Changes In Microbial Community Structure During The Processing Of Black Garlic And Its Effect On Quality Formation

Black garlic is a deep-processed garlic product formed by processing fresh garlic under high temperature and controlled humidity conditions,with black appearance,soft and elastic texture,sweet and sour taste,unique flavor and remarkable biological activity.Previous studies have found that non-enzymatic browning reactions(mainly Maillard reaction),degradation of macromolecules and oxidation reactions were responsible for the formation of black garlic,there are few studies on whether microbes are involved in this process and what role they play.At present,the production of black garlic by "high-temperature maturation" suffer from high energy consumption and unstable quality and safety indexes,and it remains to be studied whether microbes can be used to accelerate the process and improve the product quality.This study followed route of "analysis of microbial community structure-characterization of endophytic strains-influence on the formation of black garlic quality-potential mechanisms".First,the microbes in black garlic processing were investigated by culture-dependent approaches combined with multi-locus phylogenetic tree and Illumina high-throughput sequencing technology to clarify the structure and change patterns of microbial communities.Subsequently,the growth features and fermentation characteristics of the dominant endophytes isolated from black garlic processing were analyzed to elucidate their influence on the processing process and quality formation during the natural fermentation of black garlic.Finally,the production conditions,isolation and purification methods,enzymatic characteristics and the effect on the Maillard reaction of fructan hydrolase secreted by endophytic bacteria were studied to reveal the mechanism of microbial influence on quality formation of black garlic.The main findings are as follows.1.Isolation and identification of endophytic bacteria from garlic during black garlic processingSeven kinds of Bacillus species were identified from garlic and black garlic,respectively,according to morphological characteristics,physiological and biochemical properties and phylogenetic analysis.Bacillus subtilis and B.methylotrophicus were the dominant strains in garlic and black garlic.The total bacteria and endophytes showed a trend of a sharp decrease and then a rapid rise,finally a slow decrease during black garlic processing.Based on the phenotypic characterization combined with the gyr A-rpo B-16 S r DNA multi-locus phylogenetic tree,a total of 77 garlic endophytes were successfully identified,with B.subtilis,B.methylotrophicus,B.amyloliquefaciens and B.licheniformis being the dominant strains during black garlic processing by culture-dependent approaches.Therefore,the garlic endophytes were present in the processing of black garlic.2.Microbial community structure and change pattern in black garlic processingThe Illumina Mi Seq sequencing technology was performed to sequence the bacterial 16 S r DNA V3�V4 hypervariable region of black garlic processing samples,producing a total of1,247 OTUs from 20 samples.Alpha diversity of the samples underwent a gradual increase with increasing processing time,reaching a maximum on Day 8 and Day 10 and then decreasing slightly.Taxonomic analysis indicated that bacterial community was classified into45 phyla and 1125 distinct genera,which was mainly dominated by four phyla(Proteobacteria,Firmicutes,Actinobacteria,Deinococcus-Thermus)and four genera(Thermus,Corynebacterium,Streptococcus,Brevundimonas).It should be noted that Bacillus also had a higher proportion.Pearson correlation analysis revealed that processing temperature and chemical composition of black garlic(e.g.allicin,reducing sugars,total phenols,and total acids)significantly affected the microbial community structure.3.Growth features and fermentation characteristics of the dominant garlic endophytes and their influence on the formation of black garlic qualityFour dominant Bacillus species selected from the garlic endophytes isolated during black garlic processing had similar growth curves and exhibited some heat tolerance(more than 7Lg CFU/m L of vegetative cells at 50 �C)and a wide p H growth range of 5�9.Allicin and5-HMF produced during black garlic processing resulted in a decrease in the growth density of 7.45%�69.73% and 17.78%�30.68% for the four strains,respectively,where B.subtilis was highly tolerant.All four strains were found to possess neutral protease(7.70�31.91 U/m L),alkaline protease(3.32�16.72 U/m L)and fructan hydrolase(0.65�0.81 U/m L)activities,with B.subtilis having the strongest enzyme secretion capacity.In the simulated natural fermentation of black garlic,the inoculation of B.subtilis,B.methylotrophicus and B.amyloliquefaciens accelerated the browning rate and enhanced the browning intensity(0.81%�2.78%),while B.licheniformis slowed down the processing of black garlic.This suggested that the dominant garlic endophytes influenced the formation of black garlic quality.Compared to the control group,the reducing sugar content of black garlic samples fermented by B.subtilis,B.methylotrophicus and B.amyloliquefaciens significantly increased by27.70%�99.47%,which might be the main reason for their accelerated browning rate and enhanced black garlic quality formation.Multivariate analysis revealed that the black garlic samples at different processing times were clearly distinguished from one another,but the differences caused by inoculation of different strains were limited.4.Isolation and purification of fructan hydrolase secreted by B.subtilis and enzymatic characterizationThe type of medium,carbon source,nitrogen source,incubation temperature and p H,fructan concentration and incubation time had significant effects on the fructan hydrolase activity of four dominant endophytes.After optimization of the culture conditions,B.subtilis with the strongest fructan hydrolase-producing activity was used for enzyme production and purification.A total of three purified fructan hydrolases(E1,E2 and E3)was obtained from the fermentation supernatant of B.subtilis via gradient precipitation with saturated ammonium sulfate and purification by Sephadex G-100 gel column,with specific activities of 270.24U/mg,168.91 U/mg and 202.68 U/mg,respectively.Three purified enzymes were identified as exo-hydrolases,with molecular weights of 92 k Da,20 k Da and 10 k Da,respectively.E1 had high thermal and p H stability,the smallest Km value(42.55 mg/m L)and the largest Vmax value(15.77 U/m L),and its activity was affected by some metal ions,chemical reagents and proteases.Molecular docking analysis revealed that the interaction between exo-fructan hydrolase and fructan was achieved by hydrogen bonding,with a total of six amino acid residues within 3 ? from the fructan ligand molecule in the substrate binding pocket.5.Effect of fructan hydrolase secreted by B.subtilis on Maillard reaction and potential mechanismThe effects of three purified fructan hydrolases(E1,E2 and E3)on the rate and intensity of the Maillard reaction were investigated based on a fructan-histidine model.Compared with the control group,the browning intensity of the Maillard reaction model significantly enhanced by 3.60-,2.00-and 1.27-fold at 15 d of heat processing,and the melanoidin content greatly increased by 4.28-,1.74-and 1.33-fold,respectively.Chemical composition analysis showed that the three fructan hydrolases significantly increased the reducing sugar content of the Maillard reaction model by 0.62-to 2.69-fold and remarkably decreased the histidine content by 2.42%�40.71%,accompanied by great changes in 5-HMF level,p H,UV absorption and fluorescence spectra.Taken together,dominant garlic endophytes such as B.subtilis could promote the degradation of polysaccharides and thus accelerate the browning of black garlic through fructan hydrolase and promote the formation of black garlic quality.