Food fermentation is a method widely used in the past to extend the storage life of food. Numerous studies on fermented food have revealed that they not only have biopreservative properties but also health benefits. Lactic acid bacteria are the major group of microorganisms involved in food fermentation and the properties that influence food are primarily due to the compounds released from microorganisms such as organic acids and bacteriocins. Their health benefits are exerted through several mechanisms including inhibiting the growth of pathogenic bacteria and modifying the host immune response. A number of strains that have been investigated show different properties even between the same species thus emphasizing the importance of strain identification. To determine if some traditional fermented Swedish foods contain lactic acid bacteria, bacteria from four fermented Swedish foods (two surströmming and two sausages) were isolated using MRS broth. Bacterial isolates were examined for their colony and cell morphology and Gram staining and were found to be predominantly Gram-positive cocci or rods. 16S rRNA PCR amplifications of selected isolates was performed using universal prokaryotic primers and sequenced. The sequencing results showed that the bacterial isolates from Oskars surströmming Filéer and Gognacs medvurst were Lactobacillus sakei and the isolate from Mannerströms surströmming was Enterococcus sp. This study showed that the traditional Swedish fermented food evaluated did contain lactic acid bacteria.

Abstract Two regions of the genome of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H were characterized, representing 14 % of the phage genome. The first region of 2497 bp contained genes encoding phage structural proteins and the second region of 2498 bp genes involved in lytic functions. The nucleotide sequences of the major capsid protein gene g34, a putative capsid morphogenesis gene (ORF178A), the gene mur encoding phage cell wall hydrolase (lysin), the gene hol (ORF107) encoding the cell membrane permeabilizing phage holin, and six other genes with unknown function were found. Identification of these genes was performed by amino acid sequencing of their encoded proteins (genes g34 and mur), by their physiological effect on E. coli (genes hol and mur), by sequence comparison (genes mur, hol, ORF178A), and by biochemical analysis of their encoded purified protein (gene mur). A promoter for the capsid protein encoding gene cluster was determined by primer extension method. A purification method suitable for large scale processing (cation exchange chromatography by expanded bed adsorption method) was developed for the phage LL-H lysin protein Mur. Purified Mur was biochemically determined as a N-acetylmuramidase, which was effective on cell walls of Lb. delbrueckii, Lb. helveticus, Lb. acidophilus and Pediococcus damnosus. Some biotechnological applications for the lysis genes hol and mur or the purified protein Mur are suggested. Mur digests E. coli cell walls inefficiently, but could still be used for lysis of E. coli. Coexpression of the phage LL-H lysin and holin genes yielded to lysis of the E. coli host only at low culture densities. Therefore, some chemicals were tested for their ability to trigger lysis of E. coli cells overexpressing the phage LL-H gene mur. Thymol was found to mimic the physiological effects of the phage holin in a bacterial growth state independent manner. An efficient lysis method utilizing intracellular production of Mur and triggering the lysis with thymol was developed.

Pediococci are Gram-positive, non-spore forming lactic acid bacteria, LAB, that are commonly used in the industrial fermentation of vegetables and meat. On the other hand species of pediococci may constitute part of the secondary microflora as non-starter lactic acid bacteria, NSLAB, responsible for cheese ripening. This study was conducted to determine and isolate pediococci from South African farm-style cheese and to morphologically and physiologically characterize the pediococci isolates to a species level. Strains of Pediococcus species isolated from South African farm-style cheese were evaluated for the production of antimicrobial peptides, pediocins, and their effect against food pathogens Bacillus cereus ATCC 1178 and Listeria monocytogenes ATCC 7644. Eight farm-style cheeses namely pasteurized young Gouda (PYG), pasteurized matured Goude (PMG) as well as pasteurized matured Parmesan (PMP); un-pasteurised or raw milk aged Bouquet (RAB), aged Gouda (RAG) and matured Gouda (RMG) as well as goat cheese Gouda (:RGG) were chosen for the isolation of Pediococcus species. LAB were cultivated on MRS agar where logarithmic counts of LAB ranged from 6.90 cfu/g to 9.40 cfu/g. Microscopic examination of selected colonies identified 110 (18%) of 606 isolates as Gram-positive, catalase negative presumptive pediococci occurring in pairs, clusters and tetrads. Presumptive pediococci were distributed among five of the eight cheeses namely PYG, PMG, RAB, RAG and RMG in numbers of 33, 21,28, 12 and 16 respectively. Physiological characterization of presumptive pediococci isolates was determined under specific growth parameters, temperature, pH and salt (w/v). These isolates were characterized as P. acidilactici, forty-nine, and P. pentosaceus, sixty-one, isolates. For the antimicrobial assay three techniques namely, the agar disc, spot and overlay methods were evaluated. Comparisons on the sensitivity or susceptibility of two Lactococcus strains, L. diacetilactis NCDO 176 and L. lactis NCDO 605, to crude extract from P. acidilactici STI was assessed. The agar disc assay technique produced more reliable results compared to the other techniques and L. diacetilactis NCDO 176 was more susceptible to crude pediocin extract produced from P. acidilactici STI. A total of fifty-two strains (47%) from both species, twenty-seven (24%) P. acidilactici and twenty-five (23%) P. pentosaceus, exerted antagonism against L. diacetilactis NCDO 176 through the action of perdiocins. Among these strains thirteen (13%) of both species, seven (6%) P. acidilactici and six (7%) P. pentosaceus inhibited B. cereus ATCC 1178 while a total of thirty-seven strains (33%) of both species where, seventeen (15%) P. acidilactici and twenty (18%) P. pentosaceus showed inhibition against L. monocytogenes ATCC 7644. Among these strains inhibition of food pathogens was variable against L. monocytogenes ATCC 7644 and low against B. cereus ATCC 1178. Comparison of the antimicrobial activity of the two Pediococcus species showed similarity in the inhibition pattern, however, more strains of P. pentosaceus exerted antagonism against L. lactis NCDO 176 and L. monocytogenes ATCC 7644 compared to P. acidilactici. / Dissertation (MSc)--University of Pretoria, 2005. / Food Science / unrestricted

