CN117264846A

CN117264846A - Tetrabipyridis halophilum with high protease activity and high production of bioactive peptide and application thereof

Info

Publication number: CN117264846A
Application number: CN202311410736.1A
Authority: CN
Inventors: 乌日娜; 武俊瑞; 刘曲; 潘国杨; 安飞宇; 姜锦惠; 陶鑫禹
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2023-10-28
Filing date: 2023-10-28
Publication date: 2023-12-22

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a tetracoccus halophilus (Tetragenococcus halophilus) with high protease activity and high production of bioactive peptide and application thereof. The tetracoccus halophilus sieve is selected from northeast traditional naturally fermented bean paste, and the preservation number is CGMCC No.27586. The activity of the strain gamma-glutamyl transferase is 7.361 +/-0.003U/mL, the DPPH free radical scavenging capacity reaches 57.32%, the hydroxyl free radical scavenging capacity reaches 56.03%, the protease activity is 342.71 +/-0.48U/mL, and the strain gamma-glutamyl transferase is higher than that of a standard strain ATCC 33315. The strain has high protease activity, high bioactive peptide production capacity, good antioxidant capacity, and freshness-increasing potential, and can be applied to the production of fermented functional foods.

Description

Tetrabipyridis halophilum with high protease activity and high production of bioactive peptide and application thereof

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to a tetracoccus halophilus with high protease activity and high production of bioactive peptide and application thereof.

Background

The bioactive peptide is peptide compound beneficial to the life activity of organism or with physiological action, and is polypeptide with relative molecular mass less than 6000Da and multiple biological functions. The molecular structure is a molecular polymer between amino acids and proteins, is small enough to be composed of two amino acids, is large enough to be formed by connecting dozens of amino acids through peptide bonds, and can be modified through phosphorylation, glycosylation or acylation. The different fermentation strains can produce various bioactive peptides, including antioxidant peptide, antihypertensive peptide, immunoregulatory peptide, antibacterial peptide, and opioid peptide, and have physiological functions of regulating antioxidant stress, cardiovascular system, immune system, nervous system and gastrointestinal system.

As early as 1978, the umami octapeptide of beef was isolated from beef, and research and utilization of umami peptide has begun. The umami peptide is small peptide with molecular weight of 150-3000Da, and can be directly extracted from natural food. The umami peptide is used as a novel green and safe food flavoring agent, can effectively improve the umami taste of food, and can adjust other taste attributes. The taste of umami peptides is mainly derived from the intermediate products of the protein synthesis and decomposition process. In recent years, various sources including umami peptides such as meats, insects, aquatic products, fungi, cereals, beans, and fermented products thereof have been found in many processed foods and raw protein hydrolysates, but at present, there are few studies on the production of umami peptides by fermentation of microorganisms through various metabolic pathways, and the search for new umami peptides is continued. Meanwhile, microbial fermentation has received much attention. The microbial fermentation method is to decompose the protein or synthesize amino acid in the material under the action of some microbe or microbial enzyme to convert the material into small flavor peptide and separate and extract to prepare delicious peptide.

The antioxidant peptide belongs to bioactive peptide, is a micromolecular bioactive substance with antioxidant function, and generally consists of 2-20 amino acids. The antioxidant peptide is taken as a small molecular natural active substance, and is concerned by students because of the characteristics of green, strong activity, easy absorption and the like, and researches show that the biological activity of the antioxidant peptide can be influenced by amino acid sequences, molecular weight and hydrophobic amino acid content. In general, the smaller the molecular weight, the higher the proportion of hydrophobic amino acids such as proline (Pro), valine (Val), tryptophan (Trp) and phenylalanine (Phe) bonded to the amino acid residues, and the higher the antioxidant activity. In recent years, antioxidant peptides have been found in unprocessed foods such as beans, meats, eggs, etc., fermented products, and hydrolysates, and synthetic production methods have been hot because of their characteristics such as "green", "natural", "safe", "easy-to-absorb", etc.

The tetracoccus halophilus belongs to gram positive bacteria, is spherical bacteria with the diameter of about 0.5-1.5 microns, can be widely used in high-salt environments, can be separated from fermented foods, such as soybean paste, soy sauce, pickle and the like, and can be fermented to produce organic acids such as lactic acid, acetic acid, pyruvic acid and the like, so that the foods have sour taste and special flavor. The secretion of enzymes to produce a number of metabolic pathways that in turn produce flavour substances may play a vital role in the development of the flavour quality of soy sauce. The results of the application of tetracoccus halophilus to fermented soy sauce prove that the taste of the soy sauce can be improved. Tetranectias is one of the core flora affecting soy color and flavor, and the enzymes produced catalyze a series of metabolic pathways that produce flavor substances. Proteases are used as a major enzyme in the brewing process of soybean paste, and they degrade proteins in raw materials into small molecule peptides and amino acids, and therefore are often used as one of the indicators for measuring flavor levels.

