre (AOAC 993.43 and 985.29) was

performed by Maxxam Analytics (Mississauga, ON) and the amount of potato (in grams)

required to feed subjects 50g of avCHO (defined as total carbohydrate minus fibre) was

calculated. Energy and macronutrient composition content of potato test meals can be found in

table 3.2.

The study was a randomized block design. Group A and B each tested four novel potato

varieties with varying starch profiles and fibre content. In addition to the potato meals, subjects

also tested white bread (reference food) and potato chips (positive control). The amount of

potato consumed varied depending on the variety and ranged from 227-365g per test meal; each

meal contained 50g of avCHO. Prior to cooking, potatoes were washed thoroughly, cut into 1”

cubes (with skin) and the appropriate amount weighed out individually. Potatoes were served to

28

subjects in two ways; boiled for 13mins (1tsp table salt dissolved in 750mL water) and

consumed immediately (freshly cooked) or prepared as above, cooled at room temperature

(24°C) for 30mins, stored at 4°C for 24-72h and consumed cold. Subjects were allowed to add

table salt to their test meals if they desired. White bread was used as the reference food for GI

calculation and was tested three times throughout the study period (beginning, half-way point

and completion). White bread was made onsite with an automatic bread maker as previously

described (Wolever et al., 2003). White bread was used instead of glucose because subjects tend

to prefer it over a glucose drink (Wolever, 2006). A baked potato chip meal containing 50g of

avCHO (determined using the nutrition label on the package) was used as a positive control to

validate between- group comparisons.

29

Table 3.2 Energy and Macronutrient Composition of Test Meals

Test Meals Weight

(g)

Energy

(kcal)

Fat

(g)

Protein

(g)

Total

CHO

(g)

Fibre

(g)

Available

CHO (g)

Variety1

298 259 0 9.8 54.8 4.8 50.0

Variety2

272 255 0 9.2 54.6 4.6 50.0

Variety3

365 265 0 10.1 55.7 5.7 50.0

Variety4

338 270 0 10.6 57.3 7.3 50.0

Variety5

227 250 0 7.1 55.4 5.4 50.0

Variety6

316 241 0 6.4 53.9 3.9 50.0

Variety7

309 256 0 9.2 54.7 4.7

50.0

Variety8

240 259 0 9.3 55.2 5.2

50.0

Baked Potato

Chips (Lays®)

66

264

3.3

5.3

54.1

4.1

50.0

White Breadb

104

245

1.7

7.5

52.8

2.8

50.0

*Potatoes were weighed raw with skin

aThe following analyses were performed by Agri-Food Laboratories, Guelph, ON: moisture

AOAC 93.15 (not shown), protein: AOAC 990.03, ash: AOAC 942.05 (not shown), fat: AOAC

920.39 and carbohydrate: by difference. Analysis for total dietary fibre (AOAC 993.43 and

985.29) was performed by Maxxam Analytics, Mississauga.

bTypical portion size- actual weight varied slightly due to moisture variations.

30

3.2.3 Protocol

The study was conducted at Glycaemic Index Laboratories in Toronto, Ontario from

January 2010 through March 2010. Subjects were asked to visit the laboratory on 12 separate

occasions; each test took approximately 2hrs to complete. Prior to their visit, subjects were

asked to refrain from eating, drinking and engaging in strenuous exercise for10-14hrs. Each test

was separated by a 48h washout period. Subjects arrived at the laboratory between 8-10am on

the morning of the test. Prior to consuming their test meal, subjects were weighed and two

fasting capillary blood samples were collected five min apart (-5 min and 0 min) via finger prick

using an ascensia microlet lancet device (Bayer Diagnostics, New York). Subjects then

consumed their test meal plus a beverage of their choice (250ml of water or 220mL of tea or

coffee with or without 30mL of 2% milk and low-calorie sweetener, if desired) type and volume

of beverage remained consistent throughout the study. All subjects consumed their test meal

within 20 min. Six additional blood samples were drawn at 15, 30, 45, 60, 90, and 120mins after

starting the meal. Capillary blood samples were placed into tubes containing fluoro-oxalate (to

prevent clotting), stored at -20°C and analyzed within 72hr using a glucose-oxidase automatic

analyzer (Yellow Spring Instruments, 2300 Stat). Fasting blood glucose (FBG) was taken as the

mean of two fasting blood samples (-5 min, 0 min).

3.2.4 Calculation of Glycaemic Index

The incremental areas under the blood glucose response curves (AUC) were calculated

geometrically for each test meal using the trapezoidal rule ignoring the area below fasting (FAO,

1998). To calculate the GI, the AUC for each test food (potato and potato chip) was expressed as

a fraction of the mean AUC for three white bread tests taken by the same subject. The resultant

values were then multiplied by 0.71 in order to represent them on the glucose scale (GI of

31

glucose= 100). Both glucose and white bread have been used as the reference food for GI

determination. However, because it can be confusing to have two sets of GI values it has been

suggested that all GI values be converted to the glucose scale (glucose= 100).(Wolever, 2006)

The glycaemic response of white bread is 71% of that elicited by glucose and thus all values

obtained using white bread as the reference food must be multiplied by 0.71 to express them on a

glucose scale. (Wolever, 2003) All values in the present study are represented on the glucose

scale. The GI for each test food is determined by taking the mean GI value obtained from 10

individual subjects.

3.2.5 Statistical Analysis

Results are expressed as means ± SEM. In order to pool data from both groups, a

positive control test meal of potato chips was conducted and a two-tailed, un-paired t-test was

used to assess the probability of significant differences.

Statistical analysis was conducted on blood glucose profiles for each variety (V1-V8) and

treatment (fresh cooked, cold). To examine the differences between treatments, data were

analyzed using a three-factor analysis of variance (ANOVA) to examine the effect of time,

variety, treatment and time x variety x treatment interaction. For AUC and GI values two-factor

ANOVA was conducted to examine the effect of variety, treatment and variety x treatment

interaction. After demonstration of significant heterogeneity, Tukey‟s post-hoc test was used to

determine significant differences between individual mean blood glucose concentrations at each

time point, mean AUC and GI. In addition to examine the differences between the GI of each

potato variety to that of white bread we conducted a one-way ANOVA.

32

Differences were considered significant if a two-tailed P ≤ 0.05. Analysis was done using

SPSS Statistical package 18.0.

