Yosep Ji

Functional properties of Lactobacillus strains isolated from kimchi

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) from kimchi, a traditional Korean fermented vegetable product generally consumed raw as a side-dish with practically every meal. Twelve mild acid producing facultatively heterofermentative Lactobacillus strains were selected for their potential as starter cultures for fermentation of kimchi, and evaluated for their functional properties. Eleven strains were identified as Lactobacillus sakei and one as Lactobacillus plantarum. The strains identified as L. sakei differed in some physiological features; of particular interest was the fact that 9 of these strains produced L(+) lactic acid from glucose in presence of acetate. All strains were able to survive gastrointestinal conditions simulating stomach and duodenum passage. In addition, they showed higher adherence to HT-29 cells than Lactobacillus rhamnosus GG, a commercial probiotic strain used worldwide. These strains also showed antimicrobial activity against a number of food-borne pathogens. Their ability to lower cholesterol was demonstrated by BSH (bile salt hydrolytic) activity, and cholesterol assimilation tests in vitro. The results suggest the probiotic potential of these strains for use in kimchi fermentation.

Modulation of the murine microbiome with a concomitant anti-obesity effect by Lactobacillus rhamnosus GG and Lactobacillus sakei NR28

The microbiota of the gastrointestinal tract (GIT) constitutes the major part of the total human microbiome and is considered to be an important regulator of human health and host metabolism. Numerous investigations in recent years have focused on the connection between the human microbiota and metabolic diseases such as obesity, type II diabetes and atherosclerosis. Yet, little is known about the impact of probiotic consumption on the GIT microbial population and the potential effect on chronic diseases. In this study, the modulation of the microbial community in the murine small intestine resulting from probiotic feeding was investigated and was found to be associated with an anti-obesity effect. Changes in the microbiota of the mouse faeces and small intestine were monitored using quantitative real-time PCR and by following the mRNA expression levels of various obesity-related biomarkers following probiotic feeding in a mouse model. Lactobacillus rhamnosus GG and Lactobacillus sakei NR28 (a putative probiotic strain isolated from kimchi) were administered at a daily level of approximately 1×10(8) viable bacteria per mouse (C57BL/6J mice) for up to three weeks. Feeding these strains resulted in a significant reduction of epididymal fat mass, as well as obesity-related biomarkers like acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 in the liver. The total number and ratio of the microbial groups, i.e. Firmicutes, Bacteroidetes, Clostridium cluster I and XIVab, and Lactobacillus spp. were modulated in the small intestine, and the Firmicutes:Bacteroidetes ratio was decreased. In contrast, no noticeable effect of probiotic feeding could be detected on the faecal microbiota, neither quantitatively, nor with regard to the bacterial groups (Firmicutes, Bacteroidetes, Clostridium cluster I and XIVab, and Lactobacillus spp.) studied.

Selection of functional lactic acid bacteria as starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler ex Sprengel.).

The purpose of this research was to find safe and suitable starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler), also known as garlic chives or Oriental garlic. This traditional herb has several functional properties and a strong flavour; its leaves are used as food material. Eighteen strains of lactic acid bacteria (LAB) were isolated from well-fermented leek kimchi. Controlled fermentation of the leek leaves was conducted with 2 strains (Weissella confusa LK4 and Lactobacillus plantarum LK8), selected as potential starter cultures on the basis of their safety properties, and on the pH, total titratable acidity (TTA), and viable cell numbers [colony forming units (CFUml(-1))] achieved during the fermentation. Microbial dynamics was also followed during fermentation by using PCR-DGGE (Denaturing Gradient Gel Electrophoresis) on DNA level. To analyse bioactive compounds such as thiols and allicin (diallyl thiosulfinates), the total flavonoid and polyphenolic contents were determined by colorimetric methods. Functional properties were assessed on the basis of anti-oxidative capacities by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, and ferric reducing antioxidant power (FRAP). W. confusa LK4 rapidly increased during the first stage of leek fermentation, and was mainly responsible for accelerated fermentation during the early period in contrast to L. plantarum LK8, a stronger acid producer during the later stages of fermentation. After 48 h fermentation, leeks fermented with W. confusa LK4 showed the highest radical scavenging effects and reducing ability. The detectable amount of allicin of fermented leeks decreased relative to the change in pH, whereas the concentration of thiols significantly increased. Total flavonoid and poly-phenolic contents changed during fermentation and showed correlation with anti-oxidant effects. We therefore suggest the suitability of W. confusa LK4 as a potential starter culture for fermentation of leeks.

Protective effects of Lactobacillus rhamnosus GG against dyslipidemia in high-fat diet-induced obese mice.