Tetranectium halophilum has certain protease production capability, and protease is used as a main enzyme in the brewing process of soybean paste, and can degrade proteins in raw materials to generate small molecule polypeptides and amino acids with the activity. The halophilic tetrazococcus promotes the utilization of glucose in bean paste by secreting proteases such as Aldose 1-epi-isomerase (EC 5.1.3.3), glucose phosphomutase (Alose 1-epi-ase and sugar-specific II), which are key saccharification microorganisms in bean paste. The protease activity value of the existing tetracoccus halophilus is low, so that the tetracoccus halophilus is poorly applied in the production practice of food, and the strain utilization rate is low. The halophilic tetracoccus with high protease has the capacity of improving the flavor of food, so that the halophilic tetracoccus with high protease activity needs to be screened out in order to improve the strain utilization rate of the halophilic tetracoccus for being applied to the production of the food.

Disclosure of Invention

The invention provides a halophilic tetracoccus and application thereof (Tetragenococcus halophilus) SNTH-3 for producing bioactive peptides with high protease activity, wherein the halophilic tetracoccus is screened from 96 parts of northeast traditional naturally fermented soybean paste in different areas of Liaoning province, has high protease activity, can improve the yield of bioactive peptides, promotes the development of the fields of umami peptides and antioxidative peptides, and improves the application value of the halophilic tetracoccus.

The invention provides a tetracoccus halophilus (Tetragenococcus halophilus) SNTH-3, wherein the preservation number of the tetracoccus halophilus is CGMCC No.27586. The invention also provides application of the tetracoccus halophilus in producing bioactive peptide.

In the technical scheme, further, the fermentation temperature of the tetracoccus halophilus is 25-40 ℃, the inoculation amount is 1-5%, the NaCl concentration is 1-10%, and the pH value is 5.5-10.5.

In the technical scheme, further, the fermentation temperature of the tetracoccus halophilus is 37 ℃, the inoculation amount is 3%, the NaCl concentration is 4%, and the fermentation pH value is 8.5.

The invention also provides application of the tetracoccus halophilus in fermented foods.

In another aspect, the invention provides a microbial preparation comprising the aforementioned tetracoccus halophilus.

In the technical proposal, further, the living bacterial load of the tetracoccus halophilus in the microbial preparation is not less than 10 ⁸ CFU/g。

The invention also provides application of the microbial preparation in fermented foods.

In the above technical scheme, further, the fermented food is soybean paste or soy sauce; preferably soybean paste, soybean paste or soybean paste.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a novel tetracoccus halophilus (Tetragenococcus halophilus) SNTH-3 with high protease activity, by fermenting the strain, the protease activity value of the supernatant of a culture medium can reach 342.71 +/-0.48U/mL, which is obviously higher than the protease activity value of a standard strain ATCC 33315, the gamma-glutamyl transferase activity is 7.361 +/-0.003U/mL, the DPPH free radical removing capability reaches 57.32%, the hydroxyl free radical removing capability reaches 56.03%, the polypeptide producing capability reaches 29.34+/-0.008 mg/mL, and the peptide producing content is obviously higher than that of the standard strain ATCC 33315. The tetracoccus halophilus SNTH-3 can be widely applied to the production of bioactive peptides, and has the advantages of short production period, high yield, safety and no toxic or side effect. The tetracoccus halophilus SNTH-3 has the capability of removing DPPH free radical and hydroxyl free radical, has important value in antioxidation, can be used for preparing fermented food, is beneficial to improving the flavor of the fermented food, improves the safety of the fermented food, and has wide application prospect.

Drawings

FIG. 1 is a diagram showing the morphology of an individual SNTH-3 cell of Tetracoccus halophilus;

FIG. 2 is a phylogenetic tree of tetracoccus halophilus SNTH-3 based on the 16S rDNA gene sequence;

FIG. 3 is a drawing of a scanning electron microscope of tetracoccus halophilus;

FIG. 4 is a transparent circle of a portion of a tetranectin halophilum protease produced by a bacterium.

FIG. 5 shows DPPH scavenging free radical Activity of Tetracoccus halophilus SNTH-3 against ascorbic acid as a positive control

FIG. 6 shows the hydroxyl radical scavenging free radical activity of Tetracoccus halophilus SNTH-3 against ascorbic acid as a positive control.

FIG. 7 is a graph showing the effect of temperature on SNTH-3 peptide production and growth of tetracoccus halophilus;

FIG. 8 is a graph showing the effect of inoculum size on SNTH-3 peptide production and growth of tetracoccus halophilus;

FIG. 9 is a graph showing the effect of salt concentration on SNTH-3 peptide production and growth of tetracoccus halophilus;

FIG. 10 shows the effect of pH on SNTH-3 peptide production and growth of tetracoccus halophilus.

FIG. 11 is a response surface plot and contour plot of fermentation temperature versus inoculum size for the effect of polypeptide content;

FIG. 12 is a response surface plot and contour plot of fermentation temperature versus NaCl concentration for polypeptide content;

FIG. 13 is a response surface and contour plot of fermentation temperature and pH impact on polypeptide content;

FIG. 14 is a response surface plot and contour plot of the effect of inoculum size and NaCl concentration on polypeptide content;

FIG. 15 is a response surface plot and contour plot of the effect of inoculum size and pH on polypeptide content;

FIG. 16 is a response surface plot and contour plot of the effect of NaCl concentration and pH on polypeptide content.

