Screening Of Planococcus Bacteria Producing Low-temperature Proteases And Its Application In Low-salt Fish Sauce Fermentation

Fish sauce is a popular fermented condiment.However,the traditional fermentation technology of fish sauce is usually characterized by long cycle,high salinity and unstable quality.In order to meet the requirements of modern industrial production,a variety of new fish sauce fermentation technologies have been developed.However,the disadvantages of high salt still exist,and the development of low-salt fish sauce has become the research direction.Fish meat is prone to spoilage under low salt conditions.Therefore,lowering temperature becomes a necessary means for low-salt fermentation.However,the fermentation activity of microorganism usually decreases under low temperature,resulting in the reduction of fermentation efficiency and the prolongation of the cycle.Therefore,finding the balance between salt consumption and fermentation temperature becomes the core issue in the development of low-salt fish sauce production technology.To find a starter culture that can grow rapidly and produce protease with high enzyme activity at low temperature has become a key issue.In this study,the traditional shrimp paste produced by low temperature fermentation technology was selected as the starting object,and low temperature protease strains were screened from it.The characteristics of their growth and protease production were preliminarily studied.4 strains were selected as the external mixed starter cultures to establish and optimize the low salt fermentation technology of fish sauce by using the processed by-product of minced fish as raw material.The effects of mixed starter cultures on the physicochemical properties and flavor characteristics of low-salt fish sauce fermentation process were analyzed.The research results provide support for the development of low-salt fish sauce.The main results are as follows:1.Screening and identification of low-temperature protease producing strains from shrimp paste with traditional flavorUsing the method of hydrolytic transparent circles,12 strains with protease activity were isolated from traditional fermented shrimp paste.Through 16 S rRNA sequence comparison,5 strains were identified as Virgibacillus(XJ 1,XJ 4,XJ 5,XJ 6,XJ 8);4 strains are Planococcus(XJ 2,XJ 10,XJ 11,XJ 12);2 strains are Ornithinibacillus(XJ 3,XJ 7);1 strain is Halobacillus(XJ 9).After rescreening by enzyme activity measurement(40°C),4 strains of Planococcus display the characteristics of producing low-temperature protease.Among them,the enzyme activity of the bacterium XJ 11 was the highest(10.07 ± 0.11 U/mL).The phylogenetic trees of Planococcus were constructed by sequence alignment,and four strains of Planococcus were identified as Planococcus maritimus XJ 2,Planococcus plakortidis XJ 10,Planococcus dechangensis XJ 11 and Planococcus rifietoensis XJ 12.4 strains were explored the growth characteristics and the enzymatic characteristics of Planococcus(XJ 2,XJ 10,XJ 11,XJ 12).The results show that the Planococcus grew well in low-temperature,low-salt,and alkaline environments.The protease produced at 40°C,pH 9.0 has the most enzyme activity,and has a certain tolerance.It provides a basis for Planococcus as starter cultures of low-salt fish sauce.2.Optimization of fermentation process of low-salt flavor fish sauce with Planococcus mixed starter cultureUsing minced catfish meat as the fermentation raw material,4 strains of Planococcus: Planococcus maritimus XJ2,Planococcus plakortidis XJ10,Planococcus dechangensis XJ11 and Planococcus rifietoensis XJ12 were mixed at a ratio of 1:1:1:1 as starter cultures of low-salt fish sauce.The optimal conditions of the low-salt fish sauce fermentation process were optimized through single factor and response surface tests: fermentation temperature is 21°C,salt addition is 8%(W/W),Planococcus mixed starter culture inoculation amount is 3%(V/W),fermentation time is 15 days,and the average value of amino acid nitrogen content was 1.26 g/100 mL.3.The effect of Planococcus starter culture on the quality of low-salt fish sauceThe fish sauce sample(group A)added with the Planococcus had a lower pH and a higher total acid content;In the final stage of fermentation,the addition of Planococcus starter culture could effectively increase the content of amino acid nitrogen in the low-salt fish sauce;After 15 days of fermentation,the content of volatile basic nitrogen(TVB-N)in the fish sauce sample(group B)without adding the Planococcus was up to 209.00 ± 1.49 mg/100 mL.However,the TVB-N content in group A(170.00 ± 1.05 mg/100 mL)was significantly lower than that in group B(P < 0.05);There was no significant difference in the histamine content in 3 different treatments(Fish sauce samples with starter culture were recorded as group A;fish sauce samples without starter culture were recorded as group B;raw chopped fish is group C)of low-salt fish sauce samples,which were all lower than the limit of histamine(40 mg/100 g)in fermented foods prescribed by EU standards;The total free amino acid content of the three differently processed low-salt fish sauce samples were 4257.69,3765.02 and 415.74 mg/100 mL respectively.After adding Planococcus starter culture,the type and content of free amino acids in fish sauce are more abundant,which gives better taste to low-salt fish sauce;Fish sauce samples inoculated with starter(Planococcus)produced stronger green,malt and nutty odor and reduces the smell of fish;According to sensory evaluation,the fish sauce samples of group A had the highest sensory score and good flavor.