Food Chemistry 158 (2014) 262–269

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Food Chemistry

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Antiradical and tea polyphenol-stabilizing ability of functionalfermented soymilk–tea beverage

http://dx.doi.org/10.1016/j.foodchem.2014.02.1190308-8146/� 2014 Published by Elsevier Ltd.

⇑ Corresponding author. Tel.: +852 2299 0836; fax: +852 2299 9914.E-mail address: npshah@hku.hk (N.P. Shah).

Danyue Zhao, Nagendra P. Shah ⇑Food and Nutritional Science, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong

a r t i c l e i n f o

Article history:Received 16 November 2013Received in revised form 6 February 2014Accepted 19 February 2014Available online 1 March 2014

Keywords:Soymilk fermentationAntioxidant capacityTea polyphenolpH stabilityHPLC

a b s t r a c t

This study examined the potential of two-step fermentation to preserve TPs in functional soy–tea bever-age. Fermented soymilk–tea (FST) was produced by culturing Streptococcus thermophilus, Lactobacillusdelbrueckii ssp. bulgaricus and Bifidobacterium longum in soymilk supplemented with tea extract (TE).Total phenolic content (TPC) and anti-radical activities were determined for FSTs and fermented soymilk(FS). A HPLC method was employed to quantify nine major tea phenolics in FST products. TPC was signif-icantly higher (p < 0.05) in FST than FS, in the order of green tea > oolong tea > black tea > soymilk. TheFSTs were effective at scavenging DPPH-radical rather than hydroxyl radical. Optimal pH to stabilizeTPs in SMT was ca. 5.7, which reduced total TP loss by ca. 40% compared with that obtained from productswith TE supplemented at the beginning of fermentation. A gradual decrease in TPs was observed duringstorage (4 �C), with more than half of total TPs remained in FST after 8 weeks.

� 2014 Published by Elsevier Ltd.

1. Introduction

Notwithstanding the ever-growing demand for having foodswith multiple health benefits, foods are fortified with health-pro-moting compounds or microorganisms. The combined features oflive microorganisms and functional components present a great po-tential for fermented functional foods to exert multiple health ben-efits, through the interactions of ingested microorganisms withintestinal microflora (the probiotic effect) or with bioactive com-pounds (the biogenic effect) (Hervert-Hernández & Goñi, 2011;Laparra & Sanz, 2010).

Tea, as the most popular flavoured beverage in the world, isconsumed by over two-thirds of the world’s population daily(http://www.statisticbrain.com/tea-drinking-statistics/). Soymilk,as a healthy milk substitute, is being accepted by an increasingnumber of people around the world. Tea and soymilk deserve tre-mendous research interest not only for the popularity, but also forthe high level of phenolic compounds derived from them, whichare reported to have anti-radical, anti-mutagenic, anti-inflamma-tory and anti-carcinogenic effects in vitro and in vivo (Cencic &Chingwaru, 2010; Hsu et al., 2011; Li, Lo, Pan, Lai, & Ho, 2013). Ithas also been found that regular intake of tea and soy enhancescardiovascular health and lowers blood pressure by reducing oxi-dative stress (Perumalla & Hettiarachchy, 2011). The pivotal

bioactive phytochemicals in tea and soymilk are tea polyphenols(TPs) and soy isoflavones (SIs), respectively. In case of SIs, theirlatent bioactivity is hindered by the conjugated glycosides. Thisproblem can be resolved by bacterial fermentation as well-illus-trated in several studies on the bioconversion of isoflavone glyco-sides to aglycones during soymilk fermentation with lactic acidbacteria (LAB) or bifidobacteria (Champagne, Thomas, Nicole, &Julia, 2010; Donkor & Shah, 2007). However, the instability of TPsin non-acidic or high-temperature environment creates great chal-lenge for TPs to function as potent antioxidants in tea drinks duringlong-term storage. Chen, Zhu, Tsang, and Huang (2001) investi-gated composition of major tea catechins in 14 popular ready-to-drink tea beverages in Hong Kong, and found that most tea bever-ages contained only trace amount of catechins, considerably lowerthan that found in freshly brewed tea. They also demonstratedgood stability of tea catechins in acid-supplemented soft drinksduring long-term storage. It appears that acids were able to stabi-lize TPs, while the artificially-acidified soft drinks may not bedesirable functional beverages for consumers. As an alternative,fermentation with LAB and bifidobacteria may be an effectiveway to maintain the functional compounds in tea extract and toproduce a novel functional tea beverage. Despite the probioticand biogenic benefits of fermented products, the decreased pH va-lue can create a compatible environment for introducing TPs sincethey are much more stable in acidic rather than neutral or alkalineenvironment while the exact pH varies from media to media(Lun-Su, Leung, Huang, & Chen, 2003).

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The combination of functional food compounds, such as TPs, SIs,and probiotic bacteria may open a new field of food research deal-ing with bioactive components. Fermentation may further enhancethe nutritional and even pharmaceutical value of the product.Several authors examined the effects of fermenting milk supple-mented with tea on microbial growth and antioxidant capacity,reporting non-inhibiting effects on LAB or bifidobacteria and eleva-tion in antioxidant capacity (Ankolekar et al., 2011; Najgebauer-Lejko, Sady, Grega, & Walczycka, 2011). However, no study hasbeen done to investigate processing conditions to reduce lossesof TPs during fermentation and cold storage. This study providesan efficient and practical approach for preserving TPs as well asfor maintaining antioxidant potency in functional fermented soy-milk–tea beverage.

