CN111034795A - Production method of yoghourt with high glutathione and S-adenosine-L-methionine content - Google Patents

Production method of yoghourt with high glutathione and S-adenosine-L-methionine content Download PDF

Info

Publication number
CN111034795A
CN111034795A CN201811184095.1A CN201811184095A CN111034795A CN 111034795 A CN111034795 A CN 111034795A CN 201811184095 A CN201811184095 A CN 201811184095A CN 111034795 A CN111034795 A CN 111034795A
Authority
CN
China
Prior art keywords
glutathione
yoghourt
lactococcus lactis
adenosine
methionine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201811184095.1A
Other languages
Chinese (zh)
Inventor
徐楚田
石朱伟
邵家琪
周可
李易潇
余承楷
张景庭
刘伟
徐志南
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Beigu Biotechnology Co ltd
Original Assignee
Hangzhou Beigu Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Beigu Biotechnology Co ltd filed Critical Hangzhou Beigu Biotechnology Co ltd
Priority to CN201811184095.1A priority Critical patent/CN111034795A/en
Publication of CN111034795A publication Critical patent/CN111034795A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/123—Bulgaricus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/231—Lactis

Abstract

The invention discloses a production method of two yoghourt with high glutathione and S-adenosine-L-methionine content. A method for preparing yogurt containing glutathione and S-adenosine-L-methionine by adding glutathione during yogurt production process is provided. In addition, the method is a new method for preparing the yoghourt with high glutathione content and S-adenosine-L-methionine through cloning the bifunctional glutathione synthetase endogenous biosynthesis glutathione in the lactococcus lactis. The invention realizes the new way supply of two liver protective factors (glutathione and S-adenosine-L-methionine) in the form of yoghourt, has industrial production potential and can generate great economic and social benefits.

