Theses and Dissertations (Consumer and Food Sciences)

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Viability and functionality of Lacticaseibacillus rhamnosus and Bifidobacterium spp. in yoghurt
Marole, Tlaleo Azael (University of Pretoria, 2025-03)
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.
South African consumer knowledge of the current paediatric food-based dietary guidelines
Van Veijeren, Rochelle (University of Pretoria, 2024-02-16)
The co-existence of undernutrition, overnutrition, and micronutrient deficiencies constitutes what is known as the Triple Burden of Malnutrition. While the perception may persist that this phenomenon predominantly affects lower-income communities within developing nations, it is crucial to acknowledge its impact on consumers across all settings. Contributing factors extend beyond resource scarcity, often related to lower-income groups, and also encompass a lack of awareness of proactive measures. Within the framework of the United Nation’s second Sustainable Development Goal (SDG) the “Zero Hunger” initiative strives to reduce the Triple Burden of Malnutrition by 2023. However, this goal will largely be missed if current consumer ignorance toward proactive measures endures. The Paediatric Food-based Dietary Guidelines (PFBDGs) are available to caregivers as an educational tool that has the potential to make targets, such as the second SDG, more realistic. Among the most vulnerable to the Triple Burden of Malnutrition are young children aged 0-5 years, whose nourishment directly depends on their caregiver’s competence toward feeding. Although there are various proactive measures in place, such as the abovementioned guidelines, caregivers often put the health of young children at risk due to overestimating their abilities. The situation is described by the Dunning-Kruger Effect (DKE), which is a cognitive bias whereby people with low capability regarding a certain domain tend to overestimate their abilities or knowledge. The DKE theory suggests that overconfidence gradually decreases as one’s accurate knowledge of a specific topic of interest increases. A threshold is eventually reached, where confidence and competence increase in a favourable direction and limitations are recognised and admitted. DKEs can be harmful, especially in the light of young, vulnerable children. Such occurrences will consequently contribute to the country’s prevalence of the Triple Burden of Malnutrition. This study therefore aims to investigate the possibility of DKEs amongst South African caregivers when it comes to young child feeding by comparing their subjective knowledge with objective knowledge (i.e. the PFBDGs). If the subjective evaluation surpasses objective criteria, a DKE is recognised, which underscores the need for consumer education of proactive measures, such as the PFBDGs, in this context. A structured, self-administered questionnaire was designed and distributed to consumers electronically through a link that could be accessed on an internet-connected device. Data was conveniently collected throughout Gauteng by strategically focusing on areas with a significant concentration of participants meeting the predefined inclusion criteria. The inclusion of the sample was driven by the prerequisites of being a caregiver of one or more children under the age of five, residing in Gauteng, and being over 18 years old. Overall findings indicate that the vast majority of consumers were largely unaware of the PFBDGs. A significant 61% of consumers reported their unfamiliarity with the PFBDGs, highlighting a crucial requirement for heightened awareness efforts. It is essential to make this proactive approach better available to ensure all consumers can fully capitalise on its benefits. Retailers bear a corporate responsibility of assisting consumers in making well-informed decisions. An effective strategy is to enhance the accessibility of the PFBDGs to consumers by incorporating direct links to the guidelines on the packaging of food intended for young children. Subjective knowledge scores were collected with a 5-point Likert scale, and consumers were asked to rate their level of knowledge out of five for each dimension of the PFBDGs. The objective knowledge of the PFBDGs was tested with a 5-point ‘true’ or ‘false’ questionnaire. This approach enabled the calculation of total mean scores i.e. subjective knowledge and percentage scores i.e. objective knowledge which received a rating ranging from poor to excellent. The outcomes facilitated a comprehensive comparison to determine whether an overall DKE existed. The investigation delved into each dimension of the PFBDGs, allowing for a detailed comparison between subjective and objective knowledge to pinpoint specific areas where DKEs may be present. Interferential statistics strengthened these results through ANOVAs and Multiple Comparison tests. This analytic approach not only facilitated the identification of possible influential factors or predictors of consumers’ subjective and objective knowledge but also enabled the precise identification of demographic groups with a heightened requirement for education on proactive measures. Overall, the subjective knowledge outcome (M = 3.2) was aligned with the objective criteria (58.9%), and both knowledge fields across all dimensions were of average quality. The key findings from the subjective knowledge assessment indicated that a significant number of respondents held an inflated perception of their knowledge. Respondents seemed to rate their knowledge of the Feeding Practices (M = 3.26) and Hygiene (M = 3.6) dimensions of the PFBDGs as higher than average. Nonetheless, a contrast emerged when comparing the subjective evaluations with the objective knowledge scores. The latter provided an accurate reflection of the consumers’ actual knowledge which was of poor quality for Feeding Practices (46.23%) and average quality for Hygiene (52.08%), suggesting DKEs within these fields. In essence, even though an overall DKE was not determined, an overestimation of knowledge was particularly identified within the Feeding Practices and Hygiene fields. The study’s outcomes inspired focus toward policymakers to adhere to their corporate responsibility by focusing education on the identified fields most in need of intervention. By tackling concern areas in young child feeding, targets such as the Zero Hunger SDG can significantly be amplified. This can lead to a reduction in the harsh effects of the Triple Burden of Malnutrition among South Africa’s youngest and most vulnerable generation. Keywords • Dunning-Kruger Effect • Objective Knowledge • Subjective knowledge • Sustainable Development Goals (SDGs) • The Paediatric Food-based Dietary Guidelines (PFBDGs) • Triple Burden of Malnutrition
