Journal of the Science of Food and griculture J Sci Food gric 5:2134 2141 (2005) DOI:.02/jsfa.2223 pplication of pure and mixed probiotic lactic acid bacteria and yeast cultures for oat fermentation ngel ngelov, 1 Velitchka Gotcheva, 1 Tsonka Hristozova 2 and Stoyanka Gargova 1 1 Department of iotechnology, University of Food Technologies, 2 Maritza lvd, 4002 Plovdiv, ulgaria 2 Institute of Microbiology, ulgarian cademy of Sciences, 2 Maritza lvd, 4002 Plovdiv, ulgaria bstract: Fermentation of a prebiotic containing oat substrate with probiotic lactic acid bacteria and yeast strains is an intriguing approach for the development of new synbiotic functional products. This approach was applied in the present work by using pure and mixed microbial cultures to ferment a heat-treated oat mash. Results show that the strains studied were appropriate for oat fermentation and the process could be completed for h depending on the strain. The viable cell counts achieved within this time were above the required levels of 7 cfu ml 1 for probiotic products. oth single lactic acid bacteria strains and mixed cultures of the same strains with yeast were found suitable for oat fermentation. However, the pure L cultures attributed better flavour and shelf life of the oat drinks. The content of the prebiotic oat component beta-glucan remained within 0.30 0.3% during fermentation and storage of the drinks obtained with each of the strains used. Thus, these products would contribute diet with the valuable functional properties of beta-glucan. lso, the viability of pure and mixed cultures in the oat products was good: levels of cell counts remained above the required numbers for probiotic products throughout the estimated shelf-life period. 2005 Society of Chemical Industry Keywords: oat fermentation; probiotics; beta-glucan; lactic acid bacteria; yeasts bbreviations L lactic acid bacteria GIT gastro-intestinal tract T titratable acidity LDL low-density lipoprotein cfu colony forming units v/v volume/volume w/v weight/volume L Lactobacillus C Candida INTRODUCTION The application of probiotic microbial strains for fermentation of cereal substrates is a rational approach for the development of functional foods, which, as part of the daily food intake, contribute to enhancing and maintaining consumers health and to improving their quality of life. The beneficial effects of foods containing probiotic bacteria on human health include improved food digestibility and bioavailability of vitamins and minerals, improved lactose metabolism, control of gastro-intestinal infections, reduction of some liver and kidney catabolic products, cancer suppression, reduction of serum cholesterol levels and immune stimulation. 1 For the efficacy of probiotic foods, high levels of viable microorganisms are recommended: 7 cfu ml 1 (g). Technological challenges associated with the introduction and maintenance of high numbers of probiotic microorganisms in foods include the form of the probiotic inoculant, ability of the probiotic culture to retain viability in the environment of the food matrix, and maintenance of the probiotic characteristics in the food product through the time of consumption. 7 Given that most probiotics worldwide are applied in dairy products, many of them are unsuitable to grow in cereal-based media. number of studies have shown that the survival of probiotic strains under the gastrointestinal conditions is strongly effected by the nature of the food carrier: its buffering capacity, ph, chemical and physical characteristics.,9 Cereals have proved to be appropriate substrates for lactic acid fermentation, and the use of probiotic strains as starter cultures could result into probiotic products, which are suitable for vegetarians, avoid lactose-intolerance problems and are a tasty contribution to the functional food family. Correspondence to: ssociate Professor Dr ngel ngelov, Department of iotechnology, University of Food Technologies, 2 Maritza lvd, 4002 Plovdiv, ulgaria E-mail: angelov@uft-bio.com Contract/grant sponsor: ulgarian Ministry of Education and Science; contract/grant number: CC 04/2000 (Received 13 July 2004; revised version received 7 December 2004; accepted 27 January 2005) Published online 7 June 2005 2005 Society of Chemical Industry. J Sci Food gric 0022 5142/2005/$30.00 2134
