Characteristics And Mechanisms Of Organic Acids Production In Lactic Acid Bacteria Strains From Hunan Traditional Fermented Vegetables And Their Application In Silage Fermentation

The global human population is constantly increasing and has necessitated the increased need for food to meet the population’s needs by 2050.As a result,there is a tremendous rise in the demand for animal protein,requiring the preservation of forages from agricultural biomass through ensiling.However,the rising demand for animal-based protein is causing a detrimental effect on the environment because it utilizes more land and generates more greenhouse gas(GHG)emissions per edible protein and field burning of abandoned crop residues and agricultural products.This has prompted a universal call to action by the United Nations’ Sustainable Development Goal(SDG)to protect the environment while trying to meet the demands of feeding the global human populace.Ensiling,or silage production,is a microbial-driven process by lactic acid bacteria(LAB)to preserve fresh forage for animal feed production.Due to variations in the constituents of different forages and the prevalence of pathogenic bacteria over native LAB,providing optimal conditions for mycotoxins and spoilage,various studies have aided the search for new silage additives/inoculants,thus,emphasizing the potential of certain LAB strains that are more efficient in ensiling.Also,inconsistency in silage quality in recent times could be interpreted by the lack of information on microbial community dynamics,gene expression,and functional metabolite changes of LAB involved in silage production.However,modern biotechnology approaches,including metagenomics,genomics,and transcriptomics,have proven useful in unraveling the molecular mechanisms of various metabolite production,including beneficial organic acids in distinct LAB strains during fermentation,thus,requiring the need to evaluate their performance in silage production.Therefore,the study aimed to identify and characterize distinct LAB strains with disparate functional properties that can produce beneficial organic acids using several cultures and molecular techniques.Besides,the key genes,enzymes,and pathways associated with organic acids biosynthesis in the LAB strains were uncovered using whole-genome sequencing and RNA sequencing(RNA-seq)technology.Furthermore,excellent LAB strains with exceptional functional and metabolic properties were utilized in crop residues and agricultural products to monitor their performance on the fermentation profile,aerobic stability,and microbial community dynamics.The main results are outlined as follows:1.This study identified excellent organic acid-producing LAB from several isolated strains of Hunan traditional fermented vegetables.Nine distinct strains belonging to four genera and five species,Lactobacillus plantarum PC1-1,YC1-2(8),YC1-1-4B,YC1-4(4),and YC2-9,L.buchneri PC-C1,Pediococcus pentosaceus PC2-1(F2),Weissella hellenica PC1 A,and Enterococcus sp.YC2-6 were selected for further analysis.Based on the results of organic acids,acidification,growth rate,antibiotic activity,and antimicrobial inhibition,PC1-1,YC1-1-4B,PC2-1(F2),and PC-C1 showed exceptional biopreservative potential.Additionally,PC-C1,YC1-1-4B,and PC2-1(F2)recorded higher(p < 0.05)growth by utilizing glucose(20 g/L)and soy peptone(10 g/L)as carbon and nitrogen sources in optimized culture conditions at 24 h and acidification until 72 h in batch fermentation,which suggests their application as starter cultures in industrial fermentation.2.Several molecular and bioinformatics approaches were used to perform wholegenome sequencing and comparative genomics of five representative LAB species,YC1-1-4B,PC2-1(F2),PC1 A,PC-C1,and YC2-6,to enhance our understanding of their different genetic functionalities,organic acid biosynthesis,and secondary metabolism.The results revealed major carbohydrate-active enzyme genes,putative operons,and unique mobile genetic elements,including plasmids,resistance genes,insertion sequences,and composite transposons involved in organic acid biosynthesis.The five LAB species,YC1-1-4B,PC2-1(F2),PC1 A,PC-C1,and YC2-6,utilized sugars primarily through the Embden-Meyerhof-Parnas(EMP),the pentose phosphate(PP),and the pentose phosphoketolase(PPK)pathways.However,YC1-1-4B and PC-C1 genomes contained more diverse genes involved in the final step of beneficial organic acids biosynthesis,including fum and asp A genes involved in the tricarboxylic acid(TCA)cycle and aspartate catabolism.Also,the two strains contain an alternative pathway for carboxylic acid synthesis by catalyzing the complete breakdown of pyruvate to acetylCo A and CO(2),linking the glycolytic pathway to the TCA cycle,and regenerating NAD+.Furthermore,the presence of phosphate acetyltransferase and L-serine dehydratase genes showed the potential adaptation of homofermentative L.plantarum and heterofermentative L.buchneri to glucose limitation during prolonged fermentation.3.High-throughput