Potential Probiotic Strains From Milk and Water Kefir Grains in Singapore—Use for Defense Against Enteric Bacterial Pathogens

Abstract

Kefir grains consist of complex symbiotic mixtures of bacteria and yeasts, and are reported to impart numerous health-boosting properties to milk and water kefir beverages. The objective of this work was to investigate the microbial communities in kefir grains, and explore the possibility of deriving useful probiotic strains from them. A total of 158 microbial strains, representing six fungal and 17 bacterial species, were isolated from milk and water kefir grains collected from a Singapore-based homebrewer. Based on 16S rRNA sequencing, isolated genera included Lactobacillus, Liquorilactobacillus, Lacticaseibacillus, Lentilactobacillus, Leuconostoc, Lactococcus, Acetobacter, Gluconobacter, Oenococcus, Clostridium, Zymomonas, Saccharomyces, Kluyveromyces, Pichia, Lachancea, Candida, and Brettanomyces. To characterize these isolates, a funnel approach, involving numerous phenotypic and genomic screening assays, was applied to identify kefir-derived microbial strains with the highest probiotic potential. Particular focus was placed on examining the pathogen inhibitory properties of kefir isolates toward enteric pathogens which pose a considerable global health burden. Enteric pathogens tested include species of Bacillus, Salmonella, Vibrio, Clostridium, Klebsiella, Escherichia, and Staphylococcus. Well diffusion assays were conducted to determine the propensity of kefir isolates to inhibit growth of enteric pathogens, and a competitive adhesion/exclusion assay was used to determine the ability of kefir isolates to out-compete or exclude attachment of enteric pathogens to Caco-2 cells. Seven bacterial strains of Lentilactobacillus hilgardii, Lacticaseibacillus paracasei, Liquorilactobacillus satsumensis, Lactobacillus helveticus, and Lentilactobacillus kefiri, were ultimately identified as potential probiotics, and combined to form a “kefir probiotics blend.” Desirable probiotic characteristics, including good survival in acid and bile environments, bile salt hydrolase activity, antioxidant activity, non-cytotoxicity and high adhesion to Caco-2 cells, and a lack of virulence or antimicrobial resistance genes. In addition, vitamin and γ-aminobutyric acid (GABA) synthesis genes, were identified in these kefir isolates. Overall, probiotic candidates derived in this study are well-characterized strains with a good safety profile which can serve as novel agents to combat enteric diseases. These kefir-derived probiotics also add diversity to the existing repertoire of probiotic strains, and may provide consumers with alternative product formats to attain the health benefits of kefir.

Screening of Bacteria Inhibiting Clostridium perfringens and Assessment of Their Beneficial Effects In Vitro and In Vivo with Whole Genome Sequencing Analysis

“…Our results showed that Bacillus had better growth curve, bile salt, and trypsin tolerance, while Lactobacillus had good acid tolerance ability. For further detecting the ability to bind epithelial cell sites of isolates, in the present study, porcine intestinal epithelial cells (IPEC-J2) and the method was used as previous study described [ 27 , 53 ]. The BA40 showed the strong ability to outcompete pathogens for epithelial cell adhesion sites.…”

“…We used agar well diffusion to test antimicrobial activity of isolates from JinHua pigs [ 27 , 28 ]. Briefly, the broth (LB or RCM) with 1.5% agar (10 mL) was poured onto the sterile Petri plates (10 × 10 mm).…”

Letter to the Editor: Focus on Zymomonas spp for the sake of clarity

“…Zymomonas as fermenting agents of plant saps is commonly used to produce alcoholic beverages. Often isolated from milk and kefir grains, Zymomonas strains may exhibit various probiotic properties (Azeredo et al, 2010;Tan et al, 2022;Tallyne de Aguiar Silva et al, 2020) including inhibition of pathogenic microorganisms (Weir, 2016) and explain some health benefits provided by fermented foods to consumers (Marco et al, 2017;Rul et al, 2022). Nevertheless, research is actively needed to sustain and achieve the probiotic potentials of this genera.…”

Oral delivery of microbiome-modulating synbiotics derived from kefir via alginate encapsulation systems

“…The health-promoting properties of kefir are likely attributable to its unique microbiota profile. The primary bacterial and fungal genera in kefir include lactobacilli, Lactococcus, Streptococcus, Leuconostoc, Acetobacter, Saccharomyces, Kluyveromyces, and Candida [21], [169]. Several species isolated from kefir have been found to exhibit probiotic properties.…”

“…Prebiotic characteristics of hydrolysed polysaccharides were then evaluated, such as the capacity to resist digestion by acidic pH and digestive enzymes typical of the human GIT. The selective substrate utilisation assay was next conducted to determine if derived polysaccharides could be selectively utilised by beneficial probiotics, specifically kefir-derived probiotics [169], but were not utilised by, or could inhibit, the growth of pathogens. Polysaccharides which demonstrated prebiotic efficacies were combined with kefir probiotics (Chapter 3) and tested in an ex-vivo model which simulates the large bowel to determine their effects on gut microbial communities and SCFA content.…”