Ming-Ju Chen

Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods.

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.

Diversity in gut bacterial community of school-age children in Asia

Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

Hypocholesterolaemic effects of milk-kefir and soyamilk-kefir in cholesterol-fed hamsters

This study aimed to evaluate the hypocholesterolaemic property of milk-kefir and soyamilk-kefir. Male hamsters were fed on a cholesterol-free or cholesterol-enriched diet containing 10 % skimmed milk, milk-kefir, soyamilk or soyamilk-kefir for a period of 8 weeks. The soyamilk, milk-kefir and soyamilk-kefir diets all tended towards a lowering of serum triacylglycerol and total cholesterol concentrations, and a reduction of cholesterol accumulation in the liver, the decrease in serum cholesterol concentration being mainly in the non-HDL fraction. The soyamilk-kefir diet led to a significant increase in the faecal excretion of neutral sterols and bile acids compared with the other two diets. The soyamilk-kefir diet also elicited a significant decrease in the serum ratio of non-HDL-cholesterol to HDL-cholesterol, compared with the control, than was the case for the other diets. These findings demonstrate that soyamilk-kefir may be considered to be among the more promising food components in terms of preventing CVD through its hypocholesterolaemic action.

Identification of Yeasts and Evaluation of their Distribution in Taiwanese Kefir and Viili Starters

The objective of the present study was to investigate yeast communities in kefir grains and viili starters in Taiwan through conventional microbiological cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DNA sequencing was used as a validity technique to ensure that all isolates within each group belonged to just one species, and to confirm the identified results of PCR-DGGE. Results indicated that a combination of conventional microbiological cultivation with PCR-DGGE and sequencing could successfully identify 4 yeast species from both types of cultures in Taiwan. Kluyveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with a distribution of 76, 22, and 2%, respectively, whereas Klu. marxianus, Saccharomyces unisporus and P. fermentans were identified in viili starters corresponding to 58, 11, and 31% of the total cell counts, respectively. Furthermore, the culture-independent method was applied to identify the yeast species using DGGE. Only 2 yeast species, Klu. marxianus and S. turicensis, were found in kefir grains and 2, Klu. marxianus and P. fermentans, in viili starters. These results suggest that in samples containing multiple species, PCR-DGGE may fail to detect some species. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under accession nos. DQ139802, AF398485, DQ377652, and AY007920.

Lactobacillus kefiranofaciens M1 isolated from milk kefir grains ameliorates experimental colitis in vitro and in vivo

Lactobacillus kefiranofaciens M1, isolated from and identified in Taiwanese milk kefir grain, has demonstrated immune-modulating activity. In the present study, we further investigated the effects of Lb. kefiranofaciens M1 on intestinal epithelial cells in vitro and on dextran sodium sulfate (DSS)-induced colitis in vivo. The possible mechanisms regarding the cytokine products and intestinal epithelial barrier restoration as well as the putative receptor for the protective effects of Lb. kefiranofaciens M1 were investigated. In vitro results indicated that Lb. kefiranofaciens M1 could strengthen the epithelial barrier function in vitro by increasing the transepithelial electrical resistance (TEER) and significantly upregulated the level of the chemokine CCL-20 at both the apical and basolateral sites. The in vivo effects of Lb. kefiranofaciens M1 on the regulation of intestinal physiology indicate that this strain could ameliorate DSS-induced colitis with a significant attenuation of the bleeding score and colon length shortening. Production of proinflammatory cytokines was decreased and that of the antiinflammatory cytokine IL-10 was increased in the DSS-treated mice given Lb. kefiranofaciens M1. The putative receptor for the protective effects of Lb. kefiranofaciens M1 was toll-like receptor 2 (TLR2), which was involved in probiotic-induced cytokine production in vitro and in attenuation of the bleeding score and colon length shortening in vivo. In this study, the kefir lactobacillus Lb. kefiranofaciens M1 clearly demonstrated an anticolitis effect. Based on these results, Lb. kefiranofaciens M1 has the potential to be applied in fermented dairy products as an alternative therapy for intestinal disorders.

The antiallergic effect of kefir Lactobacilli.

This study demonstrated that oral feeding of heat-inactivated Lactobacillus (L b.) kefiranofaciens M1 from kefir grains effectively inhibited immunoglobulin (Ig) E production in response to ovalbumin (OVA) in vivo. The pattern of cytokine production by splenocyte cells revealed that the levels of cytokines produced by T helper (Th) 1 cells increased, and those of cytokines produced by Th2 cells decreased in the heat-inactivated M1 feeding group. These findings indicated that Lactobacillus kefiranofaciens M1 in the kefir played an important role in antiallergic activities. By additional analysis using flow cytometry and microarray, the mechanism of suppression of IgE production by oral feeding of the heat-inactivated M1 probably occurs because of upregulation of the expression of Cd2, Stat4, and Ifnr leading to skewing the Th1/Th2 balance toward Th1 dominance, elevation of the CD4(+)CD25(+) regulatory T (Treg) percentage, and reduction of activated CD19(+) B cells. Downregulation of complement system and components was also involved in suppression of IgE production. Practical Application: Kefir has long been considered good for health. Its health benefits include immunoregulatory effects. However, there is a lack of knowledge concerning the immunoregulatory effects induced by kefir lactic acid bacteria (LAB). Our data clearly demonstrated the antiallergic activity of kefir LAB, Lactobacillus (L b.) kefiranofaciens M1. By additional analysis using flow cytometry and microarray, the possible mechanism of suppression of IgE production by oral feeding of the heat-inactivated M1 was also elucidated. Our findings indicated that Lactobacillus kefiranofaciens M1 may have a great potential for utilization in functional food products.

Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity

A thermally stable and alkalophilic xylanase, XynCDBFV, from Neocallimastix patriciarum was overexpressed in Escherichia coli as a recombinant protein fused to the N-terminus of oleosin, a unique structural protein of seed oil bodies. As a result of the reconstitution of the artificial oil bodies (AOBs), the immobilization of active xylanase was accomplished. Response surface methodology (RSM) was employed for the optimization of the immobilized xylanase activity. The central composite design (CCD) and regression analysis methods were effective for determination of optimized temperature and pH conditions for the AOB-immobilized XynCDBFV. The optimal condition for the highest immobilized xylanase activity (3.93IU/mg of total protein) was observed at 59 degrees C and pH 6.0. Further, AOB-immobilized XynCDBFV retained 50% of its maximal activity after 120min at 60 degrees C, and it could be easily and simply recovered from the surface of the solution by brief centrifugation, and could be reused eight times while retaining more than 60% of its activity. These results proved it is a simple and effective method for direct immobilization of xylanases.

Effects of cow's and goat's milk as fermentation media on the microbial ecology of sugary kefir grains.

In the present study, we have investigated the importance of fermentation media on grain formation and the microbial characteristics of sugary kefir. The sugary kefir grains were fermented in brown sugar, cows milk or goats milk. Using culture-dependent and culture-independent methods, we identified the microorganisms present in both the grains and filtrate and then evaluated their distribution. The structure of the grains was also observed by scanning electronic microscopy (SEM). The identification results indicated that there were remarkable changes in microbial ecological profiles of the sugary kefir grains and their filtrates when brown sugar and milk were compared as fermentation media. Three lactic acid bacteria (LAB) species (Leuconostoc mesenteroides, Lactobacillus mali and Lactobacillus hordei) were found in the grains fermented using brown sugar. However, four species, named Leu. mesenteroides, Lactococcus lactis, Bifidobacterium psychraerophilum and Enterococcus faecalis, were identified in the grains fermented using either cows or goats milk. The size and structure of the kefir grains were also significantly influenced by the culture medium. We hypothesize that the grains originally may contain many different microorganisms and the identified changes are an adaption to each specific medium during grain formation and growth. The distribution of strains thus may vary depending on the carbon and energy sources available for grain fermentation and these microbial changes will further affect the granulation and growth of the grains. This study is important to our understanding of the mechanism of kefir grain formation and growth because it explores the relationship between fermentation media and kefir microorganisms.

Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains

In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.

Investigation of microorganisms involved in biosynthesis of the kefir grain.

The purpose of this study was to understand the significance of each microorganism in grain formation by evaluating their microbial aggregation and cell surface properties during co-aggregation of LAB and yeasts together with an investigation of biofilm formation. Non-grain forming strains from viili were also evaluated as a comparison. Results indicated that the kefir grain strains, Lactobacillus kefiranofaciens and Saccharomyces turicensis possess strong auto-aggregation ability and that Lactobacillus kefiri shows significant biofilm formation properties. Significant co-aggregation was noted when S. turicensis and kefir LAB strains (Lb. kefiranofaciens and Lb. kefiri) were co-cultured. Most of the tested LAB strains are hydrophilic and had a negative charge on their cell surface. Only the kefir LAB strains, Lb. kefiranofaciens HL1 and Lb. kefiri HL2, possessed very high hydrophobicity and had a positive cell surface charge at pH 4.2. In contrast, the LAB and yeasts in viili did not show any significant self-aggregation or biofilm formation. Based on the above results, we propose that grain formation begins with the self-aggregation of Lb. kefiranofaciens and S. turicensis to form small granules. At this point, the biofilm producer, Lb. kefiri, then begins to attach to the surface of granules and co-aggregates with other organisms and components in the milk to form the grains. On sub-culturing, more organisms attach to the grains resulting in grain growth. When investigated by scanning electron microscopy, it was found that short-chain lactobacilli such as Lb. kefiri occupy the surface, while long-chain lactobacilli such as Lb. kefiranofaciens have aggregated towards the center of the kefir grains. These findings agree with the above hypothesis on the formation of grains. Taken together, this study demonstrates the importance of cell surface properties together with fermentation conditions to the formation of grains in kefir.