FACETS OF PROJECT ON PROBIOTIC YOGURT TO COMBAT LISTERIOSIS AND CANDIASIS

1. Viability of Bifidobacterium species in probiotic yoghurt as influenced by reducing agents.

The consumption of probiotic yogurt containing Bifidobacterium species could positively influence a person’s health if the viability of the probiotic species is above a threshold of 10⁸ CFU/g in yoghurt. To meet this threshold, the viability of the chosen Bifidobacterium species, namely B. animalis and B. bifidum, needs to be maintained during the shelf-life period of the yogurt. High oxygen levels threaten the viability; thus reducing the amount of oxygen present in yogurt by incorporating reducing agents should assist in maintaining viability. The most suitable reducing agent for bio-yogurt was found to be ascorbic acid that reacts with molecular oxygen and converts it to water. This lowers the oxygen levels and improves the viability of the Bifidobacterium species to the required levels, which should allow consumers to experience health benefits when consuming such a probiotic yogurt.

2. Bifidobacterium species in synbiotic yoghurt: viability and health benefits.

Studies have shown that adequate consumption of bifidobacteria in yoghurt offer many health benefits to humans including antimicrobial activity, preventing gastrointestinal infections, colon cancer and reducing serum cholesterol. However, as mentioned above, the concentration of active probiotic bacteria should be at least 10⁸ CFU/g viable cells in yoghurt before consumption to exert the health benefits to the host. Often, though, there is not an adequate amount of viable bifidobacteria present in yoghurt products before consumption. The viability of bifidobacteria in yoghurt is affected by factors in yoghurt processing, the yoghurt environment and as yoghurt passes through the gastrointestinal tract. Below a pH of 5, Bifidobacterium spp. growth slows down, but yoghurt must have a pH of <4.5 to meet legal requirements. The acidic conditions of the stomach and bile in the intestine also decrease bifidobacteria viability. As anaerobes, oxygen is another major factor affecting viability.

Approaches to improve Bifidobacterium spp. viability in yoghurt include prebiotic addition to form synbiotic yoghurt and pre-adaptation to stress before addition to yoghurt during manufacture. Prebiotics such as lactulose, inulin and oligosaccharides are consumed by probiotics to enhance their growth. When bifidobacteria are exposed to stress, they develop protective responses, which can assist with a greater tolerance to the stresses once in the yoghurt environment, thereby enhancing their viability. It is postulated that although the viability of bifidobacteria is affected by many factors, their viability in yoghurt should be enhanced through prebiotic addition and stress adaptation, which should allow the bifidobacteria in yoghurt to remain viable.

3. Oxidative stress adaptation of Bifidobacterium spp.

Oxygen is readily incorporated during yoghurt production and results in toxic and damaging reactive oxygen species that affect the viability of incorporated bifidobacteria cells. A sub-lethal oxidative stress treatment of H₂O₂ (0.01 mM; 30 min) of three Bifidobacterium spp. (B. bifidum, B. breve and B. animalis), followed by a lethal oxidative stress treatment of H₂O₂ (1 mM; 30 min) was done to induce aero-tolerance and the development of oxidative stress-adapted variants. Flow cytometric measurements were carried out on stress-adapted variants before and after exposure to lethal stress (1 mM; 30min) using SYTO 9 and Propidium Iodide in a single-parameter analysis of membrane integrity, as well as CellROX Green and Propidium iodide in a multi-parameter analysis of membrane integrity and oxidative cell state. Stress-adapted variants of B. animalis were able to retain membrane integrity under the oxidative state, indicating effective stress-adaptation. This is likely due to a genetic predisposition, with the microorganism containing the necessary mechanisms to effectively detoxify H₂O₂. Genetic expression of detoxification enzymes will be confirmed through qPCR. In future studies, survival within a yoghurt environment of stress-adapted strains will be investigated.

4. Co-culturing and point of inoculation influence yoghurt fermentation, physicochemical properties, and probiotic viability.

As implicated above, the incorporation of probiotics in yoghurt remains technologically challenging due to viability decrease during storage. In yoghurts made with cultures of Lacticaseibacillus rhamnosus strains and Bifidobacterium bifidum, the probiotic viability, fermentation kinetics, and physicochemical properties of the yoghurt were influenced by the point of inoculation and co-culturing. Post-fermentation inoculation of the probiotics resulted in lower viability at the beginning of storage. However, the viable counts were able to stabilize during storage to levels above minimum therapeutic limits (≥7 log CFU/g). When inoculated post-fermentation, probiotic strains probably experience acid shock and require time to adapt to the low pH. In contrast, pre-fermentation inoculation results in a gradual adaptation to the decreasing pH during fermentation. Regardless of co-culturing, fermentation was quicker in yoghurts incorporating L. rhamnosus leading to products with higher acidity and increased firmness. This influence of L. rhamnosus on yoghurt quality could be due to the additional utilization of galactose, which leads to increased acidity that promotes casein micelle aggregation that increases firmness.                                                                                        

In conclusion, inoculation of probiotics pre-fermentation is a better technique for retaining their viability during storage. Probiotic co-culturing influences yoghurt fermentation and physicochemical properties, especially in the presence of L. rhamnosus strains. Due to limitations in the method of quantification, there was insufficient evidence to conclude the effect of co-culturing on probiotic viability, particularly on B. bifidum. Therefore, an alternative method of quantification based on molecular techniques will be essential.