Detailed Description

The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The culture medium used in the embodiment of the invention comprises the following components:

preparing an improved MRS culture medium (g/L): 10g of peptone, 3g of sodium acetate (anhydrous), 2g of dipotassium hydrogen phosphate, 0.575g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of glucose, 2.42g of trisodium citrate, 4g of yeast extract powder, 8g of beef extract, 80 g of tween, 100g of sodium chloride and 1L of distilled water, and regulating the pH value to 7.0.

Enrichment medium (g/L): 150g of sodium chloride, 2g of natamycin, 500 mu L of crystal violet and pH value of the MRS culture medium are added to the MRS culture medium, and the pH value is adjusted to 7.0.

Solid isolation Medium (g/L): 150g of sodium chloride, 2g of natamycin, 10g of calcium carbonate and 20g of agar are added into the MRS culture medium, and the pH value is adjusted to 7.0.

Seed medium (g/L): 100g of sodium chloride was added to the basic medium, and the pH was adjusted to 7.0.

Skim milk powder medium (g/L): 110.0g of skimmed milk powder, 15g of agar, 1.0L of distilled water and pH value of 7.4.

Simulated fermented soybean paste medium: 20g of isolated soy protein, 8g of wheat flour, 0.028g of calcium chloride and 28g of deionized water are uniformly mixed, sterilized at 121 ℃ for 30min, cooled to 38 ℃, inoculated with Shanghai brewing 3.042 aspergillus oryzae, fermented for 3 days, added with 100mL of phosphoric acid buffer solution, vigorously stirred for 1h, centrifuged at 4 ℃ for 15min at 10000g/min, the supernatant is taken, 10% sodium chloride is added, sterilized at 121 ℃ for 15min, and inoculated with tetracoccus halophilus according to an inoculum size of 3%.

Soytone medium (g/L): 10g of soybean peptone, 3g of sodium acetate (anhydrous), 2g of dipotassium hydrogen phosphate, 0.575g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of glucose, 2.42g of trisodium citrate, 1g of tween 80 and 50g of sodium chloride.

ProteinsCulture medium (g/L): protein->10g, sodium acetate (anhydrous) 3g, dipotassium hydrogen phosphate 2g, heptahydrate0.575g of magnesium sulfate, 0.25g of manganese sulfate monohydrate, 20g of glucose, 2.42g of trisodium citrate, 4g of yeast extract powder, 8g of beef extract, 1g of tween 80, 100g of sodium chloride and 1L of distilled water, and the pH value is 7.0.

Soy protein isolate medium (g/L): 10g of soybean protein isolate, 3g of sodium acetate (anhydrous), 2g of dipotassium hydrogen phosphate, 0.575g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of glucose, 2.42g of trisodium citrate, 4g of yeast extract powder, 8g of beef extract, 1g of tween 80, 100g of sodium chloride, 1L of distilled water and pH value of 7.0.

The invention will be further illustrated with reference to specific examples.

Example 1: isolation and identification of tetranectiococcus halophilus

(1) Bean paste sample collection

96 parts of peasant natural fermented soybean paste are collected from 9 areas of Benxi, dandong, dalian, cucurbit island, jinzhou, liaoyang, panjin and Shenyang. The sample is naturally brewed by a traditional method of farmers, and after being uniformly stirred, the sample is split-packed by a disposable sampling tube and placed in an ice box, quickly brought back to a laboratory and placed in a refrigerator at the temperature of minus 80 ℃.

(2) Isolation of tetratecoccus halophilus

Accurately weighing 1.0 g of the sauce sample, adding the sauce sample into 9mL of sterile physiological saline, fully stirring and standing for 15 to 20min. After the dilution was diluted to a suitable multiple, 200. Mu.L of the dilution was added to the enrichment medium. The culture medium was left to stand at 36℃for 2 to 3 days, and then cultured in a solid medium for 5 to 6 days by a plating method. Single colonies similar in morphology to lactic acid bacteria colonies were selected and gram stained. Selecting a pair-shaped or tetrad spherical thallus, placing the colony in a seed culture medium for purification culture, and preserving 136 suspected tetrad micrococcus halophilus.

(3) Identification of tetranectiococcus halophiles

1) Colony morphology identification

And (3) carrying out physiological and biochemical characteristic identification experiments by referring to the lactobacillus classification identification and experimental method and the common bacteria identification manual. The 136 suspected tetrad halophiles are picked from the bacteria with smaller colony, convex, milky white, opaque, smooth and glossy surface and smooth edge; no motility and no generation of spores; gram staining was positive and cells were spherical, tetrad or paired strain 118.

2) Molecular biological identification

The 118 strains are inoculated into a modified MRS liquid culture medium according to the volume of 3% (v/v), and are subjected to stationary culture at 37 ℃ for 48-72 hours, and DNA extraction is carried out on the strains by using a bacterial genome kit.