2. Materials and methods

2.1. Microorganisms and culture conditions

Streptococcus thermophilus ASCC 1275, Lactobacillus delbrueckiissp. bulgaricus ASCC 859 (L. bulgaricus) and Bifidobacterium longumCSCC 5089 were used for the production of fermented soymilk–tea(FST). Each organism was stored at �80 �C. For activation, 10-mLaliquots of sterile MRS (with 0.05% cysteine w/v, for B. longum)were inoculated with 10% (v/v) of each organism and incubatedat 37 �C for 24 h. After the second transfer in MRS broth, the acti-vated organisms were transferred into sterile soymilk containing1% (v/v) lactose for another two transfers at 10% (v/v). Activatedcultures were used for fermentation.

2.2. Preparation of soymilk

Soymilk containing lactose (SML) was prepared as per themethod of Donkor and Shah (2007) with some modifications. Soyprotein isolate (SPI) was kindly provided by Solae DuPont ChinaHolding Co. Ltd., (Shanghai, China) containing 90% protein (dry ba-sis), 6% moisture, 1.5% fat and 5.0% ash. Soymilk containing lactosewas made by dissolving 4% (w/v) SPI and 1% (w/v) a-lactose (SigmaChemical Co., St. Louis, MO, U.S.A.) in double deionized waterpreheated to 50 �C. For cultivating B. longum, additionally 0.05%HCl-cysteine (w/v) was added to SML. Upon reconstitution, SMLwas autoclaved at 105 �C for 15 min.

2.3. Preparation of tea extract (TE)

Green, oolong and black tea (Rickshaw, Unilever, Hong Kong)were purchased from local tea retailers. Tea leave powder (2%,w/v, corresponding to the strength of ‘‘a normal cup of tea’’ accord-ing to Yam, Shah, & Hamilton-Miller, 1997) was infused in boilingdouble deionized water for 10 min. Green, oolong and black TE, re-ferred to as GTE, OTE and BTE, were produced by first suction-fil-trated through triple-layered Whatman #1 filter paper twice andthen sterilized by filtering through 0.22-lm membrane (Millipore,Bedford, MA, U.S.A.). The membrane-sterilized filtrates were col-lected in 50 mL sterile tubes and frozen at �80 �C before freeze-drying using a Virtis freeze mobile (Virtis Co., Gardiner, U.S.A.).The freeze-dried TE powder was stored at �20�C for future use.

2.4. Preparation of fermented soy-milk tea (pre-addition method),determination of viable cell count and pH values

The activated cultures were transferred into SML or SML sup-plemented with TE (SMT) at an inoculum level of 1% (v/v) for S.thermophilus and 2% for L. bulgaricus and B. longum, and incubatedat 37 �C for 24 h. This is referred to the pre-addition method.

Fermented SML or SMT were referred to FS or FST, respectively,and non-inoculated SML or SMT incubated under the same condi-tions was used as a control for FS or FST. All fermentation tubeswere wrapped with aluminium foil to minimize photodegradationof TPs.

Upon completion of fermentation, viable cell count was enu-merated for each product by spread plate method. pH values ofboth the fermented or non-fermented SML were measured usingan Orion 250A portable pH meter (Orion Research, Boston, MA).

2.5. Antioxidant capacity of fermented soymilk with or without teaextract

2.5.1. Preparation of antioxidants extracts of fermented soymilk–teaA 2 mL aliquot of fermented SMT or SML was mixed with 4 mL

of 80% methanol, votexed for 1 min and centrifuged at 5000�g for10 min at 20 �C. The non-inoculated SMT or SML incubated at 37 �Cfor 24 h was used as a control and 2 mL of a control sample wasmixed with 4 mL of water–methanol–acetic acid (20:78:2, v/v/v).The precipitate was further extracted twice with 2 mL of 80%methanol and the supernatants were combined. All supernatantswere filtered through 0.45-lm hydrophilic filter (Corning, N.Y.,U.S.A.) and were used as the antioxidants extracts (AEs) for deter-mining total phenolic content (TPC), DPPH radical scavengingactivity and hydroxyl radical scavenging activity.

2.5.2. Determination of total phenolic contentTotal phenolic content (TPC) was analysed using the Folin–

Ciocalteu (F–C) method described by Cicco, Lanorte, Paraggio,Viggiano, and Lattanzio (2009). An aliquot of 200 lL of AE wasdiluted with 200 lL DI water. Then 400 lL of the diluted extractsor blank were pipetted into 5 mL test tubes wrapped with foiland 400 lL of F–C reagent was added to each tube. Each tubewas votexed for 3 s, equilibrated for exactly 2 min, and 3200 lLof 5% (w/v) sodium carbonate solution was added. The mixturewas swirled and incubated in a water bath at 30 �C for 1 h andthe absorbance was measured at 725 nm. The concentration wasdetermined by comparing the absorbance of the AEs with a calibra-tion curve constructed using (+)-catechins (0.01–1.0 mg/mL). Theconcentration of TP was expressed as milligram of catechins (C)/100 mL of inoculated or non-inoculated SML or SMT.

2.5.3. DPPH radical scavenging activityThe efficiency of SMT to scavenge 1,1-diphenyl-2-picryl-hydrazyl

(DPPH) radical was determined as per the method of Morales-de laPeña, Salvia-Trujillo, Rojas-Graü, and Martín-Belloso (2010) withminor modifications. In brief, 0.2 mL of AE of FSM was mixed with3.8 mL of methanolic solution of DPPH (0.039 g/L). The mixturewas shaken vigorously for 1 min and kept in dark for 30 min.Absorbance was measured at 517 nm against a methanol blank.Radical scavenging ability (RSA) was expressed in terms of percentDPPH inhibition using the following equation:

DPPH RSAð%Þ ¼ ½ðA0 � AsÞ=A0� � 100

where A0 is the absorbance of (methanolic solution of DPPH radicalwithout sample extract and As is the absorbance of fermented ornon-fermented SML or SMT.