33

3.3. RESULTS

3.3.1 Blood Glucose Responses

Blood glucose profiles for potato varieties 1, 2, 3 and 4 are shown in Figure 3.1a and

varieties 5, 6, 7 and 8 shown in Figure 3.1b. There was a main effect of variety for the mean

blood glucose AUC (F=2.782, P= 0.006) and a main effect of cooling (F= 30.471, P <0.001)

with freshly cooked potatoes eliciting a significantly greater AUC than cold potatoes (147.4 ±

14.9 vs. 105.9 ± 13.7) (Table 3.3). There was no variety x treatment interaction for the AUC of

freshly cooked and cold potatoes (F=0.656, P= 0.709). Varieties 1, 2 and 3 freshly cooked

elicited a greater blood glucose response at 15, 30 and 45 min when compared to cold. There

were no significant differences in AUC between freshly cooked and cold potatoes for varieties 4

and 5. Potato variety 6 freshly cooked elicited a greater blood glucose response at 45 min.

Variety 7 freshly cooked showed a greater response at 30 and 45 min while variety 8 had a

greater blood glucose response at 30 min when compared to cold potatoes (P <0.05).

3.3.2 Glycaemic Index

Figure 3.2 shows that the GI for baked potato chips was not significantly different

between group A and group B, 78.7 ± 4.9 and 80.7 ± 7.4, respectively (F= 3.277, P= 0.551).

This allowed us to pool the data from group A and B to compare the GI for different potato

varieties. Table 2.4 shows the GI values for potato varieties 1-8 compared to white bread.

ANOVA showed the GI of V2 (35.5 ± 5) and V3 (34.0 ± 6) served cold were significantly

different (P<0.001) from the GI of WB (GI=71). Only 3 of the potato test meals (V1, V6 and

V8) were classified as high GI (≥70) when consumed freshly cooked. Four test meals were

classified as medium GI (55-69) when freshly cooked (V3-V5, V8) and two when consumed

cold (V4, V6). Six of the potato test meals (V1-V3, V5, V7, V8) were classified as low

34

GI (≤ 55) when consumed cold. Two-way ANOVA showed a main effect of variety (F=4.84, p

< 0.001) a main effect of treatment (F=36.33, P <0.001) but no significant variety x treatment

interaction (F= 1.33, P=0.235). Tukey‟s test showed that variety 3 served cold had a

significantly lower GI than freshly cooked (65 ± 6 vs. 34 ± 6; p<0.001). Although no variety x

treatment interaction was observed in our analysis we saw a wide variation in GI values with

cooling. For example, we observed a 50% reduction in GI for V3 while V4 showed no

difference, in fact, it increased slightly with cooling. With this in mind we were convinced that

the lack of interaction was not justified so we conducted a separate analysis by looking at the

percent reduction in GI upon cooling and conducted a one-way ANOVA. We showed that there

was a significant difference between varieties (F= 2.427, P= 0.027) with the reduction in GI for

V3 and V7 significantly different than that observed for V4.

35

Table 3.3 Blood glucose AUC values for potato varieties 1, 2, 3, 4, 5, 6, 7 and 8 served to

subjects (n= 10) freshly cooked and cold

AUC (mmol x min/ L)

Treatment

Mean For Variety

Fresh Cooked

Cold

Variety 1

177.9 ± 22.0

118.7 ± 22.0

148.3 ± 22.0

Variety 2

130.2 ± 14.6a

83.2 ± 11.2b

106.7 ± 12.9

Variety 3

148.0 ± 7.5a

78.0 ± 13.2b

113.0 ± 10.35

Variety 4

158.1 ± 22.5

139.2 ± 15.2

148.7 ± 18.9

Variety 5

120.9 ± 18.4

92.6 ± 13.2

106.8 ± 15.8

Variety 6

159.5 ± 12.1a

124.3 ± 10.0b

141.9 ± 11.1

Variety 7

147.9 ± 10.9a

103.5 ± 11.2b

125.7 ± 11.1

Variety 8

136.9 ± 11.3

108.1 ± 13.0

122.5 ± 12.2

Mean for Treatment

147.4 ± 14.9a

105.9 ± 13.7b

Values mare means ± SEM a b

AUC values not sharing the same letter subscripts differ significantly (P<0.05) from each

other within each row.

36

Table 3.4 GI values for potato varieties 1, 2, 3, 4, 5, 6, 7 and 8 served to subjects (n= 10) freshly

cooked and cold

Treatment

Mean for

Variety

Fresh Cooked

Cold

Variety 1

72 ± 4

53 ± 6

63 ± 5

Variety 2

52 ± 4

35 ± 5x

44 ± 5

Variety 3

65 ± 6a

34 ± 6bx

50 ± 6

Variety 4

59 ± 6

61 ± 5

60 ± 6

Variety 5

57 ± 8

45 ± 6

51 ± 7

Variety 6

81 ± 9

62 ± 6

72 ± 8

Variety 7

74 ± 6

46 ± 6

60 ± 6

Variety 8

69 ± 5

53 ± 5

61 ± 5

Mean for Treatment

66 ± 6a

49 ± 6b

Values mare means ± SEM

a bGI values not sharing the same letter subscripts differ significantly (P<0.05) from each other

within each row.

x GI values differ significantly from the GI of white bread

37

0 30 60 90 1202

4

6

8Freshly Cooked

Cold

Variety 2

a

aa

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

0 30 60 90 1202

4

6

8 Freshly Cooked

Cold

Variety 3

a

a

a

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

0 30 60 90 1202

4

6

8Freshly Cooked

Cold

Variety 4

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

0 30 60 90 1202

4

6

8 Freshly Cooked

Cold

Variety 1

a

a a

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

Figure 3.1a Blood glucose response elicited by freshly cooked and cold potatoes for varieties 1,

2, 3 and 4. Values are means ± SEM (n= 10). Letters represent significant differences (P <0.05)

between freshly cooked and cold potatoes at the same time-point.

WB V1 V2 V3 V40

50

100

150

200

250

AU

C (

mm

ol.m

in/ L

)

38

WB V5 V6 V7 V80

50

100

150

200

AU

C (

mm

ol.

min

/ L

)

0 30 60 90 1202

4

6

8 Freshly Cooked

Cold

Variety 5

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

0 30 60 90 1202

4

6

8 Freshly Cooked

Cold

Variety 6

a

Time (min)

Blo

od

glu

co

se

(m

mo

l.m

in/

L)

0 30 60 90 1202

4

6

8Freshly Cooked

Cold

Variety 7

a a

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

0 30 60 90 1202

4

6

8Freshly Cooked

Cold

Variety 8

a

Time (min)

Blo

od

glu

co

se

(mm

ol.m

in/

L)

Figure 3.1b Blood glucose response elicited by freshly cooked and cold potatoes for varieties 5,

6, 7 and 8. Values are means ± SEM (n= 10). Letters represent significant differences (P <0.05)

between freshly cooked and cold potatoes at the same time-point.

