CN113999789B

CN113999789B - Halophilic tetragenococcus for producing umami peptide and application thereof

Info

Publication number: CN113999789B
Application number: CN202111162758.1A
Authority: CN
Inventors: 乌日娜; 武俊瑞; 曹恺欣; 安飞宇; 赵越
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2022-11-15
Anticipated expiration: 2041-09-30
Also published as: CN113999789A

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides Tetragenococcus halophilus and application thereof. The halophilic tetragenococcus sieve is selected from bean paste which is traditionally fermented naturally in northeast China, is preserved to the common microorganism center of China Committee for culture Collection of microorganisms at 20 days of 2021 and 20 months, is preserved in China institute of microbiology, no. 3 of China academy of sciences, north Chen West Lu No. 1 of the Chao Yangyuan in Beijing, and has the preservation number of CGMCC No.23165. The strain can greatly improve the yield of the delicious peptide, is beneficial to reducing the production cost of the delicious peptide and promotes the wide application of the delicious peptide in the field of food.

Description

Tetragenococcus halophilus for producing umami peptide and application thereof

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to a halophilic tetragenococcus for producing delicious peptides and application thereof.

Background

The umami peptide as an umami substance proposed in recent years has good umami effect, processing property and nutritional value, becomes a research focus of food umami science and an important direction of development of an umami agent in recent years, and attracts people's attention. The umami peptide is a small molecular peptide with umami characteristics extracted from food or synthesized by amino acid, can supplement and enhance the overall taste of the food, and makes the food more harmonious, soft and rich, and the molecular mass of the umami peptide is usually 150 to 3000 Da. The taste of umami peptides is mainly derived from intermediates in the protein synthesis and decomposition processes. Compared with other peptides, the umami peptide molecules usually contain glutamic acid residues or aspartic acid residues, the molecules also interact with umami receptors on human taste buds to present certain umami characteristics, and can further supplement or enhance the overall taste of food through synergistic action or Maillard reaction on the basis of not influencing other tastes of the food such as sourness, sweetness, bitterness or saltiness, and the like, so that the flavor of the food is more harmonious, soft and rich. The umami peptide can also generate synergistic effect with other umami substances through three modes of peptide-peptide, peptide-nucleotide and peptide-cation, thereby achieving the aim of increasing the umami. In addition, the umami peptide can cover and weaken bitter taste, improve the flavor of food, and simultaneously has good processing characteristics and thermal stability.

The umami peptide is widely existed in various foods, and has various common preparation and processing methods, such as an extraction method, a hydrolysis method, a synthesis method, a microbial fermentation method and the like, the properties, the purity, the functions and the like of the umami peptide obtained by different processing methods are different, the selection of the preparation method is particularly important, and different preparation methods also have respective characteristics. In recent years, microbial fermentation has received much attention. The microbial fermentation method is to decompose raw material protein or synthesize amino acid under the action of certain microbes or microbial enzymes, so as to convert the raw materials into flavor small peptide substances, and then separate and extract the flavor small peptide substances to achieve the purpose of preparing the fresh peptide. Doukanning utilizes saccharomycetes and lactic acid bacteria secondary fermentation method to improve the flavor of soybean peptide, and finds that after fermentation, some fishy smell and bitter taste of the soybean peptide are converted into sour and refreshing killing taste, and meanwhile, the soybean peptide has certain wine aroma, and the flavor of the soybean peptide is obviously improved. The preparation of the polypeptide by microbial fermentation has the advantages of low cost, high safety, stable quality, obvious function and the like, and belongs to a one-step process for preparing the polypeptide. And the probiotics resources are rich in China, the flavor, nutrition, functions and the like of the peptides produced by different strains are greatly different, and the microbial fermentation method has great potential no matter scientific research or industrial production.