Description

Production method of yoghourt with high glutathione and S-adenosine-L-methionine content
Technical field and purification method
The invention relates to a production method of two yoghourt with high glutathione and S-adenosine-L-methionine content, belonging to the field of bioengineering. More particularly, relates to a method for preparing yoghourt with high glutathione content and high S-adenosine-L-methionine content by adding exogenous nutrition health-care molecules and modifying lactococcus lactis in the process of preparing the yoghourt.
Background
Glutathione (GSH), also known as γ -L-glutamyl-L-cysteine-glycine, is a tripeptide compound formed by the condensation of L-glutamic acid, L-cysteine and glycine. GSH exists in two major forms, namely reduced Glutathione (GSH) and oxidized glutathione (GSSG). Among them, reduced glutathione is mainly used in the body. GSH is abundant in the heart, liver, kidney, red blood cells and eye lens of the human body. Free sulfhydryl on glutathione molecule is its active group, and has strong ability of supplying proton hydrogen/electron. In the living body, glutathione has many important physiological functions: such as (1) an antioxidant effect. GSH is an antioxidant with the largest content in cells, and can remove oxygen free radicals and superoxide free radicals and protect DNA, proteins, other biological macromolecules and the like from oxidative damage mediated by active oxygen; (2) the function of detoxification. GSH can be combined with exogenous substances such as heavy metal ions, toxic compounds, etc., and transferred by glutathione-S-transferase (GST) to promote its discharge out of cells, thereby achieving detoxification effect; (3) is involved in the transport and metabolism of amino acids. Under the action of Glutamyl Transpeptidase (GT), the gamma-glutamyl of GSH and extracellular free amino acid form glutamyl-amino acid to realize the transmembrane transport of amino acid, and simultaneously GSH can participate in the metabolism of related amino acid through intracellular glutamyl cycle; (4) participate in cell signal transduction. Can maintain the balance of acetylcholine and cholinesterase in cells, and prevent cytopathic effect caused by abnormal metabolism, such as fibrosis; (5) an anti-HIV immunopotentiator. Can activate proliferated T lymphocyte, resist virus invasion, and enhance immunity.
The yoghurt, as a common dairy product, has various benefits to human bodies due to the fact that the yoghurt contains lactic acid bacteria. Research shows that the lactic acid bacteria can inhibit the breeding of harmful bacteria and reduce cholesterol; the yogurt containing Bacillus bifidus or Lactobacillus acidophilus can treat infantile diarrhea, and maintain normal operation of microorganisms in intestinal tract. Compared with unfermented milk, the yoghourt has the advantages that the milk protein and the milk fat are partially decomposed and easily digested and absorbed after fermentation; the lactose content is reduced, and the product is suitable for patients with lactose intolerance and low gastric acid content, and has health promotion effects, such as preventing diseases and delaying aging. Therefore, the yoghurt is vigorously developed and is beneficial to the health of the whole people.
The invention creatively implants the glutathione synthesis way into the lactococcus lactis, so that the glutathione which is beneficial to human bodies can be synthesized when the lactococcus lactis is used for fermenting the yoghourt, and the health-care function of the yoghourt is enhanced.
Disclosure of Invention
Aiming at the defect that the existing yoghourt does not contain or has low content of specific health-care factors, two methods are adopted to produce the yoghourt with high content of glutathione and S-adenosine-L-methionine.
The invention content of the first production method is as follows: in the production process of the common yoghourt, the high-content glutathione is contained in the final yoghourt product by adding an exogenous glutathione method and adjusting the production process to reach 3-20mg/L, and simultaneously, the methionine is added, and the SAM is efficiently converted and synthesized by using the Lactobacillus bulgaricus (Danisco L340) in the yoghourt and the SAM synthetase system in the streptococcus thermophilus, so that the final yoghourt product also contains the SAM with higher content, and the range is 1-20 mg/L. In addition, lactococcus lactis (such as NZ 9000) can also be used as the strain in the fermentation process, and similar effects can be achieved.
The invention of the second production method is as follows:
1) amplification of bifunctional glutathione synthetase genes: bifunctional glutathione synthetase Gene of Streptococcus thermophilus according to NCBI (S. thermophilus) (II)gshF) The sequence, the gene size is about 2252bp, a pair of primers is designed, an upstream primer: 5'-CCCAAGCTTGGATCCTCTAGAGTCGACCT-3', downstream primer: 5'-CATGCCATGGTTATGATTATCGATCGACTGTT-3', adding Hind III and Nco I restriction enzyme sites on the two primers respectively; carrying out double enzyme digestion on the fragment obtained by PCR amplification by using Hind III and Nco I restriction enzymes, and then recovering and storing;