39

A B0

20

40

60

80

100

Gly

caem

ic In

dex

Figure 3.2 GI of baked potato chips for group A (n=10) and group B (n=10).

Bars represent means ± SEM. There is no significant difference between the GI

of baked potato chips tested in group A compared to group B. (P≥ 0.05)

40

V1 V2 V3 V4 V5 V6 V7 V80

20

40

60

80

100 Fresh CookedCold

a

b

Gly

caem

ic In

dex

Figure 3.3 GI of potato varieties 1, 2, 3, 4, 5, 6, 7 and 8 served to subjects (n= 10)

in two ways, freshly cooked and cold. Bars represent means ± SEM. A significant

effect of treatment and variety but no variety x treatment interaction was observed.

Bars with different letters within each variety group (freshly cooked and cold) are

significantly different (P <0.05).

41

V1 V2 V3 V4 V5 V6 V7 V8-40

-30

-20

-10

0

10

a

b

a

Red

ucti

on

in

Gly

caem

ic In

dex (

%)

Figure 3.4 Percent reduction in GI upon cooling for potato varieties 1, 2, 3, 4, 5, 6, 7 and

8. Each bar represents the percent reduction in GI after cooling a freshly cooked potato.

Bars with different letters are significantly different from each other (P <0.05).

42

3.4 DISCUSSION

It has been suggested that people reduce their intake of potatoes because they have a high

GI, however there is no definitive evidence to support this notion. In fact, according to the 2002

international GI tables, only ~50% of potatoes tested are classified as high GI with 28%

classified as medium and 22% classified as low GI (Brand-Miller, 2002). There seems to be a

wide variation in the reported GI values for different potato varieties, from a very low 23 to a

high 111 (Soh & Brand-Miller, 1999; Foster-Powell, Holt & Brand-Miller, 2002). The

differences seen between varieties and different cooking methods (mashed, baked, fried) are

thought to be mainly due to the properties of the starch within each potato variety as this affects

the way they respond to cooking and cooling processes. In addition, starch properties are known

to be affected by several factors including their growing environment (soil nutrient composition,

sunlight, rainfall), year of harvest and time of harvest (new vs. mature harvest).

In the present study we observed that potatoes which were cooked (boiled) and then

cooled for at least 24h at 4°C tended to elicit a lower glycaemic response (AUC) than those that

were consumed freshly cooked. Not unlike some previous studies conducted by us (Fernandes et

al., 2005; Kinnear et al., 2011) and others (Henry et al., 2005) in the present study we found that

cooling reduced the GI of freshly cooked potatoes by 10-50% in 7 of the 8 novel potato varieties

tested. This is important for individuals who are looking to reduce their dietary GI, particularly

people with diabetes who need to tightly control their blood glucose levels. This reduction in GI

may be due to an increase in the amount of RS that is formed upon cooling, however this is

unlikely. In a fully gelatinized potato there is about 7% RS; this increases to around 13% with

cooling so the minimal 6% increase RS content with cooling cannot explain these rather

43

significant reductions in GI. Instead the more likely and probable explanation for these

reductions in GI is that the starch in these potatoes converts from RDS (in gelatinized starch) to

SDS when cooled. Thus, the starch available for digestion is slowly hydrolyzed and released

slowly into the circulation resulting in a lower AUC and GI.

In this study the potato varieties we tested generally had a lower GI than those readily

available on the market. Only 3 of the potato test meals (V1, V6 and V8) were classified as high

GI (≥70) when consumed freshly cooked, although they were only moderately high with the

highest GI being 81. Four test meals were classified as medium GI (55-69) when freshly cooked

(V3-V5, V8) and two when consumed cold (V4, V6). Six of the potato test meals (V1-V3, V5,

V7, V8) were classified as low GI (≤ 55) when consumed cold. As stated previously, these

potato varieties were cross-bred specifically to increase the content of the starch properties

thought to be associated with a low GI. Although we did not measure the physicochemical

properties of these particular potato varieties we believe that the cross-breeding program was

successful considering 5 of the 8 varieties were classified as low to medium GI and only 3 were

classified as high GI when freshly cooked, this is important considering people often consume

their potatoes freshly cooked.

44

3.5 CONCLUSION

The above data supports the hypothesis that not all potatoes are high GI and that when

prepared in certain ways (ie. cold) can produce a lower glycaemic response and as a result will

have a lower GI. We also demonstrated that cooling produce a wide range of effects as we

observed an increase in GI with cooling in 1 variety while reducing the GI by 10-50% in 7 other

varieties. We were not able to show that the effect of cooling is dependent on variety, however

the wide range of effects observed (0-50% reduction in GI upon cooling) in different varieties

suggests otherwise.

45

CHAPTER 4

STUDY 2- THE ROLE OF STARCH PHYSICOCHEMICAL

PROPERTIES IN DETERMINING THE GLYCAEMIC INDEX

OF POTATO

46

4.1 INTRODUCTION

In study 1 we ascertained that the GI of potato varies between varieties and that cooling

reduces the GI in some potato varieties and may do so to different extents. However, it is

unknown why different potatoes respond to cooking and cooling processes differently. There are

several factors thought to affect the GI of potatoes as stated previously, of particular interest are

the physicochemical properties of the starch (RDS, SDS, RS, phosphorous and amylose content)

found within them (Svegmark et al., 2002; Lynch et al., 2007). Research suggests that utilizing

in vitro methodology to assess starch composition and digestibility may be a more practical and

economical way of assessing the potential glycaemic impact of CHO foods. There is some

debate as to whether or not the in-vitro measurements are reflective of how they would behave

physiologically, however several studies have demonstrated this through correlation with the in-

vivo glycaemic response (Englyst et al.,1999;Englyst et al., 2003). Studies examining the

relationship between the proportion of RAG in cereals and GI show that foods which contain a

greater proportion of RAG tend to have a higher GI than foods which contain greater amounts of

SAG (Englyst et al., 2003). Moreover, other starch properties such as amylose and phosphorous

have been shown to affect starch digestibility and ultimately GI. As stated previously amylose is

resistant to gelatinization and with cooling is more readily retrograded. In the case of

phosphorous, it is known to form complexes with starch and resist its ability to gelatinize.

