CN107502620B

CN107502620B - Staphylococcus carnosus synthetic medium and preparation method and application of fermentation liquor thereof

Info

Publication number: CN107502620B
Application number: CN201710958673.1A
Authority: CN
Inventors: 高强; 唐巧巧; 张变强; 朱燕
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2017-10-16
Filing date: 2017-10-16
Publication date: 2021-07-06
Anticipated expiration: 2037-10-16
Also published as: CN107502620A

Abstract

The invention discloses a staphylococcus carnosus synthetic culture medium and a preparation method and application of fermentation liquor thereof. Wherein Staphylococcus carnosus (A)Staphylococcus carnosus) The synthetic culture medium is calculated according to the volume of 1L and consists of the following raw materials by weight: 10-50 g of anhydrous glucose, 0.1-1.5 g of lysine, 1.0-5.0 g of valine, 0.1-1.5 g of glycine, 1.0-5.0 g of arginine, 1.0-5.0 g of proline, 5-10.0 g of glutamic acid, 0.1-0.5 g of tryptophan, 0.1-1.5 g of cystine, 1.0-3.0 mg of thiamine hydrochloride, 1.0-3.0 mg of calcium pantothenate, 1.0-3.0 mg of nicotinic acid, 1.0-3.0 mg of manganese sulfate, 0.1-1.0 g of potassium dihydrogen phosphate, 0.1-0.8 g of magnesium sulfate heptahydrate, 0.005-0.010 g of ferrous sulfate heptahydrate, 1.0-5.0 g of dipotassium hydrogen phosphate trihydrate, 6-10 g of 3- (N-morpholine) propanesulfonic acid, and the balance of water; also discloses a preparation method of the staphylococcus carnosus fermentation liquid. The staphylococcus carnosus synthetic medium disclosed by the invention is reasonable in formula matching and rich in nutrition, is suitable for the growth demand of staphylococcus carnosus, and the growth amount of staphylococcus carnosus obtained by the method disclosed by the invention is increased by 5% compared with the commonly used LB (Luria-Bertani) medium.

Description

Staphylococcus carnosus synthetic medium and preparation method and application of fermentation liquor thereof

Technical Field

The invention belongs to the technical field of microbial culture medium and fermentation, and relates to staphylococcus carnosus (Staphylococcus carnosus) Synthetic culture medium and preparation method and application of fermentation liquid thereof.

Background

Staphylococcus (1)Staphylococcus) Is a gram-positive bacterium with an average diameter of 0.5-1.0 μm, which is named because of its stacked shape like grape beading and is generally not pathogenic. Representative species is Staphylococcus epidermidis: (Staphylococcus epidermidis) Staphylococcus aureus (1)Staphyloccocus aureus Rosenbach) and Staphylococcus saprophyticus (Staphyloccocus saprophyticus) And the like. Staphylococci have long been used for fermentation of dry intestines. Initially, they were considered micrococcus, but it was demonstrated that these micrococcus were misclassified, and indeed were staphylococci. Based on DNA/DNAHybridization, biochemical properties and cell wall composition, these staphylococci form a new species, designated Staphylococcus carnosus ((S. carnosus))Staphylococcus carnosus) It could be isolated from meat fermentation products and used as a fermentation starter since 1950.

Staphylococcus carnosus (S. carnosus) It is recognized worldwide as a food grade GRAS (genetically modified safe) Staphylococcus because it does not produce any hemolysin protein, toxin, clotting enzyme, protein A or clotting factor. Staphylococcus carnosus is a key species in the meat fermentation industry for developing the flavor of meat products and has been used as a starter culture for raw meat products in Europe and the like for over 80 years. During the fermentation and maturation of the dry sausage, the staphylococcus carnosus can firstly reduce nitrate into nitrite. The advantage of this reaction is that the concentration of nitrate is reduced and nitrite can combine with myoglobin (Mb) to form nitrosohemoglobin (Hb), which gives the meat a typical red color. Nitrite is then further reduced to ammonia, thereby reducing the unbound nitrite concentration. In the culture medium and the meat matrix, the staphylococcus carnosus can convert the high-iron myoglobin into the red myoglobin derivative, so that the meat product has better color. Staphylococcus carnosus also produces enzymes which hydrolyze proteins, and the breakdown products of the enzymes during fermentation of dry sausages give rise to unique flavors. In addition, they have a very low extracellular proteolytic capacity and are therefore useful as host bacteria for gene cloning technology, allowing the study of relevant metabolic pathways of such bacteria and their secretion and expression of heterologous proteins. The strains used in the research are recombinant strains of staphylococcus carnosus:Staphylococcus carnosus ATCC 51365/pBT2-ET-5R-EGFP, Staphylococcus carnosus with enhanced green fluorescent protein, improves the growth condition of strains on one hand, and prepares fermentation liquor of the strains on the other hand.