Currently, few researches on the production of the umami peptide by fermentation of various metabolic pathways of microorganisms exist, the used production strains have low yield and high cost, the industrial large-scale production requirements cannot be met, and people continue to explore the fermentation strains and the umami peptide. Tetragenococcus halophilus (Tetragenococcus halophilus) is a gram-positive bacterium, is a halophilic or hypertonic lactic acid bacterium, and has an important influence on the generation of umami active amino acids and taste-developing peptides in fermented foods. In the preparation process of the Japanese fish paste, the tetracoccus is used as an initial fermentation strain, so that the flavor substances of the fish paste are increased and the content of free amino acids is increased. Zhang Ling discovered that the addition of Tetragenococcus halophilus to fermented soy sauce affected the production of flavor contributing compounds such as glutamic acid, aspartic acid, alanine, and umami dipeptide (Suc-Glu). A large number of researches find that the tetragenococcus halophilus has the functions of improving the flavor of the fermented food and improving the quality of the fermented food.

Therefore, the method for screening the fresh-producing strain from the fresh-taste fermented food and optimizing the fermentation conditions of the fresh-taste fermented food is always a research hotspot, has important practical significance and commercial application prospect, and has potential application value in the industrial application of the biological source fresh-taste peptide, the development of the seasoning industry and the improvement of the flavor and the safety of the fermented food.

Disclosure of Invention

The invention provides Tetragenococcus halophilus and application thereof to solve the problems of the prior art. The halophilic tetragenococcus sieve is selected from bean paste which is naturally fermented in northeast China, so that the yield of the delicious peptide can be greatly improved, the production cost of the delicious peptide can be reduced, and the wide application of the delicious peptide in the field of foods can be promoted.

The invention provides a Tetragenococcus halophilus SNTH-1 strain which is preserved in 20 days in 2021 at 08 months and is preserved in China general microbiological culture Collection center at the microbial research institute of China academy of sciences No. 3 of the North Chen Xilu No. 1 of the area facing the sun in Beijing, wherein the preservation number is CGMCC No.23165.

The 16s rDNA sequence of the quadruple halophilus SNTH-1 is SEQ ID NO 1.

The invention provides an application of Tetragenococcus halophilus SNTH-1 in production of delicious peptide.

The invention provides an umami peptide which is produced by taking tetragenococcus halophilus SNTH-1 as a fermentation strain.

The invention also provides a microbial preparation which comprises the tetragenococcus halophilus SNTH-1.

The viable bacteria amount of tetragenococcus halophilus SNTH-1 in the microbial preparation is not less than 10 ⁶ CFU/g。

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

Has the advantages that:

the tetragenococcus halophilus SNTH-1 provided by the invention can produce the umami polypeptide, after being fermented for 42 hours in a 50L fermentation tank, the content of the umami polypeptide in the fermented supernatant reaches 25.35mg/mL-26.62mg/mL, the umami value measured by an electronic tongue reaches 16.22, and the umami effect is very good. The strain can also produce protease, amylase and gamma-glutamine transpeptidase, the protease activity in the fermentation supernatant is 85.42U/mL-85.48U/mL, and the amylase activity is 57.76 +/-0.04U/mL; the activity of gamma-glutamine transpeptidase in cell lysate is 6.44 plus or minus 0.01U/mL. The tetragenococcus halophilus SNTH-1 can be widely applied to the production of the delicious peptide and has the advantages of short production period, high yield, safety and no toxic or side effect. The strain can also be used for preparing fermented food, is beneficial to improving the flavor of the fermented food and improving the safety of the fermented food, and has wide application prospect.

Drawings

FIG. 1 is a colony morphology of Tetragenococcus halophilus SNTH-1;

FIG. 2 is an individual morphology of SNTH-1 cells of Tetragenococcus halophilus;

FIG. 3 is a phylogenetic tree of S.halophilus SNTH-1 based on the 16S rDNA gene sequence;

FIG. 4 is a graph showing the effect of temperature on the production and growth of peptide from Tetragenococcus halophilus SNTH-1, (A) a peptide production curve, and (B) a growth curve;

FIG. 5 shows the effect of inoculum size on the production and growth of peptide from Tetragenococcus halophilus SNTH-1: (a) a peptide production curve, (B) a growth curve;

FIG. 6 shows the effect of salt concentration on the production and growth of peptide from Tetragenococcus halophilus SNTH-1: (a) a peptide production curve, (B) a growth curve;

FIG. 7 is a graph showing the effect of pH on the production and growth of peptide from Tetragenococcus halophilus SNTH-1: peptide production curve (A) and growth curve (B).

