CN114350557B

CN114350557B - Lactobacillus fermentum and application thereof

Info

Publication number: CN114350557B
Application number: CN202111674926.5A
Authority: CN
Inventors: 李璐; 徐玉娟; 罗文珊; 余元善; 彭健; 邹波
Original assignee: Sericulture and Agri Food Research Institute GAAS
Current assignee: Sericulture and Agri Food Research Institute GAAS
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-11-07
Anticipated expiration: 2041-12-31
Also published as: CN114350557A

Abstract

The invention discloses lactobacillus fermentum and application thereof, and relates to the technical field of microorganisms. The lactobacillus fermentum is named lactobacillus fermentum (Lactobacillus fermentum) G9, and the preservation unit is: the collection date of the microorganism strain collection in Guangdong province: 2021, 11/2, deposit number: GDMCC No. 62025. The lactobacillus fermentum G9 can degrade nitrite and biogenic amine, has better acid resistance and salt resistance, can effectively reduce accumulation of nitrite and biogenic amine in the pickle fermentation process, obviously improves food safety of pickle, and has good application prospect as pickle starter.

Description

Lactobacillus fermentum and application thereof

Technical Field

The invention relates to the field of microbial fermentation, in particular to lactobacillus fermentum and application thereof.

Background

The pickle, also called pickled vegetable and fermented vegetable, is a typical traditional fermented food for Chinese nationality, and is usually prepared by taking fresh vegetables such as green vegetables, radishes, ginger, lettuce, garlic and the like as raw materials and fermenting the fresh vegetables by microorganisms such as lactobacillus and the like. After the fresh vegetables are fermented, not only the nutrients such as vitamins, amino acids, dietary fibers and the like of the fresh vegetables can be reserved, but also rich organic acids, short peptides and certain functional small molecules can be produced. However, most of pickle is naturally fermented, the fermentation process is uncontrollable, and harmful metabolites such as nitrite, biogenic amine and the like are easy to produce, so that the food safety problem is caused.

Nitrite is a generic name of nitrogen-containing inorganic compounds, widely exists in fermented vegetables, and is an important safety index of vegetable pickled products. Excessive nitrite intake in the human body can lead to poisoning, induce paralysis of the respiratory center and methemoglobin, and seriously lead to choking and death. During fermentation, various nitrate-reducing bacteria (such as bacillus subtilis and the like) reduce nitrate of vegetable raw materials into nitrite by secreting nitrate reductase, so that nitrite is generated during fermentation of the vegetables. Biogenic amines are a generic term for a class of non-volatile low molecular weight nitrogen-containing organic compounds that are prevalent in fermented foods. Proper amount of biogenic amine can regulate various normal physiological functions in human body, but excessive biogenic amine can cause adverse reaction (such as vasodilation, migraine, abnormal blood pressure, diarrhea, emesis and the like) of human body, and even damage to liver, heart, central nervous system and the like. During the fermentation process, the vegetable protein is decomposed into amino acids, and the amino acid decarboxylase produced by the microorganism decarboxylates the amino acids to biogenic amines, so that a large amount of biogenic amines are accumulated in the kimchi. In addition, nitrite and biogenic amine can react to generate strong cancerogenic substance-nitrosamine, so the problem of nitrite and biogenic amine in fermented pickle is needed to be solved.

The existing control methods for controlling nitrite and biogenic amine in food include a physical method, a chemical method and a microbial method, wherein the physical method cannot eliminate generated nitrite and biogenic amine, the physical method has limitation, the chemical method has obvious influence on the flavor of pickled vegetables, and the microbial method is considered to be the best method for controlling nitrite and biogenic amine in fermented food, but the reported article is mainly to screen strains for controlling the content of nitrite or biogenic amine, and the screening of strains capable of degrading nitrite and biogenic amine simultaneously is very difficult.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the lactobacillus fermentum provided by the invention can degrade nitrite and biogenic amine, effectively reduce accumulation of nitrite and biogenic amine in the pickle fermentation process, remarkably improve food safety of pickle, and has good application prospect as pickle starter.