2.5.4. Hydroxyl radical (�OH) scavenging activityThe activity for FS or FSTs to scavenge hydroxyl radical was

determined using the Fenton reaction method (He, Luo, Cao, &Cui, 2004) with some modifications. Briefly, the reaction mixturecontaining 0.5 mL of brilliant green (0.45 mM), 1 mL of FeSO4 solu-tion (0.5 mM), 1 mL of H2O2 (3.0%, w/v), and 0.5 mL of the antiox-idant extract of SY samples was incubated in test tubes covered inaluminium foil at 25 �C for 30 min. The absorbance value was

264 D. Zhao, N.P. Shah / Food Chemistry 158 (2014) 262–269

measured at 624 nm. The change in absorbance value indicated thescavenging ability of the AEs for hydroxyl radicals. Hydroxyl RSAwas calculated by the following equation:

Hydroxyl RSAð%Þ ¼ ½ðAs � A0Þ=ðA� A0Þ� � 100

where As is the absorbance value of the reaction mixture. A0 is theabsorbance value of the mixture in the absence of the antioxidantextracts. A is the absorbance value of the mixture in the absenceof the extracts and Fenton Reaction System.

2.6. Extraction and HPLC quantification of tea polyphenols

The extraction method was the same as described in Sec-tion 2.5.1 except that aqueous methanol was HPLC-grade (FisherScientific, Pittsburgh, PA).The non-inoculated SML or SMT incu-bated at 37 �C for 24 h was used as a control. All extracts were fil-tered through 0.45-lm hydrophilic syringe filter (Corning, NY)before loaded onto a HPLC column.

HPLC separation of nine phenolic compounds including gallicacid (GA), catechin (C), (�)-epigallocatechin gallate (EGCG),theaflavin (TF-1), theaflavin-3-gallate (TF-2) and theaflavin-3,30-digallate (TF-3) (Sigma Chemical Co., St. Louis, MO); (�)-epigallo-catechin (EGC), (�)-epicatechin (EC), (�)-epicatechin gallate(ECG) and caffeine (CF) (Wako, Osaka, Japan) were achieved witha Luna C18 (2) column (250 mm � 4.6 mm, 5 lm, Phenomenex,Torrance, CA, U.S.A.) using Shimadzu LC-10AD HPLC (Tokyo, Japan).The source of tea theaflavin standards was black tea extract (P80%theaflavins and theaflavins gallates) purchased from Sigma(St. Louis, MO, U.S.A.). A HPLC gradient elution programme wasadopted from Lun-Su et al. (2003) with some modifications. Themobile phases consisted of 2% (v/v) acetic acid in water (SolventA) and acetonitrile (Solvent B) (Fisher Scientific, Pittsburgh, PA,U.S.A).A gradient programme was applied at a flow rate of1.0 mL/min using a Shimadzu LC 10A apparatus (Shimadzu, Kyoto,Japan), equipped with a ternary pump delivery system. UV–Visdetector was set to scan from 200 to 400 nm and the detectionwavelength was 280 nm. The injection volume was 50 lL for allstandard and extract solutions.LC pump gradient for each runstarted at 5% (solvent B) and increased to 7% within 2 min. solventB was then linearly increased to 14% within 38 min, 31% for an-other 13 min before returning to the initial ratio over 4 min. Thetea catechins and theaflavins in samples were identified by com-paring absorption spectra and retention times of unknown peakswith the standards. The concentrations of TPs and caffeine werecalculated from chromatograph area and multiplied by the dilutionfactor. The analyses were carried out at least in triplicate, with thepercentage of concentration errors limited within 2%. Since caf-feine is a stable compound under the fermentation condition indi-cated above (Casal, Oliveira, & Ferreira, 2000; Ventura, Jiménez,Closas, Segura, & De la Torre, 2003) and its concentration in eachtype of tea was found to be conserved (data not shown), its concen-trations in different types of SMT, i.e., SMT-Green, SMT-Oolong orSMT-Black, were used to minimize the fluctuations in TP concen-trations in corresponding SMT due to multiple extractions.

2.7. Optimization of fermentation conditions for preserving teapolyphenols in soymilk–tea

2.7.1. pH-stability of tea polyphenols in soymilk–tea systemThe stability of eight TPs and gallic acid was examined in non-

inoculated SMT of different pH values. Green, oolong and blackSMTs were adjusted to pH 6.7, 5.7, 4.7 and 3.7 using 10 N or 2 Nacetic acid. A 2 mL aliquot of pH-adjusted SMT was collected afterincubation at 37 �C for 24 h to mimic conditions used for SMT fer-mentation with bacteria. All SMT-containing tubes were wrappedwith aluminum foil to minimize photo-degradation of TPs.

Extraction of TPs and HPLC analyses were same as described inSection 2.6.

2.7.2. pH evolution of soymilk during 24h fermentationFermentation of SML was carried out with S. thermophilus and L.

bulgaricus as described in Section 2.4. The change in pH values dur-ing 24 h fermentation of SML was recorded at various time inter-vals. Time for SML to reach pH 5.7 was taken as the fermentationtime before adding TE powder.

