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A Bacillus licheniformis producing acid protease

A technology of Bacillus licheniformis and protease, applied in the field of food microorganisms, can solve the problems of inability to grow strains, poor flavor, single function of strains, etc., and achieve the effect of improving flavor and rich flavor

Active Publication Date: 2022-04-15
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the current research, the function of the bacterial strains is relatively single, and many bacterial strains cannot grow well in a hypertonic acidic environment, and the protease activity produced by the rare strains grown in a hypertonic acidic environment Both are low, and protease cannot adapt to the hyperosmotic and acidic environment of fermented soy sauce fermented grains, and its activity is low, resulting in low amino acid nitrogen content and poor flavor.

Method used

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  • A Bacillus licheniformis producing acid protease
  • A Bacillus licheniformis producing acid protease
  • A Bacillus licheniformis producing acid protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Screening of Bacillus licheniformis

[0043] (1) Prepare sample dilution gradient and culture

[0044] Weigh 5g of Pixian Douban broad bean fermented grain raw material, add 50mL sterile saline and shake until a suspension is formed. Aspirate the sample suspension, and perform a 10-fold gradient dilution with sterile saline to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 , take 100-200 μL and spread it on LB medium containing 15% (w / v) NaCl, and culture it at 37° C. for 48 hours until a single colony appears.

[0045] (2) Line separation and purification

[0046] After taking out the plate with colonies, select a gradient plate with obvious single colonies, pick colonies with different colony shapes, carry out secondary streaking and carry out strain culture, and repeat this several times until all single colonies are purified.

[0047] (3) 16S rDNA sequence amplification

[0048] Take 1mL of bacterial solution and centrifuge at 6000rpm for 3min, pour off the supe...

Embodiment 2

[0065] Example 2: The protease produced by Bacillus licheniformis CC5 under high-salt hyperosmotic conditions has high enzyme activity under pH 3 conditions

[0066] (1) Koji making

[0067] Use Shanghai Niang 3042 Aspergillus oryzae (purchased from China General Microorganism Culture Collection Management Center, No. Zhongke 3.951) to make koji. Inoculate Aspergillus oryzae in the PDA solid medium, cultivate for 3-5 days, until there is a layer of fine spores on the surface, scrape the spores with a sterile disposable coating rod, and collect them for later use.

[0068] Accurately weigh 100g of broad beans, scald them in boiling water at 100°C for 10s, and then immediately put them in a water bath at 45°C for 3 minutes to fill the broad beans with water. Then take 30g of wheat flour, fully mix 0.003g of Aspergillus oryzae spores with wheat flour, beat evenly, then fully mix the wheat flour containing Aspergillus spores with broad beans, and carry out koji-making culture at ...

Embodiment 3

[0084] Example 3: Bean paste prepared by Bacillus licheniformis and detection of flavor substances

[0085] The specific embodiment is referring to embodiment 2, carries out koji making, bacterial strain is cultivated in high-concentration salt water, bacterial strain ferments soybean paste, after fermenting 7 days, gets the flavor substance in the soybean paste sample determination soybean paste (table 3). It can be seen from the table that the prepared soybean paste is rich in flavor substances, up to 102 kinds, among which alcohols and esters are rich in types, and the concentration of geraniol with rose fragrance can reach 48.8mg / L, which has the aroma of fried flowers and chocolate, The content of 2,5-dimethylpyrazine with a creamy smell reaches 51.1mg / L, and together with other flavor substances, it makes the flavor of soybean paste richer and fuller.

[0086] Table 3 Flavor compounds synthesized by strain fermentation

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Abstract

The invention discloses a bacillus licheniformis producing acid protease, which belongs to the technical field of food microorganisms. The Bacillus licheniformis of the present invention, isolated from the traditional Pixian Douban brewing system, has been preserved in the China General Microorganisms Collection and Management Center on March 17, 2020, with the preservation number CGMCC NO.19482. The bacillus licheniformis can tolerate environments such as high salt, and the protease produced can reach 26.81U / mg under pH 3 conditions, and can adapt to the high salt and hyperosmotic environment of sauce fermented grains (NaCl concentration is 15%~ 18%). When the strain is applied to the fermentation of soybean paste, 102 kinds of flavor substances can be produced, and there are various types of aromatic substances such as esters and alcohols, making the flavor of soybean paste richer and fuller. The bacillus licheniformis in the present invention is suitable for the preparation of bean fermented food because of its ability to decompose broad bean protein under hyperosmotic conditions and the source attribute of pure natural strains.

Description

technical field [0001] The invention relates to a bacillus licheniformis producing acid protease, which belongs to the technical field of food microorganisms. Background technique [0002] Among the many indicators to measure the quality of fermented sauce and soy sauce seasoning, the concentration of amino acid nitrogen is an important indicator. In fermented sauce and soy sauce, amino acid nitrogen mainly comes from the degradation of protein in beans. More and more active proteases can improve protein degradation and produce more amino acid nitrogen, thereby improving product quality and yield. Fermentation of broad bean grains in Pixian Douban, using broad beans, flour and salt as raw materials, adding Aspergillus oryzae for koji making ( figure 1 ). During the koji-making stage, Aspergillus oryzae fully grows and secretes protease, which degrades macromolecular proteins into polypeptides, a small amount of oligopeptides and a small amount of amino acids. After the ko...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/50A23L11/50A23L27/50A23K10/12A23K10/30C12R1/10
CPCC12N1/20C12N9/50C12R2001/10C12N1/205
Inventor 张丽杰徐岩
Owner JIANGNAN UNIV