In a first aspect of the invention, there is provided a strain of lactobacillus fermentum designated lactobacillus fermentum (Lactobacillus fermentum) G9, deposit unit: the Guangdong province microorganism strain collection center (GDMCC), deposit address: guangzhou city first middle road 100 # college 59 # building 5, date of preservation: 2021, 11/2, deposit number: GDMCC No. 62025.

In some embodiments of the invention, lactobacillus fermentum G9 is isolated from kimchi.

In a second aspect of the present invention, there is provided a microbial agent comprising lactobacillus fermentum G9 according to the first aspect of the present invention.

In some embodiments of the invention, the microbial agent comprises live cells of lactobacillus fermentum G9 cells, freeze-dried dry microbial cells, immobilized cells, liquid microbial agents, solid microbial agents, or lactobacillus fermentum G9 strain in any other form.

In some embodiments of the invention, the microbial agent further comprises other bacteria or fungi.

In some embodiments of the invention, the active ingredient of the microbial inoculum is lactobacillus fermentum G9.

In some embodiments of the invention, the microbial agent may further include at least one of a carrier, a surfactant, a stabilizer, and a pH adjuster.

In some embodiments of the invention, the dosage form of the microbial agent can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

In a third aspect of the invention there is provided the use of lactobacillus fermentum G9 according to the first aspect of the invention or a microbial agent according to the second aspect of the invention for degrading nitrite and/or biogenic amine.

In some embodiments of the invention, the nitrite comprises sodium nitrite; the biogenic amine comprises at least one of tryptamine, beta-phenethylamine, tyramine, histamine, putrescine, cadaverine, spermine and spermidine.

In a fourth aspect of the invention there is provided the use of lactobacillus fermentum G9 according to the first aspect of the invention or a microbial inoculum according to the second aspect of the invention in the manufacture of a fermented product.

In some embodiments of the invention, the fermented product is a food product, including specifically fermented milk, kimchi, fermented soya beans and vinegar.

In some embodiments of the invention, the fermentation product is kimchi.

In some embodiments of the invention, the lactobacillus fermentum G9 of the first aspect of the invention or the microbial inoculum of the second aspect of the invention is inoculated onto a feedstock for fermentation.

In a fifth aspect of the present invention, there is provided a fermentation product obtained by inoculating lactobacillus fermentum G9 of the first aspect of the present invention or the microbial inoculum of the second aspect of the present invention on a feedstock for fermentation.

In some embodiments of the invention, the fermentation product is kimchi.

In some embodiments of the invention, the lactobacillus fermentum G9 of the first aspect of the invention or the microbial inoculum of the second aspect of the invention has a viable microbial inoculum size on a feedstock of 10 ⁶ -10 ⁸ CFU/mL。

In some embodiments of the invention, the viable bacterial inoculum size of the lactobacillus fermentum G9 of the first aspect of the invention or the bacterial inoculum of the second aspect of the invention is 10 ⁷ CFU/mL。

In a sixth aspect of the invention, there is provided a method for degrading nitrite and/or biogenic amine, comprising inoculating lactobacillus fermentum G9 according to the first aspect of the invention or the microbial inoculum according to the second aspect of the invention into a feedstock for fermentation.

In some embodiments of the invention, the nitrite comprises sodium nitrite.

In some embodiments of the invention, the biogenic amine comprises at least one of tryptamine, beta-phenylethylamine, tyramine, histamine, putrescine, cadaverine, spermine, and spermidine.

In some embodiments of the invention, the feedstock comprises vegetables.

In some embodiments of the invention, the viable bacterial inoculum size of the lactobacillus fermentum G9 of the first aspect of the invention or the bacterial inoculum of the second aspect of the invention is 10 ⁶ -10 ⁸ CFU/mL。

In some embodiments of the invention, the fermentation environment contains 2% sucrose.

In some embodiments of the invention, the fermentation temperature is 25 ℃ to 33 ℃.

In some embodiments of the invention, the fermentation temperature is 28 ℃ to 31 ℃.

