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Liquid nattokinase fermentation method

A technology of nattokinase and production method, which is applied in the field of food processing and can solve the problems of no economic benefit and high energy consumption

Active Publication Date: 2018-12-14
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the chemical structure of dietary fiber in bean dregs contains a large number of hydrophilic groups and has a high bound water content, it needs to consume a lot of energy in the process of screw extrusion and crushing treatment, and there is no economic benefit.

Method used

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  • Liquid nattokinase fermentation method
  • Liquid nattokinase fermentation method
  • Liquid nattokinase fermentation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 The preparation of nattokinase crude enzyme liquid

[0029] 1. Preparation of starter

[0030] Buy Bacillus natto powder, dissolve it with sterile water, and dilute it to 10 -3 to 10 -6 Different gradient plates were streaked and cultured on LB medium containing 2% agar for 18 hours for microscopic examination. figure 1 , and then pick a single colony into LB liquid medium, activate three generations, then inoculate 3% inoculum in LB liquid medium at 120rpm, and cultivate at 37°C for 12h; the bacterial liquid is placed in a high-speed refrigerated centrifuge at 4°C, 9500r / min centrifuged for 10min; the centrifuged cells were washed twice with sterile water, resuspended, and the concentration of the bacterial suspension was adjusted to 10 7 CFU / mL, as starter culture.

[0031] 2. Preparation of fermentation medium

[0032] At room temperature, soak soybeans at a mass ratio of 1:5 to water for 13 hours, drain the water, grind the soaked soybeans with wa...

Embodiment 2

[0035] Embodiment 2 nattokinase liquid fermentation process optimization

[0036] 1. Optimization of fermentation conditions of nattokinase

[0037] Investigate the influence of the five factors of inoculum size, fermentation temperature, fermentation time, shaker speed, and solid-liquid ratio on the activity of nattokinase, and obtain the parameter range of the optimal enzyme activity of nattokinase as follows: figure 2 , and according to the Box–Behnken principle on the basis of the single factor test, the fermentation time, the rotating speed of the shaking table, and the three main factors of the inoculum amount are carried out in a response surface test design. The test factor level table is shown in Table 1, and the test results are shown in Table 2;

[0038] Through the single factor test, it can be determined that the enzyme activity reaches the highest at 37°C, which is related to the influence of temperature on the metabolism of the bacteria. When the temperature is...

Embodiment 3

[0050] Embodiment 3 Enzyme production comparison and live bacteria count of different fermentation media

[0051] Investigate the impact of the type of medium on the activity of nattokinase, the specific operation steps are as follows:

[0052] will reach 10 7 Bacillus natto starter culture with cfu / mL number of live bacteria was respectively inserted into soybean dregs medium, corn soybean meal medium, and LB medium with the same inoculum amount, and cultured in a shaker at the same temperature and at the same speed, and then each time Take the three kinds of liquid culture medium by centrifugation every 12 hours and take the supernatant to measure the activity of nattokinase. At the same time, take part of the supernatant for 10-fold gradient serial dilution, and select 2~3 suitable gradients to spread evenly on the plate. Three parallel experiments were carried out for each gradient and the average value was used to count the viable colonies; the results of nattokinase act...

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Abstract

The invention discloses a liquid nattokinase fermentation method. The method comprises the following steps: 1) adding water into bean dregs to regulate the moisture content to be 80-90%; 2) inoculating the activated bacillus natto according to an inoculum size of 3-10%; 3) performing shake cultivation at a temperature of 36-38 DEG C for 30-40 hours; 4) centrifuging to take the supernatant, filtering and degerming the supernatant to obtain crude enzymes; adding the obtained crude enzymes into ammonium sulfate until the saturability reaches 50-90%, allowing standing still at the temperature of 0-4 DEG C for 10-20 hours, centrifuging at the speed of 9000-10000 revolutions per minutes for 8-15 minutes, taking the precipitate, re-dissolving with a buffer solution, dialyzing and desalting to obtain nattokinase; mixing with a trehalose maltodextrin protective agent, and performing spray drying, thereby obtaining the nattokinase enzyme powder. The nattokinase prepared by the method disclosed by the invention can reach specific enzyme activity of 385.14 IU / mg and has excellent stability.

Description

technical field [0001] The invention belongs to the field of food processing, and in particular relates to a method for producing nattokinase. Background technique [0002] In 1987, Dr. Sumi of Japan discovered an enzyme in natto that can effectively dissolve thrombus in the heart and brain—Nattokinase (NK), and since then began the application and research of NK. Nattokinase is extracted from food natto or fermented by Natto bacteria. It is a protein with a molecular weight much smaller than UK, SK, and tPA. It is a serine protease produced by natto during the fermentation process. It can hydrolyze fibrin into Small peptides and amino acids, and has a good thrombolytic ability, and also has a certain effect on lowering blood pressure and preventing cardiovascular and cerebrovascular diseases. Currently, the thrombolytic drugs approved for medical application include: Streptokinase (SK) and Urkinase (Urkinase, UK), etc., but they all have certain defects to a certain extent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/54
CPCC12N9/54
Inventor 朴春红胡洋唐玉芳王婧崔阳迟翰盛王玉华刘俊梅于寒松代伟长
Owner JILIN AGRICULTURAL UNIV
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