Mechanism Of Protease-producing From Penicillium Citrinum Under High-salt Conditions And Its Application In Fish Sauce Fermentation

Fish sauce is a traditional delicious condiment in China. A haltolerant fungus YL-1 is screened out from the fish sauce fermentation liquid,which is identified as Penicillium citrinum after morphological, physiological and biochemical, molecular biological identificationPenicillium citrinum YL-1 were studied and found to grow in the environment of0²6%Na Cl, which has some salt tolerance. P. citrinum YL-1 can grow in the environment of 10⁴0?,30?for the optimal temperature; P. citrinum YL-1 can grow in p H4.0-11.5environment,p H8.5 for the most suitable p H;P. citrinum YL-1 can grow in the environment of 0⁶0% sugar concentration, the sugar concentration of best growth is 20%. Metal Mg2+can promote the growth of P. citrinum YL-1,which grow best in the 2% concentration.There were no hemolytic phenomenon in P. citrinum YL-1 hemolytic experiments.The method of ergosterol extraction with HPLC and method of nucleic acid with UV for P. citrinum YL-1 biomass in solid state fermentation,the maximum biomass were all close to be got in the eighth day, respectively 80.56mg/g and 78.39mg/g dry substrate. The matrix protease activity was measured in solid-state fermentation process, the results showed that the maximum protease activity appeared on the seventh day, amounting 616.21 U / g.Moisture content was highest on the eighth day, which was positively correlated with biomass. And the largest content of reducing sugar and total sugar in the matrix appeared on the third day,respectively 3.15 g / g and 16.66 g / g dry substrate.Study on the metabolism of the protease- producing strain YL-1 under salt stress based on RNA-Seq technology. There are 62 differential genes in high-salt group Vs no-salt group,2189 differential genes in high-salt group Vs mid-salt group,18 differential genes in mid-salt group Vs no-salt group. Through the KEGG pathway, there are 32 genes metabolic pathways significantly affects in high-salt group Vs no-salt group,including 22 genes up-regulated metabolic pathways, and 16 genes down- regulated metabolic pathways; thereare 245 genes metabolic pathways significantly affecting in in high-salt group Vs mid-salt group, including 205 genes up-regulated metabolic pathways, 177 genes down-regulated metabolic pathways; there are only 15 genes metabolic pathways significantly affecting in mid-salt group Vs no-salt group, which all showing down regulation of gene expression.Fatty acid degradation, glycine?serine and threonine metabolism, carbon metabolism and tricarboxylic acid metabolism are the main metabolic pathways in these metabolic pathway,which were associated with salt effect. ADH1₇ of up regulation genes IDc8084_g2 from fatty acid degradation pathway in High-salt group Vs no-salt group is alcohol dehydrogenase gene involved in the glycolytic pathway and tyrosine metabolism,indicating that glycolysis and carbon metabolism pathway can be used as the energy metabolism to maintain haltolerant microorganisms' normal life activities under high salt environment.Glycine, serine and threonine metabolism of Significant enrichment have differences in gene expression in High-salt group Vs mid-salt group. Ser A and PHGDH gene from c5647_g1?up?, c1468_g1?down? involved in serine synthesis, may be related to haltolerant serine proteases formation metabolic pathway,which also shows that there may be salt tolerance genes in differentially expressed genes. In the fish sauce, amino acid composition in the fish sauce has a great effect on the flavor of the fish sauce.It is found that the formation of flavor has a certain relationship with a variety of amino acid metabolism through KEGG metabolic pathway,involved in gene metabolic pathways including tyrosine metabolism, tricarboxylic acid metabolism, carbon metabolism, nitrogen metabolism,amino acid biosynthesis, metabolism of phenylalanine, tryptophan, arginine and proline metabolism metabolic pathways so on,in which tryptophan, arginine, tyrosine and other amino acids in the intermediate metabolism, although each has its own special process, but also a common way. And the amino acid biosynthesis metabolic pathway is the osmotic pressure regulation mechanism of microorganism under high salt environment, and the compatible substances of high concentration in the cell could resist the external osmotic pressure environment.P. citrinum YL-1 strains, protease form P. citrinum YL-1and commercial enzyme 2709 are added to fish sauce fermentation liquid of 6 months as application research. After a month for fermentation, result found that p H in control group is almost unchanged, the p Hadded microorganisms and enzymes decline significantly, amino acid nitrogen?AAN? of fish sauce fermentation liquid added protease form P. citrinum YL-1and commercial enzyme 2709 increased by 46% and 34%, respectively compared with the control group The content of AAN reached the commercial level index of fish sauce. AAN content of fish sauce fermentation liquid added protease form P. citrinum YL-1and 2709 enzyme were19.1mg/100 m L and 22.9mg/100 m L after two months for fermentation,which repectively were close to and higher than that content?19.9 mg / 100 m L? of fish sauce fermented for two years. TVB-N content of fish sauce fermentation liquid added the serine protease form P. citrinum YL-1 and 2709 enzyme were 3.75mg/100 m L and 4.32mg/100 m L,while TMA content were 6.9mg/100 m L and 7.6mg/100 m L, all close to TVB-N content 3.7mg/100 m L and TMA content 6.4mg/100 m L from fish sauce fermented for two years. The contents of amino acid adding enzyme fermentation for one month was significantly more than that of control group added nothing, but less than that of amino acids in fish sauce fermentations for the two year.And alanine and glutamate are the main component of fish sauce flavor.With the long time fermentation of fish sauce added the enzymes, bitter free amino acids in fish sauce decreased, flavor and sweetness free amino groups increased significantly. The Asp, Gly, Val, Ser four amino acids increased rapidly, indicating that the degradation of casein can produce amino acid fragments of different length coincides with the function of protease, but glutamic acid content form flavor amino acids appeared less volume growth,fermentation broth need to be fermented to promote the formation of flavour and taste substances.With the sensory evaluation, color and odor of fish sauce added Penicillium citrinum YL-1 producing protease fermentation for two months were close to color and odor of traditional fish sauce fermented for two years. Fish sause color adding 2709 enzyme was deeper than the color of fish sause for two years, smell was meat grilled flavor. Research results indicate that the addition of two salt tolerant proteases have a positive effect on rapid fermentation of fish sauce,which can shorten the fermentation time.