Recent reports suggest that gut microbiota can be a major determinant of dyslipidemia and non-alcoholic fatty liver disease (NAFLD) and its modulation by treating probiotics is a valid strategy to exert a protective effect. In this study, high-fat diet (HFD)-fed mice were orally administrated with Lactobacillus rhamnosus GG (LGG) for 13 weeks. Significant reductions in the weights of the liver, mesenteric and subcutaneous adipose tissues were observed in LGG-treated HFD-fed mice compared to LGG-non-treated controls. The serum levels of triglyceride and cholesterol were also significantly reduced in LGG-treated mice. Gut microbial composition analysis showed that shifts in the diversity of dominant gut bacteria were caused by HFD and restored by LGG treatment. A remarkable decrease of hepatic fat content was also observed in LGG-treated mice, accompanied by downregulated expressions of lipogenic and pro-inflammatory genes in the liver. LGG-treated mice had lower expression levels of genes involved in cholesterol synthesis, but conversely, higher expression levels of cholesterol efflux-related genes compared to LGG-non-treated controls. The cholesterol-lowering effect of LGG was also found to be mediated by suppression of FXR and FGF15 signaling, resulting in the upregulation of hepatic CYP7A1. Our findings confirm a therapeutic potential of probiotics for ameliorating dyslipidemia and NAFLD.

Reduction in cholesterol absorption in Caco-2 cells through the down-regulation of Niemann-Pick C1-like 1 by the putative probiotic strains Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 from fermented foods.

Hypercholesterolaemia is a major risk factor related to atherosclerosis, and it may be influenced by our diet. This study addresses the impact of Lactobacillus rhamnosus BFE5264 (isolated from Maasai fermented milk) and Lactobacillus plantarum NR74 (from Korean kimchi) on the control of cholesterol absorption through down-regulation of Niemann-Pick C1-like 1 (NPC1L1) expression. Caco-2 enterocytes were treated with the live, heat-killed (HK) bacteria, bacterial cell wall extracts and metabolites; mRNA level and protein expression were measured. Caco-2 cells showed lower NPC1L1 expression in the presence of the live test strains than the control, elucidating down-regulation of cholesterol uptake, and were compared well with the positive control, L. rhamnosus GG. This effect was also observed with HK bacteria and cell wall fractions but not with their metabolites. The potential of some Lactobacillus strains associated with traditional fermented foods to suppress cholesterol uptake and promote its efflux in enterocytes has been suggested from these data.

Lactobacillus plantarum HAC01 regulates gut microbiota and adipose tissue accumulation in a diet-induced obesity murine model

The functional features of Lactobacillus plantarum HAC01 (HAC01), isolated from fermented Korean kimchi, were studied with regard to the fat mass, immunometabolic biomarkers and dysbiosis in a diet-induced obesity (DIO) murine model. L. rhamnosus GG (LGG) served as reference strain and a PBS-treated group as control. The administration of L. plantarum HAC01 resulted in reduction of the mesenteric adipose depot, the conjunctive tissue closely associated with the gastrointestinal tract, where lipid oxidative gene expression was upregulated compared to the control group. Metagenome analysis of intestinal microbiota showed that both strains HAC01 and LGG influenced specific bacterial families such as the Lachnospiraceae and Ruminococcaceae rather than the phyla Firmicutes and Bacteroidetes as a whole. The relative abundance of the Lachnospiraceae (phylum Firmicutes) was significantly higher in both LAB-treated groups than in the control. Comparing the impact of the two Lactobacillus strains on microbial composition in the gut also suggests strain-specific effects. The study emphasises the need for deeper studies into functional specificity of a probiotic organism at the strain level. Alleviation of obesity-associated dysbiosis by modulation of the gut microbiota appears to be associated with “indicator” bacterial taxa such as the family Lachnospiraceae. This may provide further insight into mechanisms basic to the mode of probiotic action against obesity and associated dysbiosis.

Lactobacillus rhamnosus BFE 5264 and Lactobacillus plantarum NR74 Promote Cholesterol Excretion Through the Up-Regulation of ABCG5/8 in Caco-2 Cells.

The effect of two putative probiotic strains, Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74, on the control of cholesterol efflux in enterocytes was assessed by focusing on the promotion of ATP-binding cassette sub-family G members 5 and 8 (ABCG5 and ABCG8). Differentiated Caco-2 enterocytes were treated with live bacteria, heat-killed bacteria, a bacterial cell wall fraction, and metabolites and were subjected to cholesterol uptake assay, mRNA analysis, and protein analyses. Following LXR-transfection by incubation with CHO-K1 cells in DNA-lipofectin added media, the luciferase assay was conducted for LXR analysis. Treatment of Caco-2 cells with L. rhamnosus BFE5264 (isolated from traditional fermented Maasai milk) and L. plantarum NR74 (isolated from Korean kimchi) resulted in the up-regulation of LXR, concomitantly with the elevated expression of ABCG5 and ABCG8. This was associated with the promotion of cholesterol efflux at significantly higher levels compared to the positive control strain L. rhamnosus GG (LGG). The experiment with CHO-K1 cells confirmed up-regulation of LXR-beta by the test strains, and treatment with the live L. rhamnosus BFE5264 and L. plantarum NR74 strains significantly increased cholesterol efflux. Heat-killed cells and cell wall fractions of both LAB strains induced the upregulation of ABCG5/8 through LXR activation. By contrast, LAB metabolites did not show any effect on ABCG5/8 and LXR expression. Data from this study suggest that LAB strains, such as L. rhamnosus BFE5264 and L. plantarum NR74, may promote cholesterol efflux in enterocytes, and thus potentially contribute to the prevention of hypercholesterolemia and atherosclerosis.

