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Enzyme for degrading biogenic amine in soy sauce

A technology of soy sauce and multi-copper oxidase, which is applied in the field of fermentation, can solve the problems of low degradation efficiency, inability to guarantee the absolute singleness of production strains, difficulty in controlling miscellaneous bacteria, etc., and achieve the effect of high degradation efficiency

Active Publication Date: 2018-05-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Soy sauce is generally produced in a non-airtight environment, and the control of miscellaneous bacteria is very difficult, and the absolute singleness of the production strain cannot be guaranteed. Therefore, the method of controlling biogenic amines by controlling the microorganisms that produce amino acid decarboxylase is limited
At present, enzymatic degradation of biogenic amines is the most feasible, not only does not damage food nutrition, but also does not produce new toxic substances. The existing biogenic amine degradation enzymes include amine oxidase, which includes primary amine oxidase, diamine oxidase Enzyme, monoamine oxidase, among which primary amine oxidase can only degrade phenethylamine, the optimum pH is 7.0, diamine oxidase only works on diamine, especially histamine, and the optimum pH is 8.5, monoamine oxidase can degrade tyramine, Tryptamine has an optimum pH of 6.0-7.0. These enzymes have three common problems: 1) There are few types of biogenic amines; 2) The degradation efficiency is low; 3) The optimum pH is neutral, which is not suitable for acidic applications. In fermented food, enzymes applied to soy sauce need to be resistant to salt and acid

Method used

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  • Enzyme for degrading biogenic amine in soy sauce
  • Enzyme for degrading biogenic amine in soy sauce
  • Enzyme for degrading biogenic amine in soy sauce

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 High-throughput screening of bacterial strains containing multiple copper oxidase genes

[0025] 1. High-throughput screening of biogenic amine-free strains

[0026] Mix the moromi sample with sterile physiological saline, dilute it in different times and spread it on LB medium. After standing overnight at 37°C, select a suitable plate with a single colony. Add 1 mL of LB medium to each well plate in advance, pick single bacteria into a 96-well plate with a Qpix420 automatic bacteria picker, and culture them statically at 37°C for 24 hours. Then transfer to the biogenic amine detection liquid medium (the blank control is not inoculated), and observe the color change after standing at 37°C for 48 hours. If the color of the blank control does not change color, it is positive, and if it turns purple, it is negative. By using high-throughput screening technology, 70 positive strains were screened from 8000 strains.

[0027] 2. Screening of norbiometric strains ...

Embodiment 2

[0035] Degradation of biogenic amines in soy sauce by multi-copper oxidase crude enzyme in embodiment 2

[0036] Inoculate the screened bacterial strain containing the multi-copper oxidase gene to a concentration of 0.5-1mmol / LCu 2+ In the LB medium, 37°C, 220rmp / min, cultivate for 18-24h, after the cultivation is completed, centrifuge at 7000-10000r / min for 5-10min to collect the bacteria, repeat 2-3 times, and perform ultrasonic disruption. Filtrate and centrifuge the broken solution obtained, and concentrate the supernatant through a 3KD protein ultrafiltration tube to obtain a 3-5 times concentrated crude enzyme solution.

[0037] Mix 2ml of crude enzyme solution with 2mL of a certain brand of commercially available soy sauce, place the mixture in a 37°C incubator for 48h, centrifuge at 4°C and 10,000r / min for 5min to get the supernatant, and use HPLC to detect the content of biogenic amines Content Take 2ml of heat-inactivated crude enzyme solution and 2mL of commerciall...

Embodiment 3

[0040] Enzymatic properties of embodiment 3 multi-copper oxidase crude enzyme

[0041] (1) Optimum temperature for multi-copper oxidase

[0042]The crude enzyme solution and the substrate were reacted for 10 minutes in a water bath at 25, 35, 40, 45, 50, 55, 60, 70, and 80°C respectively, and the enzyme activity was determined to determine the optimum reaction temperature of the enzyme. is 100%. The result is as image 3 It is shown that when the temperature is 45-55°C, the enzyme is relatively stable, and the activity can maintain more than 90% of the enzyme activity at the optimum temperature.

[0043] (2) Optimum pH of multi-copper oxidase

[0044] Prepare buffers with different pH: citrate buffer pH 2.2-6.0, phosphate buffer pH 6.0-8.5. React at 55°C for 10 minutes at different pH, and measure the enzyme activity at different pH conditions after the reaction is over to determine the optimum reaction pH. Take the highest enzyme activity as 100%. The result is as Fig...

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Abstract

The invention discloses enzyme for degrading biogenic amine in soy sauce, and belongs to the technical field of fermentation. A sequence of multi-copper oxidase crude enzyme provided by the inventionis as shown in SEQ ID NO.1. The enzyme disclosed by the invention has different degrees of degradation functions on biogenic amine of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine and spermidine in the soy sauce, wherein the concentration of the tyramine of which the content in the soy sauce is the most is 540.71 mg / L. After adding the multi-copper oxidase crude enzymeprovided by the invention, the content of the biogenic amines is reduced by 100 mg / L within 24 hours; compared with an existing technology for reducing the content of the biogenic amine by 58.57 mg / Lby adding pure enzyme in a biogenic amine mixed solution for 24 hours, the degradation time is short, and the effect is good.

Description

technical field [0001] The invention relates to an enzyme for degrading biogenic amines in soy sauce, which belongs to the technical field of fermentation. Background technique [0002] Biogenic amines are a general term for a class of low-molecular-weight nitrogen-containing organic compounds produced in organisms, which are precursors for the synthesis of hormones, nucleotides, and proteins in organisms. The appropriate amount of biogenic amines synthesized by the organism itself has physiological functions such as promoting growth, enhancing metabolic activity, strengthening the intestinal immune system, controlling blood pressure and eliminating free radicals. However, excessive intake of exogenous biogenic amines will cause the expansion of blood vessels, arteries and microvessels, Causes adverse reactions in the organism, such as high blood pressure, headache, abdominal cramps, diarrhea and vomiting. Common biogenic amines in food mainly include putrescine, cadaverine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/75A23L5/20
CPCA23L5/25C12N9/0057C12N15/75
Inventor 方芳徐洁陈坚周景文曾伟主
Owner JIANGNAN UNIV
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