Fermentation of oats with probiotic cultures Implementation of probiotic strains into a cerealbased medium may also contribute to enhance their acid and bile resistance, as shown in previous studies with lactobacilli. It was proved that the bile tolerance of Lactobacillus reuteri, L acidophilus and L plantarum was improved by he addition of cereal extracts to the test-medium, and the extent of improvement was related to the concentration of soluble sugars and free amino nitrogen present in the cereals. Other studies show also that gelatinized starch granules are a suitable matrix for delivering probiotics into GIT, due to their ability to protect the viable microbial cells from the unfavourable conditions in the stomach and the small intestines. 11 Cereals are a major source of fibres in the diet, and the main active component of cereal fibres is betaglucan. Numerous scientific studies demonstrated the hypocholesterolaemic effect of this compound, bringing a 20 30% reduction of LDL-cholesterol. The overall effect is reduction of cardiovascular 14 disease risk. eta-glucan is also considered a prebiotic as it can support the growth of some beneficial bacteria in the colon. 13 The cereals with highest beta-glucan content are oats and barley. 15,1 ecause of its nutritional value, a few researchers have focused their studies on oats as a substrate for lactic acid fermentation. Swedish company has developed enzymatically treated oat bases davena and Martensson et al 17,1 applied them as substrates for lactic acid fermentation with dairy starter cultures. nother yogurt-like oat product, Yosa, is at the market in Finland. Marklinder and Lonner, 19 as well as Johansson et al, 20 have reported that, after appropriate processing, oats is a suitable substrate for fermentation with lactic acid bacteria. However, no information was found in the literature regarding application of yeast for oat fermentation, and no oat drinks were reported to be available at the market yet. The aim of the present work was to explore the possibility to ferment an oat substrate with probiotic lactic acid bacteria and yeast strains in order to select an appropriate starter culture for development of a synbiotic oat drink. For this purpose, the fermentation process after inoculation of the oat substrate with pure lactic acid bacteria and yeast strains or combinations of them was studied, and the viability of the cultures during storage of the fermented oat products was observed. MTERILS ND METHODS Starter cultures The probiotic strains used throughout the study were Lactobacillus plantarum 2, L casei spp paracasei 29, Candida rugosa Y2 and C lambica Y30. The strains were isolated from a cereal-based fermented drink and selected as probiotics in previous studies. 21,22 Yeast strains were maintained on MYGP-agar (Difco, US), and lactic acid bacteria (L) strains on MRS-agar (Merck, Germany). Starter cultures were obtained by overnight incubation at 30 C in MYGP and at 37 C in MRS-broth, respectively. The cultures were centrifuged (4500 g, min,4 C), washed in distilled water and re-suspended in distilled water to the original volume of the cultures. Oat substrate The oat mash used as a substrate was prepared from whole-grain oat flour ( Mina Prim, Welfare, Ltd, ulgaria) and tap water at 5.5% (w/v). 1.5% (w/v) food grade sucrose was added to the slurry. It was then heated at 95 C for min and cooled to 37 C. Fermentation and storage The oat mash was inoculated with 5% (v/v) pure cultures of strains L plantarum 2, L casei spp paracasei 29, Candida rugosa Y2 and ClambicaY30, or mixed cultures of the following combinations: Combination 1 2 + Y2; Combination 2 29 + Y2; Combination 3 2 + 29 + Y2. The cell count levels of the yeast cultures applied were 9 cfu ml 1, and those of the L cultures were of 11 cfu ml 1. Mixed cultures were formed of equal portions of the strains included to reach a total content of 5% (v/v) of the starter culture. Fermentation was carried out at 37 C for h. Storage observations were carried outat4 C for 24 days. Cell counts Samples of 1ml were submitted to decimal dilutions in sterile peptone water and agar plates were performed to assess cell counts. MYGP-agar was used for pure yeast cultures, and MRS-agar for L counts. Cell counts of the mixed cultures were assessed by plating both on DRC-agar (Difco, US), which was selective for yeasts, and on MRS-agar, supplemented with 1% natamycin, which was selective for lactic acid bacteria. Determination of ph, T and dry matter Measurement of ph was carried out by a 420, ORION ph-meter, titratable acidity (T) was determined by titrating -ml samples, diluted with 50 ml of distilled water, with 0.1 N NaOH. Phenolphthalein was used as indicator and T was expressed as N (degrees Neuman), where 1 N equals 1ml 0.1N NaOH used for titration. Dry matter content was estimated in -ml samples by an express measurement device of UltraX (IK Werke, Staafen, Germany). Determination of beta-glucan eta-glucan content was evaluated by the enzymatic eta-glucan (Mixed Linkage) ssay Kit (K-GLU) (Megazyme, Wicklow, Ireland) after degradation with lichenase and beta-glucosidase. Statistical analysis Each experiment was performed four times. Results represent the means with standard deviations. Data J Sci Food gric 5:2134 2141 (2005) 2135
