Microflora Analysis Of The Fermentation Process Of Nata De Coco And The Effect Of Co-culture On Bacterial Cellulose Synthesis

Nata de Coco is a kind of milky white translucent cellulose gel material made by microbial fermentation of coconut water as the main raw material.Bacterial Cellulose is used in the food industry.BC has high purity,less impurities,good physical properties and is widely used.At home and abroad research on mixed bacteria fermentation production of bacterial cellulose is less,and Nata de Coco species used in the actual production is a system of mixed bacteria.And to find out the influence of other microorganisms co-culture of BC synthetic function.First of all,this study tried to collected several batches of coconut fiber strains of production,the comparing with pure Y19 fermentation production of BC capacity determination.After that,the microorganisms were isolated and purified,and the strains with high BC production and the "mixed bacteria" without BC production were obtained.Through the passage activation of the refrigerated production strains,the BC synthesis and the related physicochemical components in the fermentation process of the coconuts were analyzed to select the high-yield and stable representative passage strains.Then,based on the metagenomic sequencing technology,the bacterial facies in the fermentation process of the representative passage strains were analyzed.The influence of the "mixed bacteria" isolated above on BC synthesis was verified by co-culture,so as to find the co-culture microorganisms that have synergistic or promoting effects on BC synthesis,and to provide early reference for further analyzing the mechanism of co-culture of different microorganisms to promote or inhibit BC synthesis.The main results of this study are as follows:(1)BC capacity of different batch production strains and the isolation and identification of high yield BC strainsDue to the possibility,different batches of caducea production bacteria were collected from Ben Tre in Vietnam and Hainan in China in different periods,and the results showed that BC productivity of different caducea production bacteria differed significantly.In Vietnam,Xiong,Dang,Ou and Yu were different in their ability to synthesize BC.Xiong and Yu had higher ability to synthesize BC,and the yield of BC was 11.18 g/L and 7.67 g/L,while Dang and Ou had lower ability to synthesize BC.Xia,HK,Qb and Dq have different BC synthesis abilities,among which Xia,HK and Qb have better BC synthesis abilities,and the BC production of Dq species is 9.47 g/L,9.42 g/L and 7.82 g/L,respectively,while the BC production of Dq species is 5.43 g/L after 7 days of fermentation.Four strains with high BC yield,HK8,Q2,Q8 and D8,were screened by traditional plate isolation and purification method from different batches of HK,Qb and Dq strains from Hainan Province.and their BC yield was 6.66 g/L,5.36 g/L,5.40 g/L and 3.60 g/L,respectively.The four strains were identified as Komagataeibacter nataicola,belonging to the same species as pure strain Y19(BC yield of 6.31 g/L).In conclusion,the ability of the isolated high-yielding strains to synthesize BC could not reach the level of the original production strains,which preliminarily indicated that other microorganisms in the actual factory environment may have a great influence on the microbial composition of the production strains,thus affecting the synthesis of BC.(2)The yield and physicochemical composition analysis of BC fermentation process of subcultured speciesDue to the limitation of objective factors,after the subculture of the refrigerated strains of coconuts in the laboratory,the BC production and physicochemical composition of the subcultured strains in the fermentation process of Qa,Qb B,Dq and Dh were determined.It was found that the BC production of different subcultured strains was significantly different,and no special changes were shown in the physicochemical composition.After 7 days of fermentation,the dry weight of Qa-2 and Qb-2 were 3.43 g/L and 7.0 g/L,respectively.It was found that the BC productivity of Qb strain was still higher than that of pure strain Y19 after several passage activation.(3)Microbial facies analysis of the fermentation process of the passage speciesIn order to accurately analyze the microflora of the passed species,the membrane filtration method was used to treat the natural fermentation coconut water as the medium.There was no significant difference between the BC fermentation of the passed species and that of the untreated medium.The treatment could remove the microflora in the medium to avoid the interference of the microbial flora determination in the fermentation process.In order to analyze the microorganism composition and its influence on BC synthesis in the fermentation system,the BC fermentation process of Qb passing strains was analyzed by using Qa passing strains as the control.It was found that Lactobacillus was the dominant microorganisms in the fermentation process,followed by Komagataeibacter,which accounted for about 99.9% of the total sequence relative abundance,among which the relative abundance of Lactobacillus was about 91.0%,and that of Komagataeibacter was about 8.9%.In the fermentation process,the relative abundance of the two passed species of Komagataeibacter decreased at first and then increased,and the relative abundance of the passed species Qb-2 decreased to 1.78% after 5 days of fermentation.At the species level,the top five species richness in the samples were Lactobacillus plantarum,Komagataeibacter nataicola,Lactobacillus sp.CBA3605,Klebsiella pneumoniae and Enterobacter cloacae.The sum of relative abundance of top5 strains was more than 99.99%,and the species abundance of L.plantum and K.nataicola accounted for about 99.95% of the total sequence.The preliminary results showed that after the secondary passage of Qa and Qb,the BC fermentation process basically only lactobacillus this kind of "mixed bacteria".The correlation analysis showed that the relative abundance of L.plantarum was positively correlated with the increase of BC,suggesting that L.plantarum might promote the synthesis of BC by K.nataicola.There was a negative correlation between yeast and BC increment,which suggested that the metabolic activities and metabolites of yeast might play an inhibitory role on BC synthesis.(4)Preliminary study on the synthesis of BC by co-cultureThe above results preliminarily indicated that the fermentation system for the synthesis of BC from coconut water was a reasonable mixed bacterial system.Therefore,18 isolates were obtained by traditional isolation and purification of the "mixed bacteria" that did not produce BC from Qb-3,which had undergone three passage cultures.The results showed that the co-culture of Q211 and Q216 with K.nataicola Q8 could significantly promote the synthesis of BC,and the co-culture yield of BC was 7.88 g/L and 7.74 g/L,respectively.Compared with the control group(single fermentation),Q216 had a more stable promoting effect on Q8,which was 1.26 and 1.23 times higher.L.plantarum Q216 promoted the synthesis of BC by K.nataicola Q2 and Q8,and the yield of BC was increased by more than1.2 times.The co-culture of other lactic acid bacteria in the laboratory with K.nataicola Q2 and Q8 showed that L.plantoplanum L16 and L.fermentata L20 promoted the synthesis of BC by Q2 and Q8,and increased the BC yield by 1.3 times.When yeast was co-cultured with K.nataicola Q2 and Q8,yeast did not show the ability to promote BC synthesis under the inoculation amount and culture conditions in this experiment.