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Improving the Stability of ProbioticBacteria in Model Fruit Juices UsingVitamins and AntioxidantsN.P. Shah, W.K. Ding, M.J. Fallourd, and G. Leyer

Abstract: This study examined the survival of probiotic bacteria in a model fruit juice system. Three different strainsof probiotic bacteria were used in this study: HOWARU Lactobacillus rhamnosus HN001, HOWARU Bidobacterium lactisHN001, and Lactobacillus paracasei LPC 37. The probiotic bacteria were inoculated into model juice with various vitaminsand antioxidants, namely white grape seed extract, green tea extract, vitamin B2, vitamin B3, vitamin B6, vitamin C, andvitamin E. The model juice without any additives was used as a control. Their viability was assessed on a weekly basisusing plate count method. The model juice was made with sucrose, sodium citrate, citric acid powder, and distilled water and was pasteurized before use. Our ndings showed that probiotic bacteria did not survive well in the harsh environmentof the model fruit juice. However, the model juice containing vitamin C, grape extract, and green tea extract showedbetter survival of probiotic bacteria. The model juice containing grape seed extract, green tea extract, and vitamin C hadthe same initial population of 8.32 log CFU/mL, and at the end of the 6-wk storage period it had an average viability of 4.29 log CFU/mL, 7.41 log CFU/mL, and 6.44 log CFU/mL, respectively. Juices containing all other ingredients testedhad viable counts of < 10 CFU/mL at the end of the 6-wk storage period.

Keywords: fruit juice, probiotic bacteria

IntroductionFruit juices may be an alternative vehicle for the incorporation

of probiotics as they are rich in nutrients and sugars and promotea healthy image. Furthermore, fruit juices are often supplementedwith oxygen-scavenging ingredients, such as ascorbic acid (to pro-

mote anaerobic conditions for probiotics) (Dave and Shah 1997),vitamins (to promote a healthy image), and white grape seed ex-tract and green tea extract (to promote antioxidant properties).Fruit juices are often packaged in bottles where the headspace iscontrolled and oxygen is often removed.

The food industry often refers to foods supplemented withprobiotics as functional foods. Several health benets are as-sociated with the consumption of live probiotic bacteria (Mitaland Garg 1995). These benets, include controlling of intestinalinfections, lowering of serum cholesterol levels, synthesis of B vi-tamins, lowering of blood ammonia levels, benecially inuencingthe immune system, improving lactose utilization, and providinganticarcinogenic activity (Pool-Zobel and others 1993; Gomesand Malcata 1999; Liong and Shah 2005). Because of the thera-peutic benets, there has been an increase in the incorporation of probiotic bacteria in a wider variety of food products including yo-gurts, cheese, drinks, and dietary supplements (Lourens-Hattinghand Viljeon 2001; Ong and others 2006).

MS 20090905 Submitted 9/14/2009, Accepted 3/5/2010. Authors Shah and Ding are with School of Biomedical and Health Sciences, Faculty of Health Engineering and Science, Victoria Univ., Werribee Campus, PO Box 14428, Melbourne, Victoria8001, Australia. Author Fallourd is with Danisco France, Paris, France. Author Leyer is with Danisco U.S.A., Madison, WI 53701, U.S.A. Direct inquiries to author Shah (E-mail: Nagendra.shah@vu.edu.au).

A daily consumption of high levels of probiotic bacteria is re-quired to confer health benets. The viability of probiotics shouldbe maintained in the food product until the time of consump-tion and be present in signicant numbers, at levels of at least107 viable cells per gram or milliliter of a product. Studies con-ducted have shown poor viability of probiotic bacteria, especiallybidobacteria in functional foods (Kailasapathy and Rybka 1997;Shah and Lankaputhra 1997; Schillinger 1999; Lee and Heo 2000;Talwalkar and Kailasapathy 2004; Kailasapathy 2005). For this rea-son, changes in the number of viable probiotic bacteria duringstorage of functional foods should be studied more extensively.Several factors have been claimed to affect the viability of pro-biotic bacteria in functional foods, including acid and hydrogenperoxide produced by starter cultures, high oxygen content, andoxygen permeability through the packaging materials (Shah and Jelen 1990; Shah and Lankaputhra 1997).