The objective of study 2 is to examine the relationship between the physicochemical

properties of starch and the GI of potato. To assess these relationships, we re-tested four potato

varieties previously tested in study 1 (V2, V3, V4 and V5; newly harvested). These potato

varieties were chosen for a variety of reasons including; cooling reduced their GI significantly,

47

no significant change was observed with cooling or were classified as low-GI. We were also

interested in examining the mechanism by which cold potatoes produce a lower GI than freshly

cooked potatoes. To examine the potential mechanism responsible for the lower glycaemic

response and GI elicited by cold potatoes we measured serum insulin. To examine the role of

starch properties in determining GI we assessed some physicochemical properties thought to play

a role in starch digestibility.

48

4.2 STUDY DESIGN AND METHODS- GLYCAEMIC INDEX TESTING

4.2.1 Subjects

One group of ten healthy subjects (8 males, 2 females) with a mean age of 39.3 ± 4.2, a

mean weight of 80. 9 ± 5.2 (60.9-111.4), a mean BMI= 26.4 ± 0.8 (22.3-30.9) participated in the

following study (Table 4.1). Two subjects were unable to continue with the study after 3 visits

and were replaced by two new, same sex subjects. All subjects met the inclusion criteria as

described in section 3.2.1. The study protocol was approved by the University of Toronto

Research Ethics Board and informed consent was obtained from all subjects prior to taking part

in the study.

4.2.2 Test Meals

Four novel potato varieties tested in study 1 (V2, V3, V4 and V5) were re-tested during

the present study. These potatoes were grown in a different location (Guelph, Ontario) than

those from study 1 (Lethbridge, Alberta) and were harvested at maturity in October

2010. All varieties were stored as described previously in section 3.2.2. To determine the

amount of potato required to feed subjects 50g of available carbohydrate proximate analysis was

conducted as previously described in section 3.2.2. In addition to potato meals subjects also

tested white bread three times (reference food) and potato chips. The amount of potato

consumed varied depending on the variety and ranged from 236-309g per test meal; each meal

contained 50g of avCHO (table 4.2).

49

4.2.3 Protocol

The study was conducted at Glycaemic Index Laboratories in Toronto, Ontario from

October 2010 through December 2010. The protocol for study 2 was identical to that described

in section 3.2.3 for study 1. However, for the present study six additional blood samples were

drawn at each time point (0-120min) in order to measure serum insulin (pmol/ L). Blood glucose

was measured as described in section 3.2.3. For insulin, capillary blood samples were placed

into tubes and allowed to clot for 20 minutes at ambient temperature. Samples were then spun at

3300rpm for 15mins, serum was removed and samples stored at -70°C pending analysis. Serum

insulin was measured using an ALPCO insulin EIA kit (Cat.# 80-INSHU-E10; Salem, NH).

50

4.2.4 Calculation of Glycaemic Index

The GI for all test meals was calculated as describe in section 3.2.4.

4.2.5 Statistical Analysis

Results are expressed as means ± SEM. Statistical analysis was conducted on blood

glucose and insulin profiles for each variety (V2-V5) and treatment (fresh cooked, cold). To

examine the differences between treatments, data was analyzed using a three-factor analysis of

variance (ANOVA) to examine the effect of time, variety, treatment and time x variety x

treatment interaction. For AUC and GI values two-factor ANOVA was conducted to examine

the effect of variety, treatment and variety x treatment interaction. After demonstration of

significant heterogeneity, Tukey‟s post-hoc test was used to determine significant differences

between individual mean blood glucose and insulin concentrations at each time point, mean

AUC and GI. In addition, to examine the differences between the GI of each potato variety to

that of white bread we conducted a one-way ANOVA. Simple un-paired t-test was used to

determine if there were significant differences between the GI of each variety both freshly

cooked and cold between harvest years (2009 vs. 2010).

Differences were considered significant if a two-tailed P ≤ 0.05. Analysis was done

using SPSS statistical package 18.0.

51

4.3 MATERIALS AND METHODS- IN VITRO STARCH DIGESTIBILITY

Physicochemical measurements were performed by the laboratory Dr. Qiang Liu from

Agriculture and Agri-Food Canada.

4.3.1 Materials

Potato varieties 2, 3, 4 and 5

4.3.2 Preparation of dry matter and starch

Potato dry matter and starch were obtained according to the methods of Liu, Yada and

Arul (2002) and Liu, Weber, Currie and Yada (2003). The samples were kept in air-tight plastic

bags at room temperature until further use.

4.3.3 Chemical Composition

Total starch content of potato dry matter was determined based on AACC (2000) method

76-13. Protein content of dry matter was determined using Thermoquest CE Instrument (NA

2100 Protein, Thermo-Quest Italia S.P.A., Ann Arbor, MI, USA). The instrument determines the

nitrogen content of the sample. Protein content was calculated by multiplying the nitrogen

content by a factor of 6.25. Atropine (4.84% N), DL-methionine (9.39% N), acetanilide (10.36%

N), and nicotinamide (22.956% N) were used as standards to produce a standard curve.

Apparent amylose content in potato starch was determined by iodine colorimetry according to

Williams, Kuzina and Hlynka (1970). Starch and dry matter sample (1.000 g) was weighed into

a tared porcelain crucible, and then heated one hour at 250ºC and four hours at 475ºC in a muffle

oven. After cooling, the ash was dissolved with 10.0 mL of 1.0 M HCl for 30 min, and then the

contents were transferred to a 50 mL plastic volumetric vial and brought up to volume, mixed

and allowed to stand for one hour before analysis. Total phosphorus, potassium, magnesium and

52

calcium content in starch and dry matter were determined using a Varian Vista Pro ICP-OES

(Inductively Coupled Plasma-Optical Emission Spectrometer; Mississauga, ON, Canada).

4.3.4 In vitro digestibility of cooked and cooled potatoes

In vitro starch digestibility was determined using the method of Englyst, Kingman and

Cummings (1992) with modifications (Chung, Lim & Lim, 2006). The potatoes were cooked

and cooled according to the procedure described for the GI protocol in section 2.2.2 and then put

into freezer at -80ºC before freeze drying. The freeze-dried samples were manually ground by a

mortar and pestle, and passed through a 125 µm sieve. Percentages of rapidly digestible starch

(RDS, % digestible starch at 20 min), slowly digestible starch (SDS, % digestible starch at 120

min - % digestible starch at 20 min), and resistant starch (RS, total starch % - % digestible starch

at 120 min) were calculated.