2) constructing a recombinant expression vector, namely directionally cloning a target gene into an expression vector pNZ8148 which is subjected to double enzyme digestion by the same endonuclease to obtain a recombinant expression vector pNZ-gshF containing the bifunctional glutathione synthetase, transforming the recombinant expression vector into an escherichia coli DH5 α competence, then coating the competent escherichia coli DH5 α competence on an LB plate containing 12 mg/L chloramphenicol resistance, culturing overnight, randomly selecting a colony growing on the plate, extracting a plasmid, and performing PCR identification;
3) preparation of lactococcus lactis NZ9000 electrotransformation competence: selecting a single colony of lactococcus lactis NZ9000, inoculating the single colony into 5mL of GM17 liquid culture medium, and standing at 30 ℃ for overnight culture; inoculating to 100 mL GM17 liquid medium at 1% inoculum size, and standing to OD600Adding into a precooled sterile centrifuge tube after ice bath for 10 min, centrifuging for 10 min at 5000 rpm at 4 ℃, and discarding the supernatant, wherein the temperature is close to 0.4; adding pre-cooled 25 mL of solution I (0.5M sucrose, 10% glycerol) for heavy suspension, carrying out ice bath for 15 min, centrifuging and removing supernatant; add precooled 25 mL of solution II (0.5M sucrose, 10% glycerol, 0.05M Na)2EDTA), resuspending, centrifuging after ice bath for 15 min and discarding the supernatant; adding pre-cooled 25 mL solution I for heavy suspension, carrying out ice bath for 15 min, centrifuging and removing supernatant; 1mL of the pre-cooled solution I was added to the centrifuge tube, and 50. mu.L/aliquot was dispensed into a 1.5 mL centrifuge tube and stored at-80 ℃.
3) The recombinant expression vector is used for electrically transforming lactococcus lactis NZ 9000: taking out a tube of competence from-80 ℃, melting on ice, and adding 1 mu g of recombinant expression vector pNZ-gshF; after ice-bath for 10 min, adding a2 mm Bio-Rad electric rotating cup precooled on ice, performing electric shock under the conditions of 2000V, 25 muF and 200 omega, adding 1mL of GM17 liquid culture medium, and after ice-bath for 5 min, incubating for 1.5 h at 30 ℃; taking a proper amount of culture solution to coat a solid culture medium containing 12 mg/L chloramphenicol GM17, standing and culturing at 30 ℃ until colonies grow out, extracting plasmids and verifying.
4) Induced expression of recombinant lactococcus lactis: inoculating the strain selected in step 4) into 5mL of GM17 medium, 30%oC, standing and culturing overnight to obtain a seed solution, and then inoculating the seed solution into 30 mL of GM17 culture medium 30 according to the inoculation amount of 1 percentoStanding and culturing to OD600About 0.4; nisin was added to the cells at a final concentration of 20, 50, 100 ng/ml for induction, and the cells were collected after 4 hours of induction. After ultrasonication, SDS-PAGE protein electrophoresis was carried out.
5) And (3) fermenting and synthesizing GSH and SAM by recombinant lactococcus lactis: single clones were picked in 5mL GM17 and left overnight. Transfer 2% to 50 mL GM17 medium, culture to OD at 30 ℃600At 0.4, nisin was added to the cells at a final concentration of 20 ng/mL for induction. Glutamic acid, cysteine and glycine were added at 20 mM each. After the fermentation was terminated, 1ml of 1.2M perchlorate was added to resuspend the cells, and the cells were extracted in a refrigerator at 4 ℃ for 4 hours. Centrifuging, collecting supernatant, membrane filtering, and detecting GSH and SAM content in liquid phase.
6) Fermenting the sour milk with high glutathione and S-adenosine-L-methionine content: after two-stage homogenization, raw milk enters a sterilizer for sterilization at 90 ℃ for 5 minutes. Then cooled to 30 ℃ to prepare for inoculation and fermentation. Adding lactobacillus bulgaricus (danisc L340) and recombinant lactococcus lactis into a sample, fermenting in a fermentation tank at 30 ℃, adding appropriate amount of Nisin for induction, and adding glutamic acid, cysteine and glycine as substrates for fermentation. The yogurt samples were filled into sterile containers in a sterile worktop and refrigerated at 4 ℃ in preparation for further analysis.
Drawings
FIG. 1 is a diagram of the structure of an expression plasmid,
FIG. 2 is an SDS-PAGE electrophoresis of the fermentation of recombinant lactococcus lactis,
FIG. 3 HPLC analysis of GSH in recombinant lactococcus lactis fermentation products,
FIG. 4 HPLC analysis of SAM in recombinant lactococcus lactis fermentation products.
Detailed Description
The invention is further described below by way of example:
example 1, production of high GSH and SAM containing yogurt with addition of GSH: the raw material formula is that 90-95% (weight ratio, the same below) of raw milk is added with 5-10% of white granulated sugar, then 5-30mg/L of glutathione is added, after being uniformly mixed, the mixture is heated to 65 ℃, and is subjected to two-stage homogenization (the homogenization pressure is 5/20 MPa), and then the mixture enters a sterilizer for sterilization at 90 ℃ for 5 minutes. Cooling, inoculating Lactobacillus bulgaricus (Danisco L340) and Streptococcus thermophilus at an inoculum size of 0.2-1.0% and 0.1-1.0%, respectively, and then inoculating at 30%oC~43oCFermenting at the temperature for about 4-14 hours. After fermentation, the content of GSH in the yogurt is 3-29 mg/L, the content of SAM is 0.5-10mg/L, and the number of cells of two bacteria is 109More than ml.
Example 2, production of high GSH and SAM containing yogurt with addition of GSH: the raw material formula is that 90-95% (weight ratio, the same below) of raw milk is added with 5-10% of white granulated sugar, the raw milk and the white granulated sugar are uniformly mixed, heated to 65 ℃, subjected to two-stage homogenization (the homogenization pressure is 5/20 MPa), and then fed into a sterilizer for sterilization at 90 ℃ for 5 minutes. Cooling, adding 5-30mg/L glutathione and methionine, inoculating Lactobacillus bulgaricus (Danisco L340) and Streptococcus thermophilus at an inoculation amount of 0.2-1.0% and 0.1-1.0%, respectively, and inoculating at 30%oC~43oCFermenting at the temperature for about 4-14 hours. After fermentation, the content of GSH in the yogurt is 3-29 mg/L, the content of SAM is 1-20mg/L, and the number of the two bacteria is more than 109/ml.
Example 3, the raw material formula is 93% (by weight, the same below) raw milk, 7% white granulated sugar is added, after being mixed uniformly, the mixture is heated to 65 ℃, and after two-stage homogenization (the homogenization pressure is 5/20 MPa), the mixture enters a sterilizer to be sterilized at 90 ℃ for 5 minutes. Then cooling, adding 5-30mg/L glutathione respectivelyPeptides and methionine were inoculated with Lactobacillus bulgaricus (Danisco L340) and Streptococcus lactis NZ9000 in amounts of 0.2-1.0% and 0.1-1.0%, respectively, and then incubated at 30%oC~43oCFermenting at the temperature for about 4-14 hours. After fermentation, the content of GSH in the yogurt is 5-29 mg/L, the content of SAM is 1-20mg/L, and the number of the two bacteria is more than 109/ml.
Example 4 construction of pNZ-gshF engineering bacteria
1) A pair of primers is designed according to the sequence of the multiple cloning site of a vector plasmid pNZ8148 and the sequence of the bifunctional glutathione synthetase (gshF) of streptococcus thermophilus provided above NCBI according to the design principle of the primers, and is used for amplifying the full-length sequence of the gshF gene. Wherein Hind III and Nco I restriction endonuclease sites are added to the two primers respectively;
an upstream primer:
5’- CCCAAGCTTGGATCCTCTAGAGTCGACCT -3’
a downstream primer:
5’- CATGCCATGGTTATGATTATCGATCGACTGTT -3’
wherein the restriction sites are underlined
2) Obtaining the DNA of the gene of glutathione synthetase by using polymerase chain reaction: the reaction procedure was as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds; renaturation at 58 ℃ for 30 seconds; extension 72oC, 2 min, 30 cycles, and final reaction extension time of 10 min. Taking the polymerase chain reaction product to carry out 1.0% agarose gel electrophoresis, and adopting a gel recovery kit purchased from qiagen company to recover the DNA of the alcohol oxidase in the agarose gel;
3) adding the recovered glutathione synthetase gene fragment and the digested plasmid fragment into a 200 mu l small centrifuge tube according to the concentration ratio of 4: 1, connecting overnight at 16 ℃ by using T4 ligase, transforming 200 mu l of DH5 α competent cells prepared by a CaCl2 method into the recovered glutathione synthetase gene fragment and the digested plasmid fragment, adding 1ml of LB culture medium preheated by 37 ℃ into a 37 ℃ shaking table, oscillating for 1 hour at the rotating speed of 180rpm, centrifuging the bacterial liquid at the rotating speed of 4000rpm for 1 minute, discarding 1ml of supernatant, lightly blowing the residual solution by a pipette, uniformly mixing, coating the mixture on an LB plate containing chloramphenicol, and culturing the LB plate in a 37 ℃ incubator for more than 12 hours;
4) randomly picking a single colony in the plate, extracting a plasmid after small-amount amplification, and performing double enzyme digestion identification by using Hind III and Nco I to obtain a recombinant expression vector pNZ-gshF containing glutathione synthetase;