2.7.3. Long-term stability of tea polyphenols in fermented soymilk–tea(post-addition method) during cold storage

As pH of SML reached ca. 5.7 after the recorded time periods, TEpowder was added to the semi-fermented SML to complete the24 h fermentation. This is referred to the post-addition method.To assess the long-term TP stability in FST, all products were storedat 4 �C for 8 weeks and the remaining TP content was measuredweekly. Sterile SMTs were also stored at 4 �C and examined weeklyfor TP content. Extraction of TPs and HPLC analyses were the sameas described in Section 2.6.

2.8. Statistical analysis

For each assay, three independently samples were prepared.The data was subjected to one-way analysis of variance (ANOVA)(Turkey and Games–Howell tests) by SPSS 20.0 (IBM SPSS Statis-tics, IBM Corp., Somers, NY). Results with a p < 0.05 were consid-ered statistically significant. Correlations among anti-radicalactivities and total phenolic content were evaluated by Pearsoncorrelation test. R values were the average Pearson correlationssignificant at p < 0.01.

3. Results and discussion

3.1. Bacterial growth and acidity change during fermentation

The growth of S. thermophilus and L. bulgaricus was promoted inFSTs while the growth of B. longum was inhibited (data not show).The pH values of SMTs fermented with S. thermophilus or L. bulgar-icus were below 4.5 in the order of SMT-Green > SMT-Oo-long > SMT-Black > SML (Table S1). However, SMTs fermentedwith B. longum obtained significantly higher pH values as com-pared with the corresponding FS (p < 0.05). One possible reasonto account for such diversity among bacteria was that tea phenolicsmay exert antimicrobial effect towards certain bacteria by bindingto surface proteins, damaging membrane lipids and generatinghydrogen peroxide (Gaudreau, Champagne, Remondetto, Bazinet,& Subirade, 2012; Yi, Zhu, Fu, & Li, 2010), while they may promotethe growth of some bacteria by creating more anaerobic environ-ment, which depends on bacterial strains and phenolic contents(Ankolekar et al., 2011; Tabasco et al., 2011). Based on the growthof bacteria and final pH values, S. thermophilus and L. bulgaricuswere chosen for further study on long-term stability of TPs in FST.

3.2. Change in total phenolic content (TPC) during SML or SMTfermentation

Total phenolic content determined by Folin–Ciocalteu (F–C)method is presented in Table 1. There was a dramatic increase inTPC of the FMT products as expected since tea extract is a richsource of phenolic compounds, with green tea possessing thegreatest amount, followed by oolong and black tea (Bansal et al.,2013). Thus, there was a trend for both TPC and total tea phenolics(TTP), i.e., SMT-green > SMT-oolong > SMT-black, despite differ-ences in bacterial strains. It is noteworthy that all SMTs fermented

Table 1Total phenolic content (TPC) determined by Folin–Ciocalteu method and total tea phenolics (TTP) by HPLC method.

Total phenolic content (mg CE/mL) (Folin–Ciocalteu method) Total tea phenolics (mg CE/mL) (HPLC method)

SML SMT-Green SMT-Oolong SMT-Black SMT-Green SMT-Oolong SMT-Black

Control 0.28 ± 0.02a 2.03 ± 0.04b 1.58 ± 0.02c 1.05 ± 0.03d 1.76 ± 0.05a 1.38 ± 0.03b 0.99 ± 0.07c

S. thermophilus 0.28 ± 0.01a 2.42 ± 0.09b 2.01 ± 0.04c 1.37 ± 0.01d 2.19 ± 0.10a 1.85 ± 0.08b 1.46 ± 0.04c

L. bulgaricus 0.30 ± 0.02a 3.61 ± 0.16b 2.84 ± 0.06c 1.77 ± 0.07d 2.90 ± 0.07a 2.78 ± 0.14b 2.23 ± 0.09c

B. Longum 0.30 ± 0.01a 3.55 ± 0.05b 2.82 ± 0.05c 1.68 ± 0.04d 2.80 ± 0.11a 2.65 ± 0.11b 2.10 ± 0.06c

Values are expressed as means ± SEM (n = 4) of four independent fermentation products. Different letters (a–d) in the same row of TPC/TTP section indicate significantdifference at a level of p < 0.05.Non-inoculated soymilk containing lactose (SML) or soymilk–tea (SMT) incubated at 37 �C for 24 h was used as the control.Total phenolic content and total tea phenolics are both expressed as (+)-catechin equivalent (CE)/100 mL of product extract. (+)-Catechin was used as the standard forcalibration in Folin–Ciocalteu assay.

D. Zhao, N.P. Shah / Food Chemistry 158 (2014) 262–269 265

with bacteria showed higher TPC, compared with the control (non-inoculated SMTs incubated under the same condition). In addition,TPC of L. bulgaricus-fermented SMT was the highest among all fer-mented products. For the convenience of comparison between TPCand TTP as shown in Table 1, concentrations were all converted tocatechin equivalent (CE). Results showed that TPC was higher thanTTP on the whole, except for those of fermented SMT-black ex-tracts. As many researchers have suggested, F–C assay actuallymeasures total antioxidant content, including all reducing agentssuch as ascorbic acid, a-tocopherol, genistein and daidzein inSMT, rather than TPs alone. Therefore, TPC was expected to behigher than TPP for all samples. Nonetheless, the exception ofSMT-black is possibly due to the variations in the polyphenol com-position of different types of tea (Komes, Karlovic, KovacevicGanic,Horzic, & Glavaš, 2007). Green and oolong tea contain more mono-meric phenols and flavonoids, while black tea contains more poly-merized components, i.e. theaflavins and thearubigins (Li et al.,2013). F–C assay is based on electron transfer from phenolic com-pounds tophosphomolybdic/phosphotungstic acid complexes inalkaline condition to form blue complexes with maximum absor-bance in the range of 700–760 nm. However, the response of phen-olics in the F–C assay varies for compounds with different chemicalstructures (Parejo et al., 2002) as the redox process can be affectedby steric hindrance of bulky structures and the easiness of electrontransfer.