Green Fluorescent Protein (GFP) is of great value in research. EGFP is a GFP mutant line. Currently, the most widely used are GFP mutants: enhanced Green Fluorescent Protein (EGFP) is formed by base modification and artificial modification on the basis of Green Fluorescent Protein (GFP), wherein phenylalanine (Phe) at the 64 th position is replaced by leucine (Leu), serine (Ser) at the 65 th position is replaced by threonine (Thr), and the emitted fluorescence intensity is more than 6 times higher than that of GFP, so that EGFP is more suitable to be used as a reporter gene to study gene expression, regulation, cell differentiation, protein localization and transportation in organisms and the like.

Although the titer is high, the quantitative and qualitative analysis of experimental results is influenced because the yeast extract has complex components and different production places and batches have differences, and when protein is separated, the bands of hybrid protein are more and the separation and purification of other substances are greatly influenced.

The composition of the synthetic culture medium is definite, and the main factors influencing the growth of the strains are determined through the research on the components of the synthetic culture medium, so that the research on the mechanisms of thallus growth, genetic breeding and fermentation metabolism, transcriptomics, metabonomics and the like is facilitated.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide a construction method of a recombinant plasmid pBT 2-ET-5R-EGFP.

The invention aims to solve the second technical problem of providing a staphylococcus carnosus synthetic culture medium.

The third technical problem to be solved by the invention is to provide a preparation method of staphylococcus carnosus fermentation liquor.

The fourth technical problem to be solved by the invention is to provide the application of the staphylococcus carnosus fermentation liquor in improving the growth of staphylococcus carnosus.

The specific description is as follows:

in order to solve the first technical problem, the invention discloses a construction method of a recombinant plasmid pBT2-ET-5R-EGFP, which is characterized by comprising the following steps:

shuttle vector pBT 2-ET-5R-EGFP: the pBT2 plasmid is used as a skeletonKpnI andNhei one is inserted between cloning sitestat-egfpPreparing a fusion gene; the promoter of the EGFP fusion gene is staphylococcus carnosus ATCC 51365eftuThe promoter sequence (GenBank Accession No: 7551602) of the gene and the signal peptide adopt that of staphylococcus carnosus ATCC 51365efeBA tat signal peptide sequence (GenBank Accession No: AM 295250) of the gene, and a connecting peptide linker containing 5 continuous arginine sequences (5R) is added at the N-terminal of the subsequent EGFP gene (GenBank Accession No: AF 302837); wherein the base sequence from the tat signal peptide start codon to the EGFP protein is shown in figure 5, the base sequence is shown in SEQ ID number 1, and the amino acid sequence is shown in figure 6 and is shown in SEQ ID number 2.

Plasmid pBT2-ET-5R-EGFP the base sequence from the tat signal peptide to EGFP:

ATGACACAAGACAAGCATGAAGGTAATGAAGTTTCACGTCGTTTTTTTCTGAAAATGTTAGGTATCGGCGGTGCAGGCGCAGTCATCGGTGCTAGTGGAGTCGGCGGCATTTTTTCTTTCAAGCTTCGTCGCCGTCGCCGTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATGAGCTAGC

plasmid pBT2-ET-5R-EGFP the amino acid sequence from the tat signal peptide to EGFP:

MTQDKHEGNEVSRRFFLKMLGIGGAGAVIGASGVGGIFSFKLRRRRRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKAS

in order to solve the second technical problem, the staphylococcus carnosus synthetic culture medium disclosed by the invention is prepared from the following raw materials in parts by volume of 1L:

10-50 g of anhydrous glucose, 0.1-1.5 g of lysine, 1.0-5.0 g of valine, 0.1-1.5 g of glycine, 1.0-5.0 g of arginine, 1.0-5.0 g of proline, 5-10.0 g of glutamic acid, 0.1-0.5 g of tryptophan, 0.1-1.5 g of cystine, 1.0-3.0 mg of thiamine hydrochloride, 1.0-3.0 mg of calcium pantothenate, 1.0-3.0 mg of nicotinic acid, 1.0-3.0 mg of manganese sulfate, 0.1-1.0 g of potassium dihydrogen phosphate, 0.1-0.8 g of magnesium sulfate heptahydrate, 0.005-0.010 g of ferrous sulfate heptahydrate, 1.0-5.0 g of dipotassium hydrogen phosphate trihydrate, 6-10 g of 3- (N-morpholine) propanesulfonic acid, and the balance of water.