ngelovet al was submitted to one-way analysis of variance (NOV) with a least significant difference of 95%. RESULTS ND DISCUSSION Criteria for ending the oat fermentation were, on one hand, ph values between 4.0 and 4.5, which suppress the development of contaminating microflora and are acceptable in terms of organoleptic properties, and, on the other hand, organoleptically appropriate T values of up to 2.5 3.0 N, with an optimum around 1.5 N. 23,24 Therefore, the above values of ph and T were accepted as limiting for the duration of the fermentation process. pplication of pure cultures for oat fermentation Results from oat fermentation with pure cultures applied are presented in Fig 1. Most intensive acid formation was registered for strain L plantarum 2, with ph of the oat medium reaching values below 4.5 after h. When using strains Lcaseispp paracasei 29 and C rugosa Y2, ph values below 4.5 were registered after h, and for strain ClambicaY30 at the th hour of the fermentation. These results comply with those reported by Holzapfel, 24 who observed faster acid formation by strains L plantarum than with other Lactobacillus strains during fermentation of heatprocessed cereal substrates. When fermenting a heat-processed oat substrate supplemented with barley malt with pure cultures L plantarum and Lcasei, Marklinder and Lonner 19 registered ph values of 4.1 4. at the th hour of the process. Compared to these values, the lower acid formation observed throughout the present study might be attributed to the nature of the strains applied, as well as to the different composition of the oat substrates. The final ph values registered in the present work correspond with those reported by Martinez-naya et al 25 during fermentation of wheat dough with L plantarum. Titratable acidity of 1.5 N was reached at the th hour when using strain 2, and at the th hour when using strain 29 with initial values 0. and 0.7 N, respectively. When using the pure yeast strains C rugosa Y2 and ClambicaY30, T values of 1.5 and 1.4 N respectively were registered at the th hour (the initial T value for both strains was 0.4 N). The increase of T observed throughout the present study was statistically insignificant (p > 0.05). These results are much different than the observations of Hounhouigan et al. 2 The authors fermented a maize mash with pure cultures of L fermentum, L brevis and L salivarius, and similar levels of T were obtained after 24 h of fermentation. For the same period, when using yeast strains S cerevisiae and Ckrusei, they reported final T levels of 0.5 0.%, which are three times lower than the values estimated in the present work. The differences may be due to the different raw materials, as well as the specific fermentation properties of the different strains studied. The high cell counts of the starter cultures ensured high initial levels of cell counts in the oat medium: 7 cfu ml 1. t the end of the fermentation, highest increase of cell counts was obtained for strains L plantarum 2 and C rugosa Y2: 2.1 and 2.71 log.0 3.0 ph 5.5 5.0 4.5 4.0 T, N 2.0 1.0 3.5 0 2 4 0.0 0 2 4 2 29 Y2 Y30 C 0 2 4 Figure 1. Fermentation of heat-treated oat mash with pure cultures of lactic acid bacteria and yeasts. Changes of ph (), titratable acidity () and viable cell counts (C). 213 J Sci Food gric 5:2134 2141 (2005)