The PCR upstream primer 27F and downstream primer 1492R were synthesized by Shanghai Bioengineering Co.

27F：AGAGTTTGATCCTGGCTCAG(5'---3')；

1492R：GGTTACCTTGTTACGACTT(5'---3')。

The PCR reaction conditions were: preheating at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min for 30s, and circulating for 30 times; keeping at 72 ℃ for 10min, and preserving at 4 ℃.

The PCR product was sampled to Shanghai Bioengineering Co. And comparing the sequence obtained by sequencing with sequences in NCBI database, and matching by using BLAST algorithm to determine 118 strains of bacteria as tetracoccus halophilus. The phylogenetic tree is constructed by using MEGA 10 software, and the phylogenetic tree is evaluated and analyzed by adopting a Neighbor-Joining method to determine the phylogenetic and genetic relationship of tetracoccus halophilus.

(4) Preliminary screening of protease production capacity of tetracoccus halophilus

The isolated tetracoccus halophilus was cultured in modified MRS medium to the second generation, and the OD value was adjusted to ensure the same concentration of each group of bacteria. The bacterial liquid was transferred to a modified MRS liquid medium at an inoculum size of 3%, cultured for 60 hours, and subjected to 3 parallel experiments. The skim milk agar medium was perforated by a punch having a diameter of 1 cm. Care should be taken to avoid loosening of the medium during punching. After the cells were mixed with the medium, 200. Mu.L of the fermentation broth was injected into the vessel. The dishes were placed in a 37℃incubator and incubated for 72 hours. Finally, the diameter and depth of the transparent rings were recorded.

The protease transparent ring can reflect the activity of the protease of the strain to a certain extent, and has the representativeness of the preliminary screening condition.

The results are shown in FIG. 4: observing and measuring the growth condition of 118 tetrabipyridyl halophiles strains on a skim milk culture medium, degrading casein around the bacterial liquid, and selecting 74 strains with protein transparent rings in places where other bacterial liquids cannot permeate, wherein the circles are the tetrabipyridyl halophiles SNTH-3.

(5) Determination of halophilic tetranectin protease Activity

Protease activity of tetracoccus halophilus in a soybean matrix fermentation environment was determined using a microbial protease ELISA assay kit. The OD was measured at a wavelength of 450nm using an enzyme-labeled instrument, and the sample activity was calculated by a standard curve, operating according to the instructions.

Proteases are used as a major enzyme in the brewing process of soybean paste, and they degrade proteins in raw materials into small molecule peptides and amino acids, and therefore are often used as one of the indicators for measuring flavor levels.

Inoculating 74 strain of tetracoccus halophilus selected by primary screening into culture medium simulating different fermentation environments, respectively soybean peptone culture medium, modified MRS culture medium, and proteinThe protease activity of tetranectin halophilum in four fermentation environments is comprehensively measured by the culture medium and the soy isolate protein culture medium.

The results show that: the average value of the protease activity of the tetracoccus halophilus SNTH-3 is highest and reaches 318.64 +/-0.58U/mL.

(6) Determination of the Activity of the gamma-glutamyl transpeptidase of Tetracoccus halophilus

The gamma-GT enzyme activity of the tetracoccus halophilus in the soybean matrix fermentation environment is measured by using a gamma-glutamyl transpeptidase (gamma-GT) activity detection kit, and the method is operated according to the specification and mainly comprises the following operation steps: a. centrifugally collecting thalli; b. crushing by ultrasonic waves; c. the enzyme activity was measured spectrophotometrically.

gamma-GT, also known as gamma-glutamyl transferase, also plays an important role in the development and enhancement of umami taste during fermentation. First, the gamma-GT enzyme facilitates the conversion of glutamine (Gln) in the fermentation system to the umami amino acid glutamic acid, which is one of the important amino acid residues that make up the umami peptide.

The results show that: in the soybean peptone culture medium, the activity of SY2-1 gamma-GT in the 74 pre-screened tetracoccus halophilus is highest and reaches 4.649 +/-0.007U/mL, the activity value of SNTH-3 is ranked second and reaches 4.478 +/-0.005U/mL, and the activity values of the two are similar. In proteinsIn the culture medium, the activity of SNTH-3 gamma-GT is highest and reaches 9.069 +/-0.071U/mL, in the modified MRS culture medium, the activity of SNTH-3 gamma-GT is highest and reaches 4.581 +/-0.031U/mL, and the activity of the strain SNTH-3 is highest and reaches 7.361 +/-0.003U/mL by combining the activity conditions of gamma-GT of three fermentation culture media.

(7) Determination of polypeptide production capacity of tetracoccus halophilus

The BCA protein concentration determination kit is used for determining the content of the polypeptide in the culture solution of the fermentation medium of the tetracoccus halophilus, the operation is carried out according to the specification, the A562 nm is determined by adopting a microporous enzyme-labeling method, and the polypeptide concentration is calculated according to a standard curve.