Preferably, the pH of the staphylococcus carnosus synthetic medium is = 7.2-7.5.

Preferably, 40g of anhydrous glucose, 1.0g of lysine, 3.0g of valine, 1.0g of glycine, 2.0g of arginine, 2.0g of proline, 7.0g of glutamic acid, 0.4g of tryptophan, 1.0g of cystine, 2mg of calcium pantothenate, 2mg of nicotinic acid, 2mg of thiamine hydrochloride, 2mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3g of dipotassium hydrogen phosphate trihydrate, and 8g of 3- (N-morpholine) propanesulfonic acid.

In order to solve the third technical problem, the preparation method of the staphylococcus carnosus fermentation liquid comprises the following steps:

s1, seed culture

The formula of the seed culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; pH = 6.5-8.0; inoculating the single colony into a 250mL triangular flask filled with 50mL of seed culture medium, continuously culturing for 12h in a 37 ℃ shaking table at a constant temperature of 180rpm, then inoculating the bacterial liquid into a 250mL triangular flask filled with 50mL of seed culture medium according to the inoculation amount of 1%, and continuously culturing for 8h in a 37 ℃ shaking table at a constant temperature of 180rpm to obtain a staphylococcus carnosus liquid seed liquid.

S2, preparing a staphylococcus carnosus synthetic culture medium

The formula of the synthetic culture medium is as follows: 10-50 g of anhydrous glucose, 0.1-1.5 g of lysine, 1.0-5.0 g of valine, 0.1-1.5 g of glycine, 1.0-5.0 g of arginine, 1.0-5.0 g of proline, 5-10.0 g of glutamic acid, 0.1-1.5 g of cystine, 0.1-0.5 g of tryptophan, 1.0-3.0 mg of thiamine hydrochloride, 1.0-3.0 mg of calcium pantothenate, 1.0-3.0 mg of nicotinic acid, 1.0-3.0 mg of manganese sulfate, 0.1-1.0 g of potassium dihydrogen phosphate, 0.1-0.8 g of magnesium sulfate heptahydrate, 0.005-0.010 g of ferrous sulfate heptahydrate, 1.0-5.0 g of dipotassium hydrogen phosphate trihydrate, and 6-10 g of 3- (N-morpholine) propanesulfonic acid.

S3 liquid fermentation culture

Transferring the liquid seeds into the synthetic culture medium according to the inoculation amount of 0.5-1.5%, and continuously culturing for 12h in a shaker at the temperature of 37 ℃ and at the speed of 180rpm to obtain the fermentation liquor containing the green fluorescent protein.

Preferably, in step S1, the seed culture medium formula is: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride, and the pH is = 6.5-7.5; inoculating 1.0% of the obtained bacterial liquid into a 250mL triangular flask filled with 50mL, and performing shake culture at 37 ℃ and 180rpm for 12 h;

preferably, in step S3, liquid seeds are transferred to the synthetic medium at an inoculum size of 0.5% to 1.5%, and continuously cultured in a shaker at 180rpm at 37 ℃ for 12 h.

In order to solve the fourth technical problem, the invention discloses application of a staphylococcus carnosus fermentation liquid in improving the growth of staphylococcus carnosus. The experimental results show that: the growth amount of the staphylococcus carnosus obtained by the preparation method of the staphylococcus carnosus fermentation liquor is improved by 5 percent compared with that of a commonly used LB (Luria-Bertani) culture medium, and the research on transcriptomics and metabonomics is greatly facilitated in the later period.

Any range recited herein is intended to include the endpoints and any number between the endpoints and any subrange subsumed therein or defined therein.