3.3. Anti-radical ability of soymilk–tea

The anti-radical ability of FS and FST is illustrated in Fig. 1.Although no single method can depict the overall antioxidant

Fig. 1. Radical scavenging ability (RSA) of fermented soymilk–tea extracts against DPPHlongum. Non-inoculated soymilk containing lactose (SML) incubated at 37 �C for 24 h wasindicates significant difference at p < 0.05 compared with the control in the same group

capacity of a product, we determined RSA of two well-known freeradicals to estimate the antioxidant capacity of FSTs: DPPH�, a sta-ble free radical commonly used in antioxidant capacity assays, and�OH, the most biologically active free radical formed in vivo underhypoxic conditions (Michiels, 2004).

As shown in the histogram (Fig. 1) on the left, dramatic eleva-tion in DPPH RSA was observed when TE was added to soymilk,without significant difference among tea types (p < 0.05).Najgebauer-Lejko et al. (2011) also reported a considerableincrease in DPPH-scavenging ability with tea addition to yogurt.Increases in DPPH RSA are also highly correlated with both TPC(r = 0.88, p < 0.01) and TTP (r = 0.94, p < 0.01), which implied thattea phenolics are the major contributors to DPPH radical inhibition.In fact, TPs have been reported to be efficient DPPH radical scav-engers resulting from their potent hydrogen donating power(Von Gadow, Joubert, & Hansmann, 1997).When comparing bacte-rial-fermented SMTs with non-inoculated control, RSA of all typesof FST is higher than that of the control. This is especially signifi-cant for fermented SMT-Oolong and SMT-Black (p < 0.05), althoughno significant difference was observed among bacterial strains.This may also be related to the ability of bacteria to metabolizepolymerized flavan-3-ol gallates to release gallic acid (Tabascoet al., 2011), a well-known powerful antioxidant with three vicinalhydroxyl groups attached to the phenolic ring in ortho positionwith each other(Sroka & Cisowski, 2003).

Compared with the high DPPH RSA values (95�97%) of FST ex-tracts, hydroxyl RSA values were much lower (16–34%). Generally,fermented products exhibited more effective RSA (p < 0.05) thancontrol in the order of B. longum > L. bulgaricus > S. thermophi-lus > control; among SMTs, SMT-Black exhibited the highest RSA.

and hydroxyl radical. ST = S. thermophilus; LB = L. delbrueckii ssp. bulgaricus; BL = B.used as the control. Data are expressed as means ± SEM (n = 4). Bars with asterisk (*).

266 D. Zhao, N.P. Shah / Food Chemistry 158 (2014) 262–269

After fermentation, interestingly, FS by itself also showed relativelyhigh RSA (30–40%) and supplementation of TE did not further en-hance the scavenging efficiency of �OH. Hydroxyl radical is prone toreduce double bonds and to fit into available ortho position in thephenolic rings of some polyphenols (Lipinsk, 2011). Nonetheless,most TPs lack reducible double bond or available ortho positionin their phenolic rings. Only isoflavones, such as genistein anddaidzein, possess phenol rings with only one para position occu-pied. Thus, isoflavones are probably the major hydroxyl radicalscavengers (Zielonka, Gebicki, & Grynkiewicz, 2003), rather thanTPs, and fermentation enhanced RSA since non-conjugated isoflav-one aglycones were released as a result of bacterial glycosidaseactivity (Matsuda et al., 1994; Tsangalis, Ashton, McGill, & Shah,2002).

3.4. Stability of tea phenolics in fermented soymilk–tea

To assess the stability of TPs in SMT system, several treatmentswere carried out and results are depicted in Fig. 2. The TTP in SMT-Green, SMT-Oolong and SMT-Black were 5.24, 4.92 and 2.71 mg/mL of FST (Table 2), respectively.

3.4.1. Influence of autoclavingBy autoclaving SMTs at 105 �C for 15 min, green tea catechin

(GTC) decreased on an average by 44% in three types of SMT, whilefor black tea theaflavin (BTF), the losses were as high as 76% inSMT-Oolong and SMT-Black. Similar studies also reported instabil-ity of TPs at high temperatures (Ananingsih, Sharma, & Zhou, 2013;Chen et al., 2001). In our study, theaflavins were found to be evenmore sensitive to thermal treatment. Hence, instead of autoclaving,TE was sterilized by filtering through 0.22-lm membrane beforecombining with soymilk.