Detailed Description

The screening method of the present invention is not limited to the examples, and any known method capable of achieving the screening purpose is possible, and the screening description of the examples is only illustrative of the present invention and is not limiting the scope of the present invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention.

The culture medium used in the embodiment of the invention and the formula thereof are as follows:

MRS medium (g/L): 10g of peptone, 3g of sodium acetate (anhydrous), 2g of dipotassium 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 and 1g of Tween 80.

Enrichment medium (g/L): 150g of sodium chloride, 2g of natamycin and 500 mu L of crystal violet are added into an MRS culture medium, and the pH value is adjusted to be 7.

Solid isolation medium (g/L): 150g of sodium chloride, 2g of natamycin, 10g of calcium carbonate and 20g of agar are added into an MRS culture medium, and the pH value is adjusted to be 7.

Soy protein substrate medium (g/L): 20g of soybean peptone, 3g of sodium acetate (anhydrous), 2g of dipotassium phosphate, 0.575g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of glucose, 2.42g of trisodium citrate, 80 g of tween and 100g of sodium chloride, and the pH value is adjusted to be 7.

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

EXAMPLE 1 isolation and screening of the strains

1. Screening samples

The bean paste is naturally fermented in the village of Pingzhou in the town of the province of the Liaoyang city for half a year.

2. Primary screening for tetracoccus

Taking 1g of a soybean paste sample, adding 9mL of 0.9% sterile normal saline, fully and uniformly mixing, and soaking for 15min; then 1mL of sample liquid is inoculated in an enrichment medium, and after static culture for 1-2d at 37 ℃, the sample liquid is cultured on a solid separation medium for 5-6d by adopting a plate coating method.

Selecting single bacterial colony which is small in bacterial colony, has obvious calcium dissolving ring, is milk white and opaque and has the shape similar to that of the lactobacillus bacterial colony, and performing gram staining. The microscopic observation shows that 12 strains of the thallus in the shape of a pair or a tetragon are named as LY9-1, LY9-2, 8230, LY9-12 respectively.

And respectively carrying out purification culture on the single colonies of the 12 tetragenococcus strains for later use.

3. Rescreening of fresh Tetragenococcus

(1) Fermentation culture of strain

The 12 tetrads obtained from the primary screening were inoculated into MRS medium and cultured at 37 ℃ for 24 hours. After the culture is finished, the viable bacteria amount in the fermentation liquor reaches 1.0 multiplied by 10 ⁶ CFU/mL。

The fermentation broth was centrifuged at 8000rpm at 4 ℃ for 20min, and the supernatant was taken.

(2) Determination of the polypeptide-producing ability:

1mL of diluted fermentation supernatant was added to 4mL of biuret reagent. The mixture was shaken well and left at room temperature (24 ℃ -26 ℃) for 30min, and then the absorbance value was measured at 540 nm. 0, 2, 4, 8, 10, 12, 16 and 20mg/mL Gly-Gly-Tyr-Arg tetrapeptide standard solutions were set as standard curves with blank medium as blank control. The assays were performed in triplicate and the results are reported as milligrams of peptide per ml of fermentation supernatant. The calculation formula is as follows:

polypeptide content (mg/mL) = N (a-0.0006)/0.0141.

Wherein A is the absorbance value of the sample; n is the dilution factor of the sample; 0.0006 is the intercept of the standard curve; 0.0141 Is the slope of the standard curve.

