Method for preparing nattokinase by liquid fermentation method

A technology of nattokinase and liquid fermentation, which is applied in the field of bioengineering and medicine, can solve the problems of unbalanced nutrient components of the medium, insufficient consideration of raw material safety, and not very vigorous microbial growth, so as to ensure quality and safety, The effect of protecting nutrients and meeting the needs of growth and metabolism

Pending Publication Date: 2017-07-28
HENAN UNIVERSITY OF TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic method used to produce nattokinase is to use soybeans as raw materials, soak, cook, cool down, inoculate and then ferment in an open environment, but the internal bacteria of soybeans are not easy to grow, and the yield will naturally not be too high; CN102586141B Disclosed is a liquid fermentation method using soybean cooking water as the main raw material and supplemented with two kinds of inorganic salts as the medium. In this case, the utilization rate of raw materials is inevitably too low; The method for nattokinase; CN106085991A discloses a method for preparing nattokinase by solid-state fermentation, which provides a method for preparing nattokinase with peanut meal as the main raw material; although the above-mentioned patents provide various The selection of raw materials has made a certain contribution to the improvement of enzyme

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A method for preparing nattokinase by a liquid state fermentation method, comprising the following processing steps:

[0042] ⑴Activation of Bacillus natto

[0043] Under a sterile environment, use an inoculation loop to pick a loop of the preserved pure Bacillus natto strain and inoculate it on the slant medium of a conventional test tube. Take a well-grown single colony and spread it on the slope of a new test tube, and culture it in a 36-38°C incubator for 24-32 hours to obtain activated Bacillus natto strains;

[0044] ⑵ Preparation of fermentation medium

[0045] Among them, the required fermentation medium formula (g / L) is: soybean whole powder 100, glucose 25, sucrose 20, glycine 38, alanine 28, serine 27, lysine 29, aspartic acid 25, group amino acid 23, leucine 18, soybean protein peptide 3.0, biotin 0.5, Vc 2.0, Ve 2.0, Vb 5 3.0, niacin 2.0, niacinamide 2.0, taurine 2.0, L-cysteine ​​or its hydrochloride 2.0, L-cystine 2.0, heparin sodium 0.3, arginine 2.0...

Embodiment 2

[0061] The same as the steps of Example 1, the difference is:

[0062] a. Process step (2) The composition of the fermentation medium in the preparation of the fermentation medium is (g / L): whole soybean powder 10, glucose 40, sucrose 3, glycine 76, alanine 1, serine 56, lysine 3 , aspartic acid 50, histidine 2, leucine 38, soybean protein peptide 0.05, biotin 1.8, Vc 0.05, Ve 4.0, Vb 5 0.05, niacin 4.0, niacinamide 0.05, taurine 4.0, L-cysteine ​​or its hydrochloride 0.05, L-cystine 4.0, heparin sodium 0.02, arginine 4.0, glutamic acid 0.02, spit Wen-804.0, sodium caseinate 0.8, coenzyme Q 10 0.02, MgSO 4 ·7H 2 O 6.0, CaCl 2 0.2, balance: deionized water, pH value = 7.4;

[0063] b. Process steps (1) and (3) The method conditions for the activation of Bacillus natto strains and seed preparation are as follows: in a sterile environment, use an inoculation loop to pick 1 loop of the preserved pure Bacillus natto strains in a routine Streak inoculation on the slant medi...

Embodiment 3

[0068] The same as the steps of Example 1, the difference is:

[0069] a. Process step (2) The composition of the fermentation medium in the preparation of the fermentation medium is (g / L): soybean whole powder 180, glucose 2, sucrose 45, glycine 2, alanine 49, serine 5, lysine 58 , aspartic acid 5, histidine 46, leucine 1, soybean protein peptide 6.0, biotin 0.01, Vc 4.0, Ve 0.04, Vb 5 6.0, niacin 0.0, niacinamide 4.0, taurine 0.05, L-cysteine ​​or its hydrochloride 4.0, L-cystine 0.04, heparin sodium 3.0, arginine 0.03, glutamic acid 4.0, spit Wen-800.05, sodium caseinate 18.0, coenzyme Q 10 4.0, MgSO 4 ·7H 2 O 0.3, CaCl 2 4. Balance: deionized water, pH=6.8;

[0070] b. Process steps (1) and (3) The method conditions for the activation of Bacillus natto strains and seed preparation are as follows: in a sterile environment, use an inoculation loop to pick 1 loop of the preserved pure Bacillus natto strains in a routine Streak inoculation on the slant medium of the t...

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PUM

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Abstract

The invention discloses a method for preparing nattokinase by a liquid fermentation method. The method comprises strain activation, culture medium preparation, seed preparation, inoculation, temperature change fermentation and post-treatment. The method combines strain activation, culture medium preparation, seed preparation, inoculation, fermentation and post-treatment. The whole process is carried out in a clean or sterile environment. The method does not produce escape, overflow, dropping and leakage and satisfies GMP requirements. Compared with the traditional method, the method provided by the invention utilizes an ingenious multi-component medium design and provides a good material basis for growth of nattokinase production bacteria. Through use of a surfactant, nattokinase production and secretion are significantly improved. Through use of the temperature change fermentation method and process, a nattokinase yield is improved by 6-38%. The method has reasonable and advanced processes, is suitable for expanding reproduction, significantly improves a yield and product quality, is environmentally friendly and can be widely used for research and development of nattokinase and other related products.

Description

technical field [0001] The invention belongs to the field of bioengineering and technology, and in particular relates to a method for preparing nattokinase by a liquid state fermentation method. Background technique [0002] Natto is the traditional fermented soybean food that Japanese people love most. Nattokinase (NK) is a kind of protease with various biological activities prepared by fermenting soybean. It has been studied extensively and deeply. Modern scientific research has shown that its structure is a linear amino acid chain without space folding, and its characteristic substrate is fibrin. The fibrinolytic activity is greater than that of plasmin, and the molecular weight is much smaller than that of urokinase and lumbrokinase, and can be absorbed by the intestine. According to reports, nattokinase can effectively dissolve thrombus, and also has various physiological functions such as reducing blood viscosity, inhibiting platelet coagulation, lowering blood fat an...

Claims

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Application Information

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IPC IPC(8): C12N9/54C12R1/07
CPCC12N9/54
Inventor 王卫国赵永亮杜灵广管景帅陶宜辰林强胡晓伟王丽洁苑旺古亚楠张仟伟王卫
Owner HENAN UNIVERSITY OF TECHNOLOGY
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