| Shrimp paste is a traditional fermented food in China’s coastal areas.Most of the shrimp paste products in the domestic market use open natural fermentation.The fermentation cycle is long.The production process is dominated by manual work and experience.In addition,to enhance storage,a large amount of salt is added to the shrimp paste during the production process,which makes the product increasingly salty,and it can only be eaten as a condiment in a small amount,which limits the production of shrimp paste to a large extent.sale.Therefore,how to effectively reduce the salt content,shorten the fermentation period,improve the probiotic characteristics,increase the unique flavor,and improve the quality of shrimp paste while retaining the advantages of traditional shrimp paste is an urgent problem to be solved.In this study,Jiangsu opossum shrimp paste was used as the research object,and metagenomic sequencing technology was used to analyze the microbial community structure and fermentation pathway of naturally fermented shrimp paste and to manually screen and establish an ideal metabolic pathway to ensure the nutrition,flavor and safety of shrimp paste.Networks,back-annotation and targeting of associated microbiota in a network of ideal metabolic pathways.In addition,combined with culturable technology to isolate and obtain representative strains in the abovementioned key microbial flora,further verification and correction of the whole genome sequencing and safety assessment of the candidate strains finally locks the leading high-quality shrimp paste fermentation,safe microbial bacteria as a resource.On this basis,a single-strain/multi-strain collaborative fermentation scheme was developed for the preparation of shrimp paste to achieve the purpose of rapid and low-salt fermentation of shrimp paste on the basis of improving the quality and safety of shrimp paste.The main findings are as follows:1.Analysis of the fermentation pathway of Jiangsu opossum shrimp paste and research on the diversity of key functional microbial floraThe microbial diversity and related metabolic pathways in Jiangsu opossum shrimp paste were analyzed by metagenomic analysis.The results showed that the shrimp paste was dominated by bacteria(72.65%),followed by eukaryotic microorganisms(10.85%)and archaea(4.24%).At the family level,the top 10 are all bacteria,and Enterococcus(37.85%)and Vibrio(31.27%)have absolute dominance;at the genus level,Tetragenococcus(30.47%)is the most dominant genus,followed by Vibrio(6.60%)and Staphylococcus(4.34%).The metabolic process of protein,fat,carbohydrate and intermediate products dominated by microorganisms in shrimp paste was clarified,and from the perspective of nutrition,flavor and safety of shrimp paste,22 functional enzymes related to protein metabolism,fat metabolism and glucose metabolism were identified.These enzymes include carboxypeptidase,aldehyde dehydrogenase,alcohol dehydrogenase and fructokinase.Based on such functional enzymeencoding genes,50 species of 35 genera related to shrimp paste nutrition,flavor and safety have been identified,including Haloplanus natans,Tetragenococcus halophilus,Salinivibrio costicola,Virgibacillus halodenitrificans,Pediococcus acidilactici and Tetragenococcus muriaticus.2.Research on the key microbial resources of Jiangsu opossum shrimp paste fermentationThe strains of Jiangsu opossum shrimp paste were isolated based on culturable technology,and the isolates included 66 strains belonging to genus Staphylococcus,Enterococcus,Tetragenococcus,Pediococcus,Streptococcus,Virgibacillus,Oceanobacillus,Salinivibrio and Haloplanus.For the isolated bacterial strains P.acidilactici M1-1,V.halodenitrificans L4-16,T.halophilus subsp.flandriensis M7-2,T.muriaticus M7-3,Salinivibrio.costicola subsp.costicola L4-5 and Staphylococcus.nepalensis L5-1,as well as the archaeal strains H.natans H5-20 and H.salinus H5-10,a total of 8 strains were analyzed by whole genome sequencing,and the results showed that Salinivibrio.costicola subsp.costicola L4-5,Staphylococcus.nepalensis L5-1 and H.salinus H5-10 can produce lysine decarboxylase,tyrosine decarboxylase,tryptophan decarboxylase and agmatase,which may produce biogenic amines such as cadaverine,tyramine,tryptamine and putrescine,which are not suitable for directly fermented shrimp paste.The strains P.acidilactici M1-1,T.halophilus subsp.flandriensis M7-2,T.muriaticus M7-3 and V.halodenitrificans L4-16 have multiple genes associated with sugar metabolism and fat metabolism,which can better degrade the lipids in shrimp paste.H.natans H5-20 can produce monoamine oxidase,which can degrade tyramine.The safety and growth performance evaluation showed that these 5 strains did not have hemolysis or biofilm formation and were sensitive to most antibiotics.The bacterial strains showed better growth performance when the salinity was 10%,while the archaea strain showed better growth performance when the salinity was 18%.Finally,the five strains were identified as safe microbial strain resources that could lead to the high-quality fermentation of natural shrimp paste for the preparation of single-strain/multi-strain synergistic fermentation of shrimp paste.3.Multi-strain synergistic fermentation of Jiangsu opossum shrimp paste and its quality evaluationThe results of the related indices of shrimp paste fermented by P.acidilactici M1-1、V.halodenitrificans L4-16、T.halophilus subsp.flandriensis M7-2、T.muriaticus M7-3 and H.natans H5-20 showed that the TVB-N contents were 99.550 mg/100 g,102.550 mg/100 g,119.313 mg/100 g,135.958 mg/100 g and 59.064 mg/100 g,respectively;the AAN contents were 0.947 g/100 g,1.004 g/100 g,0.962 mg/100 g,1.125 mg/100 g and 0.748 g/100 g,respectively;the biogenic amine contents were 404.65 mg/kg,36.06 mg/kg,118.72 mg/kg,93.61 mg/kg and 89.93 mg/kg,respectively;and the types of volatile compounds were 57,61,68,84 and 76,respectively.Compared with the results of natural fermentation(TVB-N content is 213.44 mg/100 g;AAN content is 1.032 g/100 g;total biogenic amine content is 1249.57mg/kg;the types of volatile compounds is 77),single-strain fermentation can preliminarily solve the problem of unclear microorganisms in natural shrimp paste.Disadvantages of high salt and long fermentation cycle.The bacterial strain T.halophilus subsp.flandriensis M7-2and T.muriaticus M7-3 fermented shrimp paste had the advantage of a high content of AAN and a low content of TVB-N,and the archaeal strain H.natans H5-20 also had biogenic aminereducing properties.These two bacteria were co-fermented with the archaea H.natans H5-20,the M7-2+H group(T.halophilus subsp.flandriensis M7-2+ H natans H5-20)and M7-3+H group(T.muriaticus M7-3 + H natans H5-20)after synergistic fermentation,the TVB-N content was 109.4 mg/100 g and 102.8 mg/100 g;the AAN content was 1.104 g/100 g and0.973 g/100 g,and the total biogenic amine content was 103.59 mg/kg and 126.44 mg/kg;98and 69 kinds of volatile compounds may contribute to flavor.As the result,at 10% salt concentration,compared with single-strain fermentation,the shrimp paste using T.halophilus subsp.flandriensis M7-2 + H.natans H5-20 or T.muriaticus M7-3 + H.natans H5-20 cofermentation scheme showed a better positive response in total amine content,ANN content,and volatile compound species.Synergistic fermentation can improve the flavor of shrimp paste,ensure the safety of shrimp paste,and reduce the salt in shrimp paste.It can shorten the fermentation period and achieve low-salt,fast and safe fermentation.This study can provide a safe and efficient low salt fermentation method for shrimp paste.On the one hand,it can promote the utilization of low value shrimp and the industrial production and development of high-quality shrimp paste in China.On the other hand,it can provide reference for improving traditional food in China and ensuring the dietary health of residents. |
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