The results show that the polypeptide yield of 12 tetrads obtained by the primary screening of the invention is the highest LY9-1 strain, and the polypeptide content in the fermentation supernatant is up to 17.68mg/mL.

(3) Electronic tongue (SA 402B electronic tongue) measurement:

the electronic tongue is equipped with 5 test sensors and 2 reference sensors, wherein the AAE, CT0, CA0, AE1 and C00 test sensors are used for testing umami, salty, sour, astringent and bitter taste respectively.

1) Sensor activation

Activation of the test sensor: and (3) after the Ag/AgCl electrode is taken out from the test sensor, the sensor is reassembled after the internal solution is added, and the sensor is placed into the reference solution for activation for 24 hours for later use. Activation of the reference sensor: after removing the electrode from the reference sensor, the internal solution was added, the sensor was reassembled and placed in 3.33M KCl solution for activation for 24h for future use.

2) Determination of samples

After the self-checking of the electronic tongue system is completed, uniformly pouring a sample into two special beakers for the electronic tongue, soaking 6 sensors such as AAE, CT0, CA0, AE1 and C00 in an anion or cation solution to clean the sensors, then sequentially cleaning the sensors in

reference solutions

1 and 2, then cleaning the sensors in a reference solution 3 to obtain a reference solution potential Vr, then soaking the sensors in a sample cup to obtain a sample potential Vs, and evaluating basic values such as delicate flavor, sour flavor, salty flavor, bitter flavor, astringent flavor and the like through a potential difference of Vs-Vr; after the

reference solutions

4 and 5 are respectively washed, the solution 6 is soaked for testing aftertaste, the potential Vr is detected, and the aftertaste of the delicate flavor, the bitter flavor or the astringent flavor of the sample can be detected through the potential difference of the Vr' -Vr. Each sample is repeatedly detected for 4 times, and in order to reduce system errors, the components of reference solutions 1-6 are completely the same in the data of 3 times of measurement after the original data are selected.

The results show that: the LY9-1 strain in 12 strains of tetragenococcus obtained by primary screening has the best umami effect, and the umami value of fermentation supernatant reaches 15.06.

EXAMPLE 2 identification of LY9-1 Strain

1. Colony morphology identification

The LY9-1 strain is small and convex in colony, is milky and opaque, and has a smooth and glossy surface and a flat edge; no motility and no generation of spores; gram staining is positive, cells are spherical, tetrad or paired. The colony morphology and cell morphology are shown in FIGS. 1 and 2.

2. Molecular biological identification

(1) And (3) extracting bacterial DNA:

and sucking 100 mu L of the bacterial suspension of the LY9-1 strain into the sterilized MRS liquid culture medium, and culturing for 24h in a shaking incubator at 37 ℃. The genome of this strain was extracted according to the procedure on the bacterial genome kit (Solarbio D1600).

(2) And (3) PCR amplification:

PCR forward primer 27F and reverse primer 1492R were designed and synthesized by Shanghai bioengineering, inc.

27F：AGAGTTTGATCCTGGCTCAG(5'---3')；

1492R：GGTTACCTTGTTACGACTT(5'---3')。

The PCR reaction conditions were: preheating at 95 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min for 20s, and circulating for 36 times; keeping the temperature at 72 ℃ for 8min, and keeping the temperature at 4 ℃.

(3) And (3) recovering a PCR product:

the PCR product was recovered using AxyPrep DNA gel recovery kit (Boyao, ASJ 0013), and the detailed procedures were performed according to the kit instructions.