4.3.5 Statistical Analysis

All samples were tested in duplicate in each analytical technique. Percentages for RDS,

SDS and RS were converted into grams per serving (amount consumed in vivo GI test) for each

potato variety. Simple linear regression was used to assess the relationships between the

physicochemical properties amylose, phosphorous and in vitro digestibility products RDS, SDS

and RS and the GI of potato varieties for 2, 3, 4 and 5 consumed freshly cooked and cold.

Differences were considered significant if a two-tailed P ≤ 0.05. Analysis was done using

SPSS statistical package 18.0.

53

4.4 RESULTS

4.4.1 Blood Glucose Responses

Blood glucose profiles for potato varieties 2, 3, 4 and 5 are shown in figure 4.1. There

was no main effect of variety for the mean blood glucose AUC (F=1.739, P= 0.157) and no

variety x treatment interaction for the AUC of freshly cooked and cold potatoes (F=0.656, P=

0.709). However, a main effect of cooling (F= 6.729, P= 0.010) was observed with freshly

cooked potatoes eliciting a significantly greater AUC than cold potatoes (154.1 ± 28.0 vs. 107.3

± 18.6) (table 4.3). Varieties 2 and 5 freshly cooked elicited a greater blood glucose response at

30 and 45 min when compared to cold (P <0.05). Variety 3 freshly cooked elicited a greater

blood glucose response at 30 min when compared to cooled potatoes (P <0.05). Variety 5

consumed freshly cooked caused a greater rise in blood glucose at 15 mins when compared to

cooled potatoes (P <0.05). There were no significant differences between the blood glucose

response elicited by variety 4, both freshly cooked and cold.

4.4.2 Insulin Responses

Serum insulin profiles for potato varieties 2, 3, 4 and 5 are shown in figure 4.3. There

was no main effect of variety for the mean insulin AUC (F=1.61, P= 0.184) and no variety x

treatment interaction for the AUC of freshly cooked and cold potatoes (F= 0.44, P= 0.730).

However, a main effect of cooling (F= 6.24, P=0.012) was observed with freshly cooked

potatoes eliciting a significantly greater insulin AUC than cold potatoes (434.7 ± 96.5 vs. 275.7

± 61.6) (table 3.3). Variety 3 cold elicited a significantly lower insulin response than freshly

cooked (P<0.05). Figure 4.5 shows the relationship between the GI and II. There was a

significant positive relationship between the GI and II for all potato meals (freshly cooked and

cold) (r2= 0.59, P= 0.03).

54

4.4.3 Glycaemic Index

Table 4.4 shows the GI values for potato varieties 2, 3, 4 and 5 compared to white bread.

ANOVA showed the GI of V2 (38 ± 5) and V5 (39 ± 4) served cold were significantly different

(P< 0.001) from the GI of WB (GI=71). Only 2 of the potato test meals (V4 and V5) were

classified as high GI (≥70) when consumed freshly cooked. Only two test meals were classified

as medium GI (55-69) when freshly cooked (V2 and V3) and one when consumed cold (V4).

Three of the potato test meals (V2, V3 and V5) were classified as low GI (≤ 55) when consumed

cold. ANOVA showed a main effect of variety (F=4.84, P < 0.001), a main effect of treatment

(F=36.33, P <0.001) and a variety x treatment interaction (F= 1.33, P=0.235). Tukey‟s test

showed that variety 5 served cold had a significantly lower GI than freshly cooked (71 ± 7 vs. 39

± 4, P<0.001). A comparison between like potato varieties harvested in 2009 versus those

harvested in 2010 showed that there were no significant differences in GI for variety 2 fresh

cooked (F= 1.790, P= 0.335) and cold (F=1.050, P= 0.634), variety 3 fresh cooked (F= 1.96,

P=0.277) cold (F= 1.40, P= 0.508), variety 4 fresh cooked (F=2.040 , P= 0.3087) and cold (F=

1.146, P= 0.842), and variety 5 fresh cooked (F= 2.27, P= 0.197) and cold (F= 2.09, P= 0.672).

55

4.4.4 Starch Physicochemical Properties and GI

Figure 4.6 shows the relationship between the amounts of RDS (in grams) found in a

portion of potato containing 50g of available carbohydrate and the GI of cooked and cooled

potatoes. There is a significant positive relationship between RDS content and the GI (r2= 0.85,

P= 0.001). Figure 4.7 shows the relationship between SDS content (g) and the GI of freshly

cooked and cooled potatoes. There was a significant inverse relationship between SDS content

and GI (r2= -0.60, P= 0.02). Figure 4.8 shows the relationship between the amount of resistant

starch (RS) in a portion of potato containing 50g of available carbohydrate and the GI freshly

cooked and cold potatoes. No significant relationship (r2= -0.19, P= 0.28) was observed between

RS content (g/ serving) and the GI of freshly cooked and cold potatoes. Figure 4.9 shows the

relationship between percent amylose for each potato variety and the GI of freshly cooked and

cold potatoes. A significant inverse relationship was observed between percent amylose content

and the GI of freshly cooked and cold potatoes (r2= -0.98, p= 0.006). Figure 4.10 shows the

relationship between the GI of freshly cooked and cold potatoes and phosphorous content. A

significant inverse relationship between the GI of freshly cooked potatoes and phosphorous

content (r2= -0.97, P<0.01). No significant relationship was observed between the mean GI for

freshly cooked and cold potatoes and phosphorous content (r2=0.03 , P= 0.839).

56

Table 4.1 Subject Characteristics

Variable mean ± SEM Range

n (male: female) 8:2 -

Age (y) 39.3 ± 4.2 25-61

Weight (kg) 80. 9 ± 5.2 60.9-111.4

Height (m) 1.74 ± 0.03 1.57-1.93

BMI (kg/ m2) 26.4 ± 0.8 22.3-30.9

57

Table 4.2 Energy and macronutrient composition of test meals

Test Meals Weight

(g)

Energy

(kcal)

Fat

(g)

Protein

(g)

Total

CHO (g)

Fibre

(g)

Available

CHO (g)

Variety 2 236 241 0 6.4 53.8 3.8

50.0

Variety 3 279 246 0 7.5 53.1 3.1

50.0

Variety 4 309 246 0 7.3 54.2 4.2

50.0

Variety 5 259 241 0 7.4 54.9 4.9

50.0

Baked

Potato

Chips

66 264 3.3 5.3 54.1 4.1 50.0

White

Bread

104 245 1.7 7.5 52.8 2.8 50.0

*Potatoes were weighed raw with skin

aThe following analyses were performed by Agri-Food Laboratories, Guelph, ON: moisture

AOAC 93.15 (not shown), protein: AOAC 990.03, ash: AOAC 942.05 (not shown), fat: AOAC

920.39 and carbohydrate: by difference. Analysis for total dietary fibre (AOAC 993.43 and

985.29) was performed by Maxxam Analytics, Mississauga.

bTypical portion size- actual weight varied slightly due to moisture variations.