Influence of gastrointestinal stress on autoinducer-2 activity of two Lactobacillus species

Quorum sensing is a bacterial communication signalling system that regulates the expression of certain target genes with autoinducers in a cell density-dependent manner. The universal luxS-mediated quorum sensing using the autoinducer-2 (AI-2) signal is present in a wide variety of bacteria with only sparse information on probiotic lactobacilli. Effective probiotics should exhibit tolerance and adaptation to stress conditions typical of the GIT. Adhesion to human intestinal epithelial cells and competitive exclusion of pathogens are also considered important. The AI-2 signal system plays an important role in the response of probiotic lactobacilli to the surrounding environment. Intraspecies-related changes in quorum signalling in the GIT were determined by monitoring the AI-2 activity of two strains each of Lactobacillus rhamnosus and L. plantarum under various stress conditions. Modulation of the AI-2 activity of all the strains was induced by stress responses to pH, bile acid, temperature, osmotic pressure and starvation, and was both species- and strain-specific. AI-2 inhibition correlated with a reduction in the stress-related genes of L. rhamnosus. We therefore suggest that AI-2 quorum signalling of probiotic lactobacilli may represent one way of adapting to the hosts ecosystem and of interacting within the intestinal environment.

Impact of bubble size on growth and CO2 uptake of Arthrospira (Spirulina) platensis KMMCC CY-007.

Optimisation of cyanobacterial cell productivity should consider the key factors light cycle and carbon source. We studied the influence of CO2 bubble size on carbon uptake and fixation, on basis of mRNA expression levels in Arthrospira platensis KMMCC CY-007 at 30°C (light intensity: 40μmolm(-2)s(-1); 1% CO2). Growth rate, carbon fixation and lipid accumulation were examined over 7days under fine bubble (FB) (100μm Ø) bulk bubble (BB) (5000μm Ø) and non-CO2 (NB) aeration. The low affinity CO2 uptake mRNA (NDH-I4 complex) was stronger expressed than the high affinity NDH-I3 complex (bicA and sbtA) under 1% CO2 and FB conditions, with no expression of bicA1 and sbtA1 after 4days. The high affinity CO2 uptake mRNA levels corresponded to biomass, carbon content and lipid accumulation, and increase in NDH-I3 complex (9.72-fold), bicA (5.69-fold), and sbtA (10.61-fold), compared to NB, or BB conditions.

Long-term fermented soybean paste improves metabolic parameters associated with non-alcoholic fatty liver disease and insulin resistance in high-fat diet-induced obese mice

Recently, Korean traditional fermented soybean paste, called Doenjang, has attracted attention for its protective effect against diet-related chronic diseases such as obesity and type 2 diabetes. Long-term fermented soybean pastes (LFSPs) are made by fermentation with naturally-occurring microorganisms for several months, whereas short-term fermented soybean pastes (SFSPs) are produced by shorter-time fermentation inoculated with a starter culture. Here, we demonstrate that administration of LFSP, but not SFSP, protects high-fat diet (HFD)-fed obese mice against non-alcohol fatty liver disease (NAFLD) and insulin resistance. LFSP suppressed body weight gain in parallel with reduction in fat accumulation in mesenteric adipose tissue (MAT) and the liver via modulation of MAT lipolysis and hepatic lipid uptake. LFSP-treated mice also had improved glucose tolerance and increased adiponectin levels concomitantly with enhanced AMPK activation in skeletal muscle and suppressed expression of pro-inflammatory cytokines in skeletal muscle and the liver. LFSP also attenuated HFD-induced gut permeability and lowered serum lipopolysaccharide level, providing an evidence for its probiotic effects, which was supported by the observation that treatment of a probiotic mixture of LFSP-originated Bacillus strains protected mice against HFD-induced adiposity and glucose intolerance. Our findings suggest that the intake of LFSP, but not SFSP, offers protection against NAFLD and insulin resistance, which is an effect of long-term fermentation resulting in elevated contents of active ingredients (especially flavonoids) and higher diversity and richness of Bacillus probiotic strains compared to SFSP.