effect of tea extract on lactic acid bacterial growth, their cell surface characteristics and...

��

Effect of tea extract on lactic acid bacterial growth, their cell surface charac-teristics and isoflavone bioconversion during soymilk fermentation

Danyue Zhao, Nagendra P. Shah

PII: S0963-9969(14)00309-3DOI: doi: 10.1016/j.foodres.2014.05.004Reference: FRIN 5246

To appear in: Food Research International

Received date: 2 September 2013Revised date: 24 April 2014Accepted date: 3 May 2014

Please cite this article as: Zhao, D. & Shah, N.P., Effect of tea extract on lactic acid bacte-rial growth, their cell surface characteristics and isoflavone bioconversion during soymilkfermentation, Food Research International (2014), doi: 10.1016/j.foodres.2014.05.004

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

http://dx.doi.org/10.1016/j.foodres.2014.05.004

http://dx.doi.org/10.1016/j.foodres.2014.05.004

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPTEFFECT OF TEA ON LAB PROPERTIES AND ISOFLAVONE IN FERMENTED SOYMILK-TEA

1

Effect of tea extract on lactic acid bacterial growth, their cell surface

characteristics and isoflavone bioconversion during soymilk fermentation

Danyue Zhao and Nagendra P. Shah1

Food and Nutritional Science - School of Biological Sciences, the University of

Hong Kong, Pokfulam Road, Hong Kong SAR, China

1Corresponding author: Prof. Nagendra P. Shah

Tel: +852 2299 0836 Fax: +852 2299 9914

Email: [email protected]

ACC

EPTE

D M

ANU

SCR

IPT


2

Abstract (word count: 226)

In this study, the influences of tea extract (TE) supplementation on selected

lactic acid bacteria (LAB) growth, their cell surface characteristics and soy

isoflavone bioconversion were investigated. Soymilk-tea (SMT) was prepared by

combining TE (green, oolong or black tea) with soymilk containing lactose (SML).

Bacterial growth was studied by inoculating four LAB into soymilk or SMT

individually. FT-IR spectroscopy and cell surface hydrophobicity (SH) assay were

performed to monitor the changes in bacterial cell surface characteristics due to

TE supplementation. HPLC analysis was used for evaluation of isoflavone

bioconversion in fermented soymilk and SMT. Results showed that TE promoted

the growth of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and L.

paracasei but inhibited the growth of L. acidophilus and Streptococcus

thermophilus at a concentration of 2% (weight of TE powder/volume of soymilk).

Viabilities of all LAB in fermented SMT were maintained at above 7 log CFU/mL

during four-week refrigeration (4°C). FT-IR spectra indicated major changes in

the membrane fatty acids, proteins and cell wall polysaccharides after TE

treatment. Changes in SH provided further information on the modification of

bacterial cell surface by TE. Inhibition of deglycosilation was observed in

fermented SMT for L. acidophilus and L. paracasei, both of which exhibited strong

ability to transform isoflavone glycoside in SML, while isoflavone bioconversion

was enhanced for L. bulgaricus. Our study provides some practical significance

for developing novel functional soyfood by combining gut health-promoting

bacteria, soy isoflavones and tea polyphenols.

Key words: Soy; tea; lactic acid bacteria; FT-IR spectroscopy; isoflavone

bioconversion; cell surface hydrophobicity.

ACC

EPTE

D M

ANU

SCR

IPT


3

1. Introduction

In recent years, beneficial effects of consuming tea and soyfoods have been

studied extensively. Tea and soyfoods are rich in flavonoids, with tea polyphenols

(TP) and soy isoflavones being the major contributors, respectively (Hsu et al.,

2011). These bioactive phytochemicals possess potent antioxidant,

antimutagenic, anti-inflammatory and anti-microbial properties and the

synergistic effect of tea and soy in lowering risks of several cancers and

cardiovascular diseases has been established both in vitro and in vivo

(Holzbeierlein et al., 2005; Hsu et al., 2011; van Dam et al., 2013; Yang et al.,

2000). Thus, it is desirable to increase the daily intake of flavonoids, especially

from natural sources.

The complicated microbial-flavonoid interactions are highlighted in studies

involving dietary intervention of flavonoid on intestinal health. Tea polyphenols

are reported to exhibit inhibitory effects on the survival of certain bacteria,

mainly pathogenic ones(Ankolekar et al, 2011; Cui et al., 2012; Lee et al., 2006).

On the other hand, studies also showed that TP supplementation, at certain

concentrations, exerts positive effect on intestinal microbiota (Jaziri et al., 2009;

Najgebauer-Lejko et al., 2011). Recently, Gaudreau et al. (2012) suggested that

the growth-promoting effect of green tea extract on L. helveticus might result

from the reduced redox potential and antioxidant capacity of TPs. Moreover,

reports showed that dietary polyphenols can be metabolized by probiotic

organisms to release aromatics and small phenolic acids, which alter the

bioactivity and bioavailability of flavonoids (Lee et al., 2006). Lactic acid bacteria

(LAB) are a major group of bacteria that participate in the fermentation process

and they are reported to act on polyphenols, including deglycosylation,

ACC

EPTE

D M

ANU

SCR

IPT


4

de-esterification, demethylation, dehydroxylation and decarboxylation activities,

enhancing the intestinal absorptivity of these phenolic compounds (Aura, 2008;

Tabasco et al., 2011). Soy yogurt (SY) prepared by fermenting soymilk has been

widely studied for the deglycosylation of isoflavone glucosides into aglycones by

LAB (Otieno & Shah, 2007). However, the production and function of bacterial

β-glycosidase are influenced by several factors including incubation temperature,

pH of the media, sugar composition (Jurado et al., 2004; Osiriphun & Jaturapiree,

2009) and enzyme-polyphenol interaction (He et al., 2007).

Microbial cell surface composition also plays a part in microbial-flavonoid

interaction. Bacterial cell surface hydrophobicity (SH) refers to the dislike of cells

for water (Geertsema-Doornbusch et al., 1993). SH is highly related to the initial

attachment of bacteria to gut lumen and is generally considered as one of the

criteria for selecting probiotics (Bustos et al., 2012). For evaluation of SH, several

biochemical methods have been developed, among which the most commonly

employed is microbial adhesion to hydrocarbons (MATH) (Rosenberg, 2006).

High antioxidant potency and medicinal properties of dietary flavonoids make

flavonoid-rich products more attractive to consumers and food manufacturers

(van Dam et al., 2013). Fermentation of soymilk supplemented with TE can

integrate TPs and probiotic organisms, conferring health benefits including

balance of intestinal microbial composition (Parkar et al., 2008), enhancement of

antioxidant capacity (Najgebauer-Lejko et al., 2011) and promotion of the TP

stability under acidic conditions (Suzuk et al., 2003). To the best of our

knowledge, no study has been carried out to examine soymilk-tea (SMT)

fermentation and the subsequent changes in microbial growth and cell surface

characteristics. In this study, we investigated the effect of TE on selected LAB

ACC

EPTE

D M

ANU

SCR

IPT


5

properties and the influence on isoflavone bioconversion during SMT

fermentation.

