Viability assessment and bioactive metabolites profiling in yoghurt incorporating Lacticaseibacillus rhamnosus and Bifidobacterium spp. during shelf-life

The main aim of this study was to develop a probiotic yoghurt incorporating the co-cultures of Lacticaseibacillus rhamnosus GG and Bifidobacterium spp. with improved viability sustenance and functionality during shelf life. The first phase of this study investigated the effect of pre- and post-fermentation inoculation and co-culturing on the viability of Bifidobacterium spp. and L. rhamnosus in yoghurt. The viability was determined using culture-based techniques on various selective media. Probiotic inoculation post-fermentation could not improve the viability of Bifidobacterium spp. and L. rhamnosus strains in mixed-probiotic species yoghurts. The standardised selective culture media namely, de Man, Rogosa and Sharpe (MRS)-pH 5.4 and MRS-NNLP were not selective against L. rhamnosus strains and thus, resulted in overestimation of Lactobacillus delbrueckii subsp. bulgaricus and Bifidobacterium spp. viability respectively. In the second phase of this study, a quantitative PCR (qPCR) method coupled with propidium monoazide (PMAxx), and novel species-specific tuf gene primers was developed to selectively enumerate L. rhamnosus, Bifidobacterium spp., and yoghurt starter cultures in mixed-species probiotic yoghurt. The method was optimised for PMAxx concentration and specificity and evaluated for efficiency and applicability. The results of this study demonstrated the high selectivity, improved lead time, and reliability of PMAxx-qPCR over the culture-dependent method.

The third phase of the study determined the bioactive peptide and short-chain fatty acid (SCFA) profiles in mixed-species yoghurt incorporating Bifidobacterium spp. namely, B. bifidum ATCC 11863, B. breve ATCC 15700, B. animalis subsp. animalis ATCC 25527 and L. rhamnosus GG using ultra-performance liquid chromatography coupled to electrospray ionisation quadrupole-time of flight mass spectrometry (UPLC-ESI-QTOF-MS), and gas chromatography-mass spectrometry (GC-MS), respectively. Novel short peptides (<10 amino acids) derived from minor milk serum proteins with potential antimicrobial properties were identified in different yoghurt samples. L. rhamnosus GG positively influenced the production of both bioactive peptides and SCFA in yoghurt. The fourth phase of this study utilised Raman spectroscopy coupled with principal component analysis (PCA) and second-order derivative spectral analysis for structural characterisation of exopolysaccharides (EPS) isolated from Bifidobacterium spp. and L. rhamnosus GG. This study revealed unique EPS structural features that are likely involved in the functional properties of probiotics in yoghurt.