CN116042483A

CN116042483A - Enterococcus faecium FSUH-1 capable of producing gamma-aminobutyric acid and application thereof

Info

Publication number: CN116042483A
Application number: CN202310126244.3A
Authority: CN
Inventors: 钟先锋; 李连娇; 黄桂东; 王阿利; 任红; 郑剑玮; 杨新泉; 关晓仪; 孙哲; 钟楷杰; 蔡曼婷; 邱湘湘; 万可
Original assignee: Foshan University
Current assignee: Foshan University
Priority date: 2023-02-17
Filing date: 2023-02-17
Publication date: 2023-05-02
Anticipated expiration: 2043-02-17
Also published as: CN116042483B

Abstract

The invention discloses a enterococcus faecium FSUH-1 strain capable of producing gamma-aminobutyric acid and application thereof, and belongs to the technical field of microorganisms. The enterococcus faecalis FSUH-1 was deposited at 20 days 4 of 2022 with the microorganism strain collection of Guangdong province under the accession number GDMCC No:62397. the strain can resist 35% sodium chloride and 30 g/L pig bile salt, has good gastrointestinal fluid resistance, can inhibit the growth of escherichia coli and staphylococcus aureus, and can produce GABA on an optimized fermentation medium and a sodium chloride-containing medium. The enterococcus lactate and the GABA produced by the invention have application potential in microecologics such as inhibiting animal intestinal pathogenic bacteria and regulating intestinal flora, and probiotic fermentation products.

Description

Enterococcus faecium FSUH-1 capable of producing gamma-aminobutyric acid and application thereof

Technical Field

The invention relates to a enterococcus faecium FSUH-1 strain capable of producing gamma-aminobutyric acid and application thereof, belonging to the field of microorganisms.

Background

Gamma-aminobutyric acid (gamma-Aminobutyric acid, GABA) is an important central nervous system inhibitory neurotransmitter, has physiological activities of reducing blood pressure, treating epilepsy, preventing obesity, controlling asthma, improving brain functions and the like, and has wide application prospects in the fields of foods, cosmetics, medicines, feeds and the like. The preparation method of GABA mainly comprises the following steps: the plant enrichment method, the chemical synthesis method, the microbial fermentation method and the like, wherein the microbial fermentation method has the characteristics of low cost, high yield, good safety, mild conditions and the like, and the produced GABA is easier to realize large-scale production and commercialization.

Lactic acid bacteria are normal flora in human and animal intestinal tracts, and have physiological functions of promoting growth, regulating gastrointestinal normal flora, maintaining microecological balance, improving gastrointestinal functions, improving food digestibility and biological titer, reducing serum cholesterol, controlling endotoxin, inhibiting growth of putrefying bacteria in intestinal tracts, improving immunity of organisms and the like. Enterococcus lactis was first isolated from cheese in 2012 (DOI 10.1099/ijs.0.030825-0), is a lactic acid bacterium generally considered safe, and has been allowed to breed animals as a feed additive [ bulletin of national department of agriculture, no. 2045, catalog of feed additives (2013) ], but the current research on enterococcus lactis on GABA production is less, limiting its application in the fields of fermentation industry, livestock breeding, and the like.

Disclosure of Invention

The invention provides a strain of enterococcus lactisEnterococcus lactis) FSUH-1 was deposited at 20/4/2022 with the collection of microorganism strains, accession number GDMCC No:62397.

the enterococcus lactate is [ (]Enterococcus lactis) FSUH-1 is separated from a soy sauce mash sample, and the strain is identified as enterococcus lactis according to colony appearance form, microscopic examination, 16S rDNA sequencing result and biochemical identification, named as enterococcus lactis FSUH-1 and deposited with the Guangdong province microorganism strain deposit center with the deposit number of GDMCC No:62397.

the enterococcus lactate is [ (]Enterococcus lactis) FSUH-1 maintains 3 h in artificial gastric juice with pH of 3, survival rate of 118.46%, 4 h in artificial intestinal juice, survival rate of 98.90%, 3 h in artificial pig bile salt with pig bile salt concentration of 30 g/L, and survival rate of 74.54%.

The enterococcus lactate is [ (]Enterococcus lactis) FSUH-1 has salt tolerance properties and can tolerate a salt concentration of 35%.

The invention provides a microbial agent,the microbial agent contains the enterococcus lactisEnterococcus lactis) FSUH-1 or its fermentation broth or its intracellular and extracellular metabolites.

In one embodiment of the invention, the microbial agent is enterococcus lactis @Enterococcus lactis) The viable count of FSUH-1 is at least: 1X 10 ⁶ CFU/mL。

In one embodiment of the invention, the microbial agent is enterococcus lactis @Enterococcus lactis) The viable count of FSUH-1 is 1×10 ⁶ CFU/mL~1×10 ¹² CFU/mL

The invention also provides a product, which contains the enterococcus lactateEnterococcus lactis) FSUH-1 or a microbial agent containing the same.

In one embodiment of the invention, the product is a chemical.

In one embodiment of the invention, the product is a bacteriostatic agent.

In one embodiment of the invention, the product is capable of inhibiting staphylococcus aureus and/or escherichia coli.

In one embodiment of the invention, the product is an enterococcus lactis strainEnterococcus lactis) The viable count of FSUH-1 is at least: 1X 10 ⁶ CFU/mL。

In one embodiment of the invention, the product is an enterococcus lactis strainEnterococcus lactis) The viable count of FSUH-1 is: 1X 10 ⁶ CFU/mL ~ 1×10 ¹² CFU/mL。

The invention also provides the enterococcus lactisEnterococcus lactis) Application of FSUH-1 or the microbial agent in preparing GABA or products containing GABA.