2. Materials and methods

2.1 Microorganisms and culture conditions

Streptococcus thermophilus ASCC 1275, L. delbrueckii ssp. bulgaricus ASCC

859 (L. bulgaricus) (ASCC collection, Werribee, Australia), L. acidophilus CSCC

2400 and L. paracasei CSCC 279 (CSCC collection, Werribee, Australia) were used

for the production of soy yogurt. Each organism was previously stored at −80°C.

For activation, 10-mL aliquots of sterile MRS were inoculated with 10 % (v/v) of

each organism and incubated at 37°C for 20 h. After the second transfer in MRS

broth, the activated organisms were transferred into sterile soymilk containing 1%

(v/v) lactose (SML) for another two transfers at 10% (v/v). This was used as

activated culture for making SY.

2.2 Tea extract preparation

Green tea (Zhuyeqing tea, Sichuan Province, PRC), oolong tea (Iron Budda,

Fujian Province, PRC) and black tea (Dianhong, Yunnan Province, PRC) were

purchased from local tea retailers. Tea leaves were ground into powder and then

infused in boiling double deionized water for 10 min at a concentration of 2%

(w/v), corresponding to the strength of “a normal cup of tea” (Yam et al., 1997).

Green, oolong and black tea extracts (TE) produced after twice suction filtration

through triple-layered Whatman #1 filter paper are referred to as GTE, OTE and

BTE, respectively.

2.3 Soy yogurt preparation and determination of pH values

2.3.1 Preparation of soymilk

Soymilk containing lactose (SML) was prepared as per the method of Donkor

ACC

EPTE

D M

ANU

SCR

IPT


6

and Shah (2007) with some modifications. Soy protein isolate (SPI) was kindly

provided by Solae DuPont China Holding Co. Ltd. (Shanghai, China) containing 90%

protein (dry basis), 6% moisture, 1.5% fat and 5.0% ash. Soymilk containing

lactose was made by dissolving 4% (w/v) SPI and 1% (w/v) α-lactose (Sigma

Chemical Co., St. Louis, MO) in double deionized water preheated to 50°C. Upon

reconstitution, SML was autoclaved at 105°C for 15 min.

2. 3.2 Preparation of soymilk-tea (SMT)

2. 3.2.1 Membrane filtration method

Tea extracts were filtered by vacuum suction through 0.22 μm membrane

(Millipore, Bedford, MA, U.S.A.) to sterilize the extracts. The filtrates were frozen

at -80°C and then freeze-dried using a Virtis freeze mobile (Virtis Co., Gardiner,

U.S.A.). Soymilk containing lactose was autoclaved at 105°C for 15 min and

cooled to room temperature before adding freeze-dried tea filtrate powder and

produced three types of SMT1: soymilk-tea green 1 (STG1), soymilk-tea oolong 1

(STO1) and soymilk-tea black 1 (STB1).

2.3.2.2 Autoclave method

Soy protein isolate (4%, w/v) and α-lactose (1%, w/v) were dissolved in TE at

50°C before autoclaving at 105°C for 15 min to produce three types of SMT2:

soymilk-tea green 2 (STG2), soymilk-tea oolong 2 (STO2) and soymilk-tea black 2

(STB2). Remaining (%) tea phenolic compounds and caffeine in SMTs after

autoclaving were monitored by HPLC and results are displayed in Supplementary

Material. All SMTs were stored in amber containers at 4°C and prepared fresh

weekly.

2.3.3 Preparation of soy yogurt

The starter cultures were transferred into SML or SMT at an inoculum level of

ACC

EPTE

D M

ANU

SCR

IPT


7

1% (v/v) for S. thermophilus, L. acidophilus and L. paracasei, and 2% for L.

bulgaricus, and incubated at 37°C for 16 h, 18 h, 18 h, 24 h, respectively. The pH

values of soy yogurt samples were measured using an Orion 250A portable pH

meter (Orion Research, Boston, MA). Fermentation was terminated by cooling

samples immediately in ice bath (4°C). Soymilk containing lactose without TE

incubated at 37°C was used as a control. Viable cell count was enumerated and

pH was determined followed by isoflavone analysis. Samples were analyzed

directly after 7, 14, 21 and 28 days of cold storage.

2.4 Viable cell count

Viable cell counts of LAB were determined in triplicate using pour plate

method in MRS agar. Plates were incubated at 37°C for 24 h in an anaerobic jar

(BD GasPak™, Sparks, MD, U.S.A.) with BD GasPak™ EZ kit (Becton-Dickinson,

Sparks, MD, U.S.A.).

2.5 Fourier transform infrared (FT-IR) spectroscopy

To evaluate the effect of the presence or absence of GTE, OTE and BTE in the

culture medium on the molecular structure of lipids in the phospholipid bilayer,

polysaccharides and cell surface protein of bacterial membrane, FT-IR

spectroscopy analysis was performed. An inoculum at 1% (v/v) for S.

thermophilus, L. acidophilus, L. paracasei and 2% (v/v) for L. bulgaricus in 20 mL

test tubes in MRS broth with or without supplementation with TE in MRS. The

ratio between each type of tea powder and MRS broth was 1:50 (w/v).

Fermentation was carried out for the same period of time as those for SY

production, i.e. 16 h for S. thermophilus, 18 h for L. acidophilus and L. paracasei,

and 24 h for L. bulgaricus. Cells were harvested aseptically by centrifugation at

4500 xg for 20 min at 4°C and washed twice with sterile double deionized water

ACC

EPTE

D M

ANU

SCR

IPT


8

(SDW). The cell mass of each sample was weighed and adjusted to approximately

100mg/mL with SDW and 50 μL homogenized bacterial suspension was placed

on a CaF2 plate and oven-dried for 20 min at 50°C to form a transparent bacterial

film. FT-IR spectra were recorded from 4000 to 900 cm−1 at a resolution of 2

cm−1 using a Bruker FT-IR TENSOR 27 spectrometer (Billerica, Massachusetts,

U.S.A.). Spectra were collected from three independent fermentation batches.

Each spectrum represented an average of 20 scans. Spectral processing was

performed using Opus software (version 6.5, Bruker Optics GmbH), with 2nd

derivatization of the spectra bands at 9 smoothing points. Wavenumbers

assigned to signature bands were obtained from the second derivative of spectra.

2.6 Cell surface hydrophobicity assay

Hydrophobicity assay was carried out as per microbial adhesion to

hydrocarbons (MATH) method of Rosenberg (1991) with some modifications.