5) preparation of lactococcus lactis NZ9000 electrotransformation competence: selecting a single colony of lactococcus lactis NZ9000, inoculating the single colony into 5mL of GM17 liquid culture medium, and standing at 30 ℃ for overnight culture; inoculating to 100 mL GM17 liquid culture medium according to 1% inoculum size, standing and culturing until OD600 is close to 0.4, ice-cooling for 10 min, adding into a precooled sterile centrifuge tube, centrifuging at 4 deg.C and 5000 rpm for 10 min, and removing supernatant; adding pre-cooled 25 mL of solution I (0.5M sucrose, 10% glycerol) for heavy suspension, carrying out ice bath for 15 min, centrifuging and removing supernatant; adding precooled 25 mL solution II (0.5M sucrose, 10% glycerol, 0.05M Na2 EDTA) for resuspension, carrying out ice bath for 15 min, centrifuging and discarding the supernatant; adding pre-cooled 25 mL solution I for heavy suspension, carrying out ice bath for 15 min, centrifuging and removing supernatant; 1mL of the pre-cooled solution I was added to the centrifuge tube, and 50. mu.L/aliquot was dispensed into a 1.5 mL centrifuge tube and stored at-80 ℃.
6) The recombinant expression vector is used for electrically transforming lactococcus lactis NZ 9000: taking out a tube of competence from-80 ℃, melting on ice, and adding 1 mu g of recombinant expression vector pNZ-gshF; adding a2 mm Bio-Rad electric rotor precooled on ice after ice-bath for 10 min, performing electric shock under the conditions of a Bio Rad Gene Pulser electric rotor instrument of 2000V, 25 muF and 200 omega, adding 1mL of GM17 liquid culture medium, and incubating for 1.5 h at 30 ℃ after ice-bath for 5 min; taking a proper amount of culture solution to coat a solid culture medium containing 12 mg/L chloramphenicol GM17, standing and culturing at 30 ℃ until colonies grow out, extracting plasmids and verifying.
4) Induced expression of recombinant lactococcus lactis: inoculating the strain screened in the step 4) into 5mL of GM17 culture medium, standing and culturing at 30 ℃ for overnight to obtain a seed solution, and then inoculating the seed solution into 30 mL of GM17 culture medium at 30 ℃ according to the inoculation amount of 1% to perform standing and culturing until the OD600 is about 0.4; nisin was added to the cells at a final concentration of 20, 50, 100 ng/ml for induction, and the cells were collected after 4 hours of induction. After ultrasonication, SDS-PAGE protein electrophoresis was carried out.
Example 5 recombinant lactococcus lactis production of high GSH and SAM containing yogurts: the raw material formula is that 90-95% (weight ratio, the same below) of raw milk is added with 5-10% of white granulated sugar, the raw milk and the white granulated sugar are uniformly mixed, heated to 65 ℃, subjected to two-stage homogenization (the homogenization pressure is 5/20 MPa), and then fed into a sterilizer for sterilization at 90 ℃ for 5 minutes. Cooling, adding 5-30mg/L cysteine and methionine, inoculating Lactobacillus bulgaricus (Danisco L340) and recombinant lactococcus lactis with inoculum concentration of 0.2-1.0% and 0.1-1.0%, respectively, and inoculating at 30%oC~43oCFermenting at the temperature for about 4-14 hours. After fermentation, the content of GSH in the yogurt is 1-70 mg/L, the content of SAM is 1-20mg/L, and the number of the two bacteria is more than 109/ml.
Example 6 recombinant lactococcus lactis production of high GSH and SAM containing yoghurts: the raw material formula is that 90-95% (weight ratio, the same below) of raw milk is added with 5-10% of white granulated sugar, the raw milk and the white granulated sugar are uniformly mixed, heated to 65 ℃, subjected to two-stage homogenization (the homogenization pressure is 5/20 MPa), and then fed into a sterilizer for sterilization at 90 ℃ for 5 minutes. Cooling, adding 5-30mg/L glutamic acid, cysteine and methionine, inoculating Lactobacillus bulgaricus (Danisco L340) and recombinant lactococcus lactis with inoculum size of 0.2-1.0% and 0.1-1.0%, respectively, and inoculating at 30%oC~43oCFermenting at the temperature for about 4-14 hours. After fermentation, the content of GSH in the yogurt is 1-70 mg/L, the content of SAM is 1-20mg/L, and the number of the two bacteria is more than 109/ml.
Example 7 recombinant lactococcus lactis production of high GSH and SAM containing yogurts: the raw material formula is that 90-95% (weight ratio, the same below) of raw milk is added with 5-10% of white granulated sugar, the raw milk and the white granulated sugar are uniformly mixed, heated to 65 ℃, subjected to two-stage homogenization (the homogenization pressure is 5/20 MPa), and then fed into a sterilizer for sterilization at 90 ℃ for 5 minutes. Cooling, adding 5-30mg/L glutamic acid, cysteine, glycine and methionine, respectively, inoculating Lactobacillus bulgaricus (Danisco)L340) and recombinant lactococcus lactis in amounts of 0.2-1.0% and 0.1-1.0%, respectively, and then at 30%oC~43oCFermenting at the temperature for about 4-14 hours. After fermentation, the content of GSH in the yogurt is 1-70 mg/L, the content of SAM is 1-20mg/L, and the number of the two bacteria is more than 109/ml。