The starting materials of the present invention are commercially available, unless otherwise specified, and the equipment used in the present invention may be any equipment conventionally used in the art or may be any equipment known in the art.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a staphylococcus carnosus (Staphylococcus carnosus) The culture medium and the preparation method of the fermentation liquid thereof. Book (I)The staphylococcus carnosus synthetic culture medium is reasonable in formula matching and rich in nutrition, is suitable for the growth of staphylococcus carnosus and the secretion requirement of green fluorescent protein, and the growth amount of the staphylococcus carnosus obtained by the invention is increased by 5% compared with the commonly used LB (Luria-Bertani) culture medium.

Drawings

FIG. 1 shows the effect of cystine concentration on the growth of Staphylococcus carnosus strains;

FIG. 2 shows the effect of glutamic acid concentration on the growth of Staphylococcus carnosus cells;

FIG. 3 shows the effect of tryptophan concentration on the growth of Staphylococcus carnosus cells;

FIG. 4 shows the effect of glycine concentration on the growth of Staphylococcus carnosus cells;

FIG. 5 shows the base sequence of shuttle vector pBT2-ET-5R-EGFP from the start codon of the tat signal peptide to the EGFP protein;

FIG. 6 shows the amino acid sequence of shuttle vector pBT2-ET-5R-EGFP from the start codon of the tat signal peptide to the EGFP protein;

FIG. 7 shows the shuttle vector pBT2-ET-5R-EGFP plasmid map.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention. For example, chloramphenicol, ampicillin, and other test materials used were purchased from conventional biochemical manufacturers;

staphylococcus carnosus used (Staphylococcus carnosus) ATCC 51365 strain sources: https:// www.dsmz.de/transactions/tails/culture/dsm-20501. html. Reference documents: schliefer, K.H., Fischer, U.S. Description of a New specifices of the GenusStaphylococcus: Staphylococcus carnosus. International Journal of Systematic Bacteriology, 1982, 32(2):153–156；

plasmid origin pBT 2: http:// www.biofeng.com/zaiti/qita/pBT2. html. Reference documents:Bruckner R. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiology Letters, 1997, 151(1):1-8。

a synthetic culture medium for staphylococcus carnosus comprises the following components in percentage by weight: 10-50 g of anhydrous glucose, 0.1-1.5 g of lysine, 1.0-5.0 g of valine, 0.1-1.5 g of glycine, 1.0-5.0 g of arginine, 1.0-5.0 g of proline, 5-10.0 g of glutamic acid, 0.1-0.5 g of tryptophan, 0.1-1.5 g of cystine, 1.0-3.0 mg of thiamine hydrochloride, 1.0-3.0 mg of calcium pantothenate, 1.0-3.0 mg of nicotinic acid, 1.0-3.0 mg of manganese sulfate, 0.1-1.0 g of potassium dihydrogen phosphate, 0.1-0.8 g of magnesium sulfate heptahydrate, 0.005-0.010 g of ferrous sulfate heptahydrate, 1.0-5.0 g of dipotassium hydrogen phosphate trihydrate, and 6-10 g of 3- (N-morpholine) propanesulfonic acid.

A preparation method of staphylococcus carnosus fermentation liquor comprises the following steps:

s1, seed culture: the formula of the culture medium is (g/L): tryptone 10, yeast extract 5, sodium chloride 10; conditions are as follows: initial pH = 6.5-8.0, and shake culturing is carried out at 37 ℃ and 180rpm for 12 h;

s2, preparing a culture medium for growing staphylococcus carnosus strains, wherein the formula of the culture medium is (L): 10-50 g of anhydrous glucose, 0.1-1.5 g of lysine, 1.0-5.0 g of valine, 0.1-1.5 g of glycine, 1.0-5.0 g of arginine, 1.0-5.0 g of proline, 5-10.0 g of glutamic acid, 0.1-0.5 g of tryptophan, 0.1-1.5 g of cystine, 1.0-3.0 mg of thiamine hydrochloride, 1.0-3.0 mg of calcium pantothenate, 1.0-3.0 mg of nicotinic acid, 1.0-3.0 mg of manganese sulfate, 0.1-1.0 g of potassium dihydrogen phosphate, 0.1-0.8 g of magnesium sulfate heptahydrate, 0.005-0.010 g of ferrous sulfate heptahydrate, 1.0-5.0 g of dipotassium hydrogen phosphate trihydrate, and 6-10 g of 3- (N-morpholine) propanesulfonic acid;

s3, liquid fermentation culture: transferring the liquid seed solution into the synthetic culture medium according to the inoculation amount of 1.0%, and continuously culturing for 12h at 37 ℃ by a shaking table at 180rpm to obtain the fermentation liquor containing the green fluorescent protein.