The invention also provides a preparation method of GABA, which comprises the steps of mixing the enterococcus lactis with the amino acid solutionEnterococcus lactis) FSUH-1 is inoculated into a reaction system containing sodium glutamate for reaction, and the GABA is prepared.

In one embodiment of the present invention, the reaction conditions in the reaction system are: the third generation of the enterococcus acidilactici FSUH-1 with the inoculation amount of 1% (v/v) is inoculated into a fermentation medium, and the enterococcus acidilactici FSUH-1 is subjected to stationary culture at the constant temperature of 37 ℃ for 72 h.

In one embodiment of the invention, the fermentation medium formulation is: glucose 25 g/L, yeast extract 6.25 g/L, soy peptone 6.25 g/L, mnSO ₄ ·H ₂ O0.16 g/L, tween-80 2 mL/L, sodium glutamate 10g/L, and adjusting pH to 5+ -0.2.

The invention also provides the enterococcus lactateEnterococcus lactis) Application of FSUH-1 or the microbial agent in preparing products for inhibiting staphylococcus aureus and/or escherichia coli.

The invention also provides an enterococcus lactis strainEnterococcus lactis) Application of FSUH-1 in preparing medicines or health products for inhibiting enteropathogenic bacteria and feed additives.

In one embodiment of the invention, the enteropathogenic bacteria are staphylococcus aureus and escherichia coli.

The invention also provides an enterococcus lactis strainEnterococcus lactis) Application of FSUH-1 and its metabolite in preparing intestinal flora medicine or health product is provided.

The invention also provides an enterococcus lactis strainEnterococcus lactis) Growth curve of FSUH-1.

The invention also provides a strain of enterococcus lactisEnterococcus lactis) A method for producing GABA by fermenting FSUH-1.

In one embodiment of the invention, the method is: firstly, activated enterococcus lactis is treatedEnterococcus lactis) FSUH-1 is inoculated into a seed culture medium according to a certain proportion to be cultured, seed liquid is obtained, and then the seed liquid is inoculated into a fermentation culture medium according to a certain proportion to be fermented and cultured.

In one embodiment of the invention, the seed medium comprises the following components: glucose 25 g/L; yeast extract 6.25 g/L, soy peptone 6.25 g/L, mnSO ₄ ·H ₂ O0.16 g/L; tween-80 2 mL/L.

In one embodiment of the invention, the culture conditions of the seed medium are: stationary culture at 37℃for 8 h and initial pH 5.0.

In one embodiment of the invention, the inoculation ratio is 1%; the components of the fermentation medium are as follows: glucose 25 g/L, yeast extract 6.25 g/L, soy peptone 6.25 g/L, mnSO ₄ ·H ₂ O0.16 g/L, tween-80 2 mL/L.

In one embodiment of the invention, the fermentation medium is grown at 37℃and allowed to stand at 72℃ 72 h.

In one embodiment of the invention, the fermentation medium has an initial pH of 5.

In one embodiment of the invention, the fermentation medium is supplemented with sodium glutamate in an amount of 10 g/L.

In one embodiment of the invention, the inoculation ratio is 1%.

In one embodiment of the present invention, the fermentation medium has a GABA yield of 1.84 g/L under the above conditions.

In one embodiment of the present invention, the seed medium is inoculated at an inoculum size of 1% or more and cultured at 25-45℃for 5 h or more.

In one embodiment of the present invention, the culture medium is inoculated at an inoculum size of 1% or more, and the culture medium is subjected to stationary culture at 25 to 45℃for 24 to h or more.

Advantageous effects

(1) The strain enterococcus faecalis of the inventionEnterococcus lactis) FSUH-1 is selected from fermented soy sauce mash, can grow normally in MRS liquid culture medium with 35% sodium chloride salt concentration, and OD after 24 h culture _600nm Reaching 0.175, before-inoculation OD _600nm The strain was 0.083, and the problem of reduced viable bacteria due to higher salt concentration was avoided.

(2) The invention obtains the enterococcus acidi FSUH-1 for producing GABA, which can produce GABA in a fermentation culture medium, cultures for 72 hours in the fermentation culture medium with 10g/L sodium glutamate as a substrate, and the GABA yield reaches 1.87 g/L, and the yield reaches 14.57 g/L after the optimization of the fermentation culture medium, thereby improving the yield by about 6.8 times.

(3) The enterococcus lactate provided by the invention still produces GABA after being cultured by an optimized fermentation culture medium containing 5% sodium chloride, and the yield reaches 13.22+/-0.25 g/L.

(4) The invention relates to enterococcus lactisEnterococcus lactis) FSUH-1 maintains 3 h in artificial gastric juice with pH value, the survival rate reaches 118.46%, and maintains 4 h in artificial intestinal juice, and the survival rate reaches 98.90%.

(5) The enterococcus lactate FSUH-1 disclosed by the invention is kept at 3 h under the condition of 30 g/L of pig bile salt concentration, the survival rate is 74.54%, and the pig bile salt concentration is 100 times of the average bile salt concentration of 0.3 g/L in an animal body.

(6) The strain enterococcus faecalis FSUH-1 provided by the invention has inhibition effect on both escherichia coli and staphylococcus aureus, wherein the diameter of a bacteriostasis zone for escherichia coli is 13.67+/-0.33 mm, and the diameter of a bacteriostasis zone for staphylococcus aureus is 10.04+/-0.81 mm.

(7) The enterococcus acidilactici FSUH-1 has good adhesion, wherein the surface hydrophobicity is 40.24%, and the self-agglomeration capacity of 24 h reaches 89.64%, which indicates that the strain has the capacity of colonizing in intestinal tracts.

(8) The enterococcus lactis provided by the invention has a high growth speed, and can reach a growth log phase within 4-6 h.