In some embodiments of the invention, the temperature of the fermentation is 30 ℃.

In some embodiments of the invention, the pH of the fermentation environment is from 3.5 to 7.0.

In some embodiments of the invention, the pH of the fermentation environment is from 4.5 to 7.0.

In some embodiments of the invention, the pH of the fermentation environment is from 5.0 to 7.0.

In some embodiments of the invention, the salt concentration of the fermentation environment is 0-6%. The salt is NaCl.

In some embodiments of the invention, the salt concentration of the fermentation environment is 0-4%. The salt is NaCl.

In some embodiments of the invention, the salt concentration of the fermentation environment is 4%. The salt is NaCl.

The beneficial effects of the invention are as follows:

the invention provides a lactobacillus fermentum G9 which is derived from pickle, is a probiotic, has no harm to animals and human bodies, and can be used for producing fermented foods and the like. The lactobacillus fermentum G9 has better acid resistance and salt resistance, can grow normally in an environment with the pH value of 4.5-7.0 and the salt concentration of 1-4 percent, and can still grow better when the pH value is reduced to 4.0 or the salt concentration is increased to 6 percent. Meanwhile, the lactobacillus fermentum G9 does not produce nitrite and biogenic amine, and can effectively degrade the nitrite and biogenic amine, and in a simulated pickle fermentation system, the degradation rate of the lactobacillus fermentum G9 on the nitrite and biogenic amine is 61.42 percent and 13.69 percent respectively in 48 hours. The lactobacillus fermentum G9 can effectively reduce the accumulation of nitrite and biogenic amine in the pickle fermentation process, and the content of the nitrite and the biogenic amine is always lower than the national safety limit standard and is always lower than the content of the nitrite and the biogenic amine in the control group within 15 days of fermentation. The lactobacillus fermentum G9 is utilized to ferment the pickle, so that the food safety of the pickle is obviously improved, the pickle has application prospect as a starter, and a foundation is laid for solving the problem of the food safety of the fermented pickle and developing the starter special for the fermented pickle.

The invention provides a microbial inoculum, which contains the lactobacillus fermentum G9, can effectively degrade nitrite and biogenic amine, and has application prospect as a starter.

The invention provides a method for degrading nitrite and/or biogenic amine, which is characterized in that the lactobacillus fermentum G9 or the microbial inoculum is used for fermenting raw materials, the method is simple to operate, low in cost and easy to realize, and the accumulation of nitrite and biogenic amine in the fermentation process can be effectively reduced.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a morphology feature map of a portion of colonies of the primary screening in the example of the present invention (A, B, C: morphology feature map of a portion of colonies of the primary screening);

FIG. 2 is a graph showing the growth of Lactobacillus fermentum G9;

FIG. 3 shows the results of salt and acid tolerance test of Lactobacillus fermentum G9 (A: salt tolerance test of Lactobacillus fermentum G9; B: acid tolerance test of Lactobacillus fermentum G9);

FIG. 4 shows the results of the test for the tolerance of Lactobacillus fermentum G9 to nitrite and biogenic amine (A: nitrite; B: biogenic amine);

FIG. 5 is a graph showing the effect of Lactobacillus fermentum G9 inoculation on the nitrite and biogenic amine content of kimchi (A: nitrite; B: biogenic amine).

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.

The reagents, methods and apparatus employed in the examples which follow are all conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer.

MRS liquid medium: 10g of peptone, 10g of beef extract powder, 4g of yeast powder, 2g of triamine citrate, 5g of sodium acetate and MgSO ₄ ·7H ₂ O 0.2g，MnSO ₄ ·4H ₂ 0.05g of O, 2g of dipotassium hydrogen phosphate, 20g of glucose and 1.08g of tween-80, and adding distilled water to fix the volume to 1L.

MRS solid medium: agar 15g was added to the MRS liquid medium.

