Screening Of Low-salt Fish Sauce Fermentor Strains And The Effect Of Different Fermentation Methods On Their Quality

Fish sauce is a popular food consumed in Southeast Asia for its unique flavour and nutrition.The traditional fermented fish sauce is generally produced through spontaneous fermentation addition to 20%-30% salt,and the highly salted environment in fermentation process raises concerns,such as adverse health effects,difficulty in controlling the fermentation process and a strong odor that limits consumer acceptability.Therefore,there is an increasing demand for low-salt fermented fish sauce with improved flavor.In this study,the strains with high enzyme activities were screened and multiple growth characteristics were analyzed.The effects of the isolated strains on low-salt fermentation of coarse fish(Hemiculter leucisculus)and the fermentation process optimization were explored.Two types of fermentation with simultaneous and sequential inoculum mixed bacteria were selected to produce lowsalt fermented fish products with a characteristic flavor.The study will provide a theoretical basis for the fermentation of low-salt fish sauce.The main research results are as follows:1.Screening and identification of acid-produced,proteolytic and lipolytic isolates and their growth conditions and enzymatic assayUsing the traditional culture method,one acid-produced and two proteolytic and lipolytic strains were isolated from the traditional fermented fish sauce.Through morphological observation and 16 S r RNA molecular biology,the acid-produced strain was identified as Rummeliibacillus stabekisii(m7),and two enzyme-producing strains were identified as Virgibacillus halodenitrificans(DZ4-9)and Bacillus velezensis(DZ6-11).The factors influencing the growth conditions and enzymatic properties of the strains were investigated,including salt concentration,temperature and p H.The results showed that the isolates grew well in salt concentration less than 10%,25-35°C and p H 6.0-8.0.Additionally,strain m7 had a strong acid production.The protease activity could reach up to 9.67 U/m L and 9.92 U/m L respectively;and the lipase had a higher enzyme activity at 40°C,p H 7.0 and salinity more than 3%,which were produced by strains DZ4-9 and DZ6-11.2.Optimisation of process conditions for monobacterial fermentation of fish sauceUsing small fish as raw material,and the content of total volatile base nitrogen and amino acid nitrogen as evaluation indexes,the monobacterial fermentation conditions were optimized through single factor experiment and orthogonal design.The optimal conditions for strain m7 were determined as follows: fermentation temperature is 30 °C,salt addition is 9 % and inoculum level is 9 %;for strain DZ4-9: fermentation temperature is 30°C,salt addition is 9% and inoculum level is 11%;for strain DZ6-11:fermentation temperature is 30°C,salt addition is 9% and inoculum level is 7%.Under these conditions,the fermentation broth has the highest amino nitrogen content and no significant undesirable odour.3.Effect of mixed bacterial fermentation on the quality and volatile flavour components of low-salt fish sauceThe selected strains were applied according to two different fermentation methods based on steering sequential and temporally deferred inoculum of three strains(R-2V-5B),and ternary simultaneous inoculum(RVB).The effect of sequential and simultaneous fermentations on amino acid nitrogen,total volatile base nitrogen,TCAsoluble peptides,p H and total acid content and non-enzymatic browning of low-salt fish sauce were evaluated,and compared to conventional low-salt fermentation(control)lasting 60 days.The results showed that RVB and R-2V-5B fermentations had a higher total acid content,a lower total volatile base nitrogen content,and the soluble peptide content increased firstly and then decreased compared to control fermentation.Moreover,the mixed bacterial fermentation could promote the formation of the brown colour of fish sauce.Sensory evaluation and electronic nose were used to analyse the volatile components of the fermentation broth.The results showed that the main flavour of RVB and R-2V-5B groups were fresh,sour and fishy,with significantly lower response values for W1W(sulphide sensitive)and W1S(alkane sensitive)than the control group.The quantitative analysis of the volatile compound content showed that the RVB and R-2V-5B groups were dominated by alcohols,ketones,acids and phenols,with 2-undecanone,phenol,butyric acid and acetophenone being the key flavour substances that confer the fish sauce its characteristic flavour.4.Effect of mixed bacterial fermentation on non-volatile metabolites in low-salt fish sauceThe LC-MS technique combined with metabolomics was used to analyse the effect of mixed bacterial fermentation on non-volatile metabolites.The results showed a significant increase in the content of various amino acids and long-chain fatty acids,and a significant decrease in the content of oligopeptides and biogenic amines in the RVB and R-2V-5B groups after 30 days of fermentation.The mixed bacterial fermentation enhanced the type and content of oligopeptides and short-chain fatty acids,ensuring at the same time lower level of long-chain fatty acids and biogenic amines.Sequential and simultaneous fermentations not only could shorten the fermentation time,but also create a unique flavour of fish sauce,mainly by participating in tryptophan biosynthesis,pyrimidine metabolism,arginine biosynthesis,arginine and proline metabolism pathways.