Survival of oxidative stress-adapted Bifidobacterium spp. in yoghurt
Thomashoff, Ursula Louise (University of Pretoria, 2024-07-12)
Bifidobacterium species are prominent constituents of the gut microbiota of healthy humans whose presence in the gut is linked with several health benefits. The supplementation of Bifi- dobacterium spp. as a probiotic through foods such as yoghurt is considered to be an effective way of sustaining a healthy gut microbiome and preventing gut dysbiosis. However, the ability to maintain the viability of Bifidobacterium spp. above the recommended therapeutic dose during the production and storage of yoghurt remains challenging due to its susceptibility to oxidative stress. This study aimed to investigate the effect of oxidative stress adaptation treatments on the physiological responses of three Bifidobacterium species, B. bifidum, B. breve and B. animalis subsp. animalis. The study also sought to isolate stress-adapted Bifidobacterium spp. variants and subsequently investigated their survival and viability during yoghurt manufacturing and storage shelf-life. Cultures of B. bifidum, B. breve and B. animalis were subjected to a sublethal (0.4 mM) hydrogen peroxide (H2O2) treatment followed by exposure to lethal (1 mM) H2O2 treatments across three successive generations. Membrane integrity and intracellular oxidation states of the H2O2-treated cells were evaluated using flow cytometry (FC) and fluorescent staining with SYTO 9 (S9) coupled with propidium iodide (PI), and CellROX® Green (CRG), respectively. The H2O2 treatments improved membrane integrity in B. breve and B. animalis, but increased intracellular oxidation states in all three Bifidobacterium species. Furthermore, the H2O2-treated cells were subjected to a lethal H2O2 challenge(30 min; 1 mM H2O2) before combined FC analysis of membrane integrity and intracellular oxidation states using CRG with PI. Results showed that the H2O2 treatment had no effect on B. breve while improving the membrane integrity retention of B. bifidum, indicating potential adaptive changes that mitigated oxidative damage. B. animalis had the most distinct response in maintaining membrane integrity in an oxidised intracellular state, potentially reflecting the species’ intrinsic oxidative stress tolerance. The morphological and ultrastructural characteristics and stress responses of stress-adapted Bifidobacterium cells were examined us- ing scanning and transmission electron microscopy (SEM and TEM). B. bifidum consistently expressed extracellular vesicles (EVs), affecting the cell surface texture and possibly indicating disrupted cell division and granule formation – features that were enhanced following the lethal H2O2 challenge. Further adaptations and responses observed in Bifidobacterium spp. included cellular elongation, compaction of intracellular components, thinning of its cell envelope and surface texturing. B. breve also underwent cytoplasmic compaction for protection, whereas prominent circumferential rings observed on B. animalis enhanced cell aggregation and stress resistance. Finally, the adapted Bifidobacterium spp. were evaluated for their viability during yoghurt fermentation and storage, and their storage was compared to that of unadapted cells over 28 days. Although the viability of B. bifidum and B. breve declined during yoghurt storage, the stress adaptation resulted in better survival for both species during fermentation, suggesting that the stress adaptation may not be sufficient to protect the species against the combined effects of oxidative and acid stress during yoghurt shelf-life. Consistent with its known intrinsic stress tolerance, B. animalis maintained stable viability counts during yoghurt fermentation and storage. Bacterial viability was also determined using a novel propidium monoazide-quantitative polymerase chain reaction (PMAxx-qPCR) method. Interestingly, this culture-independent technique showed that all three Bifidobacterium spp. remained above the probiotic minimum level (6 log CFU/g) throughout storage. The results suggested a significant loss of culturability for some Bifidobacterium species and the potential transition into a viable but non-culturable (VBNC) state. Thus, the PMAxx-qPCR method may be a feasible option for accurate probiotic viability quantification that can account for cells in a VBNC state. The study shows that exposing B. bifidum, B. breve, and B. animalis subspp. animalis to sublethal- and subsequent lethal H2O2 treatments result in variants that are less susceptible to ROS-induced damage. Furthermore, the study confirms that stress adaptation is a promising method to enhance the viability of Bifidobacterium spp. during yoghurt manufacturing and storage, maintaining recommended probiotic levels throughout the product’s shelf life. This enhanced survival, attributed to an active oxidative stress response induced by adaptation treatments, suggests that oxidative stress adaptation is a feasible method to improve the survivability and functional stability of some probiotic Bifidobacterium spp. in yoghurt. This approach not only supports the maintenance of the minimum recommended probiotic levels (6 log CFU/g viable cells) throughout production and storage but also potentially extends the probiotic shelf-life of the yoghurt.

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