Example 1

Strain: staphylococcus carnosus (Staphylococcus carnosus) ATCC 51365/pBT2-ET-5R-EGFP was constructed by the team of the present inventors by the following method:

Plasmid pBT2-ET-5R-EGFP the base sequence from the tat signal peptide to EGFP:

example 2

The used fermentation synthesis medium consists of (L): 40g of anhydrous glucose, 0.4g of lysine, 0.4g of valine, 0.4g of glycine, 2.0g of arginine, 2.0g of proline, 0.4g of glutamic acid, 0.4g of tryptophan, 0.4g of cystine, 1.0g, 1.5g, 2.0g and 3.0g of calcium pantothenate, 2mg of nicotinic acid, 2mg of thiamine hydrochloride, 2mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3g of dipotassium hydrogen phosphate trihydrate and 8g of 3- (N-morpholine) propanesulfonic acid.

First of all, the deposited Staphylococcus carnosus: (Staphylococcus carnosus) ATCC 51365/pBT2-ET-5R-After single strain is obtained by activating the EGFP strain, inoculating the single strain into a 250mL triangular flask filled with 50mL seed culture medium, continuously culturing for 12h at constant temperature of 180rpm in a 37 ℃ shaking table, then inoculating the strain liquid into the 250mL triangular flask filled with 50mL seed culture medium according to 1% inoculation amount, continuously culturing for 8h at constant temperature of 180rpm in the 37 ℃ shaking table, obtaining staphylococcus carnosus liquid seeds, and cleaning and suspending the staphylococcus carnosus liquid seeds; transferring the liquid seeds into the high-yield liquid culture medium according to the inoculation amount of 1%, and continuously culturing for 12h in a shaker at 37 ℃ at the constant temperature of 180rpm to obtain the fermentation liquor of the staphylococcus carnosus strain active substances, wherein the growth amount of the staphylococcus carnosus in the synthetic culture medium is only 37.3% of that in the fermentation liquor of an LB (Luria-Bertani) culture medium, as shown in figure 1.

Example 3

The composition of the fermentation synthetic medium is (g/L): 40g of anhydrous glucose, 0.4g of lysine, 0.4g of valine, 0.4g of glycine, 2.0g of arginine, 2.0g of proline, 0.4g of cystine, 0.4g of tryptophan and 0.4g of glutamic acid, wherein the glutamic acid is 0.4g, 1.0g, 3.0g, 5.0g and 7.0g respectively, 2mg of calcium pantothenate, 2mg of nicotinic acid, 2mg of thiamine hydrochloride, 2mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3g of dipotassium hydrogen phosphate trihydrate and 8g of 3- (N-morpholine) propanesulfonic acid.

First of all, the deposited Staphylococcus carnosus: (Staphylococcus carnosus) ATCC 51365/pBT2-ET-5R-After activation of EGFP strain to obtain single colony, the single colony was inoculated into 2 mL of seed mediumContinuously culturing in a 50mL triangular flask at a constant temperature of 180rpm in a 37 ℃ shaking table for 12h, then inoculating the bacterial liquid into a 250mL triangular flask filled with 50mL of seed culture medium according to the inoculation amount of 1%, and continuously culturing in a 37 ℃ shaking table at a constant temperature of 180rpm for 8h to obtain staphylococcus carnosus liquid seeds, and cleaning and suspending; transferring the liquid seeds into the high-yield liquid culture medium according to the inoculation amount of 1%, and continuously culturing for 12h in a shaker at 37 ℃ at the constant temperature of 180rpm to obtain the fermentation liquor of the staphylococcus carnosus strain active substances, wherein the growth amount of the staphylococcus carnosus in the synthetic culture medium is only 52.8% of that in the fermentation liquor of an LB (Luria-Bertani) culture medium, as shown in figure 2.