Claims (2)

1. The method for producing the yoghourt containing the glutathione and the S-adenosine-L-methionine (SAM) by adding a proper amount of the Glutathione (GSH) in the manufacturing process of the yoghourt is characterized by comprising the following steps:
1) adding glutathione (with concentration range of 5-30 mg/L) into yogurt fermentation medium formula by using traditional yogurt strains (Lactobacillus bulgaricus and Streptococcus thermophilus) to prepare yogurt with double liver protection factors; 2) lactobacillus bulgaricus and lactococcus lactis are adopted, and glutathione (with the concentration range of 5-30 mg/L) is added into a fermentation medium formula to prepare the yoghourt with the double-liver protection factor.
2. The novel method for preparing the yoghourt with high glutathione content and S-adenosine-L-methionine content by cloning the bifunctional glutathione synthetase endogenous biosynthesis glutathione in the lactococcus lactis is characterized by comprising the following steps of:
1) construction of recombinant lactococcus lactis: designing a primer for PCR amplification according to a gene (gshF) sequence of the bifunctional glutathione synthetase of the streptococcus thermophilus on NCBI, and cloning a target gene to an expression vector pNZ8148 to obtain a recombinant expression vector pNZ-gshF; introducing the recombinant plasmid into lactococcus lactis by an electrotransformation method to obtain recombinant lactococcus lactis capable of endogenously biosynthesizing glutathione;
2) fermenting the sour milk with high glutathione and S-adenosine-L-methionine content: homogenizing raw milk for two sections, and then sterilizing in a sterilizer; and then cooling to 30 ℃ for preparing inoculation and fermentation, adding lactobacillus bulgaricus (Danisco L340) and recombinant lactococcus lactis into the sample, fermenting in a fermentation tank at the temperature of 30 ℃, and adding a Nisin inducer and substrates such as glutamic acid, cysteine, glycine and the like for fermentation.
CN201811184095.1A 2018-10-11 2018-10-11 Production method of yoghourt with high glutathione and S-adenosine-L-methionine content Pending CN111034795A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811184095.1A CN111034795A (en) 2018-10-11 2018-10-11 Production method of yoghourt with high glutathione and S-adenosine-L-methionine content

