| Fermentation broth of Kombucha is a traditional acidic beverage tea with many sorts of microbes such as yeast, lactic acid bacteria and acetic acid bacteria. Fermentation broth of Kombucha have many nutrient ingredients such as nutrient elements, live microorganisms and metabolites. The composition and concentration of microbial varies with different Kombucha. This study was explored systematically isolation and identification of predominant microbes, microbes phase, and changes of metabolites in fermentation, the optimization conditions of protein poly-acrylamide gel electrophoresis (PAGE) and anti-microbes mechanism in Hunan kombucha.The main results are as the follows:1 Isolation and identification of microbes from kombuchaPredominant microbes from kombucha were isolated and identifyied. they were as the follows: six strains of acetic acid bacteria including two strains of A.liquef aciens, a strain of A.aceti, two strains of A.diazot- rophicus, a strain of A.metha nolica. four strains of yeast such as Candida Krusei,Debaryomyces hansenii,Schizosaccharomyces pombeLinder ,Saccha-romyces cerivisiae. Lactic acid bacteria mainly as Lactobacillus A and Lactobacillus P. A.diazot- rophicus and A.metha nolica were found foremost from kombucha.2 Study on the microbes phase of kombuchaMicrobe cell quantities in fermented broth were more than microbe pellicle during the fermented kombucha. The quantity of acetic acid bacteria in fermented broth was seven times more than microbe pellicle at the sixth day after the fermented kombucha, and the quantity of yeast was twice more than that of microbe pellicle. The growth curve of acetic acid bacteria was similar with that of yeast. The quantity of lactic acid bacteria in fermented broth was lower than that of yeast and acetic acid bacteria. The logarithmic growth phase of growth curve of acetic acid bacteria was later than that of yeast and acetic acid bacteria.Acetic acid bacteria and yeast isolated from the Kombucha were cultured, respectively. This had found that they had typical growth curve. But the change of microbes quantity had not regularization mixed culture acetic acid bacteria and yeast, this microbes quantity was larger than that of Kombucha, and the pH value of fermented broth of acetic acid bacteria and yeast was higher than that of Kombucha.3 Forming microbe pellicle of the Kombucha and change of substances in fermented solutionForming microbe pellicle of the Kombucha had nearly correlated with sugar metabolism. The content of sugar in fermented broth was as main factor influenced forming microbe pellicle. The forming microbe pellicle quantity was markedly improved by added appropriate concentration of alcohol, yeast extract and peptone.Enlarged surface area of fermented broth was also beneficial to forming films, but it had not forming films when carbon source was only alcohol. The pH value of fermented broth was negative relativity with the quantity of forming films.The change of soluble solid content in fermented broth was decreased as the time of fermented Kombucha prolonged. The pH value was fast decreased at the early stage, and following held stabile. Total content of free amino acid was decreased from 70.494μg/mL to 27.439μg/mL. Many sorts of free amino-acid was brought when fermentation was mixed yeast Y3, acetic acid bacteria C64, lactic acid bacteria LA and lactic acid bacteria LP. The total epicatechin isomers, contents of EGCG,GCG and ECG were decreased respectively, but the contents of EGC, DL-C and EC were increased a little.4 Extraction of predominant microbes combination having anti-microbes activities in kombuchaThe combination of Schizosaccharomyces pombe Linder Y3, Acetic acid bacteria C64, Lactic acid bacteria LA and LP was the combination predominant microbes combination produced primarily substances of anti-microbes. Acetic acid was removed from fermented broth of acetic acid bacteria, which enhanced the growth of indicator bacteria. The strain of acetic acid bacteria C64 having weak enhancement was selected. Schizosaccharomyces pombe hinder Y3 and lactic acid bacteria had restrained the growth of indicator bacteria. The strains of lactic acid bacteria LA and lactic acid bacteria LP having strong inhibitory effect to E. coli and S. aureus were selected. The anti-microbes effect of fermentation was mixed acetic acid bacteria C64 and yeast Y3 was the best combination of microbes phase in kombucha. 5 the optimization conditions of PAGE for proteins in fermented broth of kombuchaThe optimization conditions of PAGE for proteins in fermented broth of kombucha were studied. The results were that the sample protein solution of PAGE was got after some optimization conditions such as regulated the pH6.0 of fermented broth, added 2% insoluble PVP into fermented broth, surged fermented broth 20min, centrifugated 15min at 4000 revolution per minute. Dialysised fermented broth 3 days in MD34 dialytic envelope and concentrated fermented broth at 4℃. The optimization electrophoresis conditions of PAGE were 11% separation gel.6 Anti-microbes mechanism of kombuchaThe fermented broth of kombucha played a decisive role in the anti-microbial activity because of the effect of acidic substances, anti-microbial proteins and tea poly-phenols. E. coli and S. aureus were choosed as indicator bacteria in this study. Different concentration of tea extractive substances, the pH value of fermented broth, dialytic solution of fermented broth of kombucha, and the anti-microbial effects after the dialytic solution treated by tyrpsin,catalase and proteinase K respectively, were studied. More than 1.0% tea solution concentration ,the fermented broth of kombucha restrained E. coli and S. aureus. The dialytic solution of the fermented broth of kombucha restrained indicator bacteria. The effects against E. coli and S. aureus after the dialytic solution treated by tyrpsin, catalase and proteinase K was markedly lowered. These had demonstrated that the anti-microbial proteins existed in the fermented broth of kombucha.
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