Fermentation of oats with probiotic cultures orders, respectively. When fermenting oat substrate with different L strains, Martensson 4 reported cell count increase of about 1. log orders. The lower increase in this case may be due to the differences of the oat substrate, fermenting properties of the strains used and/or the higher dry matter content of the substrate, which is about two times higher that the dry matter of oat mash prepared in the present study (data not presented). t the th hour, the cell counts of strains L plantarum 2 and L casei spp paracasei 29 were 9 and 4 cfu ml 1, respectively, and for the yeast strains 7 9 (Y2) and 2. 9 cfu ml 1 (Y30). These levels did not change during the next 2 h of fermentation, which shows that the fermentation time needed to achieve maximal cell count values should be h. The cell concentrations obtained are above the levels required for probiotic products ( 7 cfu ml 1 ), which would ensure a sufficient number of living cells reaching the colon in order to be effective. The effect of the L and yeast strains on the beta-glucan content of the oat substrate during fermentation was also investigated. Results are presented in Table 1. The content of beta-glucan in the oat mash before fermentation with pure L cultures and yeasts was within 0.31 0.3%. fter h of fermentation it was estimated that beta-glucan content in the oat mash has not changed significantly with any of the strains tested. These results confirm the fact, that most Lactobacillus species and yeasts do not ferment beta-glucan, which is known to be fermented by bifidobacteria mainly. 7 Martensson 4 observed the same when fermenting an oat medium with pure and mixed L cultures and decrease of beta-glucan concentration was found only when ifidobacterium bifidum was used. The maintained content of beta-glucan in the oat mash when applying Table 1. eta-glucan content before and after fermentation and storage of heat-treated oat mash with pure cultures of lactic acid bacteria and yeasts eta-glucan content (%) Fermentation Storage Strain 0 h h 0 days 24 days L plantarum 2 0.3 ± 0.03 0.34 ± 0.04 0.34 ± 0.04 0.31 ± 0.05 L casei spp paracasei 29 0.32 ± 0.0 0.2 ± 0.05 0.32 ± 0.02 0.34 ± 0.07 C rugosa Y2 0.34 ± 0.02 0.31 ± 0.02 0.31 ± 0.0 0.27 ± 0.04 C lambica Y30 0.31 ± 0.07 0.35 ± 0.0 0.35 ± 0.0 0.32 ± 0.03 Results represent the means and standard deviations of four independent measurements. 2.5 5 2.0 ph T, N 4 1.5 3 0 4 1 20 24 1.0 0 4 1 20 24 11 9 2 29 Y2 Y30 C 7 0 4 1 20 24 Figure 2. Refrigerated storage of the oat drink after fermentation with pure cultures of lactic acid bacteria and yeasts. Changes of ph (), titratable acidity () and viable cell counts (C). J Sci Food gric 5:2134 2141 (2005) 2137
ngelovet al the selected L and yeast strains for fermentation would precondition its functional effect as an LDLcholesterol-reducing and bifidogenic factor when the oat drinks are consumed. Results from the studies on viability of the probiotic strains throughout refrigerated storage of the fermented oat products are presented in Fig 2. Changes of ph for strains L plantarum 2 and L casei spp paracasei 29 were statistically insignificant (p > 0.05) throughout the observed storage period. Less ph decrease (0.1) was observed for strain C rugosa Y2, and about twice as much (0.3) for strain ClambicaY30. In general, none of the four strains applied produced a significant change of ph during the observed time. Changes of T remained statistically insignificant for 20 days storage. fter this time, significant increase (p < 0.05) of T was registered for strains 2 and 29. These results show that the appropriate storage period for the fermented oat products is 20 days. During the observed storage period, smaller changes of the viable cell counts were registered for the L strains: 0.77 log order for strain 2 and 0.0 log order for strain 29. For both strains, changes were insignificant. Cell counts of yeast strains decreased with 0. and 1.0 log orders for strain Y2 and strain Y30, respectively. It should be pointed out that for 24 days refrigerated storage the number of viable cells in the oat products remained above cfu ml 1. These levels should provide a sufficient number of viable cells in the colon in order to ensure the probiotic effect of the oat products. Results obtained correlate with those of Martensson et al, 17,1 who reported good survivability of L strains during refrigerated storage of fermented oat products. They found cell concentrations of cfu ml 1 on the 25th day, and of 5 cfu ml 1 on the 30th day of storage. During storage, beta-glucan content of the fermented oat products was also monitored. Results are presented in Table 1. Consistent with observations on beta-glucan during fermentation, storage studies show that its content remained unchanged within 0.27 0.35% in all oat products, fermented with the pure microbial cultures. These results confirm that beta-glucan is not fermented by the L and yeast strains applied for oat fermentation in the present work. Results from the