Improving the Stability of Probiotic Bacteria in Model Fruit Juices Using Vitamins and Antioxidants

N.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 strains of probiotic bacteria were used in this study: HOWARU Lactobacillus rhamnosus HN001, HOWARU Bidobacterium lactis HN001, and Lactobacillus paracasei LPC 37. The probiotic bacteria were inoculated into model juice with various vitamins and antioxidants, namely white grape seed extract, green tea extract, vitamin B2, vitamin B3, vitamin B6, vitamin C, and vitamin E. The model juice without any additives was used as a control. Their viability was assessed on a weekly basis using 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 environment of the model fruit juice. However, the model juice containing vitamin C, grape extract, and green tea extract showed better survival of probiotic bacteria. The model juice containing grape seed extract, green tea extract, and vitamin C had the 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 tested had viable counts of <10 CFU/mL at the end of the 6-wk storage period.

M: Food Microbiology & Safety

Keywords: fruit juice, probiotic bacteria

Introduction
Fruit juices may be an alternative vehicle for the incorporation of probiotics as they are rich in nutrients and sugars and promote a healthy image. Furthermore, fruit juices are often supplemented with oxygen-scavenging ingredients, such as ascorbic acid (to promote anaerobic conditions for probiotics) (Dave and Shah 1997), vitamins (to promote a healthy image), and white grape seed extract and green tea extract (to promote antioxidant properties). Fruit juices are often packaged in bottles where the headspace is controlled and oxygen is often removed. The food industry often refers to foods supplemented with probiotics as functional foods. Several health benets are associated with the consumption of live probiotic bacteria (Mital and Garg 1995). These benets, include controlling of intestinal infections, lowering of serum cholesterol levels, synthesis of B vitamins, lowering of blood ammonia levels, benecially inuencing the immune system, improving lactose utilization, and providing anticarcinogenic activity (Pool-Zobel and others 1993; Gomes and Malcata 1999; Liong and Shah 2005). Because of the therapeutic benets, there has been an increase in the incorporation of probiotic bacteria in a wider variety of food products including yogurts, cheese, drinks, and dietary supplements (Lourens-Hattingh and 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, Victoria 8001, 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 required to confer health benets. The viability of probiotics should be maintained in the food product until the time of consumption and be present in signicant numbers, at levels of at least 107 viable cells per gram or milliliter of a product. Studies conducted have shown poor viability of probiotic bacteria, especially bidobacteria 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 reason, changes in the number of viable probiotic bacteria during storage of functional foods should be studied more extensively. Several factors have been claimed to affect the viability of probiotic bacteria in functional foods, including acid and hydrogen peroxide produced by starter cultures, high oxygen content, and oxygen permeability through the packaging materials (Shah and Jelen 1990; Shah and Lankaputhra 1997). Commercially available fruit juices are acidic and often contain preservatives that may kill or inhibit the growth of the fastidious probiotic organisms. Commercially available juices have various sugars at various levels and vitamins and minerals. More importantly, sugars are added to juices. All these ingredients will have an inuence on the growth and activity of probiotic bacteria. In order to avoid these variabilities, it is important to examine the stability of probiotic bacteria in a model juice system. Antioxidants can be dened as substances that interfere with the normal oxidation process in oils and fats, thus delaying the onset of oxidation. Previous studies have demonstrated that damage to cell membrane occurs during long-term storage and some antioxidants were reported to protect membrane lipids against such damage (Teixeira and others 1995). It is known that addition of ascorbic acid (vitamin C) can also have a protective effect on probiotic cells during storage, presumably because it is an oxygen scavenger, thus
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Improving the stability of probiotic bacteria . . .

promoting a more favorable anaerobic environment (Teixeira and others 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 extract on the viability of probiotic bacteria in a model juice to understand the role of these ingredients on their survival and for developing novel functional fruit juices. and the viscosity was measured at 20 C. The turbidity was measured using a LKB ultraspec III, UV/Vis spectrophotometer set at 600 nm (Pharmacia, Stockholm, Sweden). Absorbance readings were taken once a week for up to 6 wk.