The results show that: all the test strains have the capability of producing the polypeptide, but the capability of producing the polypeptide is greatly different among different strains. SNTH-3 has the strongest polypeptide production capacity (18.41+ -0.001 mg/mL) in Aspergillus oryzae culture medium simulating soybean paste fermentation process, and three culture mediums (soybean peptone culture medium, modified MRS culture medium, protein)Culture medium) has the strongest polypeptide production capacity of SNTH-3, which reaches 22.14+/-0.008 mg/mL.

(8) Factor analysis

1) KMO and Bartlett sphere test

The results are shown in table 1: KMO values of 0.710, values greater than 0.5, indicate some correlation between variables, whereas in Bartlett sphere test, sig values of 0.000 < 0.005, indicate that the data satisfies the overall normal distribution. Thus, it can be concluded that the flavor index data of different tetrazococcus halophilus are suitable for factor analysis.

Table 1KMO and Bartlett sphere test

2) Taste data factor analysis of different tetracoccushalophiles

The data were factor analyzed using spss software, and the results are shown in table 2, with the first factor feature root being 2.694, which contained 89.803% of the total variance of the original 3 variables; the first 2 factors accumulated variance contribution rate is 98.234%, which indicates that taking the first 2 common factors contains substantially all variable information.

TABLE 2 eigenvalue and variance contribution ratio

A linear regression equation of 2 common factors was constructed from fi=ui×xi (i=1, 2, 3):

F1＝0.860X1+0.668X2-0.765X3

F2＝-0.504X1-0.257X2+1.495X3

a factor score model was constructed based on the function f= 0.57999/0.98234 x f1+0.40235/0.98234 x F2 and a composite score was calculated.

The results show that: the strain with the highest factor comprehensive score is SNTH-3, and has the fresh-increasing and antioxidant potential and the capability of producing bioactive peptide.

In summary, the tetracoccus halophilus SNTH-3 is determined to be the optimal strain, the protease activity value reaches 342.71 +/-0.48U/mL, the gamma-glutamyl transferase activity reaches 7.361 +/-0.003U/mL, and the polypeptide production capacity reaches 27.14+/-0.008 mg/mL.

Example 2

(1) Identification of colony morphology of tetracoccus halophilus SNTH-3

The tetracoccus halophilus SNTH-3 colony is small, convex, milky white, opaque, smooth and glossy in surface and flat in edge; no motility and no generation of spores; gram staining was positive and cells were spherical, quadruple or paired. The cell morphology is shown in FIG. 1.

(2) Molecular biological identification

16s rDNA sequence of tetracoccus halophilus SNTH-3, SEQ ID NO 1, was prepared as described in example 1, as follows: AGGGGGGGGGCCTATACATGCAAGTCGAACGCTGCTTAAGAAGAAACTTCGGTTTTTTCTTAAGCGGAGTGGCGGACGGGTGAGTAACACGTGGGGAACCTATCCATCAGCGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATATGGCTTTTTTTCACCTGAAAGAAAGCTCAAAGGCGCTTTACAGCGTCACTGATGGCTGGTCCCGCGGTGCATTAGCCAGTTGGTGAGGTAACGGCTCACCAAAGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGCAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTCAGCAAAGAACAGGAGAAAGAGGAAATGCTTTTTCTATGACGGTAGCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTGATTTAAGTCTGATGTGAAAGCCCCCAGCTCAACTGGGGAGGGTCATTGGAAACTGGATCACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGACTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCGCCCTAGAGATAGGGTTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTGAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGCAAGCCAAGCCGCAAGGCCTAGCGAATCTCTGAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATCCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCAAAGTCGGTGCGGCAACCCTTAGGGAGCCCAGCCCGACG.

SEQ ID NO. 1 was aligned with sequences in the NCBI database and matched using the BLAST algorithm to determine tetracoccus halophilus.

The phylogenetic tree is constructed by using MEGA 10 software, and the phylogenetic tree is evaluated and analyzed by adopting a Neighbor-Joining method to determine the phylogenetic and genetic relationship of tetracoccus halophilus. As shown in FIG. 2, the phylogenetic tree of the SNTH-3 strain has the highest homology with tetracoccus halophilus (Tetragenococcus halophilus).

The applicant has preserved tetracoccus halophilus SNTH-3 in 2023 and 6/08 at China general microbiological culture Collection center (CGMCC, address: north Chen West Lu No. 1, 3 of the area of Charpy, beijing, and post code 100101 of the institute of microorganisms, china academy of sciences) with a preservation number of CGMCC No.27586.

Example 3: halophilic tetracoccus scanning electron microscope analysis

And (3) observing and analyzing the structure of the tetracoccus halophilus SNTH-3 by using a cold field emission scanning electron microscope technology. 200mL of modified MRS medium was prepared, enrichment culture was performed at an inoculum size of 3% for 56 hours, and finally centrifugation was performed at 3000g/min for 10 minutes at 4℃and the centrifuged cells were washed 3 times with phosphate buffer solution. The sample was fixed with a 2.5% glutaraldehyde electron microscope special fixing solution for 4 hours. The samples were then washed with phosphate buffer 4 times for 20 minutes each. Samples were dehydrated using 30%, 50%, 70%, 90%, 100% ethanol gradient and the dehydrated samples were immersed in isoamyl acetate for 15 minutes. And finally, drying the sample by using a carbon dioxide critical point dryer, and observing and analyzing by using a cold field emission scanning electron microscope technology.