Example 4

The used fermentation synthesis medium consists of (L): 40g of anhydrous glucose, 1.0g of lysine, 3.0g of valine, 0.4g of glycine, 2.0g of arginine, 2.0g of proline, 1.0g of cystine, 7.0g of glutamic acid, and 0.4g, 1.0g, 1.5g, 2.0g and 3.0g of tryptophan, 2mg of calcium pantothenate, 2mg of nicotinic acid, 2mg of thiamine hydrochloride, 2mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3g of dipotassium hydrogen phosphate trihydrate and 8g of 3- (N-morpholine) propanesulfonic acid.

First of all, the deposited Staphylococcus carnosus: (Staphylococcus carnosus) ATCC 51365/pBT2-ET-5R-After single strain is obtained by activating the EGFP strain, inoculating the single strain into a 250mL triangular flask filled with 50mL seed culture medium, continuously culturing for 12h at constant temperature of 180rpm in a 37 ℃ shaking table, then inoculating the strain liquid into the 250mL triangular flask filled with 50mL seed culture medium according to 1% inoculation amount, continuously culturing for 8h at constant temperature of 180rpm in the 37 ℃ shaking table, obtaining staphylococcus carnosus liquid seeds, and cleaning and suspending the staphylococcus carnosus liquid seeds; transferring the liquid seeds into the high-yield liquid culture medium according to the inoculation amount of 1%, and continuously culturing for 12h in a shaker at 37 ℃ at a constant temperature of 180rpm to obtain the fermentation liquor of the staphylococcus carnosus strain active substances, wherein the growth amount of the staphylococcus carnosus in the synthetic culture medium is only 85.9% of that in the fermentation liquor of an LB (Luria-Bertani) culture medium, as shown in figure 3.

Example 5

The composition of the fermentation synthetic medium is (g/L): 40g of anhydrous glucose, 1.0g of lysine, 3.0g of valine, 0.4g of tryptophan, 2.0g of arginine, 2.0g of proline, 1.0g of cystine, 7.0g of glutamic acid, 0.4g, 1.0g, 2.0g, 3.0g and 4.0g of glycine, 2mg of calcium pantothenate, 2mg of nicotinic acid, 2mg of thiamine hydrochloride, 2mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3g of dipotassium hydrogen phosphate trihydrate and 8g of 3- (N-morpholine) propanesulfonic acid.

First of all, the deposited Staphylococcus carnosus: (Staphylococcus carnosus) ATCC 51365/pBT2-ET-5R-After single strain is obtained by activating the EGFP strain, inoculating the single strain into a 250mL triangular flask filled with 50mL seed culture medium, continuously culturing for 12h at constant temperature of 180rpm in a 37 ℃ shaking table, then inoculating the strain liquid into the 250mL triangular flask filled with 50mL seed culture medium according to 1% inoculation amount, continuously culturing for 8h at constant temperature of 180rpm in the 37 ℃ shaking table, obtaining staphylococcus carnosus liquid seeds, and cleaning and suspending the staphylococcus carnosus liquid seeds; transferring the liquid seeds into the high-yield liquid culture medium according to the inoculation amount of 1%, and continuously culturing for 12h in a shaker at 37 ℃ at a constant temperature of 180rpm to obtain a fermentation liquid of the staphylococcus carnosus strain active substances, wherein the growth amount of the staphylococcus carnosus in the synthetic culture medium is 105% of that in the fermentation liquid of an LB (Luria-Bertani) culture medium, as shown in figure 4.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention. For example, chloramphenicol, ampicillin, and other test materials used were purchased from conventional biochemical manufacturers.

Sequence listing

<110> Tianjin science and technology university

<120> preparation method and application of staphylococcus carnosus synthetic culture medium and fermentation liquor thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 867

<212> DNA

<213> Artificial sequence ()

<400> 1

atgacacaag acaagcatga aggtaatgaa gtttcacgtc gtttttttct gaaaatgtta 60

ggtatcggcg gtgcaggcgc agtcatcggt gctagtggag tcggcggcat tttttctttc 120

aagcttcgtc gccgtcgccg tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 180

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 240

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 300

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 360

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 420

caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 480

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 540

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 600

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 660

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 720

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 780

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 840

gacgagctgt acaagtaatg agctagc 867

<210> 2

<211> 287

<212> PRT

<213> amino acid sequence ()