(9) The enterococcus lactate provided by the invention has high safety, the indole reaction is negative, the hemolysis is avoided, the results of lysine decarboxylase and ornithine decarboxylase are negative, and the enterococcus lactate has sensitivity to tetracycline, vancomycin, ampicillin and norfloxacin. The invention can provide strain resources for health-care microecological preparations and feed additives, and has good application value.

Preservation of biological materials

Enterococcus lactis strainEnterococcus lactis) FSUH-1, taxonomic designationEnterococcus lactisHas been deposited at the collection of microorganisms and cell cultures of Guangdong province at 20/4/2022 under the accession number GDMCC No:62397 the preservation address is building 5 of Guangdong national institute of science and microbiology, guangzhou City, first, china, no. 100, university, 59.

Drawings

Fig. 1: HPLC diagram of fermentation broth sample.

Fig. 2: HPLC profile of GABA standard samples.

Fig. 3: enterococcus lactate FSUH-1 microscopy results.

Fig. 4: enterococcus lactate FSUH-1 colony morphology.

Fig. 5: enterococcus lactis FSUH-116SrDNA sequence evolutionary tree diagram.

Fig. 6: enterococcus lactate FSUH-1 growth curve.

Fig. 7: enterococcus lactate FSUH-1 salt tolerance results.

Fig. 8: enterococcus lactate FSUH-1 hemolysis results.

Fig. 9a to 9h: enterococcus acidilactici FSUH-1 single factor experiment results; wherein FIG. 9a shows the effect of different pH on GABA yield, FIG. 9b shows the effect of different substrates on GABA yield, FIG. 9c shows the effect of different sodium glutamate concentrations on GABA yield, FIG. 9d shows the effect of different carbon sources on GABA yield, FIG. 9e shows the effect of different sucrose concentrations on GABA yield, FIG. 9f shows the effect of different nitrogen source species on GABA yield, FIG. 9g shows the effect of different nitrogen source concentrations on GABA yield, FIG. 9h shows the effect of different PLP concentrations on GABA yield.

Detailed Description

The following detailed description is merely illustrative of specific details of individual exemplary embodiments of the invention and is not intended to be limiting thereof; it will be appreciated by those skilled in the art that: any person skilled in the art may modify the steps or schemes based on the present invention, or may equally replace some or all of the techniques, and all the techniques may be combined with the technical schemes that are considered in the art, which falls within the protection scope of the present invention.

The strain staphylococcus aureus referred to in the following examples is staphylococcus aureus ATCC 25923, and the indicator strain escherichia coli in the following examples is: ATCC 25922.

Arginine decarboxylase, lysine decarboxylase, ornithine decarboxylase, amino acid decarboxylase control, peptone water, agar, all of which are referred to in the examples below were purchased from Guangdong CycloKjeldahl microorganism technologies, inc., and 0.5% glycerol fermentation tubes (lactic acid bacteria) were purchased from Shandong Tulipa bioengineering, inc.

The following examples relate to the following media:

MRS liquid Medium (g/L): 10.0 parts of casein enzyme digests, 10.0 parts of beef extract powder, 4.0 parts of yeast extract powder, 2.0 parts of triammonium citrate, 5.0 parts of sodium acetate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 2.0 parts of dipotassium hydrogen phosphate, 20.0 parts of glucose, 1.08 parts of tween-80, and the final pH value is 5.7+/-0.2;

MRS solid Medium (g/L): 10.0 parts of peptone, 5.0 parts of beef extract powder, 4.0 parts of yeast extract powder, 20.0 parts of glucose, 2.0 parts of dipotassium hydrogen phosphate, 5.0 parts of sodium acetate, 2.0 parts of tri-ammonium citrate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 15.0 parts of agar, 1.0 part of tween-80 and the final pH value of 6.2+/-0.2;

LB solid medium (g/L): peptone 10.0, sodium chloride 5.0, glucose 1.0, yeast extract powder 5.0, and final pH of 7.0+ -0.2

Seed medium (g/L): glucose 25, yeast extract 6.25, soy peptone 6.25, mnSO ₄ ·H ₂ O0.16; tween-80 ml, final pH 5.0±0.2;

fermentation medium (g/L): glucose 25, yeast extract 6.25, soybean peptone 6.25, mnSO4.H2O 0.16, sodium glutamate 10, tween-80 2 ml, and final pH of 5.0+ -0.2.

The solutions involved in the examples below were formulated as follows:

the preparation of the simulated artificial gastric fluid is prepared by referring to the method mentioned in Chinese pharmacopoeia: taking 1 mol/L dilute hydrochloric acid 16.4. 16.4 mL, adding 800 mL of distilled water and 10g of pepsin, transferring to a volumetric flask, shaking uniformly, adding water to a constant volume of 1000 mL, adjusting the pH to 3.0, and filtering and sterilizing by using a microporous filter membrane with the specification of 0.22 mu m for later use.

The preparation of the simulated artificial intestinal juice is prepared by referring to the method mentioned in Chinese pharmacopoeia: dissolving potassium dihydrogen phosphate 6.8 and g in 500 mL distilled water, and regulating pH to 6.8 with 0.1 mol/L NaOH solution; pancreatin 10g is weighed, dissolved by adding a proper amount of distilled water, transferred to a volumetric flask to be fixed to 1000 mL, and filtered and sterilized by a microporous filter membrane with the specification of 0.22 mu m for later use.

The artificial pig gall salt solution comprises the following components: trypsin 1 g/L, pig bile salts 30 g/L or 20 g/L or 10g/L, sodium chloride 15 g/L, final pH 7.0.

The activation method of the strain involved in the following examples is as follows:

enterococcus lactate FSUH-1 taken out at-20deg.C was inoculated into a test tube containing 10 mL MRS medium at an inoculum size of 2% (v/v), and subjected to stationary culture at 37deg.C for 12 h, and the procedure was repeated for three generations.