Statistical analysis and sampling The data for viability were analyzed using SPSS software (SPSS version 15.0 Inc., Chicago, Ill., U.S.A). All data were analyzed Materials and Methods using one-way analysis of variance (ANOVA) and the signicant difference was set at the 5% signicance level. For the enumeration Bacterial strains and their cultivation of probiotic organisms, experiments were independently replicated A total of 3 probiotic bacteria including HOWARU L. rhamno3 times (n = 3). Data regarding pH, Brix, viscosity, and turbidity sus HN001, HOWARU B. lactis HN001, and L. paracasei Lpc 37 are presented as an average of 3 replicates (n = 3). were obtained from Danisco (Madison, Wis., U.S.A). All probiotic strains have proven health benets, and are available commercially. Cultures were maintained individually and grown in MRS Results and Discussion broth (Oxoid Ltd., Hampshire, United Kingdom). MRS broth for Viability of probiotic bacteria in model juice growing Bidobacterium spp. was supplemented with lter sterilThe viable count of probiotic bacteria stored in model juice ized 0.05% w/v L-cysteine-hydrochloride (Sigma Chemical Co., for 6 wk is shown in Table 1. The control model juice showed Sydney, Australia) to create a more favorable anaerobic environ- an average initial viable count of 8.32 log CFU/mL. A steady ment. All probiotic bacteria used had their identity conrmed loss of viability was detected and at week 5 the average viabilusing biochemical methods as described by Shah and Lankaputhra ity of probiotic bacteria was 2.44 log CFU/mL, and at week 6 (1997). all strains tested showed a viability count of < 10 log CFU/mL. Production of model juice and the addition of vitamins and antioxidants One liter of model juice was made with by combining, 0.1 g trisodium citrate, 0.135 g citric acid powder, and 9.268 g saccharose and 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 M HCl. The model juice was pasteurized in a water bath at 85 C for 10 min before being stored in sterilized glass bottles at 4 C for 18 h before use. Samples of approximately 5 mL were aseptically taken for microbial analysis at weekly intervals. For the preparation of model juices with various vitamins and antioxidants, various concentrations of vitamins and antioxidants were added to the model juice prepared as previously described. The following concentrations were used for the various vitamins and antioxidants: 0.00056% vitamin B2, 0.0072% (w/v) vitamin B3, 0.0008% (w/v) vitamin B6, 0.001% (w/v) 0.024% vitamin C, 0.001% (w/v) grape extract, and 0.001% (w/v) green tea extract. The concentrations were chosen based on the recommended daily allowance and the level used in this study covers 100% of the recommended daily allowance (RDA) in a 250 mL portion. Enumeration of bacteria The enumeration of probiotic bacteria was performed using the methods described by Shah and Lankaputhra (1997). The enumeration of the probiotic bacteria in the fruit juice was preformed on a weekly basis over a period of 6 wk, using MRS agar and incubation conditions of 37 C for 72 h under anaerobic conditions. pH and Brix measurements The 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 (Hanna Instruments, Singapore) was used to measure the pH. Viscosity and turbidity measurements Viscosity was measured using a Physica MCR 301 rheometer (Anton Paar, Tokyo, Japan). Samples were taken once a week
The control model juice had an intitial pH of 3.8; however, the pH 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 at the end of the storage period the average viscosity was 1.13 cP. Similarly, the initial turbidity was 0.09 and it increased to an average of 0.23 at the 6-wk storage period. Results from the control model juice indicated that the juice did not support the growth of probiotic bacteria. Other studies have shown similar declines in
Table 1 The inuence of model juice on the viability of probiotic bacteria. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log CFU/mL 8.24 0.92a 7.73 0.59a 6.71 0.12a 6.31 0.02a 5.67 0.04a 3.31 0.12a <10 CFU/mL pH

Brix

Viscosity (cP) 1.08 1.08 1.12 1.14 1.16 1.16 1.15 1.08 1.09 1.09 1.11 1.12 1.12 1.11 1.08 1.09 1.09 1.12 1.16 1.16 1.14