Four L. acidophilus B strains were inoculated in each tea media or MRS broth

(control) at 1% (v/v) for S. thermophilus, L. acidophilus, L. paracasei and 2% (v/v)

for L. bulgaricus and were incubated at 37°C for 16 h (for S. thermophilus), 18 h

(for L. acidophilus and L. paracasei) or 24 h (for L. bulgaricus). The strains were

harvested by centrifuging at 12,000 xg for 5 min at 4°C, washed with 50 mM

potassium phosphate buffer (pH 7.0) and resuspended in the same

buffer. Absorbance at 560 nm was adjusted to approximately 0.8 with the buffer;

0.6 mL xylene or n-hexadecane (Sigma Chemical Co., St. Louis, MO) was added to

3 mL of bacterial suspensions and vortexed for 120 s. The mixture was allowed

to separate for 20 min before the absorbance of the aqueous phase was

measured at 560 nm using a BIO-RAD Smartspec Plus spectrophotometer

(Hercules, CA, U.S.A.). The decrease in the absorbance of the aqueous phase was

ACC

EPTE

D M

ANU

SCR

IPT


9

taken as a measure of bacterial adhesion to xylene (SHx%) or

n-hexadecane(SHh%) using the equation below:

SH%= [(Ao − A)/Ao] × 100%;

Where Ao and A are the absorbance before and after extraction with xylene or

n-hexadecane. Each measurement was performed at least in triplicate and the

experiment was repeated twice with independent bacterial cultures.

2.7 Extraction and HPLC quantification of isoflavones

The SY samples or non-fermented SML were frozen at -80°C for 24 h before

freeze-drying for two days using a Virtis freeze mobile (Virtis Co., Gardiner,

U.S.A.). The isoflavone content was determined using the modified method of the

AOAC official method “extraction, saponification and liquid chromatography”

method 2001.10 (C18 reversed phase column). In brief, 1g of dried sample was

mixed with 20 mL of 80% methanol. The container was wrapped with aluminum

foil and shaken in 65°C water bath in dark for 2 h. When cooled to room

temperature, 0.8 mL of 2M NaOH was added to the mixture and shaken at room

temperature for 10 min before 0.5 mL glacial acetic acid was added. The mixture

was filtered and 8 mL of the filtrate was diluted to 10 mL with 50% methanol in a

15-mL centrifugal tube. The solution was centrifuged at 4500 xg for 5 min and

filtered through 0.45-μm hydrophilic syringe filter (Corning, NY) before loaded

onto HPLC column.

2.7.1. Establishment of calibration curves of isoflavones

Standard stock solutions at a concentration of 1 mg/mL were prepared by

dissolving pure daidzin, glycitin, genistin, daidzein, glycitein, genistein (Wako,

Osaka, Japan) in 100% methanol. Stock solutions were stored in dark at 4°C. The

working solutions for calibration curve establishment were prepared by serial

ACC

EPTE

D M

ANU

SCR

IPT


10

dilutions from stock solution with methanol-water (50-50) to six concentration

levels, among which the highest concentrations for daidzin, glycitin, genistin,

daidzein, glycitein, genistein were 20μg/mL, 10μg/mL, 20μg/mL, 20μg/mL,

20μg/mL, 20μg/mL. Mean peak area (n=3) of individual analyte was plotted

against concentrations to establish calibration equations.

2.7.2. HPLC conditions

The column for analysis was an Ascentis C18 column (250 mm x 4.6 mm, 5 μm)

from Supelco (Bellefonte, PA, U.S.A.). A linear gradient was applied with a

mixture of two solvents: 88% water, 10% methanol, 2% acetic acid (solvent A)

and methanol (solvent B) at a flow rate of 1.0 mL/min using a Shimadzu HPLC

apparatus LC 10A (Shimadzu, Kyoto, Japan). UV-Vis detector was set to scan from

200 to 500 nm and the detection wavelength was 260 nm. The injection volume

was 20 μL for each extract solution. LC pump gradient for each run (A%:B%)

started at 90:10 and linearly decreased to 40:60 within 25 min, then held for 5

min before returning to 90:10 to reach initial conditions. The isoflavones in

samples were identified by comparing absorption spectra and retention times of

unknown peaks with isoflavone standards. Concentrations of isoflavone

glucosides (IG) (genistin, glycitin, and daidzin) were converted to isoflavone

aglycone equivalents using the following equation:

IAe = ;

where IAe was the concentration of isoflavone aglycone equivalent, MWia was

the molecular weight of aglycone, MWig was the molecular weight of glucoside,

IG was the concentration of isoflavone glucoside. The reduction of IG was

calculated as:

Reduction of IG (%) = (IAe(SML)- IAe(SMT))/ IAe(SML) 100.

ACC

EPTE

D M

ANU

SCR

IPT


11

2.8 Statistical analysis

For each assay, three independently soy yogurt samples were prepared. The

data was subjected to one-way analysis of variance (ANOVA) (Tukey and

Games-Howell tests) by SPSS 20.0 (IBM SPSS Statistics, IBM Corp, Somers, NY).

Results with a p<0.05 were considered statistically significant.

3. Results and discussion

3.1 Preliminary evaluation on product attributes

Fermented SMTs exhibited significant differences in color, viscosity, texture

(Fig. 1) and smell. Soy yogurts made from SML, GTE, OTE, and BTE had

predominant milky white, greyish purple, greyish yellow and dark brown color,

respectively. Compared with control SY, those with added TE had reduced sour

odor, replaced by pleasant tea aroma. In terms of viscosity, SY made from SMT

prepared by the autoclave method appeared to be thicker than those with SMTs

obtained by the membrane filtration method, whereas, control SY showed the

highest viscosity. Fermented SMTs prepared by the autoclave method were

homogenous and stable, while products made from SMTs using membrane

filtration method were unstable and separated into two layers, with upper as the

transparent and pigment-depleted layer, and lower as the thick, chalky and

pale-colored layer. The de-coloration may be due to the sorption ability of soy

protein for pigments in TE (Roopchand et al., 2012). The different sterilization

methods, i.e. membrane filtration and autoclaving, contributed to the variations

in TE composition and results obtained from viable cell count, FT-IR, cell surface

hydrophobicity and HPLC analysis of isoflavone content, which will be discussed

below.

3.2 Effect of tea supplementation on the acidity and bacterial growth in soy yogurt

ACC

EPTE

D M

ANU

SCR

IPT


12

The pH values determined for different types of SY are shown in Table 1 and

cell populations are presented in Fig. 2. In general, the acidity and microbial

population were significantly different (p<0.05) between fermented SMTs and

the control SY. The initial pH levels of the SMTs prepared by the membrane

filtration method were higher than that of SML by 0.01-0.08 unit, while those of

SMTs made by the autoclave method were lower by 0.13-0.18 unit. Upon

fermentation, acidity increased and pH levels reached 4.4-4.8 for the controls,

whereas the pH of fermented SMTs dropped to as low as 4.2, with STG2, STO2

and STB2 groups generally possessing higher acidity than STG1, STO1 and STB1.

Among the fermented products, L. paracasei acidified the SYs to the lowest pH

levels while S. thermophilus had the weakest acidification ability, which also

correlated with its survival in the SMT media. In the control SY, cell number

increased from the initial population of ~6.5 log CFU/mL to ~8.5 log CFU/mL.

The supplementation of TE promoted the growth of L. bulgaricus and L. paracasei,

increasing cell population by 0.09-0.44 and 0.04-0.21 unit, respectively. This

could possibly be attributed to the reduction in redox potential and protection of

membrane from undesirable oxidation by tea antioxidants (Dave, & Shah, 1997;

Gaudreau et al., 2012). However, all types of SMT reduced the population of S.

thermophilus significantly (p<0.05), by 0.28-0.46 unit. L. acidophilus was not

significantly affected by GTE and OTE but was inhibited by BTE.