The detection method involved in the following embodiment is:

detection of GABA content:

GABA was detected by HPLC.

Sample pretreatment: centrifuging 1 mL fermentation broth at 8000 r/min for 5 min, boiling supernatant at 100deg.C for 10 min, centrifuging at 12000 r/min, collecting 200 μl of supernatant, adding 400 μl of o-phthalaldehyde derivatizing agent, adding 1 mL boric acid buffer solution, reacting for 5 min, filtering with 0.22 μm filter membrane, and immediately introducing sample.

Mobile phase a:20 mmol/L sodium acetate buffer, mobile phase B:100% acetonitrile;

mobile phase a: mobile phase b=80: 20 (v: v)

Drawing a GABA standard curve: 5.000 GABA standard mother liquor of g/L is weighed to prepare standard solutions with concentration gradients of 0.5, 1, 2, 3 and 4 g/L and 5, the peak area is measured by adopting a high performance liquid chromatography after the standard solutions are derived, and a GABA standard curve is drawn by taking the GABA concentration as an abscissa and the peak area as an ordinate.

Chromatographic conditions: agilent high performance liquid chromatograph, column temperature 40 ℃, detection wavelength 338 nm, sample injection amount 20 μl, mobile phase A sodium acetate, mobile phase B acetonitrile, detection time 14 min, flow rate 1.0 mL/min, chromatographic column: ecosil 120-5-AQ PLUS 5 μm 4.6X1250 mm.

Preparation of the reagent:

(1) Boric acid buffer solution, 24.7. 24.7 g boric acid, 1. 1L ultrapure water, and final pH of 10.4.

(2) Mobile phase a: sodium acetate 2.72 g, triethylamine 200. Mu.L, glacial acetic acid 200. Mu.L, ultrapure water 1L, final pH 7.3, and passing through 0.22 μm filter membrane for ultrasonic degassing.

(3) Mobile phase B:100% acetonitrile.

(4) Preparing a derivatizing agent: the phthalaldehyde 10 mg, beta-mercaptoethanol 10. Mu.L, acetonitrile 2.5 mL were weighed and either prepared as is or placed in a refrigerator at 4℃for 2 days with a brown bottle.

Example 1: separation and screening of enterococcus faecalis FSUH-1

(1) Separating, screening and preserving

Weighing 25 g sauce mash sample, adding 225 mL sterile physiological saline, diluting bacterial solution with PBS to 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 ，10 ^-5 、10 ^-6 The diluted sample solutions with different gradients of 0.1 and mL are uniformly coated on an MRS solid culture medium, and are cultured in a constant temperature incubator at 37 ℃ for 24-48 hours, and two parallel diluted sample solutions are arranged. Single bacterial colonies with obvious boundaries are selected and streaked and purified on an MRS solid culture medium for 2-3 times to obtain single bacterial strains, and after the single bacterial strains are cultured by the MRS liquid culture medium for 16-18 h, bacterial liquid and 50% sterilized glycerol are added in the following ratio of 1:1 is added into a sterilizing EP pipe according to the volume ratio, and is placed in a refrigerator at the temperature of minus 80 ℃ for preservation after being evenly mixed.

(2) Preliminary screening of GABA producing Strain

Inoculating the activated strain into seed culture medium, standing at 37deg.C for culturing to OD _600nm 0.5 to 0.6, and inoculating the strain into a fermentation medium containing 10g/L sodium glutamate according to an inoculum size of 1%, and performing stationary culture at 37 ℃ for 72 h. Shaking, taking 1 mL of fermentation liquor, and centrifuging at 10000 rpm for 10 min for standby.

The paper chromatography is adopted for primary screening: drawing a straight line at a position 2-cm away from the bottom of the filter paper by using a Xinhua No. I filter paper with a height of 15-cm, taking a pencil at a position 2-cm away from the straight line as a sample application position, taking 2 mu L of supernatant sample application by using a pipette or a microsyringe, controlling the diameter of the point after sample application to be 0.3-mm, repeatedly applying sample application after a sample is dried, fixing the filter paper, placing the filter paper in a chromatography cylinder filled with a developing agent for developing 2-h, taking out the filter paper when the front edge of the developing agent reaches 1-2 cm away from the top end of the filter paper, placing the filter paper in a fume hood for 10 min after the filter paper is slightly dried, drying and developing the filter paper in an oven at 80 ℃ for 30 min, and taking sodium glutamate of 1 g/L and GABA standard sample of 1-g/L as a control; the GABA yield of the strain FSUH-1 was initially measured by a paper chromatography-microplate reader method and found to be 1.74 g/L.

The steps for measuring GABA by using a paper chromatography-enzyme-labeled instrument are as follows: shear with cutting edgeThe GABA color spots are developed, the color spots are eluted in 5 mL eluent (75% alcohol: 0.6% copper sulfate=38:2), after the elution is completed, the absorbance value is measured by an enzyme-labeled instrument at 512 nm wavelength, and the GABA content in the fermentation broth is calculated according to the standard curve and the absorbance value of the sample. Paper chromatography-microplate reader standard curve function is y=03836x+0.07856, r ² =0.9975。

(3) HPLC (high Performance liquid chromatography) re-screening

According to the method, the product identification and further quantitative detection can be carried out by an ultraviolet detector of a high performance liquid chromatography method according to the reaction of GABA and phthalic dicarboxaldehyde to produce the derivative with ultraviolet absorption, the detection result is shown as a figure 1, the retention time of the product GABA of the strain FSUH-1 fermentation liquor is consistent with the retention time of a GABA standard substance under the same HPLC detection condition, the retention time is about 9.0 min, and the final yield of the enterococcus faecalis FSUH-1 is 1.87+/-0.12 g/L measured by an HPLC method according to the calculation of a GABA standard curve of the HPLC method. GABA standard samples are shown in FIG. 2. The HPLC method for determining GABA standard curve functions is as follows: y= 7496.5x-1317.6, r ² =0.9995。

Example 2: identification and preservation of enterococcus faecalis FSUH-1

(1) Bacterial strain gram staining

The activated strain FSUH-1 was subjected to three-region streaking to obtain a purified single colony, the purified strain obtained in example 1 was stained with a sterile inoculating loop according to the instructions for use of the gram staining solution kit of Changdybikerman biotechnology Co., ltd, the morphology of which was observed by microscopic examination, the result of microscopic examination of the strain FSUH-1 was shown in FIG. 3, and the morphology of the colony was shown in FIG. 4.

