Screening And Identification Of Metabolites That Promote Bacterial Cellulose Synthesis In Purebred Inoculated Pre-fermented Coconut Water

Bacterial Cellulose（BC）is coconut fruit and coconut fiber fruit,it is a high-purity glucose polymer synthesized by cellulose fermentation with cellulose-producing bacteria（such as Komagataeibacter）.It has excellent performance and is widely used.In recent years,research hotspots of BC have focused on replacing expensive synthetic culture media with agricultural and industrial wastes as carbon sources.Among them,the study of using coconut water as a raw material for BC production has a long history and has achieved large-scale production and commercial application.Long-term production practice proves that coconut water is the most important and successful raw material for BC production.Especially,fresh coconut water（NCW）was exposed to the air for several days（pre-fermentation）to become fermented coconut water and then formulated into a medium for BC production,which is several times,ten times or even twenty times higher than the output of NCW.It even exceeds the BC yield after adding various synergistic factors to the basic medium（such as HS medium）.This study found that some yeast isolated from natural pre-fermented coconut water was inoculated into NCW for the pre-fermentation process,and the coconut water obtained had the same effect on promoting BC synthesis as the natural pre-fermented coconut water in the laboratory.Motivated by this fact,the high-performance liquid chromatography-mass spectrometry（UPLC-MS/MS）detection technology and the multivariate statistical analysis of the metabolomics method were used to analyze and screen the differential metabolites between pre-fermented coconut water and NCW inoculated with purebred.The effect of some metabolites on the synthesis of BC by K.nataicola Y19 was verified by adding compounds exogenously.It is intended to identify the key differential metabolites that have a significant promotion effect on BC synthesis.The main results are showed as followings:（1）Effect of inoculation with pure fermented coconut water on BC synthesisInoculating a single strain and its combined strains isolated from natural pre-fermented coconut water into NCW for pre-fermentation,compared with natural pre-fermentation,the yield of BC synthesized by pre-fermented coconut water medium inoculated with 25 strains of yeast,9 strains of Lactobacillus and 30 groups of combined bacteria was studied.The results showed that the BC yield of pre-fermented coconut water culture medium of yeast strains was generally higher than that of Lactobacillus and combination bacteria strains.The BC yield of 1-day pre-fermentation of pure yeast strains was higher than that of 3d,5d,and 7d.The 74-1,1-7-1,21-1,142-2,Y33 and 84-3pre-fermented BC yields can reach 7-8 g/L for 1 day,which is about 14 times that of NCW in the negative control fresh coconut water.This preliminary indicates that the BC yield of pre-fermented coconut water inoculated with some yeast purebreds can be comparable to that of natural pre-fermented coconut water in a laboratory environment.It is speculated that the screening of differential metabolites based on coconut water pre-fermented by these strains is feasible.In this study,the strains 84-1 and 84-3,which have the highest promotion amplitude and stable promotion effect after multiple experiments,were selected for subsequent research.（2）Membrane filtration method explores the reasons for promoting BC synthesis in pre-fermented coconut waterOn the basis of not destroying the components by heating,the bacteria in the pre-fermented coconut water were separated from the supernatant by membrane filtration and sterilization,and it was investigated whether the bacteria liquid or bacteria in the pre-fermented coconut water had a promoting effect on BC synthesis.The results showed that the BC production of the culture medium of the supernatant group was equivalent to that of the untreated pre-fermented coconut water,and there was no significant difference（P>0.01）.Although the BC production of the bacteria precipitation group has a promoting effect compared with the control group NCW,the promotion degree is significantly weaker than that of the supernatant group.The reason for the promotion may be that the bacteria fragments provide the nutrients such as the growth factors required for BC fermentation synthesis.The results revealed that during the pre-fermentation of coconut water,the metabolites of the bacteria played the most important role in promoting BC synthesis.It is speculated that the pre-fermentation process of coconut water produces metabolites that have important regulatory effects on BC synthesis.（3）Re-identification of the preferred strains 84-1 and 84-3Because the selected strains 84-1 and 84-3 had different identification conclusions,they were again identified by ITS sequencing.The results of NCBI Blast comparison showed that strain 84-1 and Saccharomyces cerevisiae YJM681 had the closest parental relationship with 99%homology,The strain 84-1 was initially identified as Saccharomyces cerevisiae.Strain 84-3 was recently related to Saccharomyces cerevisiae（baker’s yeast）,strain 84-3 was initially identified as Saccharomyces cerevisiae.（4）Screening of metabolites that promote BC synthesis in pre-fermented coconut waterBased on UPLC-MS/MS detection technology,S.cerevisiae 84-1 and S.cerevisiae84-3 purebreds were inoculated with coconut water pre-fermented for 1 and 7 days（samples are designated Y84-1-1d,Y84-1-7d,Y84-3-1d and Y84-3-7d）.The results showed that a total of 296 metabolites were identified.Through cluster analysis and principal component analysis,NCW could be clearly separated from the other four groups of pre-fermented coconut water samples.The prediction parameters of the OPLS-DA model of each comparison group show that the model is reliable.The variable metabolites were screened using the VIP value and the Fold Change value.The Venn diagram was used to significantly compare the comparison groups.Cross-screening of sexually different metabolites.The screening threshold was set to VIP>1 and Log₂FC≧8 and Log₂FC≦0.125.Finally,the control group and the treatment group（NCW vs Y84-1-1d,NCW vs Y84-3-1d,NCW vs Y84-1-7d and NCW vs Y84-3-7d）17 common significant metabolites were screened out from the four groups,from the treatment group（Y84-1-1d vs Y84-1-7d and Y84-3-1d vs Y84-3-7d）four common significant metabolites were screened out in the two groups.At the same time,from a macro and indirect perspective,conventional methods were used to monitor the changes in the physicochemical components of the coconut water pre-fermentation process（1-7d）.The results showed that changes in ethanol and organic acid content had the greatest effect on Y19 synthesis of BC.（5）Validation of the effect of adding some metabolites on the synthesis of BC by K.nataicola Y19The six differential metabolites screened by metabolomics and the three common metabolite components that promote BC synthesis in combination with indirect physicochemical tests were verified by the method of external addition to NCW medium The results are used as reference)to identify its effect on the regulation of BC synthesis by K.nataicola Y19.The results showed that 0.5%（w/v）L-（-）-3-phenyllactic acid,0.25%（w/v）,and 0.5%（w/v）N-α-acetyl-L-glutamine,0.25%（w/v）and 0.5%（w/v）serine and0.5%（w/v）3-（4-hydroxyphenyl）propionic acid have a significant promotion effect on the synthesis of BC,1%（w/v）Serine、2-indolecarboxylic acid and cyclic AMP have a very significant inhibitory effect on BC synthesis.When each concentration（0.25-2.0%）of ethanol,acetic acid,and lactic acid was added,they showed a promotion effect,but the promotion range showed a"dose-effect dependence"relationship.The BC output showed the largest increase at 1%addition,which was 7.5,10,7.7 times of the control.This shows that it is necessary to find substances that regulate the BC synthesis pathway of K.nataicola not only from different types of metabolites,but also from multiple dimensions such as common metabolite concentrations.