Viability of starter organisms in control and fermented SMTs prepared by

autoclave method and acidity evolution during 28 days of refrigerated storage at

4°C are depicted in Fig. 3. All four L. acidophilus B remained viable above the

required level of 7 log CFU/mL during the 4 weeks of cold storage and pH levels

further dropped as excepted. It was found that decrease in S. thermophilus cell

ACC

EPTE

D M

ANU

SCR

IPT


13

population in fermented SMT was significant (p<0.05), with the greatest

reduction by 15.12% in week 4-STB sample compared with that of day 0,

suggesting the potential bactericidal effect of TE on S. thermophilus. The decrease

in pH of L. paracasei-fermented products was the most evident among all

samples, indicating TE supplementation supported the production of lactic acid

and other organic acids during post-fermentation period (Donkor et al., 2006),

especially L. paracasei. Lower acidity may inhibit the growth of spoilage

microorganisms that survived pasteurization as well as delay the degradation of

phenolics in TE and hence may prolong shelf life (Sun-Waterhouse et al., 2012).

3.3 Effect of tea supplementation on FT-IR spectral features of LAB cells

Variations in wavenumber assigned to characteristic functional groups of

bacterial cells are displayed in Table 2 and the normalized spectra of S.

thermophilus and L. bulgaricus incubated in MRS (control) or MRS containing TE

are shown in Fig. 4 A and B, respectively. Information provided by FT-IR

spectroscopy enables the examination of biochemical signatures and functional

structures of bacterial cells including cell membrane fatty acids (FA), cytoplasmic

proteins, nucleic acids and cell wall polysaccharides (Al-Qadiri et al., 2008;

Alvarez-Ordóñez et al., 2011). Compared with the control, the frequency shifts of

major peaks and changes in the areas of spectral bands reflected how tea

components affected LAB cells differently. In general, TE altered spectral

features associated with FA, ester groups of phospholipids, proteins, and

polysaccharides for all LAB studied.

The spectral bands corresponding to features of cell membrane FA

(3000-2800 cm-1) is characterized by the stretches of methyl and methylene

groups (Alvarez-Ordóñez et al., 2011). In the spectra, intensities of peaks at

ACC

EPTE

D M

ANU

SCR

IPT


14

~2962 cm−1 (C-H asymmetric stretch of CH3), ~2925 cm−1 (C-H asymmetric

stretch of CH2), and ~2874 cm−1 (C-H symmetric stretch of -CH3) were

diminished for cells of the four bacteria cultivated in all TE-MRS media as

compared with the control. In particular, such phenomenon was most obvious

for S. thermophilus cells grown in BTE-MRS media (Fig. 4A), with right-shifted

peaks for methyl group stretches and peaks significantly leveled-off near 2874

cm−1. In addition, the C=O stretches of ester groups of phospholipids at ~1740

cm-1 varied for S. thermophilus and L. acidophilus cells while no significant change

was found for L. bulgaricus and L. paracasei cells. These variations indicated the

varying degrees of influence of different TEs, especially BTE, on cell membrane

FA and lipid, possibly resulting from changes in acyl chain composition and the

hydrophilic head groups of the lipid bilayer that lead to structural changes of FA

and phospholipids within the membrane. In fact, the membrane components of

bacterial cells may be changed in response to environmental stress and

membrane stability, permeability, and fluidity can be altered consequently

(Denich et al., 2003; Mohan et al., 2010; Yi et al., 2010). On the contrary, the

growth pattern of S. thermophilus and L. bulgaricus cells in SMT implied that TE

components, possibly TP, gallic acid and caffeic acid, which are the major

phenolic components in tea (Li et al., 2013), may have altered membrane lipid

composition and packing arrangement after the treatments in an innocuous or

positive way for L. bulgaricus but destructive for S. thermophilus (Denich et al.,

2003; Khalil, 2010).

Appreciable changes occurred in regions representing amide band

deformation of cell surface proteins as well. There was no evident shift in peak

wavenumbers for amide I band at ~1647 cm−1 for all bacterial cells, but a major

ACC

EPTE

D M

ANU

SCR

IPT


15

decrease in the peak intensity for all TE-treated S. thermophilus cells,

TE1-treated L. acidophilus cells, and GTE and BTE-treated L. paracasei cells.

Amide II bands (~1550 cm−1) of S. thermophilus, L. acidophilus and L. paracasei

cells appeared at higher frequencies and amide III band (~1240 cm−1) for S.

thermophilus shifted to the right while L. paracasei band to the left. For L.

bulgaricus cells, although peak frequencies were similar to those of the control,

absorbance of the bands varied with respect to different media. Cell viability has

been implied to associate with the dislocation or deformation of key protein or

enzymes on cytoplasmic membrane, resulting from the binding or intercalation

of flavonoids (Cushnie & Lamb, 2011). This may explain the difference in

bacterial growth in our study (Fig. 2 & 3).

In the region corresponding to the C-O-C ring vibration of bacterial envelope

polysaccharide (1200-900 cm−1), variations in the peak intensities of all

TE-adapted cells were obvious compared to the control, implying an alteration in

the structure of lipopolysaccharide anchored in the outer membrane or the

production of bacterial exopolysaccharides (EPS) (Maeyama et al., 2005;

Al-Qadiri et al., 2006). The appreciable higher intensities of peaks in this range

for all TE-treated S. thermophilus cells is possibly due to the excretion of capsular

EPS for the protection of cell from unfavorable conditions in the media. This

phenomenon is in accordance with the limited cell growth in SMT (Fig. 1) and the

reduced SH% of S. thermophilus cells in TE-MRS media (Fig. 4 A & B) as EPSs are

mostly found hydrophilic polymers (Broadbent et al., 2003; Zisu & Shah, 2003).

3.4 Effect of tea supplementation on cell surface hydrophobicity

As illustrated in Fig. 5, four strains exhibited varying degrees of cell surface

hydrophobicity reflected by the partitioning of cells between water and xylene or

ACC

EPTE

D M

ANU

SCR

IPT


16

n-hexadecane. In general, S. thermophilus exhibited the highest adherence to

both solvents in the control MRS among all bacteria tested, with higher affinity

towards xylene (65%) than hexadecane (42%). For cells incubated in TE-MRS

media, SH was modified to different extents, whereas no clear relationship was

found for changes between SH% and tea type. Nevertheless, regardless of the TE

added, the adherence of L. paracasei was always higher towards xylene (an

electron-donating solvent) than hexadecane (a nonpolar solvent), revealing the

electron-accepting nature of L. paracasei cells, while L. acidophilus exhibited the

opposite property, showing higher affinity towards hexadecane. For L. bulgaricus,

all cells were found to have low SHx% (0.65-7.38%) and SHh% (3.16-6.68%),

implying its low hydrophobicity. However, it's worth noting that MATH appeared

to be of low discriminating power towards hydrophilic strains and may not be

accurate in determining the SH differences of L. bulgaricus cells treated with

various TEs (Rosenberg, 2006). Compared with cells grown in MRS, adherence to

both solvents decreased for S. thermophilus and L. acidophilus cells cultured in all

types of TE-MRS media, whereas changes were irregular for L. bulgaricus and L.

paracasei cells.