(4) 16S rDNA sequencing and sequence alignment

The positive PCR product was sequenced by supheipai Senno Biotech GmbH to obtain the 16s rDNA sequence of LY9-1 strain SEQ ID NO:1 as follows:

GGCGGGGGCGCTATAATGCAGTCGACGCTGCTTAAGAAGAACTTCGGTTTTTTCTTAAGCGGAGTGGCGGACG GGTGAGTAACACGTGGGGAACCTATCCATCAGCGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATATGGCTTT TTTTCACCTGAAAGAAAGCTCAAAGGCGCTTTACAGCGTCACTGATGGCTGGTCCCGCGGTGCATTAGCCAGTTGGTG AGGTAACGGCTCACCAAAGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGC CCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGCAAGTCTGACCGAGCAACGCCGCGTGAGT GAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTCAGCAAAGAACAGGAGAAAGAGGAAATGCTTTTTCTATGACGGTA GCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATT GGGCGTAAAGCGAGCGCAGGCGGTGATTTAAGTCTGATGTGAAAGCCCCCAGCTCAACTGGGGAGGGTCATTGGAA ACTGGATCACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACAC CAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGTAGCAAACAGGATTAGATACC CTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGTTAACGCATTAA GCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCGCCCTAGAGATAGGGTTTCCCC TTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGA GCGCAACCCTTATTGTTAGTTGCCAGCATTGAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGC GGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGCAAGCC AAGCCGCAAGGCCTAGCGAATCTCTGAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGG AATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATCCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGA GAGTTTGTAACACCCAAAGTCGGTGCGGCAACCCTAGGGAGCCAGCCGCCTAAGTGATCAATTA。

the alignment of SEQ ID NO:1 in the NCBI database using the BLAST tool with the existing sequences in the GenBank database showed that the similarity between SEQ ID NO:1 and Tetragenococcus halophilus (Tetragenococcus halophilus) was the highest.

Further, a phylogenetic tree was constructed using MEGA 7.0. As a result, as shown in FIG. 3, the LY9-1 strain according to the present invention has the highest homology with Tetragenococcus halophilus.

In conclusion, the colony morphology, the physiological and biochemical characteristics and the molecular biological identification result of the LY9-1 strain are combined, and the LY9-1 strain is a Tetragenococcus halophilus and is named as Tetragenococcus halophilus SNTH-1.

The applicant has already deposited the Tetragenococcus halophilus SNTH-1 (Tetragenococcus halophilus SNTH-1) in 20.08.2021 in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, address: no. 3 of West Lu 1 of Beijing, institute of microbiology, japan, postal code 100101) with the preservation number of CGMCC No.23165.

EXAMPLE 3 determination of the enzyme-producing ability of Tetragenococcus halophilus SNTH-1

1. And (3) protein enzyme activity determination:

activating the tetragenococcus halophilus SNTH-1 strain, inoculating the tetragenococcus halophilus SNTH-1 strain into an MRS liquid culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃.

Preparing a protease screening culture medium (10 g of casein, 3g of yeast powder and 5g of Na) ₂ HPO ₄ ·12H ₂ O,50g of NaCl, 0.05g of bromothymol blue, 20g of agar and 1L of distilled water, and adjusting the pH value to 7), and measuring the protease producing capacity of the strain by adopting an Oxford cup method. Putting the sterilized Oxford cup on a protease screening culture medium, sucking 10 mu of a Tetragenococcus halophilus SNTH-1 bacterial solution, adding the solution into the Oxford cup, culturing in a constant-temperature incubator at 37 ℃ for 24 days, and observing whether a transparent ring is generated.

The casein hydrolysis circle diameter (D) and the colony diameter (D) were measured with a vernier caliper, and the enzyme-producing ability of the strain was judged from the ratio of the area of the transparent circle to the area of the colony. Further, the enzyme activity of protease is determined according to a Folin phenol method, the protease in the fermentation supernatant can hydrolyze a casein substrate under certain temperature and pH conditions to generate amino acid containing phenolic groups, the Folin phenol reagent can be reduced under alkaline conditions to generate molybdenum blue and tungsten blue, and the enzyme activity is calculated by spectrophotometry: the amount of enzyme that hydrolyzes casein per minute to produce 1. Mu.g of tyrosine in 1mL of enzyme solution was 1 enzyme activity unit, expressed in U/mL.