3.4.2. Influence of 24 h fermentation or incubationThe effect of fermentation on the stability of GTC and BTF was

tested during 24 h incubation at 37 �C, using the non-inoculatedSMT as a control. Results (Fig. 2) showed that significantly lessreduction in the contents of GTC and BTF in bacterial fermentedSMTs was observed than that in the non-inoculated controls(p < 0.05), implying that fermentation may protect TPs from degra-dation or epimerization to some degree. The reason for such a phe-nomenon can be the decrease in pH as fermentation progressedsince TPs have been reported to be unstable in aqueous solutionwith a pH > 6.0 (Ananingsih et al., 2013). Lun-Su et al. (2003) deter-

Fig. 2. Tea phenolic content of soymilk–tea (SMT) with different treatments determineepigallocatechin gallate and epicatechin. Black tea theaflavin (BTF) consists of theaflaviAutoclaved: SMT autoclaved at 105 �C for 15 min; Non-inoculated: membrane-sterilizefermented with S. thermophilus/L. delbrueckii ssp. bulgaricus/B. longum at 37 �Cfor 24 h. D

mined that EGC and EGCG disappeared completely in phosphatebuffer (pH 7.4) after incubation for 3 h and 6 h, respectively. Thus,introduction of TE at the beginning of fermentation where the pHof media was ca. 6.4 should lead to degradation or epimerization ofTPs during the first several hours of incubation before the mediabecame acidic enough to retain TPs. Overall, L. bulgaricus-fer-mented SMT exhibited the highest percentages of remaining GTCand BTF, followed by B. longum and S. thermophilus-fermentedproducts. Such discrepancy may be attributed to difference in inoc-ulum dose and ability of one bacterium to acidify the media to acertain degree to stabilize TPs.

3.4.3. pH-dependent stability testThe remaining TTP amount in SMT of varying pH values after

24 h incubation at 37 �C is shown in Fig. 3. As shown in the figure,tea phenolics in 3 types of SMT all demonstrated pH-dependentstability. TTP content in SMT of pH 6.7 was the lowest among 4pH levels tested. For SMT-Green, the greatest amount of remainingtea phenolics (ca. 90%) was in samples with pH 5.7, while forSMT-Oolong and SMT-Black, it was pH 3.7 that led to the least lossof TTP (ca. 76%). However, there was no significant differenceamong SMTs at pH 5.7, 4.7 and 3.7, regardless of the types of TEadded (p < 0.05).

With reference to remaining TP contents after fermentation(Fig. 2), it is desirable to carry out fermentation in two steps in or-der to preserve most of TPs: soymilk media may be acidified to aproper pH during the first several hours, followed by supplement-ing tea powder and finishing fermentation in 24 h. To select a prop-er pH, final product attributes should be taken into account as well.Acid production by bacteria leads to gelation of soy protein nearthe isoelectric point, which is ca. 5.6 (the exact value may varyamong different varieties) (Grygorczyk & Corredig, 2013). Preli-minary study showed that when TE was introduced at pH 5.6 orbelow, the gel network formed by the precipitated soy protein sub-units was destroyed and the final products exhibited weak gelstructure. Hence, TE powder could be introduced to the SMT sys-tem when the pH of the semi-fermented SML dropped to 5.7, sothe media is acidic enough to stabilize TPs while soy protein is stilluncoagulated. The acidity of S. thermophilus and L. bulgaricus-fer-mented SML was monitored until the pH reached 5.7 in order todetermine the time required before adding TE powder. For S. ther-mophilus and L. bulgaricus, the time was 13 h and 7 h, respectively.Upon completing 24 h fermentation, the final products gave athick, yogurt-like look and final pH determined was lower than

d by HPLC method. Green tea catechin (GTC) consists of epigallocatechin, catechin,n, theaflavins gallate and theaflavins digallate. Original: membrane-sterilized SMT;d SMT incubated at 37 �Cfor 24 h; ST/LB/BL-fermented: membrane-sterilized SMTata are expressed as means of samples (n = 4).

Table 2Changes in concentrations of individual phenolic compound in fermented soymilk–tea (SMT) during 8-week cold storage (4 �C).

Concentration (mg/mL) GA EGC C EGCG EC ECG TF1 TF2 TF3 Total (%)

Original SMT-Green 0.65 1.73 0.20 1.33 0.92 0.41 N.D. N.D. N.D. 5.24Original SMT-Green 0.65 1.73 0.20 1.33 0.92 0.41 N.D. N.D. N.D. 5.24

SMT-Oolong 0.36 1.49 0.15 1.10 0.75 0.31 0.37 0.28 0.12 4.92SMT-Black 0.55 0.60 0.10 0.15 0.13 0.07 0.55 0.40 0.18 2.71

4-Week cold storage SMT-Green 0.38 0.24 0.10 0.4 0.53 0.15 N.D. N.D. N.D. 1.81 (34)SMT-Oolong 0.18 0.15 0.06 0.33 0.39 0.09 0.07 0.05 N.D. 1.31 (27)SMT-Black 0.29 N.D. N.D. N.D. N.D. N.D. 0.18 0.12 N.D. 0.68 (25)

S. thermophilus-fermented SMTSMT-Green Pre-added 0.44 0.59 0.12 0.52 0.43 0.17 N.D. N.D. N.D. 2.26 (43)

Post-add 0.61 1.56 0.18 0.84 0.80 0.33 N.D. N.D. N.D. 4.31 (82)Week 1 0.62 1.47 0.16 0.78 0.76 0.30 N.D. N.D. N.D. 4.08 (78)Week 2 0.59 1.42 0.15 0.74 0.70 0.27 N.D. N.D. N.D. 3.86 (74)Week 3 0.58 1.40 0.16 0.72 0.68 0.26 N.D. N.D. N.D. 3.79 (72)Week 4 0.56 1.37 0.15 0.70 0.67 0.26 N.D. N.D. N.D. 3.71 (71)Week 8 0.54 1.35 0.14 0.65 0.62 0.25 N.D. N.D. N.D. 3.55 (68)