According to Schär-Zammaretti and Ubbink (2003), cell wall constituents such

as phosphate, carboxylate groups, and proteins impart bacteria with variable

surface charge and hydrophobicity. These properties are involved in the initial

attachment of an organism onto intestinal lumen, as such complicated behavior

has been shown to be the integrated result of electrostatic and van der Waals

interactions and surface hydrophobicity (Bustos et al., 2012). Reduction in SH

decreases the potential of bacterial adhesion and hence is unfavorable for

salutary bacteria to improve gut health (Kiely & Olson, 2000; Rosenberg et al.,

ACC

EPTE

D M

ANU

SCR

IPT


17

1980). Moreover, the extracellular substances excreted by bacteria, such as

bacterial polysaccharides, either attaching to the outer surface of cells or in the

surrounding medium have been implicated to influence adhesion (Bustos et al.,

2012; Lebeer et al., 2008).

3.5 Effect of tea supplementation on isoflavone bioconversion in soy yogurt

Figure 6 presents the changes in isoflavone contents in fermented SML with or

without TE addition in terms of reduction rate of IG. As shown, regardless of the

media, L. acidophilus possessed the highest ability to hydrolyze IG (68-78%),

followed by L. paracasei (43-58%), L. bulgaricus (21-38%) and S. thermophilus

(13-21%). Such variations in bacterial deglycosylation of isoflavone agreed with

previous studies (Pyo et al., 2005; Tsangalis et al., 2002), resulting from different

ability of bacteria in the production of β-glucosidase and β-galactosidase. Tea

extract had an adverse effect on isoflavone biotransformation during SMT

fermentation compared with the control except for L. bulgaricus. It was also

found that for the same type of TE, i.e. GTE, OTE or BTE, reductions of IG in

fermented SMTs prepared by the membrane filtration method were generally

less than those using the autoclave method, indicating that changes in TE

components due to types of tea extracted and sterilization method might have

some influence on isoflavone bioconversion as well. In addition, TPs have been

shown to bind and precipitate enzymes including peroxidase, trypsin (Huang et

al., 2004), decarboxylase (Bertoldi et al., 2001), α-amylase and pepsin (He et al.,

2007). This suggests that TE may also bind and denature bacterial

deglycosylating enzymes, thus altering isoflavone biotransformation. The actual

intervention mechanism may be clarified by measuring enzyme activity using

non-spectroscopic methods in order to avoid the interference of pigments in TE.

ACC

EPTE

D M

ANU

SCR

IPT


18

The higher reduction rate of IG found in L. bulgaricus-fermented SMTs, compared

with control SY, coincided with the increased cell population in soymilk

supplemented with tea, implying the beneficial effects of TE on the production of

bacterial enzyme that contributed to higher isoflavone bioconversion rate.

4. Conclusion

In this study, novel SYs were produced from soymilk supplemented with

various types of tea extract. Results obtained from microbiological and

biochemical analyses indicated that tea supplementation significantly influenced

bacterial proliferation and cell surface characteristics (including cytoplasmic

fatty acids, proteins and cell wall polysaccharides), which were dependent on

both TE type and strain. In particular, black tea extract exerted more obvious

impact on cell surface characteristics as showed by FT-IR spectra. The

growth-promoting effects of TE supplementation in soymilk for L. bulgaricus and

L. paracasei, and the satisfactory viability maintained after refrigeration

suggested the possibility of integrating health-promoting bacteria, dietary

flavonoids and tea flavor into SY. For industrial manufacture of SY containing

dietary flavonoids, the type and appropriate concentration of flavonoids and

bacterial strains should be taken into consideration. Nevertheless, the

complicated and unpredicted microbial-flavonoid interactions still pose

controversies over how flavonoids influence bacterial growth and whether

flavonoid-probiotic combination is always better than consuming flavonoids and

probiotic organisms separately. Therefore, more focus is needed to investigate

changes in cell surface composition due to addition of flavonoids and the

microbial-flavonoid interaction mechanism.

5. Acknowledgments

ACC

EPTE

D M

ANU

SCR

IPT


19

The authors are thankful to Solae DuPont China Holding Co. Ltd. for

providing a sample SPI and Mr. Victor Yeung in the School of Biological Sciences,

the University of Hong Kong, for his technical support.

Reference

Al-Qadiri, H.M., Lin, M., Cavinato, A.G., & Rasco, B.A. (2006). Fourier transform

infrared spectroscopy, detection and identification of Escherichia coli O157:H7

and Alicyclobacillus strains in apple juice. International Journal of Food

Microbiology, 111, 73-80.

Al-Qadiri, H.M., Al-Alami, N.I., Al-Holy, M.A. & Rasco, B.A. (2008). Using Fourier

transform infrared (FT-IR) absorbance spectroscopy and multivariate analysis to

study the effect of chlorine-induced bacterial injury in water. Journal of

Agriculture and Food Chemistry, 56, 8992-8997.

Alvarez-Ordóñez, A., Mouwen, D.J.M., López, M. & Prieto, M. (2011). Fourier

transform infrared spectroscopy as a tool to characterize molecular composition

and stress response in foodborne pathogenic bacteria. Journal of Microbiological

Methods, 84, 369-378.

Ankolekar, C., Johnson, D., Pinto, M.S., Johnson, K., Labbe, R. & Shetty, K.

Inhibitory potential of tea polyphenolics and influence of extraction time against

helicobacter pylori and lack of inhibition of beneficial lactic acid bacteria. Journal

of Medicinal Food, 14, 1321-1329.

Aura, A.M. (2008). Microbial metabolism of dietary phenolic compounds in the

colon. Phytochemical Review, 7, 407-429.

Bertoldi, M., Gonsalvi, M., Voltattorni, C.B. (2001). Green tea polyphenols: novel

irreversible inhibitors of dopa decarboxylase. Biochemical and Biophysical

Research Communications, 284, 90-93.

ACC

EPTE

D M

ANU

SCR

IPT


20

Broadbent, J.R., McMahon, D.J., Welker, D.L., & Oberg, C.J. (2003). Biochemistry,

Genetics, and Applications of Exopolysaccharide Production in Streptococcus

thermophilus: A Review. Journal of Dairy Science, 86, 407-423.

Bustos, I., Garcia-Cayuela, T., Hernandez-Ledesma, B., Pelaez, C., Requena, T. &

Martinez-Cuesta, M.C. (2012). Effect of Flavan-3-ols on the Adhesion of Potential

Probiotic Lactobacilli to Intestinal Cells. Journal of Agricultural and Food

Chemistry, 60, 9082-9088.

Cui, Y., Oh, Y.J., Lim, J., Youn, M., Lee, I., Pak, H.K., Park, W., Job, W., & Parka, S.

(2012). AFM study of the differential inhibitory effects of the green tea

polyphenol (−)-epigallocatechin-3-gallate (EGCG) against Gram-positive and

Gram-negative bacteria. Food Microbiology, 29, 80-87.

