Isolation,Identification Of Microorganisms And Screening Of Optimal Strains Combination For Degradading Tannins From Chinese Gall Leaven Samples During Its Processing

Processed Chinese gall leaven is a traditional Chinese herbal made from the natural fermentation of the gall,tea and wine lees.It is found that Processed Chinese gall leaven could be used to moisten lung,resolve phlegm,relieve heat,and treat chronic cough,hematochezia,hemorrhoidal bleeding,skin eczema and so on.The main raw material of processed Chinese gall leaven is gallnut that contains a large amount of tannins with great hepatotoxicity,while the gallic acid produced by fermentation exhibits a variety of activities including antibacterial,antiviral,trypanosomiasis,hypoglycemic and lipid-lowering activities.The traditional concoction method of Chinese gall leaven is mixed solid fermentation by a variety of natural microorganisms,mostly mold and bacteria.The quality control of processed Chinese gall leaven is limited because the concentration of gallic acid in the Chinese gall leaven matrix is not easily regulated,due to the impure microorganisms of the natural fermentation strains,the lack of specificity,and the fluctuations in the number and specie of bacteria involved in the fermentation.Therefore,it is necessary to isolate,identify the key microflora,and to explore a preliminary best strain combination in the process of modern processed Chinese gall leaven concoction,which will help establish the processing technology of modern identified hybrid fermentation of Chinese gall leaven,and provide the strain foundation for industrialized production of it.In this paper,the classic methods of microbial separation and classification were used to isolate the microorganisms from the traditional processed Chinese gall leaven.The preliminary identification was carried out combined with the morphological characteristics,physiological and biochemical characteristics,and 16S rRNA/18S rRNA molecular biological characteristics of the strains.Meanwhile,the strains’antibacterial activities,protease production,amylase capacity,and tannin transformation characteristics were investigated preliminarily.Further,microorganisms with the ability to degrade tannins were chose as screening strains for single strain fermentation and mixed strain fermentation,in order to seek the single strain as well as the best mixed strains combination with the ability to enhance the ability of degrading tannins to gallic acid solely or synergistically.The main results as follows:1.17 strains were isolated and purified from the samples prepared by the traditional method of processing.After getting rid of the same strains,these strains were identified as:7 bacterial strains including Bacillus licheniformis,Bacillus subtilis,Bacillus cereus,Bacillus licheniformis,Bacillus Bacteria,Bacillus megaterium strain,Bacillus streptoceli and Bacillus spp.;7 yeast strains including Pichia viridis,Kluyveromyces strain,Kluyveromyces strain,Ferbin yeast,Omecoda,Abnormal Wilkhamomyces,and Abnormal Pichia pastoris;and 3 fungal strains including Penicillium fungi,Melaleuca mucor,and Aspergillus niger.The pathogenic strains were not isolated in the samples.2.Antibacterial activities of these strains were screened.The results showed that strains HMB3,HMB4,HMB7and HMY3 were active against Staphylococcus aureus.Meanwhile,the strains HMB3 and HMB7 were active against E.coli.Only the bacteria from the processed Chinese gall leaven could produce extracellular proteases.All the strains had no ability to produce extracellular amylase.3.13 strains with degrading tannins were obtained by primary screening,including 5 strains of bacteria(HMB1,HMB2,HMB3,HMB5,HMB6),5 strains of yeast(HMY1,HMY2,HMY5,HMY6,HMY7)and 3 strains of filamentous fungi(HMF1,HMF2,HMF3).4.Single-bacterial fermentation and mixed-bacterial fermentation were carried out for the microorganisms capable of degrading tannins,respectively.HPLC method was used to detect gallic acid or tannin in the fermentation broth.The best mixed bacterial combinations HMB2,HMB5,HMY1,and HMY2 with the ability to synergistically degrade tannin-produced gallic acid were obtained by screening.After the optimization of the best mixed bacteria combination in the solid state fermentation process,we conclude that:at a substrate concentration of 50%,inoculum size of 1.2 mL,bacterial and yeast inoculation ratio of 1:2,and incubation at 30℃ for 66 h,the amount of gallic acid in the sample of traditional processed was 0.52 g per gram of Chinese gall leaven,,while after the solid-state fermentation of the best mixed strain combination was about 0.59 g,which increased by about 11%.Importantly,the antibacterial activity against Staphylococcus aureus after the best mixed strain combination fermentation was better than that of traditional processed Chinese gall leaven.The optimal bacterial and yeast strains combinations HMB2,HMB5,HMY1,and HMY2 were obtained by screening,providing a foundation for controlled fermentable strains for the modern industrial production of processed Chinese gall leaven and reducing the possibilities of toxin residues caused by mold contamination during fermentation process.