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Separation, purification and application of a hydrolyzed milk allergen β-lactoglobulin protease

A technology for producing protease and seeds, applied in the biological field, can solve the problems of limited β-lactoglobulin desensitization effect, influence on milk flavor, complex hydrolysis process, etc., and achieve stable protease properties, short fermentation period and simple fermentation medium. Effect

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the enzymes mainly used to eliminate allergens in the market are mainly animal protease, plant protease, subtilisin, pepsin, trypsin, etc., but their desensitization effect on β-lactoglobulin is limited, and desensitization under optimal conditions The allergy effect is only below 50%. Studies have found that when these enzymes are used in combination, the hydrolyzate produced by hydrolyzing whey protein is less allergic than that produced by a single enzyme hydrolysis, and the best desensitization effect can reach 70%. or so, but the enzymatic hydrolysis process of several different sources is complicated and costly, and may also produce bitter polypeptides, affecting the flavor of milk

Method used

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  • Separation, purification and application of a hydrolyzed milk allergen β-lactoglobulin protease
  • Separation, purification and application of a hydrolyzed milk allergen β-lactoglobulin protease
  • Separation, purification and application of a hydrolyzed milk allergen β-lactoglobulin protease

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Effect test

Embodiment 1

[0056] The separation and purification of embodiment 1 bacillus subtilis

[0057] The strains used in this invention were screened from Mashan Ranch in Wuxi, and more than 3,000 wild strains were obtained from soil and water, and then 30 strains capable of degrading protein were obtained by screening with fat-free milk medium ( figure 1 ), and then directly react the crude enzyme produced by the strain with β-lactoglobulin, and finally determine that Strain 7 (S7) has the best effect on β-lactoglobulin, and then through 16S rDNA and functional gene gyrA sequencing and calibration, determine S7 is Bacillus subtilis, which was preserved in the China Center for Type Culture Collection on September 27, 2016, with the strain preservation number M2016532, the preservation address is the Wuhan University Collection Center, and the classification is named Bacillus subtilis.

Embodiment 2

[0058] The mensuration of embodiment 2 bacillus subtilis performance

[0059] After the activation of bacterial strain S7, the inoculation amount of 2% (v / v) was transferred to a 250mL Erlenmeyer flask with 50mL of AM medium in each bottle, and cultured on a shaker at 220r / min at 37°C. Samples were taken every 4h in the early stage. Take a sample every 2h, measure the OD value at 600nm wavelength with a spectrophotometer, take time as the abscissa, OD 600 The value is the ordinate, draws the growth curve of Bacillus subtilis S7, and does three groups of parallel experiments ( figure 2 ). It can be found that the strain can reach a stable phase in about 12 hours and grow very rapidly.

Embodiment 3

[0060] Production and separation and purification of embodiment 3 protease

[0061] (1) Inoculate a single colony of Bacillus subtilis S7 into AM liquid medium with an inoculation needle, and cultivate it at 37°C and 220rpm for 12h;

[0062] (2) Inoculate the logarithmic phase seed liquid in the AM liquid medium in the PDA liquid medium according to the inoculum size of 2% by volume, and cultivate the medium for 12h at 37° C. at 220 rpm;

[0063] (3) Centrifuge the fermented liquid, collect the supernatant of the fermented liquid to be the crude enzyme liquid, and the enzyme activity is 37.32 U / mL.

[0064] (4) Ferment and cultivate Bacillus subtilis S7 to obtain the crude enzyme liquid, and use a gradient precipitation of ammonium sulfate acid with a mass fraction of 60% to precipitate the crude enzyme liquid, then resuspend the precipitate with pH 8.0, 50 mM phosphate buffer, dialyze, and use pH 8.0, 50mM phosphate buffer to equilibrate Q Sepharose fast-flow ion exchange co...

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Abstract

The invention discloses an isolation and purification of a hydrolyzed milk allergy source beta-lactoglobulin protease and an application thereof, and provides a Bacillus subtilis S7 producing a hydrolyzed milk allergen source beta-lactoglobulin protease. The protease produced by the fermentation and purifying of the Bacillus subtilis S7 can effectively degrade the beta-lactoglobulin in the milk protein, the protease output has high yield, the fermentation method is simple, a fermentation medium is simple, the fermentation cycle is short, and the sensitization can be effectively eliminated.

Description

technical field [0001] The invention relates to the separation, purification and application of a hydrolyzed milk allergen β-lactoglobulin protease and belongs to the field of biotechnology. Background technique [0002] Milk is rich in protein and is an important nutrient for human growth, but eating milk can also cause allergic reactions in the human body. Milk allergy is mainly a type I hypersensitivity reaction mediated by specific IgE antibodies, which can cause respiratory tract, digestive tract, skin or systemic allergy, mainly manifested as vomiting, abdominal pain and diarrhea and other gastrointestinal discomfort, and some children also develop angioedema, Symptoms such as urticaria, rhinitis, asthma, and allergic syncope can also damage the small intestinal mucosa and liver function, seriously affecting the absorption of milk protein by infants and young children. [0003] Milk protein is mainly composed of casein and whey protein, and α-lactalbumin and β-lactogl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/54C12R1/125
Inventor 张娟郑天飞堵国成陈坚汤恒杨佩珊马浩
Owner JIANGNAN UNIV
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