Research On Nattokinase Activity Determination Method And Safety Of Natto Products

Natto is a food made from high-quality soybeans and fermented by Bacillus subtilis natto.Various active substances such as nattokinase are produced during the fermentation process.In the industry,the quality of the product is mainly evaluated by the level and characteristics of the enzyme activity.After consulting a large number of documents,we summarized the existing methods for measuring the activity of nattokinase at home and abroad.Through the experimental comparison of several methods,the agarose-fibrin plate method was selected as the target method for research.Natto is fermented by pure strains.Product safety and the safety of fermentation strains are also inseparable from the production process.Generally speaking,the fermentation process is mature,the conditions are controllable,and have high safety guarantees.However,the fermentation strains produce toxic Pollution of harmful metabolites and miscellaneous bacteria inevitably poses a threat to product safety.This thesis takes the commercially available natto and its products as the research object,Mainly research the nattokinase activity determination method and the safety of natto products,The main findings are as follows:1.Study on Methodology and Condition Optimization of Agarose-Fibrin Plate MethodBased on the fact that there is no unified method for measuring nattokinase activity at home and abroad,and to reduce the cost of nattokinase activity determination,by comparing the advantages and disadvantages,accuracy and market application value of existing nattokinase activity determination methods,choose the agarose-fibrin plate method for methodological and condition optimization research.This study compared several linear models of this method.Among them,the linear model established with the logarithm of the concentration and the area of the dissolved circle as the abscissa and ordinate has stronger correlation,and the calculated objective value is closer to the actual enzyme activity of nattokinase.The intra-day and inter-day precision of samples with different concentration gradients were studied,and the relative standard deviation（RSD）was less than 10%.The recoveries of the three parallel experiments of sample addition were 86.95%,85.48%,and87.22%respectively,all of which were above 85%,Shows that the method has high precision and accuracy.The optimization results showed that,the substrates of Beijing Bolsi Biotechnology Co.,Ltd.are more suitable for the research of nattokinase activity.5 mm is the optimal thickness of the plate,and the optimal incubation time is 17.5 h.2.Study on the enzyme activity characteristics of nattokinaseIn this study,the optimal reaction temperature of nattokinase is 50℃,the relative enzyme activity is still as high as 70%after treatment at 45℃for 4 hours,the half-life of enzyme activity at temperatures below 50℃is≥1 h,and treatment at 80℃is 10 min will be inactivated,the relative enzyme activity can still reach 86%after 5 times of repeated freezing and thawing.The optimal reaction p H is 8.5,and the optimal p H range is 6.0-9.0.It is still active at p H 3.0 and p H 11.0,but the activity is very low;after 5 mmol/L of Mn²⁺,Mg²⁺,and Ca²⁺are treated with nattokinase,the activity will be reduced obviously enhanced,of which the promotion effect of Mn²⁺is the most significant,and will increase with time.Nattokinase activity has no obvious change after K⁺and Na⁺treatment,and it is not limited by time and concentration.Three concentrations（5 mmol/L,10 mmol/L,20mmol/L）of Zn²⁺,Cu²⁺,Fe³⁺can inhibit the activity of nattokinase,Among them,the inhibitory effect of Cu²⁺and Zn²⁺on nattokinase will be significant with the increase of concentration and the extension of time,but it will not be inactivated.3.Isolation and Identification of Strains in Commercial Natto ProductsTo determine the total number of colonies on commercially available natto and its products,the study found that 8 kinds of fermented natto were all detected with viable bacteria,and the total number was between 1×10⁷-2×10⁹CFU/g;3 kinds of natto freeze-dried powder detected viable bacteria,and the total number was between 1×10⁷-2×10⁸CFU/g;3 types of natto candies were detected with viable bacteria,and the total number was between 2×10⁵-3×10⁷CFU/g.Natto is fermented from pure strains,so the microbial community in natto products is relatively single,and no colonies of enterobacteria pathogens are observed.The morphology of the viable bacteria colony of each product was basically the same.14 target bacteria were isolated.After Gram staining,physiological and biochemical identification and 16S r DNA molecular means,it was identified that the 13 dominant bacteria of the product were all Bacillus subtilis.4.Drug susceptibility test of strainsAccording to the CLSI M100 antimicrobial susceptibility testing standard,the tolerance of 14 strains to 8 common antibiotics was analyzed.Studies have shown that among the 14 Bacillus subtilis strains,only the SD-7 strain did not show resistance to any kind of antibacterial drugs,and other bacteria showed varying degrees of resistance.14strains are highly sensitive to Ceftriaxone and Gentamicin,indicating that none of them carry Ceftriaxone and Gentamicin resistance genes.SD-1,SD-2,SD-4,SD-8,SDF-4,SDP-5 strains showed high resistance to Ceftazidime,SD-6,SDF-2,SDP-3 showed mild resistance.SDF-2 and SDP-1 showed high tolerance to Ciprofloxacin,while SD-3 showed mild resistance.The MIC value of Meropenem to SD-2 and SDP-5 is 8μg/m L,showing mild resistance.SD-2,SD-3,SD-7,SDF-2,SDP-1 are sensitive to Chloramphenicol,and other strains are mildly resistant.Because the CLSI does not specify the MIC value of Ampicillin against Bacillus bacteria,the drug concentration of 8μg/m L has no inhibitory effect on the strain,and it is impossible to determine whether the strain is resistant.5.Detection of biogenic amine content in natto and its productsThe standard curve of 7 biological amine standards was established by liquid chromatography,and the biological amine content of 20 commercially available natto and its products were detected.The results showed that the 7 biological amine standards could be separated well.And the linearity of the standard curve is good（R²are all>0.9963）.After the samples are processed 6 times in parallel,the relative standard deviation（RSD）of the biogenic amine is 2.54%-8.77%,and the method recovery rate is 91-113%,7 kinds The detection limit and quantification limit range of the biogenic amine standard are0.033～0.167 mg/L and 0.112～0.558 mg/L,respectively,indicating that the selected liquid chromatograph has good precision and accuracy,and can meet the requirements of biological detection in the sample.The total content of 7 biogenic amines in 20 natto products is between 2.21-200.91 mg/kg.