Commercially available fruit juices are acidic and often containpreservatives that may kill or inhibit the growth of the fastidiousprobiotic organisms. Commercially available juices have varioussugars at various levels and vitamins and minerals. More impor-tantly, sugars are added to juices. All these ingredients will havean inuence on the growth and activity of probiotic bacteria. Inorder to avoid these variabilities, it is important to examine thestability of probiotic bacteria in a model juice system.

Antioxidants can be dened as substances that interfere with thenormal oxidation process in oils and fats, thus delaying the onset of oxidation. Previous studies have demonstrated that damage to cellmembrane occurs during long-term storage and some antioxidantswere reported to protect membrane lipids against such damage(Teixeira and others 1995). It is known that addition of ascorbicacid (vitamin C) can also have a protective effect on probiotic cellsduring storage, presumably because it is an oxygen scavenger, thus

C 2010 Institute of FoodTechnologists RM278 Journal of Food Science Vol. 75, Nr. 5, 2010 doi: 10.1111/j.1750-3841.2010.01628.x

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Improving the stability of probiotic bacteria . . .

promoting a more favorable anaerobic environment (Teixeira andothers 1995; Dave and Shah 1997).

The objective of this study was to examine the inuence of vitamins, white grape seed extract viability, and green tea extracton the viability of probiotic bacteria in a model juice to understandthe role of these ingredients on their survival and for developingnovel functional fruit juices.

Materials and Methods

Bacterial strains and their cultivationA total of 3 probiotic bacteria including HOWARU L. rhamno-

sus HN001, HOWARU B. lactis HN001, and L. paracasei Lpc 37were obtained from Danisco (Madison, Wis., U.S.A). All probi-otic strains have proven health benets, and are available commer-cially. Cultures were maintained individually and grown in MRSbroth (Oxoid Ltd., Hampshire, United Kingdom). MRS broth for growing Bidobacterium spp. was supplemented with lter steril-ized 0.05% w/v L-cysteine-hydrochloride (Sigma Chemical Co.,Sydney, Australia) to create a more favorable anaerobic environ-ment. All probiotic bacteria used had their identity conrmedusing biochemical methods as described by Shah and Lankaputhra

(1997).Production of model juice and the addition of vitaminsand antioxidants

One liter of model juice was made with by combining, 0.1 g tri-sodium citrate, 0.135 g citric acid powder, and 9.268 g saccharoseand distilled water was added to make the total volume of 1 L.The pH of the model juice was adjusted to pH 3.8 using 2.0 MHCl. The model juice was pasteurized in a water bath at 85 Cfor 10 min before being stored in sterilized glass bottles at 4 C for 18 h before use. Samples of approximately 5 mL were asepticallytaken for microbial analysis at weekly intervals.

For the preparation of model juices with various vitamins and

antioxidants, various concentrations of vitamins and antioxidantswere added to the model juice prepared as previously described.The following concentrations were used for the various vitaminsand antioxidants: 0.00056% vitamin B2, 0.0072% (w/v) vita-min B3, 0.0008% (w/v) vitamin B6, 0.001% (w/v) 0.024% vi-tamin C, 0.001% (w/v) grape extract, and 0.001% (w/v) greentea extract. The concentrations were chosen based on the recom-mended daily allowance and the level used in this study covers100% of the recommended daily allowance (RDA) in a 250 mLportion.

Enumeration of bacteriaThe enumeration of probiotic bacteria was performed using the

methods described by Shah and Lankaputhra (1997). The enumer-ation of the probiotic bacteria in the fruit juice was preformed ona weekly basis over a period of 6 wk, using MRS agar and incu-bation conditions of 37 C for 72 h under anaerobic conditions.

pH and Brix measurementsThe pH and Brix were measured at weekly intervals for all

probiotic juices. A hand-held refractometer (Atago Inc., Bellevue,Wash., U.S.A) was used to measure Brix. A pH meter (HannaInstruments, Singapore) was used to measure the pH.

