PROBIOTIC YOGHURT WITH POTENTIAL ANTI-CANDIDAL AND ANTI-BACTERIAL ACTIVITY – THE 2022 PROJECT PROGRESS REPORT.

General aim of the project: To develop an acceptable probiotic yoghurt product containing selected probiotic strains with the potential to prevent candidiasis, listeriosis and diarrhoea.

Goals in 2022:

1. To evaluate the effect of the probiotic yoghurt on the fate of Listeria monocytogenes under simulated gastrointestinal conditions.
2. To determine the effect of yoghurt processing technology (adaptation and high-pressure homogenization) in enhancing probiotic viability.
3. To develop a qPCR-based method for the enumeration of probiotics in mixed-strain yoghurt.

Results:

Probiotic cell biomass preparation: In previous trials, the initial cell concentration at inoculation was 8 log₁₀ colony forming units (cfu)/ml for Lacticaseibacillus rhamnosus and 6-7 log₁₀ cfu/ml for Bifidobacterium. By increasing culture volume, centrifugation and re-suspending the cell mass in 10 ml sterilized milk, the cell concentrations of the probiotics were increased to 10 log₁₀ cfu/ml and 9 log₁₀ cfu/ml for L. rhamnosus and B. bifidum respectively, which was highly satisfactory.

Pre- and post-fermentation addition of probiotics: Following the improved probiotic cell biomass preparation, L. rhamnosus and B. bifidum (co-cultures) were inoculated into the yoghurt fermentation at 10.0 log₁₀CFU/ml and 9.0 log₁₀CFU/ml cell respectively. The high cell concentration seemed to have a positive impact on the viability of the probiotics when added into the milk pre-fermentation. L. rhamnosus cell concentrations remained above 8 log₁₀ cfu/ml, while B. bifidum cell counts in the yoghurt remained constant at 7-8 log₁₀ cfu/ml after 14 days of storage. However, when added into the yoghurt post-fermentation, L. rhamnosus, cell counts dropped to 6 - 7 log₁₀CFU/ml immediately after addition and the B. bifidum counts to log 7 log₁₀CFU/ml. This decrease could be due to acid shock by the low yoghurt pH that induces cell death and injury on some probiotic cells at inoculation. However, after storage of the yoghurt for 14 days, the cell counts of both L. rhamnosus and B. bifidum increased again by about 1 log₁₀CFU/ml.

Probiotic viability optimization by use of reducing agents: In further experiments to improve the viability of Bifidobacterium species, reducing agents were used that are compatible with the yoghurt system. Reducing agents can act as oxygen quenchers, thereby potentially reducing the toxic effects of dissolved oxygen, which is one of the main factors that negatively influence Bifidobacterium viability. A total of 10 yoghurt preparations consisting of six preparations incorporating ascorbic acid (0 g/L 0.25 g/L and 0.5 g/L) and four preparations incorporating L-cysteine (0 g/L and 0.5 g/L) were inoculated with a cell density of 8 Log₁₀CFU/ml of B. bifidum and 9 Log₁₀CFU/ml of B. animalis before fermentation. The fermentation in the yoghurt incorporating ascorbic acid was not different from the control and achieved pH 4.5 in 4 - 5 hours. However, the fermentation in yoghurt samples incorporating L-cysteine was significantly slower, reaching a pH of 5.2-5.4 after 7 hours. Although L-cysteine is a good reducing agent that help preserve Bifidobacterium viability, it seemed that it had a negative effect on the starter culture (especially Streptococcus thermophilus), which could have caused the fermentation to be slow. Due to this negative effect, L-cysteine was not investigated further.

In the yoghurt incorporating ascorbic acid, a significant effect of the ascorbic acid (at 0.5 g/L) was observed in the viability of B. animalis at day 0 (Log 8.0 CFU/g compared to the control at Log 6.0 CFU/g). However, this effect could not be sustained for the duration of the 28-day shelf-life as the ascorbic acid content showed a progressive decline in the yoghurt. With respect to B. bifidum, ascorbic acid had no significant effect on viability as the viability remained at 7-8 Log10 CFU/g. In addition to Bifidobacterium viability, the effect of ascorbic acid on other physicochemical qualities of the yoghurt was also investigated. A significant increase in the firmness was observed in the yoghurt containing 0.5 g/L after 28 days of storage, whereas other variables, such as the water holding capacity and titratable acidity, were not significantly affected.