Cushni, T.P.T., & Lamb, A.J. (2011). Recent advances in understanding the

antibacterial properties of flavonoids. International Journal of Antimicrobial

Agents, 38, 99-107.

Dave, R.I., & Shah, N.P. (1997). Effectiveness of ascorbic acid as an oxygen

scavenger in improving viability of probiotic bacteria in yoghurts made with

commercial starter cultures. International Dairy Journal, 7, 435-443.

Denich, T.J., Beaudette, L.A., Lee, H., & Trevors, J.T. (2003). Effect of selected

environmental and physico-chemical factors on bacterial cytoplasmic

membranes. Journal of Microbiological Methods, 52, 149-182.

Dianawati, D., Mishra, V., & Shah, N.P. (2013). Effect of drying methods of

microencapsulated Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris

on secondary protein structure and glass transition temperature as studied by

Fourier transform infrared and differential scanning calorimetry. Journal of Dairy

Science, 96, 1419-1430.

ACC

EPTE

D M

ANU

SCR

IPT


21

Donkor, O.N., Henriksson, A., Vasiljevic, T., & Shah, N.P. (2006)

Effect of acidification on the activity of probiotics in yoghurt during cold storage.

International Dairy Journal, 16, 1181-1189.

Donkor, O.N., & Shah, N.P. (2007). Production of β-glucosidase and hydrolysis of

isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis,

and Lactobacillus casei in soymilk. Journal of Food Science, 73, M15-M20.

Gaudreau, H., Champagne, C.P., Remondetto, G.E., Bazinet, L., & Subirade, M.

(2012). Effect of catechins on the growth of oxygen-sensitive probiotic bacteria.

Food Research International, http://dx.doi.org/10.1016/j.foodres.2012.10.014

Geertsema-Doornbusch, G.I., Van der Mei, H.C., & Busscher, H.J. (1993). Microbial

cell surface hydrophobicity: The involvement of electrostatic interactions in

microbial adhesion to hydrocarbons (MATH). Journal of Microbiological Methods,

18, 61-68.

He, Q., Lv, Y.P., & Yao, K. (2007). Effects of tea polyphenols on the activities of

α-amylase, pepsin, trypsin and lipase. Food Chemistry, 101, 1178-1182

Holzbeierlein, J.M., McIntosh, J., & Thrasher, J.B., (2005). The role of soy

phytoestrogens in prostate cancer. Current Opinion Urol, 15, 17-22.

Hsu, A., Bruno, R.S., Lohr, C.V., Taylor, A.W., Dashwood, R.H., Bray, T.M., & Ho,

E. (2011). Dietary soy and tea mitigate chronic inflammation and prostate cancer

via NFκB pathway in the Noble rat model. Journal of Nutritional Biochemistry, 22,

502-510.

Huang, H.H., Kwok, K.C., & Liang, H.H. (2004). Effect of tea polyphenols on the

activities of soybean trypsin inhibitors and trypsin. Journal of the Science of Food

and Agriculture, 84, 121-126.

Jaziri, I., Ben, Slama, M., Mhadhbi, H., Urdaci, M.C., & Hamdi, M. (2009). Effect of

ACC

EPTE

D M

ANU

SCR

IPT


22

green and black teas (Camellia sinensis L.) on the characteristic microflora of

yogurt during fermentation and refrigerated storage. Food Chemistry, 112,

614-620.

Jenner, A.M., Rafter, J., & Halliwell, B. (2005). Human fecal water content of

phenolics: The extent of colonic exposure to aromatic compounds. Free Radical

Biology and Medicine, 38, 763-772.

Jurado, E., Camacho, F., Luzon, G., & Vicaria, J.M. (2004). Kinetic models of activity

for β-galactosidases: influence of pH, ionic concentration and temperature.

Enzyme Microbiology Technology, 34, 33-40.

Khalil, R.K.S. (2010). Influence of gallic acid and catechin polyphenols on

probiotic properties of Streptococcus thermophilus CHCC 3534 strain. World

Journal of Microbiology and Biotechnology, 26, 2069–2079.

Kiely, L.J., & Olson, N.F. (2000). The physicochemical surface characteristics

of Lactobacillus casei. Food Microbiology, 17, 277-291.

Lee, H.C., Jenner, A.M., Lowa, C.S., & Lee, Y.K. (2006). Effect of tea phenolics and

their aromatic fecal bacterial metabolites on intestinal microbiota. Research in


Lebeer, S., Vanderleyden, J., & De, Keersmaecker, S.C. (2008). Genes and

molecules of lactobacilli supporting probiotic action. Microbiology and Molecular

Biology Reviews, 72, 728-764.

Li, S., Lo, C.Y., Pan, M.H., Lai, C.S., & Ho, C.T. (2013). Black tea: chemical analysis

and stability. Food & Function, 4, 10-18.

Maeyama, R., Kwon, I.K., Mizunoe, Y., Anderson, J.M., Tanaka, M., & Matsuda, T.

(2005). Novel bactericidal surface: Catechin‐loaded surface‐erodible polymer

ACC

EPTE

D M

ANU

SCR

IPT


23

prevents biofilm formation. Journal of Biomedical Material Research Part A, 75A,

146–155.

Mohan, H., Maheswari, K.U., Bera, A.K., & Suraishkumar, G.K. (2010). Reactive

oxygen species mediated modifications in Bacillus subtilis lipid membrane to

improve protein productivities. Process Biochemistry, 45, 467-474.

Najgebauer-Lejko, D., Sady, M., Grega, T., & Walczycka, M. (2011). The impact of

tea supplementation on microflora, pH and antioxidant capacity of yoghurt.

International Dairy Journal, 21, 568-574.

Osiriphun, S., & Jaturapiree, P. (2009). Isolation and characterization of

β-galactosidase from the thermophile B1.2. Asian Journal of Food and

Agro-Industry, 2, 135-143.

Otieno, D.O., & Shah, N.P. (2007). Endogenous β-glucosidase and β-galactosidase

activities from selected probiotic micro-organisms and their role in isoflavone

biotransformation in soymilk. Journal of Applied Microbiology, 103, 910–917.

Parkar, S.G., Stevenson, D.E., & Skinner, M.A. (2008). The potential influence of

fruit polyphenols on colonic microflora and human gut health. International

Journal of Food Microbiology, 124, 295-298.

Pyo, Y.H., Lee, T.C., & Lee, Y.C. (2005). Effect of lactic acid fermentation on

enrichment of antioxidant properties and bioactive isoflavones in soybean.

Journal of Food Science, 70, S215–S220.

Roopchand, D.E., Grace, M.H., Kuhn, P., Cheng, D.M., Plundrich, N., Poulev, A.,

Howell, A., Fridlender, B., Ann, Lila, M., & Raskin, I. (2012). Efficient sorption of

polyphenols to soybean flour enables natural fortification of foods. Food

Chemistry, 131, 1193-1200.

Rosenberg, M. (1991). Basic and applied aspects of microbial adhesion at the

ACC

EPTE

D M

ANU

SCR

IPT


24

hydrocarbon: water interface. Critical Reviews in Microbiology, 18, 159-173.

Rosenberg, M. (2006). Microbial adhesion to hydrocarbons: twenty-five years of

doing MATH. FEMS Microbiology Letters, 262, 129-134.

