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Genetically engineered bacterium for increasing butanedione content and acetoin content in yoghourt and application thereof

A technology of genetically engineered bacteria and diacetyl, applied in genetic engineering, bacteria used in food preparation, applications, etc., can solve problems such as low yield and difficulty in satisfying the flavor of yogurt

Inactive Publication Date: 2021-12-07
SHANGHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is: in view of the relatively low yields of diacetyl and acetoin in yoghurt fermented by Streptococcus thermophilus and Lactobacillus bulgaricus in traditional starter cultures, it is difficult to meet the demand for yoghurt flavor. Genetically engineered bacteria for increasing the production of diacetyl and acetoin in yogurt

Method used

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  • Genetically engineered bacterium for increasing butanedione content and acetoin content in yoghourt and application thereof
  • Genetically engineered bacterium for increasing butanedione content and acetoin content in yoghourt and application thereof
  • Genetically engineered bacterium for increasing butanedione content and acetoin content in yoghourt and application thereof

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Experimental program
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Effect test

Embodiment 1

[0020] Construction of engineering bacteria producing diacetyl and acetoin:

[0021] 1. Construction of expression vector pSIP409-nox-alsS

[0022] According to the nox gene sequence of Streptococcus mutans and the alsS gene sequence in the whole genome of Lactobacillus casei TCS, in order to co-express the nox and alsS genes in a single vector, a ribosome binding site was added to the 5' end of the alsS gene, using Premier 5.0 and Vector NTI software designed primers nox-F, nox-R and alsS-F, alsS-R, and used the one-step cloning kit to connect the nox and alsS fragments to the vector pSIP409. The NcoI restriction site was introduced at the 5' end of the primer nox-F, and the XhoI restriction site was introduced at the 5' end of the primer alsS-R. After the PCR product was purified, it was ligated with the expression vector pSIP409, which was double-digested with the same restriction site, transformed into E.coli DH5α competent cells (VazymeBiotech Co., Ltd., Nanjing, China),...

Embodiment 2

[0031] Yogurt fermented by wild-type strain TCS and engineered strain TCS-ΔalsD-nox-alsS:

[0032] Inoculate the to-be-used TCS and TCS-ΔalsD-nox-alsS strains in MRS broth respectively, culture them conventionally at 37°C for 12-16 hours, collect the bacteria by centrifugation at 6000×g for 10 minutes at room temperature, wash them twice with sterile water, and use An equal volume of sterile water was resuspended, and the suspension was used as a starter culture. Use 12% (w / v) skimmed milk powder to prepare yogurt, sterilize at 95°C for 10 minutes, wait until the sterilized skim milk is cooled to about 40°C, and inoculate the bacteria suspension obtained above at a ratio of 4% (v / v). Bacteria skim milk, and IP-673 with a final concentration of 25ng / mL was added to the inducible recombinant bacterium TCS-ΔalsD-nox-alsS for induction. After that, it was left to ferment at 37° C. until the pH was about 4.6, and then placed in a refrigerator at 4° C. for 12-24 hours after ripenin...

Embodiment 3

[0034] Enzyme Activity Determination of Engineering Bacteria:

[0035] The enzyme activities of the wild strain TCS and the engineered strain TCS-ΔalsD-nox-alsS were determined, and the enzyme activities of NADH oxidase and α-acetolactate synthase were determined respectively.

[0036](1) Determination of NADH oxidase activity: NOX activity was determined using NADH oxidase activity detection kit (Solarbio Science & Technology, Beijing, China). The enzyme activity unit (U) is defined as every 10,000 bacteria in the reaction system, and the A600 change of 0.01 per minute in the reaction system is defined as an enzyme activity unit.

[0037] (2) Assay of α-acetolactate synthase activity: ALS activity was determined according to the method described by Holtzclaw (Holtzclaw and Chapman 1975) with some modifications. In 1mM thiamine pyrophosphate, 5mM MgCl 2 , 0.1mM flavin adenine dinucleotide, 100mM sodium pyruvate and 300μL crude enzyme solution. The reaction was carried out a...

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Abstract

The invention discloses a genetically engineered bacterium for increasing the content of butanedione and acetoin in yoghourt and application thereof. The genetically engineered bacterium disclosed by the invention is lactobacillus casei which contains pSIP409-nox-alsS plasmids and is expressed on lactobacillus casei of which an alpha-acetolactate decarboxylase gene (alsD) is knocked out, and the pSIP409-nox-alsS plasmids can express NADH oxidase and alpha-acetolactate synthetase and cannot express the alpha-acetolactate decarboxylase. The genetically engineered bacterium has higher enzyme activity than wild lactobacillus casei, the yield of butanedione and the yield of acetoin in fermented yoghurt are 2.38 times and 11.19 times that of a wild strain TCS respectively, a new thought is provided for biosynthesis of butanedione and acetoin through lactobacillus casei, a theoretical basis is provided for yoghurt flavor optimization production in the future, and the application prospect is wide.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for increasing the content of diacetyl and acetoin in yogurt and its application, belonging to the technical field of bioengineering. Background technique [0002] Diacetyl and acetoin are recognized key aroma substances in yoghurt, they contribute to yoghurt the taste of milk and butter, which is essential for the typical flavor of yoghurt. Diacetyl and acetoin are often used as flavor enhancers in various dairy products, coffee, wine and other foods to increase the flavor of products and also in various flavor formulations. At present, diacetyl and acetoin are mostly produced as food additives, but are rarely produced as flavor compounds in the fermentation process of yogurt. At present, there is a trend to enhance food flavor through endogenous generation rather than exogenous addition, which is more in line with the requirements of contemporary society for green natural food. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/70C12N15/60C12N15/54C12N15/53A23C9/123C12R1/245C12R1/19
CPCC12N15/746C12N15/70C12Y401/01005C12N9/88C12Y202/01006C12N9/1022C12Y106/03001C12N9/0036A23C9/1234A23V2400/125
Inventor 田怀香景艳陈臣于海燕黄娟袁海彬娄新曼田同辉胡阳
Owner SHANGHAI INST OF TECH
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