The results are shown in FIG. 3: (A) Observations were made at a magnification of 10.0k and (B) at a magnification of 20.0 k. As a result, it was found that tetracoccus halophilus was excellent in appearance at magnification of 10.0k (A) and magnification of 20.0k (B), and showed no structural variation.

Example 4: determination of antioxidant Activity of Tetracoccus halophilus

(1) Determination of DPPH radical scavenging Activity

1) Mixing the reaction systems, and reacting the mixture for 30 minutes at room temperature;

2) Absorbance was measured at 517nm and a blank control and a positive control were made using deionized water and ascorbic acid, wherein the reaction system consisted of 0.5ml of the activated SNTH-3 strain (concentrations of 2,4,6,8,10mg/ml, respectively) to be tested, 1ml of 0.25mM DPPH ethanol solution, and 1ml of water;

clearance (%) = (1- (a) _S －A _o )/A _b )×100；

Wherein As is the absorbance of the sample and the reaction solution, ao is the background absorbance of the sample, A _b Is a blank control.

The results show that the clearance rate of the tetracoccus halophilus SNTH-3 reaches 56.03% at the concentration of 10mg/mL by measuring the clearance rate of DPPH free radicals, which shows that the strain has strong capacity of clearing DPPH free radicals. It is shown that it has a strong antioxidant capacity.

(2) Determination of hydroxyl radical scavenging Activity

1) The reaction system was mixed and the mixture was reacted at 25℃for 30 minutes

2) Absorbance was measured at 510nm and deionized water and ascorbic acid were used as blank and positive controls, respectively;

wherein the reaction system consists of 1mL of 9.0mM ferrous sulfate, 1mL of 9.0mM salicylic acid, 1mL of 0.03% H ₂ O ₂ And 1mL of the activated SNTH-3 strain (concentration of 2,4,6,8,10mg/mL, respectively).

Clearance (%) = (1- (a) _S －A _o )/A _b )×100；

The results show that, as shown in FIG. 6, the clearance rate of the tetracoccus halophilus SNTH-3 reaches 54.72% at the concentration of 10mg/mL by measuring the clearance rate of the hydroxyl radical, which shows that the strain SNTH-3 has strong capacity of clearing the hydroxyl radical and strong antioxidant capacity.

Example 5 fermentation condition analysis of highly Producer active peptides of Tetracoccus halophilus SNTH-3

(1) Response surface optimization

The fermentation temperature, the inoculation amount, the NaCl concentration and the pH value are selected as experimental factors, an experimental scheme is designed by using a Box-Behnken response surface analysis method based on single factor experiments, the fermentation temperature, the inoculation amount, the NaCl concentration and the pH value are taken as experimental factors, the polypeptide content is taken as a response value, a regression model is established, the effectiveness of the regression model and the influence degree of each single factor are analyzed, the extraction factors of the high-yield active peptide are optimized, and the optimized technological conditions of the high-yield active peptide extraction are obtained

The four factors of fermentation temperature, inoculation amount, naCl concentration and pH value are selected as independent variables, independent variable value selection is carried out by taking the single factor value of the fermentation temperature, inoculation amount, naCl concentration and pH value as an intermediate value, peptide production amount is selected as a response face value, response face optimization is carried out, and a regression equation of polypeptide production content is obtained:

Y＝29.23+0.56A+0.61B+0.33C-0.49D+0.34AB-0.12AC+0.087AD+0.18BC+0.21BD-0.010CD-3.31A ² -3.00B ² -1.28C ² -1.45D ²

wherein A is fermentation temperature, B is inoculum size, C is NaCl concentration, and D is pH value

Optimizing and obtaining the maximum value of the polypeptide content under the interaction of four factors of fermentation temperature, inoculum size, naCl concentration and pH value and the corresponding fermentation temperature value, inoculum size, naCl concentration value and pH value through software Design-Expert8.0.5.0

(2) Analysis of optimal fermentation temperature

Inoculating the activated tetracoccus halophilus SNTH-3 into a soybean protein matrix culture medium according to an inoculum size of 3%, wherein the NaCl concentration is 4%; fermenting at pH 8.5, and standing at 25deg.C, 29 deg.C, 33 deg.C, 37 deg.C and 41 deg.C. The content of the polypeptide in the bacterial liquid is measured every 8 hours within 0-6d, a blank culture medium at the time of 0h inoculation is used as a control graph, and a peptide production curve is drawn, and the result is shown in figure 7.