Turbidity at 600 nm 0.09 0.17 0.21 0.18 0.27 0.26 0.21 0.08 0.19 0.26 0.27 0.21 0.24 0.21 0.02 0.17 0.23 0.25 0.27 0.26 0.28

HOWARU L. rhamnosus 3.8 11.8 3.8 11.0 3.7 10.1 3.7 9.5 3.6 9.1 3.6 9.0 3.6 9.0 HOWARU B. lactis 8.32 0.09a 3.8 11.8 a 7.34 0.91 3.8 11.1 5.24 0.84a 3.6 10.0 4.29 0.25a 3.5 9.5 3.4 9.2 3.12 0.63a a 2.01 0.52 3.4 9.2 <10 CFU/mL 3.4 9.1 8.41 0.43a 7.21 0.21a 6.68 0.73a 5.21 0.32a 5.15 0.46a 2.01 0.32a <10 CFU/mL L. paracasei Lpc-37 3.8 11.8 3.8 11.0 3.7 10.1 3.6 9.5 3.4 9.1 3.4 9.1 3.3 9.0

a Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

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the viability of probiotic bacteria in fresh orange and apple juices (Ding and Shah 2008). The increase in turbidity and viscosity was likely due to an accumulation of lysed and dying cells. The decrease in pH in the model juice may be due to bacterial growth and acid production from the probiotic bacteria throughout the shelf life. vitamin B3, vitamin B6, vitamin C, and vitamin E. In general, the viability of probiotic bacteria stored in the model juice with various vitamins showed a declining trend over the 6-wk storage period (Table 48). The most effective vitamin in preserving probiotic viability was vitamin C, which had an average count of 6.36 log CFU/mL, whereas all other vitamins tested had counts of < 10 CFU/mL at the end of the 6-wk storage period (Table 7). In
Table 3Inuence of green tea extract on the viability of probiotic bacteria in a model fruit juice. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6
a

Viability of probiotic bacteria in model juice containing grape seed extract and green tea extract Table 2 and 3 show the viability of probiotic bacteria in a model juice containing white grape seed extract and green tea extract. Both white grape seed extract and green tea extract improved the viability of probiotic bacteria in the model juice. Both had the same initial population of 8.32 log CFU/mL, and at the end of the storage period of 6 wk they had an average viability of 4.29 log CFU/mL and 7.41 log CFU/mL, respectively. Why green tea extract gave better survival of probiotics needs further investigation. Both the grape seed and green tea extract had the initial pH of 3.8; however, after the storage period of 6 wk a drop in pH was detected and the nal pH of 3.4 and 3.6, respectively, was observed (Table 2 and 3). Both grape seed extract and green tea extract made the model juice very turbid and cloudy. Grape seed and green tea extract had average absorbance values of 0.31 and 0.36, respectively. In both model juices with grape seed and green tea extract, the value of Brix declined over the 6-wk storage period, with average values of 7.0 and 6.8 Brix, respectively. The viscosity of the model juice also increased slightly, which may be due to a larger population of live probiotic bacteria (Table 2 and 3). Viability of probiotic bacteria in the model juice containing vitamins Table 4 through 8 show the viability of probiotic bacteria in the model juice containing various vitamins namely, vitamin B2,

log CFU/mL 8.24 0.13a 8.94 0.05a 8.97 0.08a 8.13 0.58a 7.51 0.61a 7.44 0.62a 7.21 0.67a 8.32 0.26a 8.74 0.23a 8.79 0.26a 7.43 0.68a 7.61 0.23a 7.54 0.64a 7.18 0.12a 8.41 0.21a 8.74 0.84a 8.79 0.12a 8.43 0.37a 8.31 0.12a 8.03 0.23a 7.86 0.54a

pH

Brix

Viscosity (cP) 1.11 1.18 1.23 1.24 1.29 1.29 1.30 1.11 1.19 1.23 1.25 1.27 1.27 1.27 1.11 1.19 1.23 1.24 1.24 1.27 1.27

Turbidity at 600 nm 0.00 0.17 0.17 0.18 0.27 0.29 0.33 0.00 0.11 0.16 0.17 0.21 0.34 0.38 0.00 0.27 0.23 0.25 0.27 0.31 0.38