<400> 2

Met Thr Gln Asp Lys His Glu Gly Asn Glu Val Ser Arg Arg Phe Phe

1 5 10 15

Leu Lys Met Leu Gly Ile Gly Gly Ala Gly Ala Val Ile Gly Ala Ser

20 25 30

Gly Val Gly Gly Ile Phe Ser Phe Lys Leu Arg Arg Arg Arg Arg Val

35 40 45

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

50 55 60

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

65 70 75 80

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr

85 90 95

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr

100 105 110

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His

115 120 125

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

130 135 140

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

145 150 155 160

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

165 170 175

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr

180 185 190

Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile

195 200 205

Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln

210 215 220

Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val

225 230 235 240

Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys

245 250 255

Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr

260 265 270

Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ala Ser

275 280 285

Claims

1. A staphylococcus carnosus synthetic culture medium is characterized by comprising the following raw materials in parts by volume of 1L:

40g of anhydrous glucose, 0.4-1.0 g of lysine, 0.4-3.0 g of valine, 0.4-4.0 g of glycine, 2.0g of arginine, 2.0g of proline, 0.4-7.0 g of glutamic acid, 0.4-3.0 g of cystine, 0.4-3.0 g of tryptophan, 2.0mg of thiamine hydrochloride, 2.0mg of calcium pantothenate, 2.0mg of nicotinic acid, 2.0mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3.0g of dipotassium hydrogen phosphate trihydrate, 8g of 3- (N-morpholine) propanesulfonic acid and the balance of water.

2. The staphylococcus carnosus synthetic medium of claim 1, wherein: the pH = 6.5-8.0 of the staphylococcus carnosus synthetic medium.

3. The staphylococcus carnosus synthetic medium according to claim 2, wherein:

40g of anhydrous glucose, 1.0g of lysine, 3.0g of valine, 1.0g of glycine, 2.0g of arginine, 2.0g of proline, 7.0g of glutamic acid, 0.4g of tryptophan, 1.0g of cystine, 2mg of calcium pantothenate, 2mg of nicotinic acid, 2mg of thiamine hydrochloride, 2mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3g of dipotassium hydrogen phosphate trihydrate and 8g of 3- (N-morpholine) propanesulfonic acid.

4. A method for preparing staphylococcus carnosus fermentation broth by using the synthetic medium of claim 1, comprising the steps of:

s1, seed culture:

the formula of the seed culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; pH = 7.5; inoculating a staphylococcus carnosus single colony into a 250mL triangular flask filled with 50mL of seed culture medium, continuously culturing for 12h in a 37 ℃ shaking table at a constant temperature of 180rpm, then inoculating the bacterial liquid into a 250mL triangular flask filled with 50mL of seed culture medium according to 1% inoculation amount, and continuously culturing for 8h in a 37 ℃ shaking table at a constant temperature of 180rpm to obtain a staphylococcus carnosus liquid seed liquid; the staphylococcus carnosus contains green fluorescent protein;

s2, preparing a staphylococcus carnosus synthetic culture medium:

the formula of the synthetic culture medium is as follows: 40g of anhydrous glucose, 0.4-1.0 g of lysine, 0.4-3.0 g of valine, 0.4-4.0 g of glycine, 2.0g of arginine, 2.0g of proline, 0.4-7.0 g of glutamic acid, 0.4-3.0 g of cystine, 0.4-3.0 g of tryptophan, 2.0mg of thiamine hydrochloride, 2.0mg of calcium pantothenate, 2.0mg of nicotinic acid, 2.0mg of manganese sulfate, 0.5g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate heptahydrate, 0.008g of ferrous sulfate heptahydrate, 3.0g of dipotassium hydrogen phosphate trihydrate and 8g of 3- (N-morpholine) propanesulfonic acid;

s3, liquid fermentation culture:

transferring the liquid seeds into the synthetic culture medium according to the inoculation amount of 0.5-1.5%, and continuously culturing for 10-12 h in a shaking table at 150-200 rpm at the temperature of 30-38 ℃ to obtain the staphylococcus carnosus fermentation liquid containing the green fluorescent protein.

5. The method of claim 4, wherein the fermentation broth comprises:

in step S1, the seed culture medium formula is: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH = 7.5; single colonies were inoculated at 37 ℃ for 12h on a shaker at 180 rpm.

6. The method of claim 4, wherein the fermentation broth comprises:

in step S3, liquid seeds were inoculated at 1% inoculum size into the synthetic medium and incubated continuously for 12h at 37 ℃ on a shaker at 180 rpm.