58

Table 4.3 Blood glucose and Insulin AUC values for potato varieties 2, 3, 4 and 5

Glucose AUC

(mmol.min/ L)

Treatment Mean for Variety

Fresh Cooked Cold

Variety 2

119.94 ± 9.75

83.02 ± 12.14

101.48 ± 10.95a

Variety 3

137.22 ± 28.47

102.25 ± 13.83

119.74 ± 21.15

Variety 4

168.83 ± 33.21

139.48 ± 26.57

154.16 ± 29.89b

Variety 5

190.50 ± 40.47a

104.42 ± 21.74b

147.46 ± 31.11

Mean for Treatment

154.12 ± 27.98a

107.29 ± 18.57b

Insulin AUC

(pmol/ L)

Treatment

Fresh Cooked Cold Mean for Variety

Variety 2

399.3 ± 73.7

277.4 ± 69.0

338.3 ± 71.4

Variety 3

437.9 ± 103.8

291.3 ± 63.0

364.6 ± 83.4

Variety 4

419.1 ± 105.2

229.1 ± 35.9

324.1 ± 70.6

Variety 5

410.9 ± 60.5a

228.3 ± 36.9b

319.6 ± 48.7

Mean for Treatment

416.8 ± 85.8 a

256.5 ± 51.2b

Values mare means ± SEM

abAUC values not sharing the same letter subscripts differ significantly (P<0.05) from each

other within each row.

59

Table 4.4 GI values for potato varieties 2, 3, 4 and 5

Glycaemic Index

Treatment

Mean for Variety Fresh Cooked Cold

Variety 2

57 ± 5

38 ± 5x

48 ± 6a

Variety 3

62 ± 6

50 ± 6

56 ± 6

Variety 4

74 ± 6

66 ± 6

70 ± 6b

Variety 5

71 ± 7a

39 ± 4bx

55 ± 6

Mean for

Treatment

66 ± 6a

48 ± 5b

Values are means ± SEM

a bGI values not sharing the same letter subscripts differ significantly (P<0.05) from each other

within each row.

x GI values differ significantly from the GI of white bread.

60

Table 4.5 GI values for potato varieties 2, 3, 4 and 5 harvested in 2009 and 2010 and served to

subjects (n= 10) freshly cooked and cold

2009 Harvest 2010 Harvest

Treatment Treatment

Fresh Cooked Cold Fresh Cooked Cold

Variety 2

52 ± 4

35 ± 5z

57 ± 5

38 ± 5z

Variety 3

65 ± 6a

34 ± 6bz

62 ± 6

50 ± 6

Variety 4

59 ± 6

61 ± 5

74 ± 6

66 ± 6

Variety 5

57 ± 8

45 ± 6

71 ± 7a

39 ± 4bz

Mean for

Treatment

58 ± 6

44 ± 6

66 ± 6a

48 ± 5b

Values are means ± SEM

a bGI values not sharing the same letter subscripts differ significantly (P<0.05) from each other

within each row and harvest year.

z GI values differ significantly from the GI of white bread.

61

Table 4.6 In vitro digestibility of cooked and cooled potatoes

Variety Samples RDS (%)

mean ± SD

SDS (%)

Mean ± SD

RS (%)

Mean ± SD

Variety 2

Cooked

45.8 ± 0.5

5.8 ± 0.8

21.2± 0.9

Cooled

43.0 ± 1.2

8.3 ± 0.7

22.5± 0.6

Variety 3

Cooked

43.4 ± 0.4

5.7 ± 0.4

23.1± 0.8

Cooled

41.2 ± 0.2

6.7 ± 0.1

23.5± 0.1

Variety 4

Cooked

47.7 ± 0.5

3.5 ± 1.3

22.5± 0.9

Cooled

46.4 ± 1.0

5.8 ± 0.7

23.3± 0.9

Variety 5

Cooked

45.7 ± 0.3

5.9 ± 0.6

22.0± 0.6

Cooled

43.2 ± 0.3

8.1 ± 0.6

24.6± 0.4

* For cooked and cooled potatoes, the RDS, SDS and RS content are based on total mass

of dry matter

62

WB V2 V3 V4 V50

50

100

150

200

250a

b

AU

C (

mm

ol.m

in/ L

)

0 30 60 90 1203

4

5

6

7

8 Freshly Cooked

Cold

Variety 2

aa

Time (min)

Ch

an

ge in

blo

od

glu

co

se

(mm

ol.m

in/

L)

Variety 3

0 30 60 90 1203

4

5

6

7

8Freshly Cooked

Cold

Time (min)

Ch

an

ge in

blo

od

glu

co

se

(mm

ol.m

in/

L)

a

0 30 60 90 1203

4

5

6

7

8Freshly Cooked

Cold

Variety 4

Time (min)

Ch

an

ge in

blo

od

glu

co

se

(mm

ol.m

in/

L)

Variety 5

0 30 60 90 1204

5

6

7

8

9 Freshly Cooked

Cold

a

a a

Time (min)

Ch

an

ge in

blo

od

glu

co

se

(mm

ol.m

in/

L)

Figure 4.1 Incremental AUC and change in blood glucose after consuming potato varieties 2, 3, 4 and 5 served to

ten subjects two ways, freshly cooked and cold. Values are means ± SEM.Letters represent significant differences

(P <0.05) between freshly cooked and cold potatoes at the same time-point. For AUC, different letters represent

significant differences between each treatment (P <0.05).

63

WB V2 V3 V4 V50

200

400

600

800

Insu

lin A

UC

(p

mo

l/ L

)

a

b

0 30 60 90 1200

100

200

300

400

500 Freshly Cooked

Cold

Variety 2

Time (min)

Ch

an

ge

in

in

su

lin (

pm

ol/ L

)

0 30 60 90 1200

100

200

300

400

500 Freshly Cooked

Cold

Variety 3

Time (min)

Ch

an

ge

in

in

su

lin (

pm

ol/ L

)

a

0 30 60 90 1200

100

200

300

400

500Freshly Cooked

Cold

Variety 4

Time (min)

Ch

an

ge

in

in

su

lin (

pm

ol/ L

)

0 30 60 90 1200

100

200

300

400

500 Freshly Cooked

Cold

Variety 5

Time (min)

Ch

an

ge

in

in

su

lin (

pm

ol/ L

)

Figure 4.2 Incremental AUC and change in insulin after consuming potato varieties 2, 3, 4 and5 served to ten

subjects two ways, freshly cooked and cold. Values are means ± SEM . Letters represent significant differences (P

<0.05) between freshly cooked and cold potatoes at the same time-point. For mean AUC, different letters represent

significant differences between each treatment (P <0.05).

