Study On The Antifungal Activity Of Lactic Acid Bacteria And Its Application In The Storage Process Of Fresh Edible Peanuts

Fresh edible peanuts refer to peanuts that are eaten directly after harvest without drying.They are popular among consumers for their convenience,high nutritional value,delicious taste and other advantages.Fresh edible peanuts have high water content and are extremely perishable when they are stored postharvest.Lactic acid bacteria(LAB)is a potential and biological anti-mildew agent,which could not only inhibit the growth of harmful molds,but also eliminate mycotoxins.It has been reported that great progress has been made in the application of LAB in controlling the contamination of grain and food by harmful molds during the storage process.However,LAB strains with good antimildew effect are still to be developed and the research data about the antimildew effect of LAB on fresh edible peanuts after harvest is still insufficient.In this study,LAB with efficient inhibitory effect on Aspergillus flavus MA(A.flavus MA),Aspergillus niger BQM(A.niger BQM)and Penicillium citrinum MB(P.citrinum MB)were firstly isolated from soil,corn,pickles and bran.Then,the antifungal active substances of LAB were investigated.At last,the cell-free supernatant(CFS)of strain P3 was applied to fresh edible peanuts to explore its impact on microorganisms and quality of fresh edible peanuts during storage process.In addition,transcriptomics and metabolomics techniques were used to investigate the defense mechanisms of strain P3 CFS in inducing fresh edible peanuts to resist fungal infection,providing a theoretical foundation for LAB as biological anti-mildew agent for fresh edible peanuts.The main conclusions were summarized as following:(1)Firstly,the isolation and identification of LAB strains with antifungal activity.A total of 71 LAB strains were isolated from soil,corn,pickle and bran samples.The inhibitory effects of 71 strains on A.flavus MA,A.niger BQM and P.citrinum MB were tested by double plate method.The results demonstrated that 17 strains had good inhibitory effects against all three molds,and the diameters of inhibition zones were greater than 30 mm.Morphological and 16 S r DNA molecular biological identification were performed on 17 strains.The strains DA1,DA2,DA4,DB3,DC5 and DC6 were identified as Weissella cibaria,the strains DB1,DB2,DB5,DB6,DB7,DB8,DB11,DB12 and P6 were identified as Pediococcus pentosaceus,and the strains P3 and P5 were identified as Lactiplantibacillus paraplantarum.(2)Secondly,the investigation on the antifungal active substances of LAB.The antifungal activity of CFS of 17 LAB strains was determined by 96-well plate method.The results revealed that the CFS of strains DA2,DA4,DB2,DB11,DB12,DC5,P3 and P5 had inhibition rates of more than 99% against A.flavus MA,A.niger BQM and P.citrinum MB.The CFS of these 8 LAB strains were treated with p H,catalase and protease K.The results revealed that when the p H was adjusted to 6.0,the antifungal rate decreased significantly(P<0.05),from99% to 12.42%-39.28%.But the antifungal rate of CFS treated with catalase and protease K was not significantly different from that of the control group(P>0.05).Therefore,it was speculated that the active substances produced by LAB in this study were mainly organic acids.The results of organic acids demonstrated that lactic acid and acetic acid were the main organic acids in CFS of 8 LAB strains.The antifungal effects of 5 organic acid on A.flavus MA,A.niger BQM and P.citrinum MB were explored.It was confirmed that acetic acid and lactic acid had better inhibitory effects on A.flavus MA and P.citrinum MB,acetic acid and phenyl lactic acid had better antifungal effects on A.niger BQM.Liquid chromatography mass spectrometer(LC-MS)was used to analyze the components of the CFS of Weissella cibaria DA2,Pediococcus pentosaceus DB12,and Lactiplantibacillus paraplantarum P3.The experimental results showed that organic acids and derivatives were the main metabolites in LAB CFS.(3)Thirdly,the application of Lactiplantibacillus paraplantarum P3 in fresh edible peanuts during the storage process.The fresh edible peanut samples were treated with Lactiplantibacillus paraplantarum P3 CFS and stored at 5℃ and 30℃.The results of morphology and color experiments showed that the addition of strain P3 CFS could effectively improve the morphology and color,delay the increase of the total number of mold colonies,and reduce the mildew rate of fresh edible peanuts during storage.The results of microbial diversity analysis indicated that Fusarium spp.were the dominant moldy pathogen on the surface of the initial fresh edible peanuts,Cladosporium spp.were the dominant moldy pathogens of fresh edible peanuts stored at 5℃,while Fusarium were the dominant moldy pathogens of fresh edible peanuts stored 30℃.The results of GC-MS indicated that 30 kinds of volatile compounds were detected in fresh edible peanuts,including 8 hydrocarbons,6esters,3 aldehydes,4 alcohols,2 ketones and 7 others.GC-IMS results indicated that 36 kinds of volatile compounds were detected,including 10 aldehydes,8 alcohols,8 ketones,7 esters,1 alkene and 1 acid.Hexanal,(+)– limonene,2-pentylfuran,and n-hexanol were the main volatile compounds,and the contents of hexanal and 2-pentylfuran in strain P3 CFS treatment group was higher than that in control group,indicating that the addition of strain P3 CFS was helpful to maintain the good flavor of fresh edible peanuts.The results of transcriptomics displayed that the addition of strain P3 CFS could activate plant mitogen-activated protein kinase signaling pathway and hormone signal transduction pathways,promote the production of ethylene,gibberellin regulatory proteins,and other substances in fresh edible peanuts,so as to enhance the resistance of fresh edible peanuts to pathogenic microorganisms and resist the invasion of pathogenic microorganisms,inhibiting the mildew of fresh edible peanuts.The results of metabolomic analysis revealed that the metabolic pathways of glycerophospholipid metabolism,pyrimidine metabolism,and arginine biosynthesis were enriched in strain P3 CFS treated fresh edible peanuts.Therefore,it is speculated that strain P3 CFS could increase the stress resistance of peanuts and resist mold infection by inducing the biosynthesis of glycerophospholipids,arginine and other substances.In conclusion,the main antimildew active substances of lactic acid bacteria are organic acids,and strain P3 CFS can effectively inhibit the mildew of fresh edible peanuts during storage process and maintain its good color and flavor,which leading its broad application prospect as an antimildew agents for fresh edible peanuts.