The results show that: the tetragenococcus halophilus SNTH-1 provided by the invention can produce protease, and the protease activity in the fermentation supernatant is 85.42U/mL-85.48U/mL.

2. And (3) measuring the content of the produced amylase:

the halophilic tetragenococcus SNTH-1 strain was streaked and inoculated into a amylase production screening medium (20 g of soluble starch, 20g of peptone, 5g of Na) ₂ HPO ₄ ，0.1g NaCl，0.1g MgSO ₄ 20g of agar and 1L of distilled water, pH 7) and incubated at 37 ℃ for 24 hours.

And observing the growth condition of the strain by adopting a flat transparent ring method. Iodine solution is dripped into the culture medium, and transparent circles appear around colonies to prove that the strain produces amylase and has the ability of decomposing starch. And observing after color development, measuring the diameter (D) of the starch hydrolysis ring and the diameter (D) of a bacterial colony by a vernier caliper, and judging the enzyme production capacity of the strain according to the area ratio of the two.

The results show that: the tetragenococcus halophilus SNTH-1 provided by the invention can produce amylase, and the amylase activity in the fermentation supernatant is 57.76 +/-0.04U/mL.

3. And (3) determining the activity of the produced gamma-glutamine transpeptidase:

the tetragenococcus halophilus SNTH-1 strain is inoculated in an MRS culture medium (g/L), the pH value is adjusted to 7, and the strain is cultured for 24 hours at the temperature of 37 ℃.

Taking 1mL of a tetragenococcus halophilus SNTH-1 bacterial liquid into a centrifugal tube, centrifuging for 1min at 10000rpm under the condition of 4 ℃, and then discarding a supernatant; according to the number of bacteria (10) ⁴ A plurality of): adding the extracting solution according to the volume (mL) of the extracting solution of 500-1000, ultrasonically crushing bacteria (ice bath, power of 20% or 200W, ultrasonic for 3s, interval of 10s, repetition for 30 times), centrifuging for 10min at 10000rpm under the condition of 4 ℃, taking supernatant, and placing on ice for testing. The measurement was carried out using a gamma-transglutaminase activity detection kit (G-CLONE), and the gamma-transglutaminase activity was measured spectrophotometrically, and the production of 1. Mu. Mol of p-nitroaniline was defined as one unit of enzyme activity per 1 ten thousand bacteria per minute by measuring the increase rate of light absorption at 405nm, at 25 ℃ or 37 ℃, in terms of the number of bacteria.

The results show that: the activity of gamma-glutamine transpeptidase in the lysis solution of the tetragenococcus halophilus SNTH-1 cell provided by the invention is 6.44 +/-0.01U/mL.

Example 4 analysis of fermentation conditions for Tetragenococcus halophilus SNTH-1

1. Analysis of optimum fermentation temperature

Inoculating the activated tetragenococcus halophilus SNTH-1 into a soy protein matrix culture medium according to the inoculation amount of 3 percent, wherein the concentration of NaCl is 4 percent; the fermentation pH is controlled at 8.0, and the fermentation is carried out by standing at 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C and 40 deg.C respectively. The content of polypeptide in the bacterial liquid and the OD value under 600nm are measured every 8h within 0-6d, the blank culture medium at the time of inoculation for 0h is used as a control for drawing, and a peptide production curve and a growth curve are drawn, and the result is shown in figure 4.

As can be seen from FIG. 4, when the fermentation temperature was 37 ℃, the peptide-producing cycle and growth cycle of Tetragenococcus halophilus SNTH-1 were advanced by 16h. When the fermentation is carried out for 56 hours, the peptide content in the bacterial liquid is the highest and reaches 18.31mg/mL-19.73mg/mL; when the fermentation is carried out for 64 hours, the OD600 of the bacterial liquid is highest and reaches 1.400-1.406.

