Viability and functionality of Lacticaseibacillus rhamnosus and Bifidobacterium spp. in yoghurt

Abstract

Yoghurt is generally perceived as a health promoting product by many health-oriented consumers around the world and thus its popularity makes it a suitable carrier of probiotics. Probiotic functionality is species- and strain-specific, while viability is a prerequisite for any probiotic functionality. The efficacy of probiotic yoghurt is affected by several factors such as probiotic viability decline during shelf-life resulting from different factors including probiotic strain or species type, interactions, and processing stress factors. 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 pre-fermentation resulted in yoghurt with lower syneresis and improved firmness than post-fermentation inoculation. L. rhamnosus strains (GG and DSM 8746), B. bifidum ATCC 11863 and B. animalis subsp. animalis ATCC 25527 sustained their viability above the minimum therapeutic levels (>10⁶ CFU/g), while B. breve ATCC 15700 viability significantly decreased below the minimum therapeutic levels at the end of 28 day shelf-life at 4°C. Probiotic inoculation post-fermentation could not improve the viability of Bifidobacterium spp. and L. rhamnosus strains in mixed-probiotic species yoghurts. In addition, MRS-pH 5.4 and MRS-NNLP media were not selective against L. rhamnosus strains and thus, resulted in overestimation of Bifidobacterium spp. and Lactobacillus delbrueckii subsp. bulgaricus viability.

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. PMAxx-qPCR showed high specificity to the target organisms in mixed-species yoghurt, quantifying only viable cells. The linear dynamic ranges were established over five to seven orders of magnitude. The assay was reliable with an efficiency of 91-99%, R2 values > 0.99, and a good correlation to the plate count method (r = 0.882). The results of this study demonstrate the high selectivity, improved lead time, and reliability of PMAxx-qPCR over the culture-dependent method, making it a valuable tool for inline viability verification during processing and improving probiotic quality assurance for processors and consumers.

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. Significantly higher degree of proteolysis was observed in yoghurt samples incorporating single probiotic species of B. bifidum ATCC 11863 and L. rhamnosus GG. Yoghurt incorporating B. bifidum ATCC 11863 contained more peptides with different bioactivities than other yoghurt samples. On contrary, B. animalis ATCC 25527 significantly enhanced the SCFA content (acetic acid) production in yoghurt compared to other Bifidobacterium spp., while L. rhamnosus GG positively influenced the production of both bioactive peptides and SCFA in yoghurt. Novel short peptides (<10 amino acids) derived from minor milk serum proteins with potential antimicrobial properties were identified in different yoghurt samples.

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. The results revealed B. bifidum ATCC 11863 as the highest EPS producer compared to other probiotics used in this study. The probiotic species EPS with different structural features could be predicted to be heteropolysaccharides for L. rhamnosus GG, B. animalis ATCC 25527 and B. breve ATCC 15700, and homopolysaccharide for B. bifidum ATCC 11863. Through Raman spectral analysis, PCA and second order derivative, the structural similarity between the yoghurt EPS and the EPS of B. breve ATCC 15700 and B. bifidum ATCC 11863 and the presence of unique bands were confirmed. Thus, indicating the ability of these probiotics to produce EPS in mixed-species yoghurt. In contrast, EPS from B. animalis ATCC 25527 and L. rhamnosus GG were structurally different from the yoghurt EPS and lacked unique bands, signifying their inability to produce EPS in yoghurt. This study revealed unique EPS structural features which are likely involved in the functional properties of probiotics in yoghurt.

The results of this study demonstrate that a reliable and highly selective PMAxx-qPCR with enhanced lead time is a valuable alternative method to culture-dependent methods. Furthermore, this study provides insights into the bioactive metabolites namely peptides, SCFAs and EPS of yoghurt incorporating L. rhamnosus GG and Bifidobacterium spp. Hence, this study provides valuable information for the dairy and probiotic industries on selection of probiotic species or strains for production of novel mixed-species yoghurts with improved viability and functionality.