(2) PCR amplification and sequence analysis of 16S rDNA gene

Bacterial DNA of the purified strain obtained in example 1 is extracted according to the specification of a bacterial genome DNA extraction kit, the purity is detected by an enzyme-labeled instrument, the bacterial DNA is used as an amplification template, a PCR amplification reaction is carried out on a PCR instrument by adopting a 16S rDNA gene universal primer, 5 mu L of the bacterial DNA is absorbed and detected by 1% agarose gel electrophoresis, and the length of the 16S rDNA sequence of the strain is about 1461 bp. PCR products were sequenced by the manufacturer (Shanghai) Inc. And carrying out homology analysis on the obtained sequence at the website https:// www.ezbiocloud.net/identification, and constructing a phylogenetic tree by using MEGAX software.

The PCR reaction system is 50 mu L, the amplification conditions are 94 ℃ for 5 min,94 ℃ for 30 s,55 ℃ for 30 s,72 ℃ for 1 min of extension, and the total cycles are 35, and the extension is carried out at 72 ℃ for 10 min.

(3) Biochemical identification of 0.5% glycerol fermentation tube (lactic acid bacteria)

Selecting enterococcus faecalis FSUH-1 single colony, inoculating into MRS liquid culture medium, culturing at 37deg.C for activation, and regulating with sterile physiological saline to thallus concentration of 1×10 ⁸ CFU/mL, 50. Mu.L of the bacterial suspension was added to an ampoule of a 0.5% glycerol fermentation tube (lactic acid bacteria), sealed with a sealing film, and cultured for 18-24 h, and the results were negative according to the identification instructions.

According to the appearance form of the bacterial colony, microscopic examination, 16S rDNA sequencing result and 0.5% glycerol fermentation tube biochemical identification result (reference DOI: 10.1099/ijs.0.030825-0), the bacterial strain is identified as enterococcus lactis, named enterococcus lactis FSUH-1 and deposited in the Guangdong province microorganism strain deposit center, and the deposit number is GDMCC No:62397. the 16S rDNA sequence evolutionary tree is shown in FIG. 5.

Example 3: characterization of enterococcus lactate FSUH-1

(1) Enterococcus lactate FSUH-1 growth curve assay

After the enterococcus acidilactici FSUH-1 strain is activated, inoculating the enterococcus acidilactici FSUH-1 strain on MRS liquid culture medium according to an inoculum size of 1% (v/v), culturing at 37 ℃ for 24 h, sampling every 2h, and measuring the strain OD _600nm And (3) drawing a growth curve of the enterococcus acidilactici FSUH-1 by taking the light absorption value as an ordinate and the time interval as an abscissa.

The results showed that enterococcus lactate FSUH-1, after being inoculated in MRS liquid medium, had a lag phase of about 2h, and then entered into a log phase of growth, the growth rate of 6 h was reduced, and 8 h reached a stationary phase of growth. The growth curve is shown in FIG. 6.

(2) Enterococcus lactate FSUH-1 in vitro gastrointestinal fluid tolerance test

Regulating activated enterococcus faecalis FSUH-1 thallus with sterile physiological saline to thallus concentration of 1×10 ⁸ CFU/mL, 1 mL bacterial liquid is added into 9 mL simulated gastric fluid (simulated intestinal fluid), sampling liquid is absorbed in 0 h and 3 h (simulated intestinal fluid is 0 h and 4 h) respectively for 10 times gradient dilution, 0.1 mL is evenly coated in an MRS solid culture medium, standing culture is carried out at 37 ℃ for 24 h, 3 independent repeated tests are designed, the gastrointestinal fluid tolerance of the bacterial strain is calculated according to formula (1) by taking the unregulated MRS liquid culture medium as a control, and the average survival rate is calculated.

Strain survival (%) = lgN ₁ /lgN ₀ ×100%（1）；

N ₁ To tolerate the survival of the strain after 3 h (4 h), N ₀ Survival of the strain at 0 h tolerance.

The enterococcus tolerating gastrointestinal fluid survival is shown in Table 1.

Table 1: enterococcus lactate FSUH-1 in vitro simulated gastrointestinal fluid tolerance results

(3) Enterococcus lactate FSUH-1 resistant pig bile salt test

Regulating activated enterococcus faecalis FSUH-1 thallus with physiological saline to thallus concentration of 1×10 ⁸ CFU/mL is prepared to obtain enterococcus acidilactici FSUH-1 bacterial suspension, 10 mL enterococcus acidilactici FSUH suspension is respectively absorbed into 250 mL artificial pig bile salt solution triangular flasks with the concentration of 90 mL pig bile salt solution of 10g/L, 20 g/L and 30 g/L respectively, 90 mL unregulated MRS culture medium is used as a control group, standing culture is carried out for 180 min at 37 ℃, viable count is carried out on the culture at 0 min and 180 min respectively, and the survival rate of enterococcus acidilactici under different pig bile salt concentrations is calculated according to a formula (1).