Schär-Zammaretti, P., & Ubbink, J. (2003). The cell wall of lactic acid bacteria:

surface constituents and macromolecular conformations. Biophysical Journal, 85,

4076-4092.

Sun-Waterhouse, D., Zhou, J., & Wadhwa, S.S. (2012). Effects of adding apple

polyphenols before and after fermentation on the properties of drinking yoghurt.

Food and Bioprocess Technology, 5, 2674-2686.

Suzuki, M., Sano, M., Yoshida, R., Degawa, M., Miyase, T., & Maeda-Yamamoto, M.

(2003). Epimerization of Tea Catechins and O-Methylated Derivatives of

(−)-Epigallocatechin-3-O-gallate: Relationship between Epimerization and

Chemical Structure. Journal of Agricultural and Food Chemistry, 51, 510-514.

Tabasco, R., Sánchez-Patán, F., Monagas, M., Bartolomé, B., Moreno-Arribas, M.V.,

Peláez, C., & Requena, T. (2011). Effect of grape polyphenols on lactic acid

bacteria and bifidobacteria growth: Resistance and metabolism. Food


Tsangalis, D., Ashto, J.F., Mcgill, A.E.J., & Shah, N.P. (2002) Enzymic transformation

of isoflavone phytoestrogens in soymilk by β-glucosidase-producing

bifidobacteria. Journal of Food Science, 67, 3104-3113.

Van, Dam, R.M., Naidoo, N., & Landberg, R. (2013). Dietary flavonoids and the

development of type 2 diabetes and cardiovascular diseases: review of recent

findings. Current opinion in Lipidology, 24, 25-33.

ACC

EPTE

D M

ANU

SCR

IPT


25

Yam, T.S., Shah, S., & Hamilton-Miller, J.M.T. (1997). Microbiological activity of

whole and fractionated crude extracts of tea (Camellia sinensis), and of tea

components. FEMS Microbiology Letters, 152, 169-174.

Yang, C.S., Chung, J.Y., Yang, G.Y., Chhabra, S.K., & Lee, M.J. (2000). Tea and tea

polyphenols in cancer prevention. Journal of Nutrition, 130, 472–478.Yi SM, Zhu

Yi, S.M., Zhu, J.L., Fu, L.L., & Li, J.R. (2010). Tea polyphenols inhibit Pseudomonas

aeruginosa through damage to the cell membrane. International Journal of Food


Zisu, B., & Shah, N.P. (2003). Effects of pH, Temperature, Supplementation with

Whey Protein Concentrate, and Adjunct Cultures on the Production of

Exopolysaccharides by Streptococcus thermophilus 1275. Journal of Dairy Science,

86, 3405-3415.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

EFFECT OF TEA ON LAB PROPERTIES AND ISOFLAVONE IN FERMENTED SOYMILK-TEA

26

Tables and figures Table 1 pH values of fermented soymilk containing lactose (SML) or soymilk-tea (STXx)1 with S. thermophilus ASCC 1275 (ST), L. acidophilus CSCC 2400 (LA), L. delbrueckii ssp. bulgaricus ASCC 859 (LB), and L. paracasei CSCC 279 (LP) in fermented soymilk with or

without tea extract at 37℃.

pH2 SML STG1 STG2 STO1 STO2 STB1 STB2

Initial 6.70±0.02 6.78±0.01 6.52±0.00* 6.73±0.01 6.57±0.00 6.71±0.01 6.53±0.01*

ST 4.43±0.01 4.60±0.04* 4.47±0.01 4.79±0.01* 4.78±0.01* 4.61±0.02* 4.52±0.01

LA 4.61±0.01 4.41±0.01* 4.24±0.02 4.50±0.02* 4.37±0.02 4.61±0.02 4.64±0.01

LB 4.78±0.01 4.55±0.02* 4.28±0.02* 4.61±0.02* 4.54±0.03* 4.49±0.03* 4.25±0.02*

LP 4.52±0.02 4.33±0.02 4.32±0.01 4.31±0.03 4.22±0.02 4.48±0.00* 4.46±0.02* 1 Soymilk-tea STXx: X=G (Green tea), O (Oolong tea), or B (Black tea); x= 1 (membrane filtration method) or 2 (autoclave method), denoting the method for preparing soymilk-tea. 2 Values are expressed as means ±standard mean for three independent fermentation products. The asterisks (*) in the same row indicate significant difference at a level of p< 0.05 compared with the control (SML).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT


27

Table 2 Assignment of functional groups in FT-IR spectra of S. thermophilus ASCC 1275 (ST), L. delbrueckii ssp. bulgaricus ASCC 859 (LB), L. acidophilus CSCC 2400 (LA) and, L. paracasei CSCC 279 (LP) treated in different types of tea extract-MRS (TE-MRS) media.

Wavenumber (cm-1)1

∼2966 ∼2929 ∼2874 ∼2852 ∼1742 1637-1655,

∼1695 ∼1550 1240-1310 1200-900

Media Assign-

ment

C-H str (asym) of

-CH3 in fatty acids

C-H str (asym)

of >CH2 in fatty acids

C-H str (sym) of

-CH3 in fatty acids

C-H str (sym)