SMT-Oolong Pre-added 0.28 0.45 0.08 0.29 0.29 0.07 0.15 0.13 0.04 1.77 (36)Post-added 0.32 1.36 0.13 0.72 0.59 0.28 0.28 0.26 0.06 3.99 (81)Week 1 0.31 1.32 0.10 0.68 0.55 0.25 0.26 0.25 0.05 3.77 (77)Week 2 0.28 1.23 0.09 0.63 0.54 0.24 0.23 0.23 0.04 3.52 (72)Week 3 0.27 1.19 0.08 0.54 0.52 0.23 0.20 0.21 0.04 3.28 (67)Week 4 0.24 1.17 0.08 0.52 0.48 0.22 0.19 0.18 0.04 3.12 (64)Week 8 0.20 1.12 0.05 0.49 0.46 0.22 0.18 0.15 0.03 2.90 (60)

SMT-Black Pre-added 0.34 0.38 0.06 0.00 0.05 0.01 0.21 0.19 0.09 1.32 (49)Post-added 0.47 0.50 0.09 0.10 0.11 0.06 0.40 0.34 0.12 2.14 (81)Week 1 0.43 0.48 0.08 0.09 0.11 0.06 0.36 0.33 0.10 2.03 (75)Week 2 0.39 0.44 0.07 0.07 0.10 0.05 0.37 0.31 0.09 1.88 (69)Week 3 0.37 0.43 0.06 0.06 0.10 0.05 0.34 0.29 0.08 1.79 (66)Week 4 0.36 0.42 0.06 0.05 0.09 0.05 0.30 0.26 0.08 1.67 (62)Week 8 0.34 0.38 0.04 0.03 0.06 0.05 0.28 0.21 0.05 1.44 (53)

L. delbrueckii ssp. bulgaricus-fermented SMTSMT-Green Pre-added 0.53 0.71 0.14 0.62 0.49 0.17 N.D. N.D. N.D. 2.67 (51)

Post-added 0.70 1.65 0.18 0.88 0.82 0.33 N.D. N.D. N.D. 4.55 (87)Week 1 0.72 1.61 0.17 0.82 0.75 0.31 N.D. N.D. N.D. 4.38 (83)Week 2 0.73 1.57 0.16 0.75 0.69 0.30 N.D. N.D. N.D. 4.20 (80)Week 3 0.71 1.55 0.16 0.71 0.67 0.28 N.D. N.D. N.D. 4.08 (78)Week 4 0.69 1.51 0.16 0.69 0.66 0.26 N.D. N.D. N.D. 3.97 (76)Week 8 0.64 1.46 0.15 0.64 0.63 0.25 N.D. N.D. N.D. 3.77 (72)

SMT-Oolong Pre-added 0.34 0.39 0.09 0.41 0.34 0.15 0.20 0.14 0.06 2.12 (43)Post-added 0.42 1.40 0.13 0.74 0.67 0.28 0.28 0.26 0.09 4.26 (87)Week 1 0.43 1.37 0.11 0.63 0.66 0.26 0.21 0.22 0.09 3.97 (81)Week 2 0.44 1.35 0.10 0.59 0.62 0.24 0.20 0.19 0.07 3.79 (77)Week 3 0.43 1.32 0.10 0.57 0.60 0.24 0.19 0.16 0.05 3.66 (74)Week 4 0.39 1.31 0.09 0.56 0.59 0.23 0.19 0.15 0.05 3.56 (72)Week 8 0.36 1.24 0.08 0.51 0.54 0.21 0.17 0.12 0.02 3.26 (68)

SMT-Black Pre-added 0.41 0.40 0.07 N.D. 0.09 0.03 0.24 0.21 0.10 1.55 (56)Post-added 0.54 0.56 0.09 0.11 0.12 0.06 0.40 0.33 0.13 2.34 (86)Week 1 0.59 0.54 0.08 0.09 0.11 0.06 0.36 0.29 0.11 2.21 (82)Week 2 0.56 0.52 0.08 0.07 0.11 0.06 0.33 0.26 0.10 2.08 (77)Week 3 0.55 0.50 0.07 0.07 0.10 0.06 0.29 0.24 0.08 1.96 (72)Week 4 0.52 0.50 0.07 0.06 0.10 0.05 0.27 0.24 0.08 1.88 (70)Week 8 0.47 0.43 0.05 0.05 0.09 0.04 0.25 0.20 0.04 1.62 (60)

Values are expressed as means of samples (n = 3).Abbreviations of phenolic compound: GA: gallic acid; EGC: (�)-epigallocatechin; C: catechin; EGCG: (�)-epigallocatechin gallate; EC: (�)-epicatechin; ECG: (�)-epicatechingallate; TF-1: theaflavins; TF-2: theaflavin-3-gallate; TF-3: theaflavin-3,30-digallate.Percentage of remaining tea phenolics (%) is expressed as means of three independent assays.N.D. = not detected.

D. Zhao, N.P. Shah / Food Chemistry 158 (2014) 262–269 267

the values for FST prepared by pre-addition method (data notshown).