64

Figure 4.3 GI of potato varieties 2, 3, 4 and 5 served to ten subjects in two ways, freshly cooked

and cold. Bars represent means ± SEM. A significant effect of treatment and variety and a

variety x treatment interaction was observed. Bars with different letters within each variety

group (freshly cooked and cold) are significantly different (P <0.05). (upper left) Percent

reduction in GI upon cooling a cooked potato. Each bar represents the difference in GI between

freshly cooked and cold potatoes for each variety. Values are means ± SEM. Different subscripts

indicate that the reduction in GI with cooling were significantly different from each other.

V2 V3 V4 V50

20

40

60

80

100 Hot

Cold a

b

Gly

ca

em

ic I

nd

ex

V2 V3 V4 V5-60

-40

-20

0

-33%

-40%

-25%

-9%

a b

Gly

caem

ic In

dex-

% D

iffe

ren

ce

65

Figure 4.4 GI of potato varieties 2, 3, 4 and 5 served to ten subjects in two ways, freshly cooked

and cold and harvested in two separate years, 2009 and 2010. Bars represent means ± SEM. No

significant differences were observed between like varieties and preparation methods between

years.

V2

09

V2

09

V2

10

V2

10

V3

09

V3

09

V3

10

V3

10

V4

09

V4

09

V4

10

V4

10

V5

09

V5

09

V5

10

V5

10

0

20

40

60

80

100

Gly

ca

em

ic I

nd

ex

66

0 20 40 60 800

20

40

60

80

100

Glycaemic Index

Ins

ulin

ae

mic

In

de

x

Figure 4.5 Relationship between glycaemic index (GI) and insulinaemic index (II)

values of 50g available carbohydrate portions of potato varieties 2, 3, 4 and 5 freshly

cooked ( ) and cold ( ). A significant positive relationship between II and GI was

observed (r2= 0.59, p= 0.03)

67

24 26 28 30 32 340

20

40

60

80

100

Rapidly Digestible Starch (g/ serving)

Gly

ca

em

ic In

de

x

Figure 4.6 Relationship between the amount of rapidly digestible starch (RDS) in

a portion of potato containing 50g of available carbohydrate and the GI freshly cooked

( ) and cold ( ) potatoes. A significant positive relationship (r2= 0.85, p= 0.001) was

observed between RDS content (g/ serving) and GI.

68

0 2 4 60

20

40

60

80

Slowly Digestible Starch (g/ serving)

Gly

caem

ic In

dex

Figure 4.7 Relationship between the amount of slowly digestible starch (SDS) in a portion of

potato containing 50g of available carbohydrate and the GI freshly cooked ( ) and cold ( )

potatoes. A significant inverse relationship (r2= -0.60, p= 0.02) was observed between SDS

content (g/ serving) and the GI of freshly cooked and cold potatoes.

69

12 13 14 15 16 170

20

40

60

80

Resistant Starch (g/ serving)

Gly

caem

ic In

dex

Figure 4.8 Relationship between the amount of resistant starch (RS) in a portion of

potato containing 50g of available carbohydrate and the GI freshly cooked ( ) and

cold ( ) potatoes. No significant relationship (r2= -0.19, p= 0.28) was observed

between RS content (g/ serving) and the GI of freshly cooked and cold potatoes.

70

30 35 40 4540

50

60

70

80

Amylose (%)

Gly

caem

ic In

dex

Figure 4.9 Relationship between percent amylose for each potato variety and GI. A

significant inverse relationship (r2= -0.98, p= 0.006) was observed between amylose

content (%) and the mean GI of freshly cooked and cold potato.

71

140 150 160 170 180 19040

50

60

70

80

Phosphorous (mg/ serving)

Gly

ca

em

ic I

nd

ex

Figure 4.10 The relationship between the GI of potato and phosphorous

content. No significant relationship between the mean GI of freshly cooked

and cold potatoes and phosphorous content (mg/ serving) was observed (r2=0.03, P= 0.839).

72

4.5 Discussion and Conclusions

Previously we determined that the effect of cooling on GI is dependent on variety and

that these effects can vary significantly between varieties. In the present study we also found

that the effect of cooling is dependent on variety with cooling significantly reducing the GI of V5

while having no significant effect in V2, V3 and V4. Only 2 of the potato test meals, V4 and V5

were classified as high GI (≥70) when consumed freshly cooked. Cooling reduced 3 of 4 potato

varieties tested to a low GI classification.

Examination of the blood glucose raising ability of freshly cooked versus cold potatoes

showed no main effect of variety but a main effect of cooling with cold potatoes tending to elicit

a lower glycaemic response than freshly cooked. The lower glycaemic response elicited by cold

potatoes may be attributed to an increase in postprandial insulin levels or may be due to an

increase in the proportion of SDS and RS often found in cold potatoes. In order to test our

hypothesis that cold potatoes do not produce a higher insulin response we measured serum

insulin. As expected the low GI of cold potatoes was not due to an increase in postprandial

insulin. In fact, ANOVA showed a main effect of cooling (F= 6.24, P=0.012) with freshly

cooked potatoes eliciting a significantly greater insulin AUC than cold potatoes (416.8 ± 85.8 vs.

256.5 ± 51.2) (table 4.3).

It is generally accepted that cooking followed by cooling reduces the GI of potato,

however it is still unknown what is responsible for this reduction. Upon cooling, some of the

starch in potato retrogrades to form a starch product which is resistant to digestion, this is

thought to be partly responsible for the reduction in glycaemic response and the lower GI often

73

seen with cold potatoes. Although an increase in RS may help reduce the glycaemic impact of a

potato meal it seems only to play a diminutive role as there is typically only a 6 percent increase

in RS content when comparing a freshly cooked gelatinized potato to a cooked and cooled

potato.(Wolever, 2006) A more plausible explanation for the decrease in GI often observed with

cooling may be that some RDS is converted to SDS; the result is a greater proportion of SDS and

a lower GI. To explore the relationship between starch properties measured in vitro and GI we

conducted in vitro starch analysis with potato dry matter and related these properties to GI values

measured in vivo. In vitro analysis was conducted using potato dry matter so in order to make it

more physiologically relevant the amounts of RDS, SDS, RS were converted from percentages to

grams per portion of potato containing 50g of available carbohydrate. Our in vitro analysis

showed that potatoes with a lower GI had significantly higher amounts of SDS. Conversely,

potatoes with higher GI values had significantly higher amounts of RDS.