study on the possibility of previously selected probiotic L and yeast strains to be used for fermentation of a heat treated oat mash show that all four of them could be used for development of synbiotic oat products. good balance between acid formation and cell counts was observed for strains L plantarum 2 and L casei spp paracasei 29. The estimated appropriate fermentation time for both strains was h at 37 C. Value differences of the parameters monitored during fermentations with either strain were not significantly different, which makes them equally appropriate for applications in fermented oat products. Of the yeast strains studied, C rugosa Y2 was chosen as more suitable for this purpose, as it showed better growth during fermentation and better viability during storage, compared with strain C lambica Y30. In addition, strain Y2 had more intensive acid formation and the fermentation process was complete for h, while for strain Y30 h were necessary. The study showed also that the heat-treated oat mash was an appropriate medium both for fermentation with the selected probiotic L and yeast strains, and for ensuring their good viability ( cfu ml 1 after 24 days) during refrigerated storage of the fermented products. On the basis of changes of ph, the shelf life of the oat drinks was estimated to be 20 days. It is also important that the content of the prebiotic oat component beta-glucan remained within 0.30 0.3% during fermentation and storage of the drinks obtained with each of the strains used. Thus, these products would contribute diet with the valuable functional properties of beta-glucan. pplication of mixed cultures for oat fermentation The next step of the study was to explore fermentation of the heat-treated oat mash with mixed cultures of the selected probiotic L and yeast strains for their potential application in functional food development. ph 5 4 Dry matter, % 5 4 3 0 2 4 2+Y2; 29+Y2; 2+29+Y2 3 0 2 4 2+Y2; 29+Y2; 2+29+Y2 Figure 3. Changes of ph () and dry matter content () throughout fermentation of heat-treated oat mash with mixed cultures of lactic acid bacteria and yeast. 213 J Sci Food gric 5:2134 2141 (2005)
Fermentation of oats with probiotic cultures Changes of ph and dry matter content during fermentations with the mixed cultures are presented in Fig 3. When mixed cultures were applied for oat fermentation, values of ph below 4.5 were reached after h. This fermentation time is 2 h less than the time necessary when pure cultures were used. The least change of ph was registered for Combination 1 (1.25), and the highest for Combination 2 (1.41). fter 24 h of fermentation of the maize product mawe with strains L brevis, L fermentum and L salivarius combined with either Saccharomyces cerevisiae or C krusei, Hounhouigan et al 2 registered ph values in the range 3.5 4.4. In our case, ph values within the same range were reached for a quarter of this time. similar study was carried out by Ketarpaul and Chauhan, 27 who fermented millet mash with yeast Saccharomyces diastaticus in combinations with Lbrevis and L fermentum. In this work, values of ph in the range 3.71 4.2 were reached after 144 h of fermentation. Differences of acid formation rates in all different cases may be attributed to the physiological properties of the strains used, to their ability to ferment the cereal-based media, as well as to the composition of the media. In the present study, the initial dry matter content of the medium is around 5.5%, which is lower than those in the other studies discussed above, 7% and % dry matter, respectively. During fermentation, dry matter content did not change significantly for neither of the mixed cultures used. Growth dynamics of the mixed cultures during fermentation is presented in Fig 4. The initial viable cell concentrations were around cfu ml 1 for each individual strain of the mixed cultures. The highest cell count increase, 3.9 log order, was registered for Combination 1, followed by Combination 3 (2.52 log order) and Combination 2 (1. log order). These results show that mixed cultures including strain L plantarum 2 are more suitable for growth in the heat-treated oat substrate. It was established that the increase of cell counts of the yeast strain C rugosa Y2 in all combinations was in the range 1.7 1.95 log orders. Differences between these values were not significant, which shows that there is no strain-specific effect of the L strains used on the growth of the yeast strain. Growth rates of 2 and 29 were not