transcriptome sequencing analyses were further performed to understand the molecular mechanisms behind the expression and regulation of organic acid biosynthesis genes in L.plantarum and L.buchneri.Several culture time-induced pathways associated with organic acid biosynthesis,including metabolic equilibriums and the expression of growth-related genes,were identified.The expression of the organic acid biosynthesis genes revealed that L.plantarum YC1-1-4B and L.buchneri PC-C1 could utilize carbon sources other than glucose to produce beneficial organic acids at different culture times.The biosynthesis pathways uncovered the significant upregulation of ldh and ack A at 12 h,which prompted the swift metabolism of lactate and acetate.Again,putative alternative pathways for converting 6-phosphogluconolactone to organic acids and succinyl Co A to succinate in the TCA cycle were heavily expressed in YC1-1-4B and PC-C1 at 12 h.Interestingly,culture time-induced pathways of organic acid biosynthesis in L.buchneri and L.plantarum were unveiled for the first time,with massive induction of differential gene expressions at 12 h,including suc C and suc D gene overlap,thus,prompting the potential for translational coupling during fermentation.4.The performance of the distinct beneficial organic acid-producing LAB,including heterofermentative L.buchneri PC-C1(Lb)and homofermentative L.plantarum YC1-1-4B(Lp),or their combination(Lp Lb)(applied at 1.0?×?109 colony forming units(cfu)per kilogram of fresh matter)on fermentation profile,aerobic stability,and microbial community dynamics of corn stover after 7d,14 d,30d,and 60 d of ensiling were studied.Higher(p < 0.05)levels of beneficial organic acids,LAB counts,crude protein(CP),and amylase-treated neutral detergent fiber(a NDF)and lower(p < 0.05)levels of p H and ammonia nitrogen were recorded in Lp Lb-treated silages after 30 d and 60 d of ensiling.Also,a significant positive correlation between Lp Lb-treated silages with Lactobacillus,Lactococcus,Pediococcus,CP,and a NDF after 60 d emphasizes a potent interaction mechanism initiated by beneficial organic acids production and further highlights the synergistic effect of incorporating L.buchneri and L.plantarum for improved aerobic stability,fermentation quality,and bacterial community and reduced fungal population in corn stover after 60 d of ensiling.5.Due to the distinction among various forage types,which could have distinct effects on silage quality,high-throughput metagenomic sequencing technology was used to consolidate the performance of homo-and heterofermentative LAB in alfalfa during ensiling.Here,L.plantarum YC1-1-4B(Lp)or P.pentosaceus PC2-1(F2)(Pp)and L.buchneri PC-C1(Lb)or their combinations(Lb Lp or Lb Pp)(applied at 1.0?×?109 cfu per kilogram of fresh matter)on the fermentation characteristics,microbial community dynamics,and functional profiles of alfalfa(Medicago sativa)silage after 7d,14 d,30d,and 60 d of ensiling.The results indicated a reduction(p < 0.05)in glucose and p H,higher(p < 0.05)LAB counts,beneficial organic acids contents,WSC,xylose,CP,and ammonia nitrogen in Lb-,Lb Pp-,and Lb Lp-inoculated alfalfa silages after 30 d and 60 d.Also,the positive correlation between the combined LAB inoculants in Lb Lp-inoculated alfalfa silages and dominant LAB genera Lactobacillus and Pediococcus with fermentation properties confirmed the efficacy of their interaction in altering the growth of pathogenic microorganisms after 30 d and 60 d.In addition,the 16 S r RNA genepredicted functional analyses further showed that the L.buchneri PC-C1 and L.plantarum YC1-1-4B combination improved carbohydrate metabolism and facilitated further degradation of polysaccharides in alfalfa after 60 d of ensiling.This reiterated the synergistic performance of the homo-and heterofermentative LAB strains in improving the fermentation characteristics and functional carbohydrate metabolism to increase organic acid production and promote the growth of dominant LAB,which suppressed the development of pathogenic microorganisms in alfalfa during prolonged ensiling.In summary,distinct LAB strains,particularly L.plantarum YC1-1-4B and L.buchneri PC-C1 that produce beneficial organic acids other than LA were successfully identified from edible Hunan traditional fermented vegetables.The distinct LAB strains contain diverse novel genes,putative alternative pathways for efficient carbohydrate metabolism and organic acid production with a promise of successful development using gene editing and metabolic engineering techniques.L.plantarum YC1-1-4B and L.buchneri PC-C1 combination showed exceptional ability to utilize various types of forage biomasses at various conditions,as can be seen in the fermentation of corn stover(with high WSC)and alfalfa(with low WSC)silages where they interacted with native LAB to improve silage microbial community,eliminated silage pathogens and,at the same time,enhancing the silage fermentation quality,chemical components and aerobic stability.