A strain of Lactobacillus helveticus with high production of lysine aminopeptidase and its application
A technology of Lactobacillus and curd, applied in the field of microorganisms, can solve the problems of ineffective targeting of hydrophobic amino acid residues, reducing the bitterness of protein hydrolyzate, and low yield of Lactobacillus helveticus, so as to reduce the formation of bitterness, improve flavor, The effect of fast growth rate
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[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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