affected by the presence of the yeast strain in the mixed cultures and remained similar to those when L were used as pure cultures. fter h of fermentation, when appropriate ph values were reached, viable cell counts in the fermented oat products were above 9 cfu ml 1 for all culture combinations. These levels are above the required 7 cfu ml 1 for probiotic products, which shows that the oat products obtained conform to this requirement. pplication of the mixed cultures for oat fermentation resulted in products with different, yeasty compounds within the taste and flavour, compared with those obtained with pure L cultures. This is attributed to the presence of C rugosa Y2. Therefore, additional studies should be carried out in order to 0 2 4 2; Y2 0 2 4 29; Y2 C 0 2 4 2+29; Y2 Figure 4. Growth dynamics of mixed cultures of lactic acid bacteria and yeast during fermentation of heat-treated oat mash:, Combination 1;, Combination 2; C, Combination 3. J Sci Food gric 5:2134 2141 (2005) 2139
ngelovet al 0 4 1 20 24 0 4 1 20 24 29; Y2 2; Y2 C 0 4 1 20 24 2+29; Y2 Figure 5. Survivability of mixed cultures of lactic acid bacteria and yeast during refrigerated storage of fermented oat products., Combination 1;, Combination 2; C, Combination 3. find out the possibilities to balance the flavour profile of the products if mixed cultures are applied for fermentation. During development of probiotic products, it is essential that viable cell concentration is kept above cfu ml 1 during product storage. Shelf life and survivability study was carried out for 24 days refrigerated storage of oat products after h of fermentation with the three culture combinations. Results are presented in Fig 5 For the period monitored, the fastest decrease of ph was observed for the combination of all three strains, when shelf life of the product was estimated to be days. When only one L strain was combined with the yeast, ph of the oat products remained above 4.0 for 1 days (data not shown). These results bring the conclusion that in terms of shelf life, mixed cultures of one L and one yeast strain are more appropriate than the combination of the three strains. During the storage period, viable cell concentration of L in the products decreased with 0.5 1.17 log orders for all mixed cultures but remained above cfu ml 1, which would provide the cell numbers necessary for probiotic effect of the products within the estimated shelf life. For the yeast strain, similar decrease (0. 1.02 log orders) of cell counts was observed, but values at the end of storage were at levels of 7 cfu ml 1, which comply with the requirements for probiotic products. The study on application of mixed cultures for oat mash fermentation shows that fermentation is completed for a shorter time of h, compared with the h when pure cultures of the same strains were used. The more intensive acid formation is also the reason for 4 days shorter shelf life of the products obtained. The initial viable cell concentrations of the products after inoculation are above the required levels of cfu ml 1 and increase to about 9 cfu ml 1 during fermentation, which makes the oat products obtained suitable for probiotics delivery. dditional studies should be made for possibilities to mask the slightly unpleasant flavour formed by the yeast strain in the culture. Storage observations show that when combinations of two strains were used, product durability was better: 1 days, compared with days when a culture of three strains was applied for fermentation. Viability of all mixed cultures in the oat products was good; levels of cell counts remained above the required numbers for probiotic products throughout the estimated shelf-life period. In conclusion, the probiotic lactic acid bacteria and yeast strains studied proved to be appropriate for fermentation of heat-treated oat mash. The appropriate acidity was reached within h depending on the strains applied, and the viable cell counts achieved for that time were above the required levels of 7 cfu ml 1 for probiotic products. The oat mash is a suitable medium for fermentation of the strains used and this approach could be applied for the development of new synbiotic functional foods. Results show that both single L strains and mixed cultures of the same strains with yeast can be used for fermentation. However, the pure L cultures contributed better flavour and shelf life to the oat drinks. 2140 J Sci Food gric 5:2134 2141 (2005)
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