Ting is a cooked fermented sorghum food that is popular amongst southern Africans for its sour taste and unique flavour. However, major challenges are associated with large-scale production and marketing of this spontaneously fermented food due to inconsistent microbiological and sensory quality. The use of starter cultures may circumvent these limitations. Prior to engaging starter cultures, detailed knowledge of the microbial diversity and dynamics during fermentation is important. Therefore, the aim of this study was to investigate microbial diversity and dynamics during sorghum fermentations, and to clarify the role of starter cultures regarding the microbiological safety and consumer acceptance of sensory characteristics of fermented ting. A culture-independent approach, based on the use of PCR-denaturing gradient gel electrophoresis (DGGE), revealed that Lactococcus lactis, Lactobacillus curvatus, Weissella cibaria and some Enterobacteriaceae were predominant at the end of spontaneous sorghum fermentations. Culture-dependent methods indicated that Lb. fermentum, Lb. plantarum, Lb. rhamnosus, E. faecalis, E. mundtii, W. cibaria and L. lactis were predominant at the end of fermentation. These results not only indicated the predominant bacteria during sorghum fermentation, but also indicated that a combined approach is required to reveal microbial diversity and dynamics during spontaneous sorghum fermentations. Based on the above results, L. lactis, Lb. fermentum, Lb. plantarum and Lb. rhamnosus were evaluated as starter cultures for production of ting. All the starter cultures were able to ferment sorghum, but the lowest pH and highest lactic acid was produced in naturally fermented sorghum inoculated with L. lactis. This fermentation showed an increase in the number of lactic acid bacteria and yeasts, whilst pathogen counts decreased. Ting from this fermented gruel, in contrast to naturally fermented sorghum, had sensory properties preferred by panelists. The results indicated that the use of L. lactis in starter cultures may result in ting with consistent and acceptable attributes. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Probiotics are defined as live microorganisms that have, if ingested, a positive impact on human and animals health. The best known probiotics are the lactic acid bacteria, among them most commonly used are lactobacilli and bifidobacteria. For therapy the minimum daily consumption of at least 100 g of milk product with a minimum 1000000 of probiotic bacteria in 1 g or 1 ml is considered. In this work, several products that have declared on the label probiotic cultures have been selected. All products were analyzed for the number of lactic acid bacteria. Two media - MRS agar and skim milk agar Modified were used for cultivation. Cultivation proceeded at 37 °C for 48 hours under aerobic and anaerobic conditions. Amount of live bacteria, which must be present in the product, indicates Ministry of Agriculture No. 77/2003 Coll.. Quantitative representation of lactic acid bacteria in all selected products comply with the requirements of legislation.

