Sheng-Yao Wang

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.

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.

Antitumor Activity of Milk Kefir and Soy Milk Kefir in Tumor-Bearing Mice

Abstract: The effects of oral administration of milk and soy milk kefirs on tumor growth in tumor-bearing mice and the mucosal immunoglobulin A response in mice were studied. Oral administration of milk and soy milk kefirs to mice inoculated with sarcoma 180 tumor cells resulted in 64.8% and 70.9% inhibition of tumor growth, respectively, compared with controls. In addition, oral administration of the two kefir types induced apoptotic tumor cell lysis. Total immunoglobulin A levels for tissue extracts from the wall of the small intestine were also significantly higher for mice fed a milk kefir or a soy milk kefir regimen for 30 days. These results suggest that milk and soy milk kefirs may be considered among the more promising food components in terms of cancer prevention and enhancement of mucosal resistance to gastrointestinal infection.

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.

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.

The Antiinfective Effects of Velvet Antler of Formosan Sambar Deer (Cervus unicolor swinhoei) on Staphylococcus aureus-Infected Mice

We assayed the effects of velvet antler (VA) of Formosan sambar deer (Cervus unicolor swinhoei) and its extracts on the anti-infective activity against pathogenic Staphylococcus aureus in vitro and in vivo in this study. In vitro data indicated that the VA extracts stimulated the proliferation of resting splenocytes and macrophages in a dose-dependent manner up to the highest concentration used (150 μg mL−1). The production of proinflammatory cytokines (TNF-α, IL-6, IL-12) by lipoteichoic acid was significantly suppressed after being cocultured with the VA extracts in a dose-dependent manner. Animal test in S. aureus-infected mice demonstrated that the numbers of bacteria determined in the kidneys and peritoneal lavage fluid of S. aureus-infected mice were significantly higher than those found in the same organs of mice pretreated with the VA samples. Moreover, the highly enhanced phagocytic activity of macrophages was further verified after in vitro treatment with the VA samples. The protective mechanisms of the VA samples might include an immune enhancer and an inflammatory cytokine suppressor.

Effects of a novel encapsulating technique on the temperature tolerance and anti-colitis activity of the probiotic bacterium Lactobacillus kefiranofaciens M1.

Lactobacillus kefiranofaciens M1 (M1) has been shown to possess many different beneficial health effects including anti-colitis activity. The purpose of this study was to develop a novel and easily scaled-up encapsulating technique that would improve the temperature tolerance of the bacterium and reduce the sensitivity of the organism to gastrointestinal fluid. A mixture of sodium alginate, gellan gum and skim milk powder was used as a coating material to entrap M1. The M1 gel was then directly freeze dried in order to dehydrate the covering and form microcapsules. The viable cell numbers of M1 present only dropped ten folds after the freeze-drying encapsulation process. The viable cell counts remained constant at 5 × 10(7) CFU/g after heating from 25 °C to 75 °C and holding at 75 °C for 1 min. The viable cell counts were reduced to 10(6) CFU/g and 10(5) CFU/g after 8-week storage at 4 °C and subsequent heat treatment with simulated gastrointestinal fluid test (SGFT) and bile salts, respectively. The effect of encapsulated M1 on the organisms anti-colitis activity was evaluated using the dextran sodium sulfate (DSS) induced colitis mouse model. An in vivo study indicated that administration of heat treated encapsulated M1 was able to ameliorate DSS-induced colitis producing a significant reduction in the bleeding score and an attenuation of inflammatory score. These findings clearly demonstrate that encapsulation of M1 using this novel technique is able to provide good protection from temperature changes and SGFT treatment and also does not affect the organisms anti-colitis activity.

Identification of lactic acid bacteria in Taiwanese ropy fermented milk and evaluation of their microbial ecology in bovine and caprine milk

The purpose of this study was to identify species of lactic acid bacteria in Taiwanese ropy fermented milk and to study their microbial dynamics during the fermentation process through conventional microbiological cultivation and PCR-denaturing gradient gel electrophoresis. Identification results indicated that Lactococcus lactis ssp. cremoris and Leuconostoc mesenteroides ssp. mesenteroides were the major lactic acid bacteria in Taiwanese ropy fermented milk. Interestingly, 3 groups were identified as Lc. lactis ssp. cremoris using 16S rDNA sequencing, but they showed different denaturing gradient gel electrophoresis patterns and assimilation of carbohydrates. In addition, the microbial dynamics study in different fermentation stages demonstrated that Lc. lactis ssp. cremoris was the most dominant bacterial species in the samples, followed by Leu. mesenteroides ssp. mesenteroides with no differences among the fermentation stages. Finally, the microbial distribution profiles showed that the microbial ecology was different in bovine, caprine, and reconstituted milk, which might further affect the characteristics of the product.