Oxidative stress pre-adaptation: Another approach to potentially improve the viability of Bifidobacterium species is oxidative stress pre-adaptation. This is a strategy to enhance tolerance to oxygen that is often inadvertently incorporated into yoghurt during processing. Three Bifidobacterium spp (B. bifidum, B. breve and B. animalis subspp. animalis) were subjected to a sub-lethal stress treatment of H₂O₂ (0.01 mM; 30 min) followed by a lethal stress treatment of H₂O₂ (1 mM; 30 min). Survivors from the first lethal stress treatment were subjected to three successive generations of exposure to H₂O₂ to isolate oxidative stress-adapted variants. While the stress-adapted variants of B. bifidum, B. breve retained viability, >90% of the cells were in a state of injury with partly damaged membranes. On the other hand, adapted B. animalis cells retained >70% intact membranes after the lethal stress challenge. With regard to the oxidative state of the cells, the variants of all three species were able to sustain a high level of oxidation (50% for B. bifidum, B. breve and >70% for B. animalis) without losing their intact membranes. Overall, the stress-adapted variants of B. animalis produced the most resilient response under oxidative stress challenge as shown by a better ability to retain membrane integrity under an oxidised state, indicating a potentially inducible effective oxidative stress response in this organism.

Re-evaluating the effect of prebiotic inulin with additional Bifidobacterium species: Yoghurts were prepared with B. animalis, B. bifidum and B. breve and the prebiotic inulin. The effects of the probiotic species, prebiotic addition and storage time on probiotic viability, pH, titratable acidity, oxidation-reduction potential (ORP), syneresis and texture were examined over a 28-day shelf-life period. Among the three Bifidobacterium spp., B. animalis was the only organism that retained viability >10⁷ CFU/g throughout storage, whereas the viability of B. bifidum and B. breve progressively declined during storage and could not be enumerated at day 28. Among the physico-chemical parameters, there were an increase in ORP and titratable acidity and a decline in pH in all yoghurt samples during storage while the texture (firmness and cohesiveness) and gel properties (syneresis) remained stable regardless of the addition of inulin or the probiotic species. In addition, inulin did not affect probiotic viability and the performance of the starter culture. Overall, the study showed that the viability of probiotic Bifidobacterium spp. in yoghurt is dependent on species. The higher survival ability of B. animalis is probably related to its ability to survive the acid and oxidative stress associated with yoghurt as its counts are not correlated to pH and ORP changes. On the other hand, the poor viability of B. bifidum was highly correlated to reductions in pH and increases in ORP. Thus, B. animalis is a superior probiotic and can remain above the therapeutic limit throughout the yoghurt shelf life.

Development of qPCR-based probiotic enumeration protocol: The previous studies had shown that the usual culture-based methods for the enumeration of mixed-strain probiotics are inadequate. In particular, the inability of these methods to selectively enumerate Bifidobacterium spp. and Lactobacillus bulgaricus in yoghurt with Lacticaseibacillus rhamnosus were observed. Hence, the focus shifted to the development of propidium monoazide (PMA)-qPCR, based on amplification of the elongation factor Tu (tuf) gene. The tuf gene is one of the housekeeping genes and encodes the elongation factor Tu which is responsible for catalyzing the binding of aminoacyl-tRNA to the ribosome during protein biosynthesis. Unlike other commonly used housekeeping genes used for identifying lactic acid bacteria, the tuf gene occurs as a single copy in a bacterial genome.                                 

Tuf gene-specific primers for Bifidobacterium spp, Lacticaseibacillus spp, and Lactobacillus delbrueckii were designed, using primer design online tools, while Streptococcus thermophilus-specific primers were obtained from the literature. The specificity of these primers was tested, using qPCR against commonly used LAB in dairy fermented products such as Lacticaseibacillus rhamnosus, L. delbrueckii subsp. bulgaricus, Bifidobacterium breve, Bifidobacterium bifidum, Lactiplantibacillus plantarum, Limosilactobacillus fermentum and S. thermophilus. The results showed that these primers can only amplify the tuf gene of the target organisms. The work now focuses on optimizing the propidium monoazide concentration that can effectively inhibit the amplification of DNA from dead cells. Propidium monoazide (PMA) is a DNA intercalating dye that works based on membrane integrity. It can only penetrate the membrane of dead or damaged cells and binds to the DNA irreversibly when exposed to high-intensity light. During PMA-qPCR only DNA from intact membrane cells is amplified. qPCR alone is unable to distinguish between viable and non-viable cells. Hence, PMA-qPCR is a viable qPCR method that fulfils the probiotic definition and requirements by quantifying only viable cells.