Amylose, due to its linear structure is more resistant to gelatinization and is more readily

retrograded with cooling. Potatoes which contain greater amylose:amylopectin are thought to

have a lower GI, this is due to the fact that amylopectin is more readily hydrolyzed because of its

highly branched structure; this coupled with reasons stated previously lead to the hypothesis that

increasing the proportion of amylose in potatoes may be beneficial for the GI of potatoes. We

found a strong significant inverse relationship between percent amylose content and the GI of

freshly cooked and cold potatoes (r2= -0.98, p= 0.006) which supports our hypothesis.

Potatoes are also known to contain an appreciable amount of phosphorous which is

thought to affect the functional properties of the starch in potato. Phosphorous is known to form

complexes with amylose and amylopectin effectively limiting the ability for the starch to

74

gelatinize. If this is correct potatoes containing a greater proportion of phosphorous would be

advantageous from a GI stand-point because of its ability to resist starch gelatinization.

Conversely, phosphorous has been positively associated with starch retrogradation suggesting

that potatoes containing a greater proportion of phosphorous will have a lower GI than potatoes

which contain lower amounts of phosphorous. In the present study phosphorous content did not

seem to be related to GI however, this may be due to the fact that with cooking, particularly

boiling, phosphorous tends to leach out into the cooking liquid. The phosphorous measurements

for this study were conducted using the dry matter of potatoes that were boiled and then freeze-

dried which may have significantly affected the amount of phosphorous.

75

CHAPTER 5

OVERALL DISCUSSION, CONCLUSIONS AND FUTURE

DIRECTIONS

76

5.1 DISCUSSION

5.1.1 Study 1

The overall objective of this thesis was to identify low-GI potatoes and to examine the

role of the physicochemical properties of starch in determining the GI of potato. In study 1 we

screened additional novel potato varieties provided to us by the Bio-Potato Network and we were

able to ascertain that the GI of potato varies between varieties and that cooling reduces the GI in

some potato varieties and may do so to different extents. Although we did not observe a

statistically significant treatment x variety interaction we found that cooling produced a wide

range of effects from no reduction in GI with cooling to a 50% reduction in GI with cooling.

The GI values for the freshly cooked varieties tested ranged from 52 to 81 for freshly cooked

potatoes and 35 to 62 for cooled potatoes; these values were generally lower than those reported

in the literature (Soh & Brand-Miller, 1999; Henry et al., 2005).

5.1.2 Study 2

The objectives of study 2 were to re-test the GI of four varieties of interest from 2009 in

2010 and to examine the role of the physicochemical properties (RDS, SDS, RS, amylose and

phosphorous content) of their starch in determining GI. As we hypothesized, potato varieties

which contained a greater proportion of their starch in the form of RDS had a higher GI than

those which contained a greater proportion of SDS. RS did not seem to have a significant impact

on the GI of potato for reasons explained in section 4.7. Amylose content was strongly

negatively related to GI which as stated previously in section 4.7 may be due to several factors

including reduced gelatinization with cooking and enhanced retrogradation with cooling.

77

Unfortunately, contrary to what we had hypothesized we were unable to see a negative

relationship between phosphorous content and GI.

The GI values for varieties 2, 3, 4 and 5 did not significantly differ between harvest years

despite being grown in two different locations (Lethbridge, Alberta vs. Guelph, Ontario). The

fact that there was no statistically significant difference between harvest years is interesting

considering factors such as soil nutrient content, rainfall and time of harvest are thought to play

an important role in starch formation and may influence GI.

In both studies we saw that cooling tended to reduce the GI of potato, however we were

unsure of the precise mechanism(s) responsible for this reduction. To explore the possibility that

cold potatoes elicit a higher insulin response, we measured postprandial serum insulin and as

expected we were able to determine that cold potatoes have a lower GI not because they increase

postprandial insulin but because overall they tend to elicit a lower glycaemic and insulinaemic

response.

78

5.2 CONCLUSIONS

In this thesis, three main objectives were identified in order to test the hypotheses. The

first objective was to screen additional novel potato varieties, determine their GI, and to confirm

our previous findings that the effect of cooling on the GI is dependent on variety. In study 1 we

identified a few potato varieties that were classified as low GI. Unlike previous findings from

our laboratory we were unable to identify a significant variety x treatment interaction but

observed significant differences in GI between varieties with cooling reducing GI by 0-50%. In

study 2 we identified a significant variety x treatment interaction, with cooling significantly

reducing the GI in some potato varieties while having no effect in other varieties.

The second objective was to examine the role of the physicochemical properties (RDS,

SDS, RS, amylose and phosphorous content) of their starch in determining GI. As expected

potato varieties with a greater proportion of their starch in the form of RDS had a high GI than

those with a greater proportion of their starch in the form of SDS. Amylose was negatively

related to GI as we hypothesized. RS and phosphorus content did not seem to be related to GI.

The third objective was to determine if the low GI of cold potatoes is due to an increase

in postprandial insulin. In keeping with our hypothesis, cold potatoes did not produce an

exaggerated increase in postprandial insulin.

79

5.3 FUTURE DIRECTIONS

The potatoes tested in the present study tended to have lower GI values than some

potatoes currently available on the market which suggest that the work done by the breeders and

starch chemists was successful, however more work is definitely needed. During our series of

tests we identified some potato varieties that were low GI when freshly cooked and cold,

however unfortunately most of these varieties were identified as poor crops that were not

agronomically sound, meaning they were not a viable product and thus would not be a candidate

to bring to the fresh potato market. Our in vitro analysis showed some interesting data which

were in line with our hypotheses, however these analyses were conducted using potato dry

matter and not fresh potato. Future in vitro analysis conducted using fresh potato samples should

be conducted as this more accurately reflects what is consumed in vivo. In addition, examining

the effect of maturity on the physicochemical properties of starch would be an important aspect

to explore. Maturity was a factor that we had planned on exploring as part of this thesis but due

to unforeseen circumstances we were unable to obtain early harvest potatoes for our analyses.

80

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