Evaluation of the growth and survival of probiotic microorganisms in commercial bio-yogurt
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A review of the literature highlighting the importance of the 'therapeutic minimum' and the survival of the probiotic bacteria in fermented milk bioproducts is given in Chapter 2. Special reference is made to the historical background of probiotics, its therapeutic value and the survival through passage in the gastrointestinal tract. In addition, technology of bio-yogurt, factors affecting the survival of probiotic bacteria in yogurt, and the media for the differential enumeration of these microorganisms in dairy products are discussed. In Chapter 3 existing media proposed for the selective enumeration of starter cultures employed in the manufacture of bio-yogurt are compared and evaluated. It is essential for comparison reasons to standardize enumeration methods for microbial analyses in order to study the incidence of the probiotic bacteria in the presence of the conventional starter cultures. The media proposed by Chr. Hansen's laboratory proved to be the most suitable for the enumeration of the different cultures. It is essential that bio-yogurts meet the criteria of a minimum of 106 cfu/rnl of probiotic bacteria until the expiry date to induce any potential therapeutic advantages for the consumer. Consequently, in Chapter 4 we evaluated samples of AB-yogurt obtained from supermarket outlets statistically based on the enumeration of viable probiotic cultures, Lactobacillus acidophilus and Bifidobacterium bifidum; as well as conventional yogurt starter cultures, Streptococcus thermophilus and Lactobacillus bulgaricus and the maintenance with respect to the 'therapeutic minimum'. Based on the data obtained, the AB-yogurts examined comply with the criteria regarding the number of viable cells of L. acidophilus, but the consumer would not have received sufficient numbers of B. bifidum cells at the time of consumption. In Chapter 5, we monitored the survival of viable cells of the probiotic cultures and starter cultures present in bio-yogurt at frequent intervals from day 1 until the expiry date at day 31 stored at 4eC and loec. B.bifidum never exceeded counts of 106 cfuZgin any of the samples and a constant decline in its numbers was observed. L. acidophilus, despite maintaining counts higher than 106 cfu/ g in the yogurt samples, also exhibited a substantial decrease in its numbers during storage. Due to the poor survival of probiotic cultures in yogurt, we incorporated a probiotic yeast species, S. boulardii as part of the starter culture in Chapter 6 and monitored its progression and survival in yogurt and milk products. Despite good growth and the survival of the yeast species until the expiry date, excessive gas and alcohol production proved, however, to be major constraints. In order to further study the effect of yeast growth on the survival of probiotic bacteria in bio-yogurt, pure cultures of Kluyveromyces marxianus, Issatchenkia orienialis, Debaryomyces harisenii and Yarrotoia lipolytica were inoculated into commercial AB-yogurt, sterile milk and pasteurised sweetened yogurt in Chapter 7. The yeast species were able to progress in the bio-yogurt reaching maximum counts exceeding 107 cfu/ g. Despite the inability of some species to utilise lactose, the yeast species utilised available organic acids, galactose and glucose derived from bacterial metabolism of the milk lactose, as well as possible free fatty acids or free amino acids present in the dairy products and thereby sufficiently contributed to the retention or enhancing of the pH values. The production of excessive gas and alcohol was major constraints in implementing Kluyveromyces marxianus, Issatchenkia orienialis. The inclusion of Y. lipolytica and D.hansenii in AB-yogurts, therefore, seemed the most promising in controlling the pH to assure the viability of the pro-biotic microorganisms. In Chapter 8, Y. lipolytica and D.hansenii cultures were inoculated into commercial plain and fruit AB-yogurt at moderate (105 -106 cfujml) and low level (10² - 10³ cfujml), directly after manufacture and with the ABT-starter before fermentation, to compare the effects that the yeast will have on viability of probiotic bacteria. Viable bifidobacteria counts remained virtually the same in the bio-yogurt inoculated with the yeast cultures during the refrigerated storage period. A rapid decrease in L. acidophilus occurred after 2 weeks storage (2-4 log cycles) in the yeast-inoculated bio-yogurt suggesting possible antagonistic action of the yeast against L. acidophilus. Addition of the yeast primarily encouraged the growth of streptococci, which had an influence on the pH of the yogurt environment. A gradual decrease in the pH of all the bioyogurt products was observed. pH was affected by enhanced growth of streptococci, utilization of organic acids by the yeasts and the fact that L. bulgaricus was excluded form the yogurt starter culture. The yogurt inoculated with D. hanserui was still acceptable and had a pleasant taste compared to the control yogurt after 30 days storage. Inclusion of yeast as part of the starter culture for bio-yogurts seems promising. In Chapter 9, possible enhancement of the growth and survival of Bifidobacteria in bio-yogurt by the addition of a prebiotic was investigated. Commercial AB-yogurt was fortified with 1, 2 and 3% of the fructooligosaccharide, neokestose, and growth and survival of bifidobacteria as well as L.acidophilus and S. thermophilus were monitored during storage at SoC over 30 days. With the addition of neokestose the viable bifidobacteria count remained significantly higher in all the yogurts when compared to traditional yogurts without the addition of neokestose. L. acidophilus reduction in viable counts did not exceed a 30% reduction; therefore neokestose also had a better survival effect on L. acidophilus. The addition of neokestose had no affect on the survival of S. thermophilus.