HOWARU L. rhamnosus 3.8 11.8 3.8 11.1 3.7 10.1 3.7 9.1 3.6 9.1 3.6 8.0 3.6 7.1 HOWARU B. lactis 3.8 11.8 3.8 11.0 3.8 10.3 3.7 9.5 3.6 8.1 3.6 7.3 3.5 6.5 L. paracasei Lpc-37 3.8 11.8 3.8 11.3 3.7 10.1 3.7 9.7 3.7 9.2 3.6 8.0 3.4 7.2

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Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

Table 2The inuence of white grape seed extract on the viabil- Table 4 Inuence of riboavine (vitamin B2) on the survival of ity of probiotic bacteria. probiotic bacteria in fruit juices. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log CFU/mL 8.24 0.26a 7.74 0.35a 6.71 0.14a 6.32 0.64a 5.67 0.14a 5.35 0.42a 5.10 0.32a 8.32 0.22a 7.31 0.03a 5.24 0.80a 4.29 0.42a 3.14 0.64a 3.01 0.24a 2.97 0.44ab 8.41 0.35a 7.21 0.18a 6.62 0.14a 5.21 0.64a 5.15 0.16a 5.01 0.15a 4.81 0.34a pH

Brix

Viscosity (cP) 1.08 1.08 1.12 1.14 1.13 1.20 1.25 1.08 1.09 1.01 1.11 1.12 1.22 1.24 1.08 1.09 1.09 1.12 1.12 1.20 1.24

Turbidity at 600 nm 0.00 0.17 0.21 0.28 0.27 0.26 0.26 0.00 0.19 0.18 0.18 0.21 0.24 0.31 0.00 0.17 0.23 0.25 0.27 0.36 0.38

Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6
a

log CFU/mL 8.27 0.30a 6.14 0.24a 3.81 0.18a 2.64 0.18a 1.34 0.17a <10 CFU/mL <10 CFU/mL

pH

Brix

Viscosity (cP) 1.08 1.08 1.09 1.09 1.09 1.09 1.09 1.08 1.08 1.09 1.09 1.09 1.09 1.09 1.08 1.09 1.09 1.09 1.09 1.09 1.09

Turbidity at 600 nm 0.00 0.07 0.09 0.09 0.09 0.10 0.10 0.00 0.07 0.08 0.09 0.09 0.11 0.11 0.00 0.07 0.07 0.07 0.09 0.09 0.09

HOWARU L. rhamnosus 3.8 11.8 3.8 11.0 3.7 10.1 3.7 9.6 3.6 9.6 3.6 8.0 3.6 7.1 HOWARU B. lactis 3.8 11.8 3.8 11.1 3.6 10.0 3.5 9.5 3.4 9.2 3.4 8.3 3.4 7.0 L. paracasei Lpc-37 3.8 11.8 3.8 11.0 3.7 10.1 3.6 9.5 3.4 9.1 3.4 8.1 3.3 7.0

HOWARU L. rhamnosus 3.8 11.8 3.7 11.6 3.7 11.6 3.5 11.4 3.5 11.3 3.5 11.3 3.5 11.3 HOWARU B. lactis 8.33 0.72a 3.8 11.8 6.21 0.23a 3.7 11.6 4.65 0.62a 3.6 11.2 a 2.21 0.25 3.6 11.2 1.21 0.61a 3.6 11.2 <10 CFU/mL 3.5 11.2 <10 CFU/mL 3.5 11.1 L. paracasei Lpc-37 8.45 0.21a 3.8 11.8 7.21 0.65a 3.7 11.6 5.21 0.32a 3.6 11.2 3.71 0.74a 3.6 10.7 2.13 0.12a 3.6 10.7 a 1.17 0.02 3.6 10.6 <10 CFU/mL 3.6 10.6

ab Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

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general, the values of viscosity, Brix, and turbidity remained fairly unchanged in the model juice containing vitamin B6, vitamin B3, vitamin B2, and vitamin E, whereas that containing vitamin C showed a steady decrease in the Brix value with an average of 8.6, suggesting that probiotic bacteria utilized the sugar present in the model juice. The viscosity and turbidity values in the model juice containing vitamin C also increased with average values of
Table 5Inuence of niacin (vitamin B3) on the survival of probiotic bacteria in fruit juices. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log CFU/mL 8.24 0.32a 7.67 0.11a 6.43 0.15a 4.24 0.15a 3.56 0.34a 1.21 0.15a <10 CFU/mL pH

