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High-yield leucine p-nitroanilide production lactobacillus helveticus strain and application thereof

A technology of Lactobacillus and curd, applied in the field of microorganisms, can solve the problems of not being able to effectively target hydrophobic amino acid residues, low yield of Lactobacillus helveticus, and reducing the bitterness of protein hydrolyzate, so as to reduce the formation of bitterness and improve the flavor , enhance the effect of metabolic capacity

Active Publication Date: 2019-03-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, at present, there are few aminopeptidases isolated from Lactobacillus helveticus strains, and they cannot effectively target hydrophobic amino acid residues such as Leu, Pro, Arg, Lys, etc. that have an impact on bitterness, and these aminopeptidases can be produced The yield of Lactobacillus helveticus is not high enough to significantly reduce the bitterness of protein hydrolyzate

Method used

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  • High-yield leucine p-nitroanilide production lactobacillus helveticus strain and application thereof
  • High-yield leucine p-nitroanilide production lactobacillus helveticus strain and application thereof
  • High-yield leucine p-nitroanilide production lactobacillus helveticus strain and application thereof

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Experimental program
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preparation example Construction

[0057] 2. Preparation of substrate solution

[0058] Dissolve 1 mM of chromogenic substrate (H-Lys-pNA) in 50 mmol L of pH 7.5 -1 in sodium phosphate buffer;

[0059] 3. Lysine aminopeptidase (Lys-pNA) enzyme activity detection

[0060] After incubating 50 μL substrate solution and 50 μL CFE at 30°C for 30 minutes, measure the absorbance at 405 nm; no positive control, no commercial enzyme, and buffer instead of CFE for the blank control;

[0061] The calculation formula of lysine aminopeptidase (Lys-pNA) enzyme activity is: Enzyme activity = (p-nitroaniline production amount * 100 * 0.001)) / (protein concentration * 50 * 0.001 * 30);

[0062] (References: Stefanovic E, Kilcawley K N, Rea M C, et al. Genetic, enzymatic and metabolite profiling of the Lactobacillus casei group reveals strain biodiversity and potential applications for flavor diversification [J]. Journal of Applied Microbiology, 2017, 122 (5 ).)

Embodiment 1

[0063] Embodiment 1: Screening and identification of bacterial strains

[0064] 1. Screening

[0065] (1) Prepare appropriate sample dilution gradient and culture

[0066] Weigh 0.5g of traditionally fermented yak koji in Xining Tibetan area of ​​Qinghai, add it to 4.5mL of sterile water, and then take 0.5mL of bacterial liquid and dilute it in 4.5mL of sterile water, so that the concentration of the sample is gradually diluted to 10 -4 , taking four dilutions of 10 to 10 4 50 μL of each bacterial suspension was spread on the MRS solid medium, cultured at 37°C for 46-48 hours, and observed in time.

[0067] (2) Line separation and purification

[0068] After taking out the plate with colonies, select a gradient plate with obvious single colonies, pick colonies with different colony shapes, and perform secondary streaking until all single colonies are purified.

[0069] (3) Gram staining and catalase test

[0070] Pick a single colony on a glass slide, after smearing, dryi...

Embodiment 2

[0086] Embodiment 2: the cultivation of bacterial strain

[0087] Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 was cultured on MRS solid medium for 48 hours, and colonies were observed and picked for microscopic examination, Gram color and growth characteristics determination.

[0088] It was observed that the colonies of Lactobacillus helveticus CCFM1035 grown on MRS solid medium were round, small, translucent and smooth.

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Abstract

The invention discloses a high-yield leucine p-nitroanilide production lactobacillus helveticus strain and application thereof and belongs to the field of microbial technology. The high-yield leucinep-nitroanilide production lactobacillus helveticus strain, namely lactobacillus helveticus CCFM1035, has the advantages of being high in growth and production speed as well as high in yield of leucinep-nitroanilide (Lys-pNA), and can effectively enhance the protein metabolism of fermented foods, reduce bitter taste of the fermented foods and improve the flavor of the fermented foods, thereby highin application significance to prepared foods such as acidified milk, acidified milk beverages, cheese, Koumiss, sour cream, cookies, vinegar and soybean sauce.

Description

technical field [0001] The invention relates to a strain of Lactobacillus helveticus with high production of lysine aminopeptidase and application thereof, belonging to the technical field of microorganisms. Background technique [0002] The content of free peptides and amino acids in cow's milk is less, and it has higher proteolytic activity, while Lactobacillus helveticus presents the characteristics of multiple amino acid auxotrophy. The ability to form flavor plays an important role in the field of fermented food. [0003] The proteolysis system of Lactobacillus helveticus mainly includes cell wall protease (Cell-envelopeproteinase, CEP), its function is to hydrolyze protein into a series of peptides, and the peptides obtained by these hydrolysis will be transported by oligopeptide transport system (Opp), dipeptide and three The peptide transport system (Dpp and DtpT) is transported into the cell, and is further hydrolyzed into amino acids or smaller peptides by intrace...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A23C9/123A23L7/104A23L27/24A23L27/50C12J1/00C12R1/225
CPCA23C9/1234A23L7/104A23L27/24A23L27/50C12J1/00C12N1/20C12R2001/225C12N1/205A23V2400/147
Inventor 赵建新刘小鸣亓燕然王鸿超张灏陈卫
Owner JIANGNAN UNIV
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