2. Analysis of optimum inoculum size

Inoculating the activated tetragenococcus halophilus SNTH-1 into a soy protein matrix culture medium according to the inoculation amounts of 1%, 2%, 3%, 4% and 5%, wherein the NaCl concentration is 4%, the fermentation pH is controlled at 8.0, and standing fermentation is carried out at 37 ℃. The polypeptide content in the bacterial liquid and the OD value under 600nm are measured every 8h within 0-6d, the white culture medium is used as a contrast drawing when the bacterial liquid is inoculated for 0h, the peptide production curve and the growth curve are drawn, and the result is shown in figure 5.

As can be seen from FIG. 5, when the inoculation amount was 3%, the peptide production curve and the growth curve of tetragenococcus halophilus SNTH-1 were the highest, indicating that the peptide production ability and the growth ability of the bacterium were the strongest. When the strain is fermented for 56 hours, the polypeptide content and OD600 in the bacterial liquid of the strain reach the highest values, namely 19.24mg/mL-19.32mg/mL and 1.436-1.438 respectively.

3. Optimum NaCl concentration analysis

Inoculating the activated tetragenococcus halophilus SNTH-1 into a soybean protein matrix culture medium according to the inoculation amount of 3%, controlling the fermentation pH to be 8.0, and standing and fermenting at 37 ℃ under the conditions that the NaCl concentration is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 13%, 15%, 17% and 20%, respectively. The peptide content in the bacterial liquid and the OD value under 600nm are measured every 8h within 0-6d, and the peptide production curve and the growth curve are drawn by taking a blank culture medium at 0h as a control drawing, and the result is shown in figure 6.

As can be seen from FIG. 6, the peptide production curve and the growth curve of Tetragenococcus halophilus SNTH-1 were highest at a NaCl concentration of 4% in the fermentation medium. The highest polypeptide content in the bacterial liquid reaches 21.75mg/mL-21.97mg/mL when the fermentation is carried out for 56 hours.

4. pH analysis of optimum fermentation

Inoculating the activated tetragenococcus halophilus SNTH-1 into a soybean protein matrix culture medium according to the inoculation amount of 3 percent, controlling the fermentation pH to be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 10.5 by 5M NaOH solution, and standing and fermenting at 37 ℃. The polypeptide content in the bacterial liquid and the OD value under 600nm are measured every 8h within 0-6d, the white culture medium is used as a contrast drawing when the bacterial liquid is inoculated for 0h, the peptide production curve and the growth curve are drawn, and the result is shown in figure 7.

As can be seen from FIG. 7, the peptide production curve and the growth curve of S.halophilus SNTH-1 were highest at a fermentation pH of 8.0. The polypeptide content in the bacterial liquid is the highest when the fermentation is carried out for 48 hours, and the polypeptide content reaches 19.63mg/mL-20.85mg/mL.

Example 5 use of Tetragenococcus halophilus SNTH-1 in the fermentative production of umami polypeptide

1. Strain activation:

taking 1 part of a preserved Tetragenococcus halophilus SNTH-1 (Tetragenococcus halophilus SNTH-1) strain from an ultralow temperature refrigerator at minus 80 ℃, inoculating the Tetragenococcus halophilus SNTH-1 strain into 50mL of MRS culture medium according to the inoculation amount of 1% (v/v) after ice melting, adjusting the pH value to be 8.3, carrying out shake flask culture for 40h at 37 ℃, and taking 0.5mL of fermentation broth to repeatedly inoculate into 50mL of MRS culture medium when the strain grows to the end of logarithmic phase growth to obtain an activated Tetragenococcus halophilus SNTH-1 strain;

2. seed liquid culture:

inoculating the activated bacterial liquid into a 5L fermentation tank, wherein 3L of enrichment medium is contained in the fermentation tank, and the fermentation tank is controlled at the temperature of 37 ℃, the rotating speed of 200rpm, the air flow ratio of 1.5 and the tank pressure of 0.1kg/cm ² Culturing for 24h under the condition of (1) to obtain a seed solution;

3. culturing in a fermentation tank:

the vigorously grown seed solution was transferred to a 50L fermentor containing 30L of soy protein substrate medium at 37 deg.C, 200rpm, air flow ratio of 1 ² Fermenting for 42 hours under the condition of (1) to obtain fermentation liquor of tetragenococcus halophilus SNTH-1.