As can be seen from FIG. 7, the peptide production cycle of tetracoccus halophilus SNTH-3 was 17h earlier when the fermentation temperature was 37 ℃. When the fermentation is carried out for 64 hours, the content of peptide in the bacterial liquid is the highest and reaches 20.06mg/mL-20.67mg/mL;

(3) Analysis of optimal inoculum size

The activated tetracoccus halophilus SNTH-3 is inoculated into a soybean protein matrix culture medium according to the inoculum concentration of 1%, 2%, 3%, 4% and 5%, the NaCl concentration is 4%, the fermentation pH is controlled at 8.5, and the fermentation is carried out at 37 ℃ in a standing way. The polypeptide content in the bacterial liquid is measured every 8 hours within 0-6 days, the white culture medium at 0 hour is used as a control graph for inoculation, and the peptide production curve is drawn, and the result is shown in figure 8.

As is clear from FIG. 8, the peptide production curve of the tetracoccus halophilus SNTH-3 was highest when the inoculum size was 3%, indicating that the peptide production capacity of the bacterium was the highest. When the strain is fermented for 64 hours, the polypeptide content in the bacterial liquid of the strain reaches the highest value, and the polypeptide content is respectively 20.31mg/mL-20.79mg/mL.

(4) Analysis of optimal NaCl concentration

Inoculating activated tetracoccus halophilus SNTH-3 into soybean protein matrix culture medium according to 3% inoculum size, controlling fermentation pH at 8.5, and standing at 37deg.C under NaCl concentration of 0%, 2%, 4%, 6%, 8%, and 10%, respectively. The polypeptide content in the bacterial liquid is measured every 8 hours within 0-6 days, the white culture medium at 0 hour is used as a control graph for inoculation, and the peptide production curve is drawn, and the result is shown in figure 9.

As can be seen from FIG. 9, the peptide production curve of the tetracoccus halophilus SNTH-3 was highest when the NaCl concentration in the fermentation medium was 4%. The highest polypeptide content in the bacterial liquid reaches 19.06-19.14mg/mL when fermenting for 64 h.

(5) Optimal fermentation pH analysis

The activated tetracoccus halophilus SNTH-3 is inoculated into a soy protein matrix culture medium according to the inoculation amount of 3 percent, the NaCl concentration is 4 percent, the fermentation pH is respectively controlled to be 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5 by 5M NaOH solution, and the fermentation is carried out at 37 ℃ in a standing way. The polypeptide content in the bacterial liquid is measured every 8 hours within 0-6 days, the white culture medium at 0 hour is used as a control graph for inoculation, and the peptide production curve is drawn, and the result is shown in figure 10.

As can be seen from FIG. 10, the peptide production curve of Tetracoccus halophilus SNTH-3 was highest at a fermentation pH of 8.5. The highest polypeptide content in the bacterial liquid reaches 18.95mg/mL-19.04mg/mL during fermentation for 64 h.

Example 6 response surface Experimental optimization

(1) Box-Behnken experiment

According to the design principle of the response surface, 4 single factors which have obvious influence on the polyphenol extraction rate, namely fermentation temperature, inoculum size, naCl concentration and pH value are selected as independent variables, the content of the produced polypeptide is a response value, four-factor three-level experiments are carried out, 29 experiment points are total, and 5 center experiment points are total. Response surface experimental factor levels are shown in table 3, and experimental design and results are shown in table 4.

TABLE 3 Table 3

TABLE 4 Table 4

(2) Regression model establishment and significance test

Performing variance analysis on the data by using response surface optimization software, and fitting the data to obtain regression equations Y=29.23+0.56A+0.61B+0.33C-0.49D+0.34AB-0.12AC+0.087AD+0.18BC+0.21BD-0.010 CD-3.31A of Y (polypeptide content), A (fermentation temperature), B (inoculum size), C (NaCl concentration) and D (pH value) ² -3.00B ² -1.28C ² -1.45D ² Analysis of variance and significance test were performed on the regression equation, and the results are shown in table 3. Determining the coefficient R according to the significance of the model ² Proofreading a certain coefficient R _adj ² And analyzing the accuracy and the credibility of the model by the results of signal-to-noise ratio, the distortion term, the variation coefficient and the like. As can be seen from Table 5, the regression model P<0.001, showing that the regression model results are very significant; the mismatching term P is 0.5447 >0.05, namely the mismatching term is not obvious, and the coefficient R is determined ² 0.9985 > 0.8 illustrates that the model can interpret 99.85% response value change; correction of the determination coefficient R _adj ² 0.997 and R ² The model has good fitting degree, the signal-to-noise ratio is 85.932, and the accuracy of the model is high when the signal-to-noise ratio is larger than a critical value 4; the coefficient of variation CV is 0.45% and less than 10%, which indicates that the experimental result has higher precision and reliability. In conclusion, the regression model has high fitting degree, small error and high reliability, and can be used for optimizing the content of the high-yield bioactive peptide.

Analysis of the P value shows that there is a very significant factor A, B, C, D, AB, A ² 、B ² 、C ² 、D ² The method comprises the steps of carrying out a first treatment on the surface of the There are significant factors AC, BC, BD; there are also insignificant factors AD, CD, from which it is known that the influence on the response value is not only a factor of the primary term but also a factor of the secondary term and the interaction term, indicating that the influence of each factor on the response value is not a linear relationship but a quadratic relationship, and that the four factors influence each other and have interaction. The influence of four factors A, B, C and D on the polypeptide content is B > A > D > C according to the F value

TABLE 5

/>

Note that the signs are very significant and significant.