Viscosity and turbidity measurementsViscosity was measured using a Physica MCR 301 rheome-

ter (Anton Paar, Tokyo, Japan). Samples were taken once a week

and the viscosity was measured at 20 C. The turbidity was mea-sured using a LKB ultraspec III, UV/Vis spectrophotometer set at600 nm (Pharmacia, Stockholm, Sweden). Absorbance readingswere taken once a week for up to 6 wk.

Statistical analysis and samplingThe data for viability were analyzed using SPSS software (SPSS

version 15.0 Inc., Chicago, Ill., U.S.A). All data were analyzedusing one-way analysis of variance (ANOVA) and the signicantdifference was set at the 5% signicance level. For the enumerationof probiotic organisms, experiments were independently replicated3 times (n = 3). Data regarding pH, Brix, viscosity, and turbidityare presented as an average of 3 replicates ( n = 3).

Results and Discussion

Viability of probiotic bacteria in model juiceThe viable count of probiotic bacteria stored in model juice

for 6 wk is shown in Table 1. The control model juice showedan average initial viable count of 8.32 log CFU/mL. A steadyloss of viability was detected and at week 5 the average viabil-ity of probiotic bacteria was 2.44 log CFU/mL, and at week 6

all strains tested showed a viability count of < 10 log CFU/mL.The control model juice had an intitial pH of 3.8; however, thepH declined to 3.3 at the end of the storage period. A similar declining trend in Brix was also observed. Initially the model juice had 11.8 Brix and at 6 wk of storage, it had 9.1 Brix(Table 1). Turbidity and viscosity values increased steadily over the 6-wk storage period. The initial viscosity was 1.08 cP and atthe end of the storage period the average viscosity was 1.13 cP.Similarly, the initial turbidity was 0.09 and it increased to an aver-age of 0.23 at the 6-wk storage period. Results from the controlmodel juice indicated that the juice did not support the growthof probiotic bacteria. Other studies have shown similar declines in

Table 1The inuence of model juice on the viability of probi-otic bacteria.

Viscosity TurbidityWeek log CFU/mL pH Brix (cP) at 600 nm

HOWARU L. rhamnosus0 8.24 0.92 a 3.8 11.8 1.08 0.091 7.73 0.59 a 3.8 11.0 1.08 0.172 6.71 0.12 a 3.7 10.1 1.12 0.213 6.31 0.02 a 3.7 9.5 1.14 0.184 5.67 0.04 a 3.6 9.1 1.16 0.275 3.31 0.12 a 3.6 9.0 1.16 0.266 < 10 CFU/mL 3.6 9.0 1.15 0.21

HOWARU B. lactis0 8.32 0.09 a 3.8 11.8 1.08 0.081 7.34 0.91 a 3.8 11.1 1.09 0.192 5.24 0.84 a 3.6 10.0 1.09 0.263 4.29 0.25 a 3.5 9.5 1.11 0.274 3.12 0.63 a 3.4 9.2 1.12 0.215 2.01 0.52 a 3.4 9.2 1.12 0.246 < 10 CFU/mL 3.4 9.1 1.11 0.21

L. paracasei Lpc-370 8.41 0.43 a 3.8 11.8 1.08 0.021 7.21 0.21 a 3.8 11.0 1.09 0.172 6.68 0.73 a 3.7 10.1 1.09 0.233 5.21 0.32 a 3.6 9.5 1.12 0.254 5.15 0.46 a 3.4 9.1 1.16 0.275 2.01 0.32 a 3.4 9.1 1.16 0.266 < 10 CFU/mL 3.3 9.0 1.14 0.28aMeans in the samecolumn followedby differentlowercaseletters aresignicantly different

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