The fermentation broth was centrifuged at 8000rpm at 4 ℃ for 20min, and the supernatant was taken. Polypeptide content determination and electronic tongue umami taste determination were performed on the supernatant from SNTH-1 fermentation of Tetragenococcus halophilus, respectively, according to the method described in example 1,

the results show that: the content of the umami polypeptide in the SNTH-1 fermentation supernatant of the tetragenococcus halophilus provided by the invention is up to 25.35mg/mL-26.62mg/mL; the electronic tongue has the fresh taste value as high as 16.22, has very good fresh taste effect and obtains unexpected technical effect.

In conclusion, the tetragenococcus halophilus SNTH-1 screened by the invention can be widely applied to the production of the delicious peptide and has the advantages of short production cycle, high yield, safety and no toxic or side effect. The strain can also be used for preparing fermented food, is beneficial to improving the flavor of the fermented food and improving the safety of the fermented food, and has wide application prospect.

Sequence listing

<110> Shenyang university of agriculture

<120> novel high-yield delicious peptide halophilic tetragenococcus and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1453

<212> DNA

<213> Tetragenococcus halophilius)

<400> 1

ggcgggggcg ctataatgca gtcgacgctg cttaagaaga acttcggttt tttcttaagc 60

ggagtggcgg acgggtgagt aacacgtggg gaacctatcc atcagcgggg gataacactt 120

ggaaacaggt gctaataccg catatggctt tttttcacct gaaagaaagc tcaaaggcgc 180

tttacagcgt cactgatggc tggtcccgcg gtgcattagc cagttggtga ggtaacggct 240

caccaaagca acgatgcata gccgacctga gagggtgatc ggccacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt cggcaatgga cgcaagtctg 360

accgagcaac gccgcgtgag tgaagaaggt tttcggatcg taaagctctg ttgtcagcaa 420

agaacaggag aaagaggaaa tgctttttct atgacggtag ctgaccagaa agccacggct 480

aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttattggg 540

cgtaaagcga gcgcaggcgg tgatttaagt ctgatgtgaa agcccccagc tcaactgggg 600

agggtcattg gaaactggat cacttgagtg cagaagagga gagtggaatt ccatgtgtag 660

cggtgaaatg cgtagatata tggaggaaca ccagtggcga aggcggctct ctggtctgta 720

actgacgctg aggctcgaaa gcgtgggtag caaacaggat tagataccct ggtagtccac 780

gccgtaaacg atgagtgcta agtgttggag ggtttccgcc cttcagtgct gcagttaacg 840

cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgacggg 900

ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960

gtcttgacat cctttgaccg ccctagagat agggtttccc cttcgggggc aaagtgacag 1020

gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgtaacgagc 1080

gcaaccctta ttgttagttg ccagcattga gttgggcact ctagcaagac tgccggtgac 1140

aaaccggagg aaggcgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200

acgtgctaca atgggaagta caacgagcaa gccaagccgc aaggcctagc gaatctctga 1260

aagcttctct cagttcggat tgcaggctgc aactcgcctg catgaagccg gaatcgctag 1320

taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccaaagtc ggtgcggcaa ccctagggag ccagccgcct 1440

aagtgatcaa tta 1453

Claims

1. A Tetragenococcus halophilus strain is characterized in that the preservation number of the Tetragenococcus halophilus is CGMCC No.23165.

2. Use of tetragenococcus halophilus according to claim 1 for the production of umami peptides.

3. A microbial preparation comprising the tetragenococcus halophilus of claim 1.

4. The microbial preparation of claim 3, wherein the viable count of Tetragenococcus halophilus in the microbial preparation is not less than 10 ⁶ CFU/g。

5. Use of the microbial preparation of claim 3 or 4 in fermented food products.