of >CH2 in fatty acids

C=O str of esters in

phosphor- lipids

amide I band

component

amide II band

component

amide III band

component

C-O-C ring vibration of polysacchar

ides3

ST Control 2 2963.4 2924.0 2874.4 2853.4 1742.9 1646.4 1551.0 1244.6 -

GTE1 2959.5* 2924.7 2873.3 2853.4 1743.4 1647.0 1553.1 1241.4* I

GTE2 2959.9* 2924.2 2873.6 2853.3 1743.4 1647.6* 1553.2* 1242.4 I

OTE1 2959.5* 2924.3 2873.4 2853.2 1743.5 1647.3 1553.9* 1243.2 I

OTE2 2960.3* 2924.4 2873.8 2853.3 1743.3 1647.8* 1553.4* 1243.2* I

BTE1 2961.4* 2924.4 ND 2853.5 1731.9 1647.5 1553.8* 1242.3* I

BTE2 2956.8* 2923.9 ND 2853.2 ND 1647.1 1553.7 1240.0 C

LB Control 2972.2 2926.0 2874.4 2853.3 1745.1 1647.4 1551.7 1240.2 -

GTE1 2959.6* 2925.4* 2874.4 2853.4 1744.7 1647.8 1553.9 1241.0 D

GTE2 2961.1* 2925.3* 2874.2 2853.1 1744.2 1647.6 1553.2 1241.7 D

OTE1 2961.9* 2925.4* 2874.3 2853.5 1745.1 1647.8 1553.5 1241.5 D

OTE2 2960.3* 2925.2* 2874.0 2853.2 1743.9 1647.7 1553.2 1241.1 D

BTE1 2960.5* 2925.3* 2873.8 2853.5 1742.0 1647.7 1553.4 1240.9 C

BTE2 2960.4* 2924.6* 2873.9 2853.2 ND 1647.4 1553.2 1240.4 I

LA Control 2962.6 2932.5 2874.1 2853.3 1745.0 1647.0 1553.1 1241.0 -

GTE1 2959.3* 2926.6* 2874.1 2853.4 1744.7 1647.1 1551.7 1241.2 C

GTE2 2959.8 2924.8* 2874.2 2853.4 1744.4 1647.4 1554.0 1241.6* I

OTE1 2961.5 2927.5* 2874.2 2853.4 1744.2 1646.8 1552.8 1241.1* D

OTE2 2960.8* 2925.2* 2874.0 2853.2 1745.0 1647.3 1554.0 1240.8 T

BTE1 2960.8* 2925.3* 2874.2 2853.5 1744.0* 1647.0 1552.5 1241.1 I

BTE2 2960.1* 2924.6* 2874.2 2853.4 1744.4* 1647.3 1552.3 1240.0 I

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT


28

LP Control 2961.9 2925.5 2874.2 2853.4 1744.9 1646.4 1551.2 1240.8 -

GTE1 2959.9* 2924.4 2874.2 2853.5 1745.5 1646.3 1554.0* 1241.8 T

GTE2 2960.4 2924.6 2874.3 2853.5 1743.9 1647.6 1553.5* 1242.5 C

OTE1 2959.9* 2925.6 2874.2 2853.3 1744.6 1647.6 1553.8* 1242.4 C

OTE2 2960.2* 2925.4 2874.3 2853.4 1743.9 1647.8 1552.9* 1243.3* I

BTE1 2959.9* 2925.3 2874.1 2853.5 1744.9 1647.2 1554.2* 1242.8* I

BTE2 2960.6 2924.2 2874.3 2853.3 1744.0 1647.0 1552.6 1242.0 C 1 Values are expressed as means for three independent assays. The asterisks (*) in the same column of each strain indicate significant difference at a level of p< 0.05 compared with the control (MRS). 2 TE-MRS media: XTEx, X=G (Green tea), O (Oolong tea), or B (Black tea); x= 1 (membrane filtration method) or 2 (autoclave method), denoting the method for preparing TE. 3 Denotation of peak intensity: C=comparative, I= increase, D= decrease, T= tiny. 4 ND= Not Detected.

ACC

EPTE

D M

ANU

SCR

IPT


29

Captions for figures

Fig. 1 Soy yogurts (SY) prepared from soymilk containing lactose (SML) or soymilk-tea (STXx), X=G

(Green tea), O (Oolong tea), or B (Black tea); x= 1 (membrane filtration method) or 2 (autoclave

method), denoting the preparation method.

Fig. 2 Cell population of S. thermophilus ASCC 1275 (ST), L. acidophilus CSCC 2400 (LA), L. paracasei

CSCC 279 (LP), and L. delbrueckii ssp. bulgaricus ASCC 859 (LB) in fermented soymilk containing

lactose (SML) with or without tea extract at 37℃. The asterisks (*) on the bars indicate significant

difference at a level of p<0.05 compared with the control (SML).

Fig. 3 Cell populations and acidity evolution during 28 days of refrigerated storage for soymilk

containing lactose (SML) or soymilk-tea media (STX) fermented with S. thermophilus ASCC 1275 (ST),

L. acidophilus CSCC 2400 (LA), L. paracasei CSCC 279 (LP), and L. delbrueckii ssp. bulgaricus ASCC 859

(LB). Soymilk-tea media: STX, X=G (Green tea), O (Oolong tea), or B (Black tea); the STXs were

prepared by the autoclave method.

Fig. 4 Normalized FT-IR spectra (4000-900 cm−1) of selected bacteria grown in tea extract-MRS

media, XTE (X=G (Green tea), O (Oolong tea), or B (Black tea)) prepared by membrane filtration

method (denoted by “1”): black line: unsupplemented MRS media, green line: MRS+ GTE, orange line:

MRS+ OTE, red line: MRS+ BTE. Panel A: S. thermophilus cells, Panel B: L. delbrueckii ssp. bulgaricus

cells.

Fig. 5 Cell adherence to xylene (SHx%) and to n-hexadecane (SHh%) of S. thermophilus ASCC 1275

(ST), L. acidophilus CSCC 2400 (LA), L. paracasei CSCC 279 (LP), and L. delbrueckii ssp. bulgaricus

ASCC 859 (LB) cells incubated in MRS with or without tea extract (TE) at 37℃. TE-MRS media XTE,

X=G (Green tea), O (Oolong tea), or B (Black tea); x= 1 (membrane filtration method) or 2 (autoclave

method), denoting the method for preparing TE. The asterisks (*) on the bars indicate significant

difference at a level of p< 0.05 compared with the control (MRS).

ACC

EPTE

D M

ANU

SCR

IPT


30

Fig. 6 Bioconversion of isoflavone glycoside (IG) to aglycone by S. thermophilus ASCC 1275 (ST), L.

acidophilus CSCC 2400 (LA), L. paracasei CSCC 279 (LP), and L. delbrueckii ssp. bulgaricus ASCC 859

(LB) in fermented soymilk containing lactose (SML) or Soymilk-tea STXx: X=G (Green tea), O (Oolong

tea), or B (Black tea); x=1 (membrane filtration method) or 2 (autoclave method), denoting the

method for preparing soymilk-tea. The asterisks (*) on the bars indicate significant difference at a

level of p< 0.05 compared with the control (SML).

ACC

EPTE

D M

ANU

SCR

IPT


31

Fig. 1

ACC

EPTE

D M

ANU

SCR

IPT


32

Fig. 2

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

ST LA LP LB

lo

g C

FU

/m

L

Initial

SML

STG1

STG2

STO1

STO2

STB1

STB2

*

* * * * * *

* * * * * * * *

* * * * *

*

*

* *

* * *

*

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT


33

Fig. 3

ACC

EPTE

D M

ANU

SCR

IPT


34

Fig. 4

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

35

Fig. 5

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

ST LA LB LP

SH

x%

A

MRS

STG1

STG2

STO1

STO2

STB1

STB2 * *

*

*

* *

* * * *

* * * *

*

*

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

ST LA LB LP

SH

h%

B

MRS

STG1

STG2

STO1

STO2

STB1

STB2

*

*

* * *

*

* * *

* * * * *

*

* * *

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

36

Fig. 6

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

ST LA LB LP

Bio

con

ve

rsio

n (

Re

du

ctio

n %

of

IG)

SML

STG1

STG2

STO1

STO2

STB1

STB2

* * * * *

*

* * *

*

* *

* *

* *

* *

* *

* *

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

37

Highlights

Soymilk-tea yogurts were prepared by fermenting soymilk supplemented

with three types of tea extract (TE) with lactic acid bacteria (LAB).

Proliferation and survival after four-week refrigeration of four LAB were

strain-dependent in soymilk-tea media.

Cell surface hydrophobicity was altered due to TE supplementation.

FT-IR spectra indicated modification of cell membrane components by TE,

especially black tea.

Bioconversion of isoflavone in fermented SMT was generally decreased as

compared with fermented soymilk without TE, except for L. delbrueckii

ssp. bulgaricus.

effect of tea extract on lactic acid bacterial growth, their cell surface characteristics and...

Documents