Study On Microbial Diversity And Metatranscriptomics Of Natural Fermented Pepper

Natural fermented pepper is a specialty and main condiment of Hunan dishes.Studies on natural fermented pepper are limited to strain breeding,aroma composition analysis,process optimization,etc.Microbial diversity in different fermentation stages of natural fermented pepper and its role are still unclear.Microbial diversity in C.annuum L.var.dactylus M and C.annuum L.var.conoides Irish before and after fermentation was studied using 454 high-throughput sequencing technology and microbial species and functions of non-rRNAs in fresh C.annuum L.var.dactylus M and natural fermented C.annuum L.var.dactylus M for 20 days were annotated using RNA-sequencing technology to reveal the microbial community composition and function.The main results were as follows:1.Bacterial diversity in C.annuum L.var.dactylus M and C.annuum L.var.conoides Irish before and after fermentation was studied using 454 high-throughput sequencing technology.The main results were as follows:?1?Bacteria in fresh C.annuum L.var.dactylus M could be assigned to 5 phyla,8 classes,15 orders,27 families,35 genera.Taxa in proportions >1% could be assigned to 17 different genera in fresh C.annuum L.var.dactylus M,mainly including Rhizobium 41.51%,Aureimonas 6.92% and Stenotrophomonas 6.92%.Bacteria in fresh C.annuum L.var.conoides Irish could be assigned to 5 phyla,7 classes,13 orders,27 families,38 genera.Taxa in proportions >1% could be assigned to 11 different genera,mainly including Aureimonas 25.62%,Rhizobium 23.97% and Lactobacillus7.82%.?2?Bacteria in natural fermented C.annuum L.var.dactylus M could be assigned to 14 phyla,26 classes,44 orders,86 families,166 genera,236 species.Taxa in proportions >1% could be assigned to 21 different genera.Weissella,Lactobacillus and Rhizobium were dominant genera.Bacteria in natural fermented C.annuum L.var.conoides Irish could be classified into 10 phyla,23 classes,33 orders,64 families,124 genera,169 species.Taxa in proportions >1% could be assigned to 14 different genera.Weissella?Lactobacillus?Aureimonas and Rhizobium were the dominant genera.2.Fungal diversity in C.annuum L.var.dactylus M and C.annuum L.var.conoides Irish before and after fermentation was studied using 454 high-throughput sequencing technology.The main results were as follows:?1?Fungi in fresh C.annuum L.var.dactylus M could be assigned to 3 phyla,12 classes,16 orders,23 families,26 genera,36 species.Taxa in proportions >0.01% could be assigned to 9 different genera: Debarvomvces 2.69%,Rhodotorula 2.39%,Trichosporon 2.28%,Cladosporium 0.77%,Mucor,0.63% Guehomyces 0.33%,Cryptococcus 0.26%,Hanseniaspora 0.26%,Candida 0.04%.Fungi in fresh C.annuum L.var.conoides Irish could be assigned to 3 phyla,15 classes,16 orders,38 families,53 genera,79 species.Taxa in proportions >1% could be assigned to 5 different genera: Trichosporon 24.11%,Rhodotorula 7.4%,Cladosporium 4.26%,Debarvomvces 3.94%,Mucor 2.51% and Cryptococcus 1.86%.?2?Fungi in natural fermented C.annuum L.var.dactylus M could be assigned to 3 phyla,13 classes,20 orders,30 families,39 genera,56 species.Taxa in proportions>1% could be assigned to 8 different genera: Hanseniaspora,Debaryomyces,Rhodotorula,Trichosporon,Cladosporium,Cryptococcus,Guehomyces and Pichia.Fungi in natural fermented C.annuum L.var.conoides Irish could be assigned to 3 phyla,17 classes,29 orders,41 families,60 genera,96 species.Taxa in proportions >1% could be assigned to 10 different genera: Hanseniaspora,Debaryomyces,Rhodotorula,Trichosporon,Cladosporium,Cryptococcus,Pichia,Meyerozyma,Mucor,Hyphopichia.3.RNA of microorganism in before and after fermentation of C.annuum L.var.dactylus M was sequenced using Illumina Hiseq4000.Non-redundant gene catalog was constructed after sequence optimization,sequence assembly and elimination of redundancy.Species were annotated on non-redundant gene catalog.The main results were as follows:?1?A non-redundant gene catalog with 5490 non redundant genes was constructed.?2?Microbial compositions of fresh C.annuum L.var.dactylus M were bacteria 88.55%,fungi 5.73% and norank 5.72%.Microbial compositions of natural fermented C.annuum L.var.dactylus M for 20 days were bacteria 99.95%,fungi 0.00015% and norank 0.0499%.?3?Bacteria in fresh C.annuum L.var.dactylus M could be classified into 1 phylum,1 class,2 orders,4 families,4 genera,10 species.Fungi in fresh C.annuum L.var.dactylus M could be classified into 1 phylum,1 class,1 order,1 family,1 genus,1 species.Bacteria in natural fermented C.annuum L.var.dactylus M for 20 days could be classified into 3 phyla,3 classes,5 orders,11 families,15 genera,61 species.Fungi in natural fermented C.annuum L.var.dactylus M for 20 days could be classified into 2 phyla,2 classes,2 orders,2 families,2 genera,2 species.?4?9 bacterial species and 1 fungal species were detected in fresh C.annuum L.var.dactylus M and owned 76.80% of the total microbial transcripts.5 unclassified species owned 23.20% of the total microbial transcripts.The dominant active microorganisms were Lactobacillus plantarum 40.15%,Staphylococcus epidermidis 38.85%,Staphylococcus lugdunensis 8.77% and Staphylococcus caprae 2.64%.?5?61 bacterial species and 2 fungal species were detected in X₂0 and owned 72.50% of the total microbial transcripts.12 unclassified species owned 27.50% of the total microbial transcripts.The active bacteria mainly included Lactobacillus plantarum 95.16% and Weissella paramesenteroides 1.66%.4.Functional annotation and differentially expressed analysis of Non-redundant genes of microorganisms in fresh and natural fermented C.annuum L.var.dactylus M were made to study gene expression,select important functional genes and screen differentially expressed genes.The main results were as follows:?1?Fresh C.annuum L.var.dactylus M had 1048 non-redundant genes.199 non-redundant genes and 176 metabolic pathways were annotated on KEGG.495 non-redundant genes and 23 classification functions were annotated on eggNOG.280 non-redundant genes were annotated on GO.C.annuum L.var.dactylus M fermented for 20 days had 4442 non-redundant genes.2325 non-redundant genes and 183 metabolic pathways were annotated on KEGG.3591 non-redundant genes and 22 classification functions were annotated on eggNOG.1463 non-redundant genes were annotated on GO.?2?Microorganisms annotated on KEGG in X₀ were Staphylococcus epidermidis and Lactobacillus plantarum.79 species in X₂0 annotated on KEGG,mainly including Lactobacillus plantarum,Weissella paramesenteroides,Lactococcus lactis,etc.?3?There were 3 nitrite metabolic pathways,18 amino acid metabolic pathways,2 lactic acid synthesis pathways and 2 metabolic pathways associated with color were annotated on KEGG?4?There were a total of 4836 differentially expressed genes between X₀ and X₂0,4026 of which up-regulated,810 of which down regulated,3079 of which significantly up-regulated,361 of which significantly down regulated.3 differentially and highly expressed genes were screened.?5?Lactobacillus plantarum?Weissella paramesenteroides and Lactococcus lactis were the key microorganisms in the natural fermented pepper and were the best compound starter in the controlled fermentation of pepper.