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811184095.1A CN111034795A (en) 2018-10-11 2018-10-11 Production method of yoghourt with high glutathione and S-adenosine-L-methionine content

Publications (1)

Publication Number Publication Date
CN111034795A true CN111034795A (en) 2020-04-21

Family

ID=70229193

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811184095.1A Pending CN111034795A (en) 2018-10-11 2018-10-11 Production method of yoghourt with high glutathione and S-adenosine-L-methionine content

Country Status (1)

Country Link
CN (1) CN111034795A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5786297A (en) * 1980-11-19 1982-05-29 Nippon Zeon Co Ltd Preparation of s-adenosyl-l-methionine
EP1034788A1 (en) * 1999-03-11 2000-09-13 Société des Produits Nestlé S.A. Lactic acid bacteria strains capable of preventing diarrhea
US20050112612A1 (en) * 2003-04-25 2005-05-26 Klaenhammer Todd R. Lactobacillus acidophilus nucleic acid sequences encoding cell surface protein homologues and uses therefore
KR20130015192A (en) * 2011-08-02 2013-02-13 순천대학교 산학협력단 The yogurt added with turbid rice-wine and preparing method thereof
CN104293699A (en) * 2014-09-17 2015-01-21 山东大学 Streptococcus thermophilus with synthetic capability of gamma-aminobutyric acid and glutathione and application of streptococcus thermophilus
CN106819108A (en) * 2017-01-04 2017-06-13 宁夏夏进乳业集团股份有限公司 A kind of brown yoghurt and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5786297A (en) * 1980-11-19 1982-05-29 Nippon Zeon Co Ltd Preparation of s-adenosyl-l-methionine
EP1034788A1 (en) * 1999-03-11 2000-09-13 Société des Produits Nestlé S.A. Lactic acid bacteria strains capable of preventing diarrhea
US20050112612A1 (en) * 2003-04-25 2005-05-26 Klaenhammer Todd R. Lactobacillus acidophilus nucleic acid sequences encoding cell surface protein homologues and uses therefore
KR20130015192A (en) * 2011-08-02 2013-02-13 순천대학교 산학협력단 The yogurt added with turbid rice-wine and preparing method thereof
CN104293699A (en) * 2014-09-17 2015-01-21 山东大学 Streptococcus thermophilus with synthetic capability of gamma-aminobutyric acid and glutathione and application of streptococcus thermophilus
CN106819108A (en) * 2017-01-04 2017-06-13 宁夏夏进乳业集团股份有限公司 A kind of brown yoghurt and preparation method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
傅瑞燕等: "构建重组乳酸乳球菌生产谷胱甘肽", 《生物加工过程》 *
朱志钢等: "重组毕赤酵母发酵生产S-腺苷甲硫氨酸培养条件优化", 《工业微生物》 *
林谦等: "嗜热链球菌双功能谷胱甘肽合成酶基因的克隆与表达", 《玉林师范学院学报》 *
汤亚杰等: "S-腺苷甲硫氨酸的研究进展", 《生物技术通报》 *

Similar Documents

Publication Publication Date Title
RU2370533C2 (en) Method of milk bacteria cultures and start culture of milk bacteria
BR112017027640B1 (en) FERMENTED MILK INOCULATED WITH BOTH LACTIC ACID BACTERIA (LAB) AND BACILLUM
US20180139977A1 (en) Method for producing fermented milk
Drider et al. Genetic organization and expression of citrate permease in lactic acid bacteria
US8741622B2 (en) Stress tolerant Bifidobacteria
JP4802216B2 (en) Bifidobacterium-containing composition and method for producing Bifidobacterium-containing composition
JPH07123977A (en) Lactic bacteria and fermented dairy product
MX2012009975A (en) Method for producing fermented food containing bifidobacterium bacteria.
CN109536427B (en) Lactobacillus engineering bacterium with improved acid stress resistance
Arioli et al. The relevance of carbon dioxide metabolism in Streptococcus thermophilus
JP2013201936A (en) Method for controlling amine formation in lactic fermentation
CN110484480B (en) Preparation and transformation method of bacillus subtilis competent cells
CN113197312A (en) Application of streptococcus thermophilus MN002 in immunoregulation product and dietary supplement
CN111034795A (en) Production method of yoghourt with high glutathione and S-adenosine-L-methionine content
CN114703117B (en) Recombinant bacillus subtilis, construction method thereof and recombinant collagenase
US20120276245A1 (en) Method for improved fermentation
WO2005097972A1 (en) Medium for lactic acid bacteria
JPH09201164A (en) Production of milk-fermented food
WO2009150888A1 (en) Milk protein degradation product, manufacturing method for milk protein degradation product and bifidobacterial growth-promoting agent
CN109666618B (en) Lactobacillus engineering bacterium with improved viability in acid stress environment
JP6412865B2 (en) Bifidobacterium breve strain specific gene
CN111394295B (en) Preparation of immobilized arginine deiminase and production thereof 14/15 N]Method for producing L-citrulline
CN112646832B (en) Method for regulating adhesion capacity of lactobacillus plantarum
WO2023008491A1 (en) Enzyme-containing composition, method for producing milk and method for producing fermented milk
CN109628366B (en) Method for improving acid stress resistance of lactic acid bacteria

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20200421