Microorganism Composition Analysis Of Kombucha By Metagenomic Technology And Preparation Of Lyophilized Its Powder

Kombucha is a kind of folk drink that is fermented by naturally occurring mixed microorganism such as acetic acid bacteria,yeast and lactic acid bacteria,and tea sugar water is used as a medium.It mainly contains two parts of bacteria liquid and bacteria membrane.The metagenomic technology was used to study the microbial flora and metabolic pathways in the fermentation process of Kombucha.This metagenomic raw data was deposited in GenBank under the accession SRP103682.Orthogonal design was used to optimize the fermentation conditions of Kombucha with 15%sucrose and 1%black tea as raw material:inoculum 3%bacteria liquid+5%bacteria membrane(g/100 mL),fermentation temperature 28℃,bottle diameter of 5 cm(liquid volume 100 mL/250 mL).Continue to cultivate for 10 days under these conditions,the total number of fungal colonies reached a maximum of 3.5×107 CFU/mL on the 3rd day of fermentation,and the brix of the fermentation broth was 14.9 Brix,pH 3.54,which was marked as stage I;the total number of bacterial colonies reached a maximum of 5.14×108 CFU/mL on the 7rd day of fermentation,and the brix of the fermentation broth was 13.9 Brix,pH 3.05,which was marked as stage Ⅱ.Microorganism flora of the Ⅰ and Ⅱ stages of Kombucha were analysised by metagenomic techniques,and 44491 and 93449 unigene were obtained respectively,which were compared with the database.At the I stage,a total of 96.18%of the sequences belonged to the procaryotae(63.46%)and the eukaryotic(32.72%).Komagataeibacter xylinus(8.91%),Komagataeibacter europaeus(8.42%),Komagataeibacter intermedius(5.29%).Gluconacetobactersp.SXCC-1(5.08%)were identified as the dominant bacterium,at the same time,Pichia kudriavzevii(12.37%).Brettanomyces bruxellensis(9.42%),Ogataea parapolymorpha(5.06%)were identified as the dominant fungi;At the Ⅱ stage,a total of 99.97%of the sequences belonged to the procaryotae(96.57%)and the eukaryotic(3.40%).Gluconacetobacter sp.SXCC-1(29.17%),Komagataeibacter medellinensis(7.84%),Komagataeibacter xylinus(7.58%)were identified as the dominant bacterium,at the same time,Brettanomyces bruxellensis(1.86%),Ogataea parapolymorpha(0.90%),Pichia kudriavzevii(0.32%)were identified as the dominant fungi.The differences between samples were analyzed by Alpha diversity index(Shannon,Chaol),Shannon reduced from 9.53 in stage Ⅰ to 8.54 in stage Ⅱ,Chao1 increased from 49427 in stage I to 91300 in stage Ⅱ.The results show:the composition of microbial flora of the samples changed greatly,the uniformity of stage Ⅰ was higher than stage Ⅱ,while the total number of species of stage Ⅱ was higher than that in stage Ⅰ.Functional annotation and metabolic pathway of metagenome data were analysied.Unigene was aligned to each database by BLAST(E-value<1.0e-5).At the Ⅰ stage,it could be divided into 327 classes metabolic pathway,in which 548 Unigene were found to be related to amino acid metabolism;At the Ⅱ stage,it could be divided into 338 classes metabolic pathway,in which 2475 Unigene were found to be related to amino acid metabolism.In addition,26 secondary metabolic pathways were also detected,which 115 key enzymes were related to secondary metabolites of Kombucha.The microbial flora of Ⅰ and Ⅱ stage were collected,which were made as direct-fed Kombucha powder by vacuum freeze drying technology under the conditions:the ratio of cryoprotectant(trehalose+glycerol 1:1)and microbial mud at 3:1,equilibrium time 40 min and freeze-drying time 24 h.The survival rate of direct-fed Kombucha powder was 78.24%.The composition of direct-fed powder fermentation broth was analyzed,choose automatic amino acid analyzer analysis shows:the content of valine and phenylalanine of the essential amino acids of the 7rd day was higher than that of the primary medium.The two new amino acids were detected on the 7th day of fermentation:histidine and arginine.Histidine is an essential amino acid for children,which can reduce renal anemia.Arginine is an semi-essential amino acid,which can prevent high blood ammonia.The organic acids were determined by high performance liquid chromatography.The total content of organic acid increased from 4.05 g/L to 10.78 g/L,of which the content of gluconate acid was 6.13g/L and the content of acetic acid was 2.80 g/L.