1.10 cP and 0.23 absorbance. Interestingly, vitamin E being a lipid soluble vitamin did not mix well with the components of model juice, thus its antioxidant potential may not have been fully utilized by the bacteria. Previous studies have shown that vitamin C can be benecial to probiotic survival during storage, presumably because it is an oxygen scavenger, thus promoting a more favorable anaerobic environment (Teixeira and others 1995; Dave and Shah
Table 7Inuence of ascorbic acid (vitamin C) on the survival of probiotic bacteria in fruit juices. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log CFU/mL 8.24 0.31a 8.37 0.15a 8.46 0.64a 7.68 0.64a 7.12 0.52a 6.51 0.55a 6.13 0.16a 8.32 0.34a 8.72 0.26a 8.35 0.15a 7.21 0.16a 7.03 0.48a 6.57 0.14a 5.81 0.11a 8.41 0.66a 8.75 0.04a 8.94 0.04a 8.61 0.08a 8.13 0.11a 7.64 0.54a 7.16 0.35a pH

Brix

Viscosity (cP) 1.08 1.08 1.09 1.09 1.09 1.09 1.09 1.08 1.08 1.09 1.09 10.9 1.09 1.09 1.08 1.08 1.09 1.09 1.09 1.09 1.09

Turbidity at 600 nm 0.00 0.07 0.06 0.07 0.07 0.08 0.08 0.00 0.07 0.07 0.07 0.07 0.08 0.08 0.00 0.08 0.08 0.07 0.08 0.09 0.09

Brix

Viscosity (cP) 1.08 1.08 1.10 1.10 1.10 1.11 1.11 1.08 1.08 1.10 1.11 1.11 1.11 1.10 1.08 1.08 1.10 1.11 1.11 1.11 1.11

Turbidity at 600 nm 0.00 0.17 0.27 0.28 0.27 0.30 0.30 0.00 0.09 0.16 0.17 0.11 0.24 0.22 0.00 0.11 0.13 0.15 0.27 0.22 0.23

HOWARU L. rhamnosus 3.8 11.8 3.7 11.6 3.7 11.6 3.5 11.4 3.5 11.3 3.4 11.3 3.4 11.3 HOWARU B. lactis 8.32 0.10a 3.8 11.8 a 7.12 0.35 3.7 11.6 6.41 0.11a 3.6 11.2 4.14 0.04a 3.6 11.2 2.11 0.09a 3.6 11.1 a 1.03 0.25 3.5 11.0 <10 CFU/mL 3.5 11.0 L. paracasei Lpc-37 8.41 0.22a 3.8 11.8 7.71 0.14a 3.7 11.6 a 6.53 0.04 3.6 11.2 5.21 0.07a 3.6 10.7 3.21 0.63a 3.5 10.5 3.5 10.5 1.27 0.14a <10 CFU/mL 3.5 10.5

HOWARU L. rhamnosus 3.8 11.8 3.6 10.2 3.6 9.7 3.5 8.9 3.5 8.7 3.5 8.7 3.5 8.6 HOWARU B. lactis 3.8 11.8 3.6 10.1 3.5 9.7 3.5 8.8 3.5 8.7 3.4 8.7 3.4 8.6 L. paracasei Lpc-37 3.8 11.8 3.6 11.0 3.6 10.6 3.5 9.9 3.5 9.9 3.4 9.7 3.3 9.7

a Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

a Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

Table 6Inuence of pyridoxine (vitamin B6) on the viability of probiotic bacteria in a model fruit juice. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log CFU/mL 8.24 0.26a 5.32 0.16a 2.12 0.65a <10 CFU/mL <10 CFU/mL <10 CFU/mL <10 CFU/mL pH