The pig bile salt solution used in this example had a composition of trypsin 1 g/L, pig bile salt 30 g/L or pig bile salt 20 g/L or pig bile salt 10g/L, sodium chloride 15 g/L, and a pH adjusted to 7.0, and an unregulated MRS medium was used as a control group.

The results of enterococcus lactate FSUH-1 tolerance to porcine bile salts are shown in Table 2.

Table 2: enterococcus lactate FSUH-1-tolerant pig bile salt results

The determination result shows that after the enterococcus faecalis FSUH-1 is kept in the artificial pig bile salt solution with the concentration of 30 g/L for 180 min, the survival rate reaches 74.54%, and the result shows that the enterococcus faecalis FSUH-1 can tolerate high-concentration pig bile salt, can pass through the gastrointestinal tract and keep activity, and can play a role in the intestinal tract.

(4) Surface hydrophobicity test

The hydrophobicity of the strain surface was determined using a hydrocarbon adsorption capacity test. The method comprises the following steps:

collecting activated enterococcus faecalis FSUH-1 bacterial solution 5 mL, centrifuging at 5000 r/min for 8 min to collect bacterial cells, preparing suspension with PBS buffer solution, and measuring OD _600nm The value is recorded as A ₀ Adding xylene with the same volume, adding no xylene in control group, vortex oscillating for 2 min, standing at room temperature for 1 h, taking out the lower water phase to determine OD _600nm Is denoted as A ₁ The replicates were run in duplicate with sterile PBS buffer as a blank. The average value of the hydrophobicity of the strain was calculated according to the following formula (2).

Hydrophobicity (%) = (a) ₀ -A ₁ ）/A ₀ ×100%（2）；

The results show that the surface hydrophobicity of the strain of the invention, enterococcus lactic FSUH-1, is 40.24%, and the results are shown in Table 3.

Table 3: surface hydrophobicity results of enterococcus lactate

(5) Self-coagulation ability test

Collecting activated enterococcus faecalis FSUH-1 bacterial liquid 5 mL, centrifuging at 5000 r/min for 8 min, preparing suspension with the same volume with PBS buffer, shaking the bacterial suspension completely, and measuring OD _600nm The value is recorded as A _a Culturing at 37deg.C for 24 h, sucking upper liquid, and concentrating at OD _600nm The absorbance was measured and designated A _b Sterile PBS buffer was used as a blank. Calculation ofSelf-setting ability of enterococcus faecalis FSUH-1.

Self-agglomeration rate/% = (a) _a -A _b ）/A _a ×100%（3）；

The results show that the self-clotting capacity of the strain of the invention enterococcus faecalis FSUH-1 is 89.64%.

The results of the self-agglomerating ability of enterococcus faecalis FSUH-1 are shown in Table 4.

TABLE 4 enterococcus lactate FSUH-1 self-coagulation test results

(6) Salt tolerance

Activating enterococcus faecium FSUH-1 with MRS liquid culture medium for three generations to growing period, inoculating enterococcus faecium FSUH-1 into 10 ml MRS liquid culture medium test tubes containing 0%, 1%, 3%, 5%, 7%, 9%, 11% sodium chloride salt concentration respectively at 2% (v/v), standing at 37deg.C for culturing 24 h, and determining OD at 0 h, 4 h, 8 h and 24 h respectively _600nm The values were observed for strain growth. The results are shown in FIG. 7.

Meanwhile, the limiting salt tolerance of the strain of the invention enterococcus faecalis FSUH-1 was determined. Activation of the strain after growth period (OD _600nm 0.6), enterococcus lactis is inoculated in an inoculum size of 2% (v/v) in an MSR liquid culture medium containing 35% sodium chloride salt concentration of 10 mL,100 mu L of the mixture is coated on an MRS solid plate after 24 h stationary culture at 37 ℃,24 h stationary culture at 37 ℃, the living bacteria condition of the MRS solid plate is recorded, and OD is measured simultaneously _600nm Values.

The results show that enterococcus lactate FSUH-1 can normally grow on MRS culture medium with 35% salt concentration and OD _600nm 0.175, OD before inoculation _600nm 0.083. The limiting salt tolerance concentration of the strain was determined in this way. The results of enterococcus lactate FSUH-1 on MRS solid plates are shown in Table 5.

TABLE 5 results of limiting salt tolerance concentration of enterococcus faecalis FSUH-1

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Note that: "+" indicates that viable colonies appear on MRS solid plates.

Example 4: safety study of enterococcus lactate FSUH-1

The method comprises the following specific steps:

(1) Indole test

Diluting activated enterococcus faecalis FSUH-1 bacteria solution with 0.85% sterile physiological saline to 1×10 ⁸ Inoculating 0.05-0.08 mL (about 1-2 drops) of bacterial suspension in CFU/mL bacterial suspension in a peptone Shui Xilin bottle, culturing at 37 ℃ for 24-h, adding 2-3 drops of indigo substrate, and immediately observing the result to obtain escherichia coli @E.coliATCC 25922) was used as a positive control, and 85% sterile physiological saline was used as a blank, and the results are shown in table 6.

Table 6: enterococcus lactate FSUH-1 indole results

Note that: "+" represents positive and "-" represents negative.

The results showed that the surface of the medium of enterococcus faecalis FSUH-1 was yellow and negative.

