Application Of Selected Lactobacillus Plantarum In Fruit Wine-making And Characterization Of Immobilized Amine-degrading Enzyme

Biogenic amines（BA）are undesirable metabolites that are easily formed during the fruit wine-making process.Histamine is a typical representative of BA,with high content in fruit wine and the most toxic to human.Our previous result demonstrated that Lactobacillus plantarum has great potential in fruit wine,because of its effectively degradation of BA.In this study,the selected Lactobacillus plantarum with the ability to efficiently degrade BA was used as the initial strain to investigate the effect on the quality and biogenic amines content of black raspberry wine of the interaction between its and yeast.In addition,the whole genome of selected Lactobacillus plantarum was sequenced to explore its structural composition and functional genes,so as to find the functional genes related to the excellent performance during the fermentation process and the key genes related to degradation of biogenic amines.Furthermore,the heterologously expressed and purified glyceraldehyde 3-phosphate dehydrogenase（GAPDH）with histamine degrading activity was immobilized to investigate the enzymatic properties of the immobilized enzyme and its practical effect of degrading histamine in fruit wine.The main results are as follows:（1）To investigate various indexes of black raspberry wines obtained by sequential inoculation（SE）and simultaneous inoculation（CO）of self-screened Lactobacillus plantarum SGJ-24,aroma-producing yeast-T.delbrueckii Biodiva and Saccharomyces cerevisiae M2,including basic characteristics,volatile components,biogenic amines content and sensory evaluation.In a parallel experiment,spontaneous MLF（SP）was undertaken as a control.The results showed that the basic composition of black raspberry wines obtained by the three fermentation methods were all in line with the requirements of national standard,but the SP sample had the highest volatile acid concentration.The volatile components in black raspberry wines were analyzed by GC-IMS,and a total of 34typical volatile compounds were identified,including esters,alcohols,acids,ketones,aldehydes,and terpenes.The total aromatic content in the CO was the highest,followed by SE,and the SP was the lowest.The content of biogenic amines in wines was determined by HPLC,SP contained the most types of biogenic amines,and the total amount of biogenic amines in SP was the highest,followed by SE and CO.The QDA method was used to evaluate the sensory quality of the wine samples,and it was found that CO was characterized by high sense of‘fruity’and‘sweet’,and had the highest overall aroma score;SE was perceived with a medium sense of‘sweet’and‘floral’,and the overall aroma score was medium,while SP obtained the lowest rating.The results showed that the CO mode was the most favorable mode for the production of volatile aroma components in wine,while it was not easy to produce volatile acids and biogenic amines.It was further indicated that Lactobacillus plantarum SGJ-24 had positive effects on enhancing aroma complexity,reducing the content of biogenic amines and volatile acids in wines.（2）The whole genome sequence of Lactobacillus plantarum SGJ-24 was performed by de novo sequencing technology,and it was found that the genome size of the strain was3,451,191 bp.The complete genome sequence of L.plantarum SGJ-24 was composed of a circular and four plasmid,with 3361 coding sequences,68 t RNAs and 16 r RNAs,respectively.Functional annotation of the whole genome of L.plantarum SGJ-24 showed that there were a large number of coding genes associated with metabolism,replication,recombination and repair,redox and carbohydrate transport metabolism.In addition,genes such as bacteriocin gene cluster,cold shock protein,citrate lyase,β-glucosidase and tannase were also annotated in the genome,which were very important for fruit wine fermentation,and further indicated that L plantarum SGJ-24 had potential application in fruit wine production.In addition,the amine oxidase and laccase related to the degradation of biogenic amines as reported in literature were not found in the genome of L plantarum SGJ-24.However,our previous study found that GAPDH had the activity of degrading histamine in L plantarum SGJ-24.Through genome sequencing,it was detected that GAPDH was located at the position of 706794-707816 bp on the chromosome,so we speculate that GAPDH may be the enzyme responsible for degrading biogenic amines in L plantarum SGJ-24.（3）Because free GAPDH had limitation in fruit wine brewing,it needs to be immobilized to improve its stability and reduce the cost before using.The magnetic Fe₃O₄nanoparticles functionalized by 3-aminopropyl triethoxysilane（APTES）were synthesized and used as carriers to immobilize GAPDH by covalent bonding method.The FT-IR and TEM characterization of particles proved that the amino carrier was successfully prepared and GAPDH was successfully loaded onto the carrier.The enzymatic properties showed that the optimum reaction temperature and p H of the immobilized enzyme（Fe₃O₄@APTES@GAPDH）were 50℃and 7.5,respectively,which were different from that of the free enzyme.However,the thermal stability and operation stability of the immobilized enzyme were obviously better than that of the free enzyme.When Fe₃O₄@APTES@GAPDH was applied to grape wine and black raspberry wines containing excessive histamine for 20 h,it was found that the degradation rate of histamine reached more than 80%,and the immobilized enzyme had no effect on the basic composition of the wines,which indicated that it had potential in the production of fruit wine.