3.4.4. Long-term stability of tea phenolics in fermented soymilk–tea(post-addition method)

To access the effectiveness of post-addition method in stabiliz-ing tea phenolics in FST products, individual tea phenolics werequantified during 8-week cold storage (4 �C) (Table 2). Comparedwith profiles of TP in FST by pre-addition method, FST by post-addition method significantly enhanced the stability of TPs inSMT (p < 0.05). As shown in Table 2, S. thermophilus and L. bulgar-icus-fermented SMT maintained 80% and 84% TTP on average,respectively, which is 37% and 35% higher than those found in

FST prepared by pre-addition method. A gradual and slight de-crease in tea phenolics was observed during cold storage, particu-larly noteworthy for EGCG, TF-1, TF-2 and TF-3.It was found thatduring the first 2 weeks, losses of TTP were more obvious thanthe following weeks, despite the type of tea. After 8 week of stor-age, more than half of TTP was maintained for all FST products.With respect to individual phenolic compounds, gallic acid wasthe only one that showed an increase in concentration after 24 hfermentation, significant for L. bulgaricus-fermented SMT-oolong(p < 0.05). Some strains of LAB have been reported to metabolizegalloylated catechins and hence release free gallic acid and othermonomeric phenolic compounds (Tabasco et al., 2011). Increasein more available hydroxyl groups and protons available for

50.5 (b)

90.8 (a)87.8 (a)85.8 (a)

41.8 (b)

70.2 (a)68.8 (a)76.4 (a)

55.6 (b)

70.4 (a)70.5 (a)76.2 (a)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

6.75.74.73.7

Totla

tea

phen

olic

(mg/

mL)

pH Level

SMT-Green

SMT-Oolong

SMT-Black

Fig. 3. Stability of green tea phenolics in three types of soymilk–tea (SMT) with varying pH during incubation at 37 �C for 24 h. Data are expressed as means ± SEM (n = 3) ofsamples. Data labels indicate percentage remaining of tea phenolic contents and the letters (a, b) above data points in the same line indicate significant difference at p < 0.05.

Fig. 4. Characteristic HPLC chromatograms showing the elution of phenolic compounds and caffeine in soymilk-oolong tea (SMT-Oolong) fermented with or without L.delbrueckii ssp. bulgaricus. Detection: 280 nm. (A) Original membrane-sterilized SMT-Oolong; (B) Non-inoculated SMT-Oolong incubated for 24 h; (C) SMT-Oolong fermentedfor 24 h with tea extract added at the beginning; (D) SMT-Oolong fermented for 24 h with tea extract added after 7 h-incubation (pH: ca. 5.7); and (E) 4-week refrigeration(4 �C) of sample D. Peak denotation: (1) gallic acid, (2) (�)-epigallocatechin, (3) (+)-catechin, (4) caffeine, (5) (�)-epigallocatechin gallate, (6) (�)-epicatechin, (7) (�)-epicatechin gallate, (8) theaflavin, (9) theaflavin-3-gallate, and (10) theaflavin-3,30-digallate.

268 D. Zhao, N.P. Shah / Food Chemistry 158 (2014) 262–269

antioxidative donation is also related to enhancement of antioxi-dant capacity (Yilmaz & Toledo, 2004). In contrast, TTP content de-creased dramatically in the sterile SMTs after 4 week of coldstorage, with only 34%, 27% and 25% left for SMT-green, oolongand black, respectively. HPLC chromatographs (Fig. 4) directlyillustrate the changes in the phenolic profile of SMT-Oolong fer-mented with L. bulgaricus during fermentation and cold storage.The most unstable phenolics in SMT were found to be EGC, EGCG,EC, TF-2 and TF-3, which almost disappeared in incubated SMTwith no bacteria inoculation (Fig. 4B). During pre-added SMT fer-mentation, although less EGCG and EC were degraded, the majorityof EGC, TF-2 and TF-3 (peak 2, 9 and 10, respectively) was not re-tained. By contrast, using the post-addition method, noticeable in-crease in the response of those easily-degraded compounds wasdetected in both S. thermophilus and L. bulgaricus-fermented SMT(average 91% remaining for EGC, 89% for TF-2, and 64% for TF-3).The higher stability of EC and ECG than those of EGC and EGCGhas been attributed to the three vicinal hydroxyl groups attachedto the aromatic rings (the pyrogallol structure) in EGCG and EGCbeing more susceptible to autoxidation, forming semiquinone free

radicals and then phenolic coupling (Bors & Michel, 2002; Yoshiokaet al., 1991). Instead, ECG and EC only possess rings with two vic-inal hydroxyl groups attached. After 4 weeks, changes in the phe-nolic profile were not obvious, although slight decreases inconcentration of individual phenolics were detected.

4. Conclusion

When processing foods containing tea extract, temperature, pHand tea-infusion media can have marked impact on the stability oftea phenolics, separately or synergistically. By means of two-stepfermentation, tea phenolic compounds can be preserved in soy-based products while maintaining their antioxidant activities andpromoting effects towards yogurt starters. The product could alsobe considered as ‘‘functional’’ as it contains beneficial componentssuch as tea polyphenols, soy isoflavones and functional bacteria(Servili et al., 2011). Based on experimental results obtained fromthis study, we may conclude that fermentation increased totalantioxidant content, anti-radical capacity and stability of tea

D. Zhao, N.P. Shah / Food Chemistry 158 (2014) 262–269 269

polyphenols. To the best our knowledge, this is the first report onpreserving major tea phenolics by means of two-step bacterial fer-mentation. Supplementing TE when pH dropped to 5.7 markedlypreserved more catechins or theaflavins for 8 weeks, comparedwith adding TE at the beginning of fermentation. We believe thatthe application of such innovative biotechnology may create moreoptions and greater potential for the functional food market.Further investigation needs to be carried out to improve thesensory attributes of FST before it could be accepted as a satisfactoryfunctional food. It will also be important to examine whether dailyintake of such functional beverages with high polyphenol contentwill influence the balance of intestinal microorganisms.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.02.119.

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