(3) Interaction analysis

From Table III, the effect of AB interactions on polypeptide content was very pronounced (P < 0.01), and the effect of AD and CD interactions on polypeptide content was not pronounced (P > 0.05), with contour and response plots as shown in FIGS. 11-16. The shape of the contour can determine the strength of the factor interaction, and the closer the shape of the contour is to a circle, the weaker the interaction. From fig. 11 to 16, it can be seen that the interaction between the fermentation temperature and the inoculum size has a significant effect on the polypeptide content, and the interaction between the fermentation temperature and the pH, and the interaction between the pH and the NaCl concentration have no significant effect on the polypeptide content.

The influence degree of the factor level change on the response value can be reflected according to the magnitude of the response surface gradient, and the larger the response surface gradient is, the larger the influence is. The polypeptide content fluctuates with the change of the interaction of the factors, and the curve diagram obtained by optimizing the response surface is shown in fig. 11-16, wherein the curve diagram shows the interaction between the remaining two factors and the influence on the polypeptide content when any two factors in the fixed A, B, C and D are at zero level. The response surface is analyzed from the interaction of each factor, and the contour map of the response surface can intuitively reflect the influence of each factor on the response value so as to find out the optimal technological parameters and the interaction among the parameters, and the center point of the minimum ellipse in the contour line and the highest point of the response surface. From the response surface and contour plot, it can be seen that the interaction of fermentation temperature with inoculum size has a significant effect on polypeptide content.

(4) Response surface optimization results

Optimal process conditions are optimized according to software Design-Expert8.0.5.0, the fermentation temperature is 37.34 ℃, the fermentation pH is 8.5, the optimal inoculation amount is 3%, the polypeptide content can reach 29.2517mg/ml when the optimal NaCl concentration is 4%, the optimal process conditions are determined to be the optimal process conditions for high-yield umami peptide under the conditions that the fermentation temperature is 37 ℃, the fermentation pH is 8.5, the optimal inoculation amount is 3%, and the optimal NaCl concentration is 4%, the optimal culture conditions are 3 times, parallel experiment results show that the high-yield umami peptide content is not greatly different from a theoretical prediction value, the effect of the polypeptide content is well predicted by a regression model, and the optimized process condition parameters are reasonable and are suitable for the culture of the high-yield umami peptide.

(5) Experiment verification

Producing polypeptide under the optimized optimal process conditions:

inoculating activated tetracoccus halophilus SNTH-3 into soybean protein matrix culture medium, controlling the fermentation pH at 8.5 and standing at 37deg.C for fermentation, wherein NaCl concentration is 4% and inoculum size is 3%. And (5) taking (bacterial liquid) every 8 hours within 0-6d, and measuring (polypeptide content).

The fermentation is carried out for 72 hours, the yield of the polypeptide is higher than that of the tetracoccus halophilus SNTH-3 before optimization, and the content of the polypeptide is 29.25mg/mL; the electronic tongue has an umami value of 20.43 and a very good umami effect, and the protease activity value of 342.71 +/-0.48U/mL is obviously higher than that of the SNTH-3 strain which is not optimized. Provides a good research basis for the subsequent deep research of the characteristics of the umami peptide producing strain T.halophilius SNTH-3 in the improved MRS culture medium and the production and application of the strain.

In conclusion, the tetracoccus halophilus SNTH-3 obtained by screening can be widely applied to the production of bioactive peptides, and has the advantages of short production period, high yield, safety, no toxic or side effect and strong oxidation resistance. The strain can also be used for preparing fermented food, is beneficial to improving the flavor of the fermented food, improves the safety of the fermented food, and has wide application prospect. The method is favorable for deep excavation of bioactive peptides in the future, and provides basic data support for further exploring the freshness production mechanism of T.halophilius and the application of T.halophilius in the fields of foods, medicines and the like in the future.

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. The tetracoccus halophilus (Tetragenococcus halophilus) SNTH-3 is characterized in that the preservation number of the tetracoccus halophilus is CGMCC No.27586.

2. Use of a tetracoccus halophilus according to claim 1 for the production of a bioactive peptide.

3. The use according to claim 2, wherein the fermentation temperature of the tetracoccus halophilus is 25-40 ℃, the inoculation amount is 1-5%, the NaCl concentration is 1-10%, and the pH value is 5.5-10.5.

4. The use according to claim 3, wherein the fermentation temperature of the tetracoccus halophilus is 37 ℃, the inoculum size is 3%, the NaCl concentration is 4%, and the fermentation pH is 8.5.

5. Use of a tetracoccus halophilus as claimed in claim 1 in fermented food products.

6. A microbial preparation comprising the tetracoccus halophilus of claim 1.

7. The microbial preparation according to claim 6, wherein the live bacteria amount of tetracoccus halophilus in the microbial preparation is not less than 10 ⁸ CFU/g。

8. Use of the microbial preparation of claim 6 in fermented foods.

9. The use according to claim 5 or claim 9, wherein the fermented food is a soybean paste, soy sauce.