(2) Enterococcus lactate FSUH-1 amino acid decarboxylase assay

Activating the strain of enterococcus FSUH-1, diluting the activated strain of enterococcus FSUH-1 with 0.85% sterile physiological saline to 1×10 ⁸ Sucking 0.05-0.08 mL (about 1-2 drops) of CFU/mL, respectively adding into an arginine decarboxylase test tube, a lysine decarboxylase test tube, an ornithine decarboxylase test tube and an amino acid decarboxylase reference tube, respectively dripping 5-8 drops of sterile liquid paraffin for liquid sealing, standing and culturing at 37 ℃ for 24 h, and according to the specification, when no positive result is found, continuously culturing for 4 days, and observing color change;

meanwhile, the escherichia coli is usedE.coliATCC 25922) was added to each of the arginine decarboxylase test tube, lysine decarboxylase test tube, ornithine decarboxylase test tube and amino acid decarboxylase control tube at the same concentrations as positive controls.

The results show that enterococcus lactis FSUH-1 ornithine decarboxylase and lysine decarboxylase are negative, arginine decarboxylase is positive, and although arginine decarboxylase is positive, the arginine decarboxylase is not reported to be harmful, only has influence on the nitrite content in a fermentation product, and has research reports (DOI: 10.3389/fmib.2016.01050), and arginine can strongly stimulate the generation of GABA. The results of the enterococcus lactate FSUH-1 amino acid decarboxylase assay are shown in Table 7.

TABLE 7 enterococcus lactate FSUH-1 amino acid decarboxylase results

Note that: "-" indicates negative, "+" indicates positive.

(3) Hemolysis test

Taking purified enterococcus FSUH-1 single colony, streaking and inoculating on a Golgi Bi Ya blood agar plate, inversely culturing at constant temperature of 37 ℃ for 24 h, and observing whether a hemolysis transparent ring appears around the enterococcus or whether grass green ring is formed by taking staphylococcus aureus (ATCC 25923) as a positive control.

The results show that, as shown in FIG. 8, the positive control group staphylococcus aureus shows a beta hemolysis transparent ring, while the periphery of the enterococcus lactis FSUH-1 does not show a transparent ring or grass green ring, and the periphery of the colony of the enterococcus lactis FSUH-1 does not show a hemolysis ring, which is gamma hemolysis. The enterococcus lactic acid FSUH-1 is not hemolytic.

(4) Antibiotic susceptibility test

According to the antimicrobial drug susceptibility test execution standard, enterococcus is subjected to drug susceptibility test by adopting a paper sheet diffusion method. After activating the strain, the bacterial liquid is diluted to 1X 10 ⁸ CFU/mL bacterial suspension is uniformly coated in MRS solid culture medium, after bacterial solution is dried, 6 antibiotic drug sensitive tablets of tetracycline, erythromycin, vancomycin, rifampin, ampicillin and norfloxacin are placed under light pressure, 3 independent repeated experiments are set, staphylococcus aureus (ATCC 25923) is used as quality control strain, and 16-18 h is cultivated at constant temperature of 37 ℃, wherein the vancomycin is preparedThe results of culturing 24 and h are shown in Table 8.

TABLE 8 results of enterococcus lactate FSUH-1 drug sensitivity test

Note that: s in brackets is sensitivity, I is mediator, and R is drug resistance.

The results show that: the strain enterococcus faecalis FSUH-1 has sensitivity to tetracycline, vancomycin, ampicillin and norfloxacin, has poor sensitivity to erythromycin and rifampin, and a drug resistance result shows that the strain can be controlled by using antibiotics.

Example 5: antibacterial activity of enterococcus faecalis FSUH-1

The double-layer agar expansion method comprises the following specific steps:

adding 10 mL of 18% agar medium on a sterilization plate, placing four oxford cups after agar solidifies, and adding a solution containing 0.8% of OD around the oxford cups _600nm LB solid solution of indicator bacteria (E.coli ATCC 25922, staphylococcus aureus ATCC 25923) at 0.3, after solidification, the oxford cup was removed with sterile forceps, and 150. Mu.L of OD was added to the well _600nm For 0.6 of enterococcus acidilactici FSUH-1, standing culture is carried out at 37 ℃ for 18 h after room temperature diffusion for 4 h, the diameter of a bacteriostasis circle is measured, three parallels are designed, and a non-inoculated MRS liquid culture medium is used as a blank control.

The result shows that the diameter of the inhibition zone of the enterococcus faecalis FSUH-1 on the escherichia coli is 13.67+/-0.33 mm, and the diameter of the inhibition zone of the staphylococcus aureus is 10.04+/-0.81 mm. The enterococcus lactate has the capability of inhibiting escherichia coli and staphylococcus aureus.

Example 6: high-yield GABA (gamma-amino acid) enterococcus lactic acid fermentation medium condition optimization

The method comprises the following specific steps:

(1) Single factor test

The following experiments are based on fermentation media, and fermentation media with pH values of 4.5, 5, 5.5, 6 and 6.5 are respectively prepared; respectively preparing a fermentation medium of 10g/L glutamic acid and a fermentation medium of 10g/L sodium glutamate by taking glutamic acid and sodium glutamate as substrates, and then preparing fermentation media with the concentration of 8, 10, 12, 14 and 16 g/L by selecting sodium glutamate; glucose, galactose, fructose, lactose, maltose and sucrose are used as carbon sources, fermentation culture mediums of different carbon sources are prepared according to the concentration of 25 g/L, and the influence of different carbon sources on GABA produced by the strain is observed; selecting fermentation culture mediums with sucrose concentrations of 5, 20, 35, 50 and 60 g/L respectively to prepare different sucrose concentrations; preparing fermentation culture mediums with different nitrogen sources by using tryptone, beef extract, yeast extract and soybean peptone according to the concentration of 6.25 g/L; selecting yeast extract and soyase peptone compound nitrogen (1:1) to prepare fermentation culture mediums with different nitrogen sources according to the ratio of 5, 15, 25, 35 and 45 g/L; preparing a fermentation medium with pyridoxal phosphate concentration of 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 g/L; the culture temperature is 37 ℃, the culture time is 72h, and the determination method is a paper chromatography-enzyme-labeling method. The test results are shown in fig. 9a to 9 h.

