Monascus Strain Screening,Identification For Meat Fermentation And Function Properties Studies Of Its Acid Protease

Monascus spp.is important edible microorganisms of our country which with a long application history,and quality and flavor of fermented food can be improved when Monascus spp.and its metabolites are used.Many metabolites such as Monascus pigments?MPs?,monacolin K,y-aminobutyric acid,dimerumic acid and some organic acid and proteinase,etc can be produced by Monascus spp..Among which,MPs could improve food colour significantly and protease is the key factor during the flavor formation of fermented food.Researches of applying Monascus spp.in fermented food are mainly focused on the plant source fermented products processing while its applications in fermented meat products are limited to the effects of metabolites from Monascus spp.such as MPs,antioxidants,etc,on qualities and functions of fermented meat products.Studies in which Monascus spp.were used as the starters of fermented meat is still few.For a better utilization of Monascus spp.and their metabolites in fermented meat process,studies of Monascus strains screening,separation,purification and enzymatic properties study of acid protease by Monascus were carried out.Affect mechanism and effects of the acid protease on mytolin degradation and qualities of fermented meat were also studied.1.Screening and identification of Monascus strain for meat fermentation12 Monascus strains of different species were inoculated in PDB&rice medium and cultured.Pigments,citrinin and acid protease activity of its fermented products were analysed.Monascus sp.MJ-1 and Monascus ruber As 3.549 which possessed high production of pigments and acid protease but no or low production of citrinin were screened as the experimental strains.Based on those results,meat processing conditions were simulated.MJ-1 and As 3.549 were inoculated in the aforementioned medium and cultured.Indexes of growth and fermentation properties,dry cell weight of mycelia,pigments production,pH value of fermentation broth and acid protease acitivity,were detected,and tolerance capabilities of the two strains in different meat processing conditions were contrasted.Growth,pigments,acid protease production fermentation properties of MJ-1 were better than that of As 3.549.The results showed that the tolerance capabilities of MJ-1 to NaCl?NaNO2 and sodium polyphosphate were significantly stronger than that of As3.549?p<0.05?.Monascus sp.MJ-1 was selected as the experimental strain combining the preliminary screening results.Morphological observation and ITS sequence analysis were used for classification and identification of MJ-1.Cluster analysis of ITS1,ITS and ITS2 sequence of MJ-1 were carried out and MJ-1 was always clustered with Monascus ruber.MJ-1 was identified as Monascus ruber combining with the results of morphological observation.2.Effects of Monascus ruber MJ-1 on qualities of fermented minced porkMonascus ruber MJ-1 was inoculated in minced pork.Variations of colour,thiobarbituric acid reactive substances?TBARs?,total volatile basic nitrogen?TVBN?,protease activity,myofibril fragmentation index?MFI?,SDS-PAGE of troponin and free amino acid in mince,were detected during the fermentation process.The content of citrinin and biogenic amines were detected by HPLC and LC-MS at the end of fermentation.The results showed that a*value,MFI value,total free amino acid and acid protease acitivity was significantly raised during the fermentation process.The TBARs value decreased significantly?P<0.05?,the TVBN value increased slowly?P>0.05?,after the fermentation reached the highest value?6.82 mg/100 g pork?,still fresh standard?less than or equal to 25 mg/100 g?.SDS-PAGE electrophoresis showed that the degradation of muscle was observed during the fermentation of pork protein.Citrinin was not detected in fermented minced pork by MJ-1.Monascus fermentation could improve the color of fermented minced pork,inhibit fat oxidation and spoilage and improve mytolin degradation and flavor formation.The sensory and physicochemical qualities of fermented minced pork were improved.Citrinin was not detected in fermented minced pork by MJ-1.Total content of biogenic amine was 79.26 mg/100 g,and the contents of biogenic amine with stronger toxicity,histamine and tyramine content were 4.12 mg/100 g and 3.68 mg/100 g respectively which were lower than the FDA standard.And these result indicated that the fermented mince possessed preferable edible quality.Therefore,we consider that it is security for using MJ-1 as the starter in fermented meat.3.Separation,purification and enzymatic characteristics study of acid protease produced by Monascus ruber MJ-1 After ammonium sulfate precipitation,DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration chromatography,molecular sieve,one kinds of acid protease were separated from Monascus ruber,MJ-1 crude enzyme,the relative activity reached 58.57 U/?g,purified 12.28 times,the activity recovery was 5.13%.SDS-PAGE electrophoresis showed that the acid protease molecular weight is 51 KDa,and the subunit is a monomeric enzyme.Sequence analysis of the acid protease by mass spectrometry,the enzyme and purple Monascus produced acid protease?Accession number BAC97797.1?homology reached 95%,with 7 peptides matching,matching score reaches 744 points.Round two chromatography results showed that the acid protease two level structure of alpha helix random coil accounted for 84.3%,accounting for 15.7%,there is no beta folding and rotary,indicating that the thermal stability of acid protease is relatively poor.This enzyme in pH 3,the strongest catalytic capacity of 40?,in 3?5 pH,temperature 30 to 50?range has good stability.Using casein as the substrate of the acid protease for dynamic analysis,to determine the enzyme Km and Vmax were divided into 1.3 mg/mL,44.05 g/mL after min,the enzyme that casein of animal source has a strong affinity for substrate.Mn²⁺,Mg²⁺,2-mercaptoethanol and DTT had obvious effect on the activation of the enzyme,Ag+,Fe³⁺,PMSF,EDTA and EGTA had significant inhibitory effect on the enzyme activity,Cu²⁺,Ca²⁺,Fe²⁺ and Na+ on the enzyme activity was not affected.4.Hydrolysis regular patten of mytolin by acid protease form M.ruber MJ-1 and the effects of the acid protease on muscle qualityBy SDS-PAGE electrophoresis of MJ-1 acid protease degradation of myofibrillar proteins and sarcoplasmic protein,combined with the results of scanning electron microscopy of the muscle enzyme treatment,degradation of the enzyme on muscle protein.Differential pressure dehydration rate of fiber acid protease,bromelain and papain treated muscle muscle compared with MJ-1 diameter,MFI value,shear force of muscle and muscle tissue index,further study on the effect of MJ-1 on muscle quality of acid protease.The results show that the proteins of 190 KDa myosin heavy chain bands in the enzymatic hydrolysis for 30 min disappeared completely,43 KDa of actin and the protein bands of 10 min in the enzyme solution begins to weaken,but not disappeared during enzymatic hydrolysis,enzymatic hydrolysis of 97.4 KDa at 10 min after the new bands appeared the enzymolysis time,and with the continuous enhancement;sarcoplasmic protein in the enzyme did not change in the process;the cross section of muscle tissue were observed under scanning electron microscope after enzymatic hydrolysis became loose,there are a large number of muscle fiber fracture phenomenon;muscle shear force,the treatment of acid by MJ-1 protease of muscle fiber diameter and MFI value of the tender the results are close to the bromelain and papain treatment groups,but the muscle pressure water loss rate was significantly higher than that of bromelain and papain treatment group?P<0.05?.These results suggest that myosin heavy chain,the acid protease degradation of myofibrillar protein in muscle actin,partial degradation,no degradation effect on sarcoplasmic protein.The enzymatic treatment of muscle tissue can significantly improve the tenderness,color and water holding capacity.