Table 8 Inuence of alpha-tocopherol (vitamin E) on the survival of probiotic bacteria in fruit juices. Week 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log CFU/mL 8.24 0.04a 7.67 0.66a 6.43 0.23a 4.24 0.23a 3.56 0.36a 1.21 0.07a <10 CFU/mL pH

Brix

Viscosity (cP) 1.03 1.03 1.04 1.04 1.04 1.04 1.04 1.03 1.03 1.04 1.05 1.05 1.05 1.05 1.03 1.03 1.04 1.04 1.04 1.04 1.04

Turbidity at 600 nm 0.00 0.07 0.06 0.09 0.11 0.11 0.11 0.00 0.09 0.10 0.10 0.10 0.10 0.11 0.00 0.07 0.06 0.07 0.07 0.06 0.06

Brix

Viscosity (cP) 1.08 1.08 1.09 1.09 1.09 1.09 1.09 1.08 1.08 1.09 1.09 10.9 1.09 1.09 1.08 1.08 1.09 1.09 1.09 1.09 10.9

Turbidity at 600 nm 0.00 0.04 0.04 0.03 0.06 0.06 0.06 0.00 0.07 0.06 0.06 0.08 0.08 0.08 0.00 0.07 0.06 0.07 0.07 0.08 0.10

HOWARU L. rhamnosus 3.8 11.8 3.8 11.0 3.8 10.1 3.7 9.5 3.7 9.5 3.7 9.5 3.7 9.5 HOWARU B. lactis 8.32 0.16a 3.8 11.8 4.31 0.16a 3.8 11.0 1.98 0.41a 3.7 10.1 <10 CFU/mL 3.7 9.5 <10 CFU/mL 3.7 9.5 <10 CFU/mL 3.7 9.5 <10 CFU/mL 3.7 9.5 L. paracasei Lpc-37 8.41 0.15a 3.8 11.8 5.32 0.26a 3.8 11.0 2.12 0.64a 3.8 10.1 1.21 0.21a 3.7 9.5 <10 CFU/mL 3.7 9.5 <10 CFU/mL 3.7 9.5 <10 CFU/mL 3.7 9.5

HOWARU L. rhamnosus 3.8 11.8 3.7 11.6 3.7 11.6 3.5 11.4 3.5 11.3 3.4 11.3 3.4 11.3 HOWARU B. lactis 8.32 0.23a 3.8 11.8 7.12 0.20a 3.7 11.6 6.41 0.54a 3.6 11.2 a 4.14 0.61 3.6 11.2 2.11 0.11a 3.6 11.1 1.03 0.43a 3.5 11.0 <10 CFU/mL 3.5 11.0 L. paracasei Lpc-37 8.41 0.21a 3.8 11.8 7.71 0.21a 3.7 11.6 6.53 0.03a 3.6 11.2 5.21 0.07a 3.6 10.7 3.21 0.27a 3.5 10.5 a 1.27 0.63 3.5 10.5 <10 CFU/mL 3.5 10.5

a Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

a Means in the same column followed by different lowercase letters are signicantly different (P < 0.05).

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1997). Overall all 3 probiotic strains tested showed similar declines in viability, indicating that none of the strains tested are better suited to the harsh acidic environment of the model fruit juice. There is no information on the role of vitamins in improving the survival of probiotics, as these supplements are used to improve the health image of the product, not necessarily for improving survival of probiotics.
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Conclusion
Fruit juice has a relatively short shelf life and ingredients such as green tea extract, vitamin C, and grape seed extract, which contain high levels of antioxidants and have oxygen-scavenging properties, may promote a more favorable environment for probiotic bacteria and may prove useful in developing a new and novel fruit juice based functional foods.

Acknowledgments
We would like to thank Danisco for providing the probiotic bacteria and ingredients used in this study. We also give our thanks to Dr. Greg Leyer and Mary Jane Fallourd for their support in this research.

References
Dave RI, Shah NP. 1997. Effectiveness of ascorbic acid as oxygen scavenger in improving viability of probiotic bacteria in yogurts made with commercial starter cultures. Int Dairy J 7:43543.

M: Food Microbiology & Safety

M282 Journal of Food Science r Vol. 75, Nr. 5, 2010