(2) Response surface test

On the basis of single factor and level tests, five factors of sucrose, yeast extract and soybean peptone complex nitrogen (1:1), pyridoxal phosphate, pH and sodium glutamate were examined by a response surface test, 2 levels were set for each factor, and 5 replicates were designed for each set of tests.

The Box Behnken Design Design response surface test was performed using software Design Expert 8.0.6, with sucrose, yeast extract and soy peptone complex nitrogen (1:1), pyridoxal phosphate, pH, sodium glutamate as variables, GABA as response values, and Design factor level test analysis results, 5 replicates each, and the Design method and response surface results are shown in table 9.

Table 9 response surface design and results

TABLE 10 regression model analysis of variance results

As shown in Table 10, the regression equation shows that the model P is less than 0.0001 and reaches a significant level, which indicates that the predicted value of the model is consistent with the actual value, the mismatch term P is 0.5631 > 0.05 and is not significant, which indicates that the model has better fitting degree with the test and has statistical significance.

The optimal conditions for obtaining the product GABA of the enterococcus acidilactici FSUH-1 by analyzing the regression equation according to Design-Expert 11 software are as follows:

sucrose 15.893 g/L, yeast extract 11.749 g/L, pH 6.47 g/L, sodium glutamate 13.762 g/L, PLP 0.316.316 g/L, and enterococcus faecalis FSUH-1 producing GABA theoretical value 14.537 g/L.

To further verify the reliability of the results of response surface optimization, 3 sets of parallel experiments were performed under the conditions of 15.90 g/L sucrose, 11.75 g/L yeast extract, 11.75 g/L soybean peptone, 13.76 g/L sodium glutamate, 0.3 g/L pyridoxal phosphate PLP, 80 mL/L Tween, 0.16 g/L manganese sulfate and 6.5 pH, and the real average value of GABA production of enterococcus acidilactici FSUH-1 was 14.57 g/L, the results of the experiments were similar to the theoretical value, and the relative error value was 0.226%, and the results prove that it is feasible and effective to optimize the fermentation medium by using the response surface. The reaction system at this time is: the reaction conditions are as follows: and (3) standing and culturing 72h at the constant temperature of 37 ℃.

Absolute error= |theoretical value-true value|.

Relative error = absolute error/true value x 100%.

Example 7: enterococcus lactate FSUH-1 in fermentation medium containing 5% sodium chloride for producing GABA

The method comprises the following specific steps:

(1) Preparing optimized fermentation medium

The optimized fermentation medium comprises the following components: 15.9 g g/L of sucrose, 11.75 g g/L of yeast extract, 11.75 g g/L of soybean peptone, 13.76 g g/L of sodium glutamate, 0.3 g g/L of pyridoxal phosphate PLP, 80 g Tween-80, 0.16 g g/L of manganese sulfate and pH of 6.5, and 5% and 10% of sodium chloride are respectively added to obtain fermentation culture media with different salt contents;

(2) Inoculating activated enterococcus faecium FSUH-1 into a saline fermentation medium according to an inoculum size of 1%, standing and culturing at 37 ℃ for 72 hours, and measuring the GABA yield in the fermentation liquid.

The results show that enterococcus faecalis FSUH-1 of the present invention does not produce GABA at a sodium chloride salt concentration of 10%, and has the ability to produce GABA at a sodium chloride salt concentration of 5%, with a yield of 13.22.+ -. 0.25 g/L.

In conclusion, the enterococcus lactis provided by the inventionEnterococcus lactis) FSUH-1 has the capability of producing GABA, has stronger gastrointestinal fluid tolerance, bile salt tolerance and adhesion capability, has the characteristics of salt tolerance, high growth speed and the like, is safe through safety identification, and can be applied to development of microecologics, probiotic fermentation products and feed additives for inhibiting animal intestinal pathogenic bacteria, regulating intestinal flora and the like by utilizing the thallus and the produced GABA.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Enterococcus lactis strainEnterococcus lactis) FSUH-1 was deposited at 20/4/2022 with the collection of microorganism strains, accession number GDMCC No:62397.

2. a microbial agent comprising the enterococcus faecium FSUH-1 or a fermentation broth or an intracellular and extracellular metabolite thereof according to claim 1.

3. The microbial agent according to claim 2, wherein the viable count of enterococcus lactis FSUH-1 in the microbial agent is at least: 1X 10 ⁶ CFU/mL。

4. A product comprising the enterococcus lactis FSUH-1 of claim 1 or the microbial agent of claim 2 or 3.

5. The product of claim 4, wherein the product is a chemical.

6. Product according to claim 4 or 5, characterized in that in the product the viable count of enterococcus lactis FSUH-1 is at least: 1X 10 ⁶ CFU/mL。

7. Use of the enterococcus faecu-1 according to claim 1 or the microbial agent according to claim 2 or 3 for the preparation of gamma-aminobutyric acid GABA or products containing gamma-aminobutyric acid GABA.

8. A method for preparing gamma-aminobutyric acid GABA is characterized in that the method comprises the steps of inoculating the enterococcus faecium FSUH-1 in a reaction system containing sodium glutamate or glutamic acid for reaction to prepare gamma-aminobutyric acid GABA.

9. The method according to claim 8, wherein the reaction conditions in the reaction system are: inoculating the third generation of enterococcus acidilactici FSUH-1 with an inoculum size of 1-10% (v/v) in a fermentation medium with pH value of 4.5-6.5, and standing at 30-42deg.C for culturing 12-108 h.

10. Use of the microbial agent of enterococcus faecium FSUH-1 according to claim 1 or of the microbial agent of claim 2 or 3 for the preparation of a product for inhibiting staphylococcus aureus and/or escherichia coli.