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Probiotic bacteria are increasingly prevalent in food and nutritional products today. These remarkable microorganisms are capable of imparting exceptional health benefits on their host, including prevention of infection by pathogens and stimulation of immune system function. Their most common mode of delivery is through dairy products (e.g. yogurt), which are also one of their preferred habitats. The interactions between probiotic bacteria and dairy systems have been studied, but are still not well discerned. There is a need for better understanding of these associations, as well as those surrounding the mode of bacterial transfer from the food product to the human gastrointestinal tract. Discoveries into the optimal means of probiotic transport to the body may lead to great advancements in both the design of probiotic foods and their exploitation in the support of human health. Much of the previous research on probiotic bacteria has explored their possible means of adherence in the intestine, as well their strengths in the promotion of human health. Studies relating to their interaction with dairy products are lacking, however, thus this work aims to elucidate some of these aspects. The primary endeavor of this thesis was to develop a technique to quantify the binding affinity of probiotic lactic acid bacteria for milk phospholipids. An additional objective was to exploit these bacteria, as well as dairy ingredients rich in bioactive molecules, in the creation of a highly nutritious food product. In these experiments, a collection of methods were used in progression in order to arrive at a novel protocol to assess binding with excellent reproducibility and simplicity. These included various membrane blotting techniques, as well as thin-layer chromatography. Essentially, phospholipids from both animal-derived standards and milk extracts were applied to a surface (e.g. PVDF membrane), and bacteria were incubated with them to allow binding reactions. The lactic acid bacteria selected for the final assays consisted of four strains of Lactobacillus, including L. reuteri (SD2112 and T-1), L. acidophilus, and L. casei (LC-10). Their adhesion to phospholipids was detected by either colorimetric or fluorescent labeling systems. To illustrate this, the final method developed was a procedure in which bacteria fluorescently stained with acridine orange were allowed to bind to dots of PVDF membrane coated with phospholipids. The results of this study showed that lactic acid bacteria undeniably exhibit selective binding affinity for phospholipids as opposed to other lipids such as triglycerides. The bacteria demonstrated significantly greater binding for a phospholipid extract from milk as opposed to individual phospholipid standards from other sources (p<0.05). Nonetheless, adhesion to all phospholipids was substantially greater than that to triglycerides. These findings, as well as the development of this method, should prove valuable in future research regarding the associations of probiotics with dairy systems. An additional purpose of this thesis was to design a dairy-based food product containing ingredient sources rich in milk bioactives. A gel-type product was created using primarily colostrum, buttermilk powder, and whey protein isolate, as well as selected strains of Lactobacillus. With the inclusion of immunoglobulin-rich colostrum, the product was analyzed alongside fluid milk and colostrum in order to quantify and compare these bioactive molecules. An enzyme-linked immunosorbent assay (ELISA) was used to complete this, and the results revealed concentrations that would be expected by the literature. Specifically, immunoglobulin G (IgG) was quantified by interpolation from a bovine IgG standard regression curve. The results showed that the concentration of IgG in the gel was nearly twice that of colostrum, and almost eight-fold higher than that of milk. This indicates that use of bioactive-rich substances, such as colostrum, in a food product may serve as a means of delivering more concentrated doses of bioactives than their respective ingredients. The research completed in this thesis is significant in that it contributes a valuable method to the elucidation of bacterial binding interactions with milk components, and also demonstrates the successful application of dairy ingredients to an innovative food product high in beneficial compounds. The insight provided by these studies could encourage further work in improving the understanding of probiotic delivery and advancing the development of bioactive-rich food products.

This study aimed to isolate and characterize in-vitro and under simulated colonic conditions, probiotic candidates isolated from food environment producing γ-aminobutyric acid (GABA), a major inhibitory neuromediator of the enteric nervous system with a potential role in modulating the immune system in many health disorders. Several lactic acid bacteria were isolated and detected for the presence of the gadB gene using PCR and GAD enzymatic assay. The most active strains with high and fast production kinetics were identified, characterized, and included Streptococcus thermophilus, Lactiplantibacillus plantarum, and Lactobacillus delbrueckii subsp. bulgaricus. The biological safety (i.e., sensitivity to antibiotics and the presence of virulence factors) and probiotic potential (i.e., resistance to gastrointestinal conditions and whole-genome sequencing) of identified bioactive strains was also confirmed in vitro. The growth, GABA production, and competitiveness of selected probiotic candidates (B. animalis, S. thermophilus, and L. bulgaricus) were investigated in the presence of human gut microbiota ex vivo in a model of a proximal colon mimicking physiological and microbiological conditions of the human large intestine. Supplementation with GABA-producing probiotic candidates did not affect the overall gut microbiota diversity over 48 h of treatment. However, we observed modulation of the microbiome composition, especially change of Bacteroides population, a key gut microbe associated with anti-depressive and anti-inflammatory activities. The level of microbiota-generated butyrate within 12 h of treatment was significantly increased compared to control. Results from this study demonstrated the probiotic potential of tested GABA-producing bacteria and their impact on gut microbiota structure and metabolism, suggesting their suitability for gut health-promoting application.

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