MMRS-A medium: 10g of peptone, 10g of beef extract powder, 4g of yeast powder, 2g of triamine citrate, 5g of sodium acetate and MgSO ₄ ·7H ₂ O 0.2g，MnSO ₄ ·4H ₂ 0.05g of O, 2g of dipotassium hydrogen phosphate, 20g of glucose and 1.08g of tween-80, adding distilled water to a volume of 1L, sterilizing and then adding 150mg of sodium nitrite.

MMRS-B medium: glucose 20g, triamine citrate 2g, sodium acetate 5g, mgSO ₄ ·7H ₂ O 0.2g，MnSO ₄ ·4H ₂ 0.05g of O, 2g of dipotassium hydrogen phosphate, 1.08g of tween-80, adding distilled water to a volume of 1L, sterilizing and adding 1g of sodium nitrate.

MMRS-C medium: glucose 20g, triamine citrate 2g, sodium acetate 5g, mgSO ₄ ·7H ₂ O 0.2g，MnSO ₄ ·4H ₂ 0.05g of O, 2g of dipotassium hydrogen phosphate, 1.08g of tween-80, adding distilled water to a volume of 1L, sterilizing, and adding 1g of tryptophan, ornithine, lysine, histidine, tyrosine and arginine respectively.

MMRS-D medium: mgSO (MgSO) ₄ ·7H ₂ O 0.2g，MnSO ₄ ·4H ₂ 0.05g of O, 2g of dipotassium hydrogen phosphate, 1.08g of tween-80, adding distilled water to a volume of 1L, sterilizing, adding 150mg of sodium nitrite, tryptamine and beta-benzeneEthylamine, tyramine, histamine, putrescine, cadaverine, spermine and spermidine each 100mg.

Isolation and screening of strains and identification thereof

1. Primary screening for isolation of strains

Fermented kimchi samples of the cities of Shaoshan, zhanjiang, guangzhou, etc. were collected, and kimchi with relatively low nitrite and biogenic amine content (nitrite content lower than 1mg/kg, biogenic amine content lower than 15 mg/kg) was selected as a screening sample. 5mL of each of the upper layer, the middle layer and the lower layer of the pickle sample fermentation broth is sampled respectively, the pickle sample fermentation broth is mixed in a 250mL sterile conical flask, 135mL of sterile physiological saline is added into the pickle sample fermentation broth, and the pickle sample fermentation broth is placed in a shaking table for 20min under the conditions of 37+/-1 ℃ and 150rpm, so as to obtain a sample homogenate. The sample homogenate is coated by gradient dilution (10 times dilution), each concentration gradient dilution is coated on an MRS solid plate, then standing culture is carried out for 24-48 hours at 37 ℃, and single colonies (shown in figure 1) with yellow periphery and transparent rings are observed and picked, wherein the colonies are characterized by colony diameters of 1-2mm, bulges, bluish white, moist and yellow periphery, and 81 strains are screened (Shaoguan: S1, S2 … S21, zhanjiang: Z1, Z2 … Z37, guangzhou: G1, G2 … G23 according to the sample source region number) in a total.

2. Rescreening and biogenic amine degradation capability test

(1) Nitrite degradation capability test

After the strain obtained by the primary screening is activated, the strain is centrifuged for 10min at 4 ℃ and 5000rpm, and the supernatant is discarded, and a precipitate (namely the thallus) is left. The cells were resuspended in 0.05mol/L PBS buffer (pH=6.5), centrifuged at 5000rpm at 4℃for 10min, the supernatant was discarded, the cells were left, and the cells were washed repeatedly. Re-suspending thallus with MMRS-A culture medium and regulating bacterial liquid OD _600nm To 0.2 to obtain bacterial suspension. MMRS-A culture medium is used as se:Sup>A control group, and the bacterial suspension is used as an experimental group. After the control group and the experimental group were cultured in a constant temperature incubator at 37℃for 48 hours, they were centrifuged at 12000rpm for 10 minutes, respectively, and the supernatants were collected. Sodium nitrite concentration in the culture solutions of the experimental group and the control group is measured by referring to spectrophotometry of national standard reference GB 5009.33-2016. The nitrite degradation capacity of each strain is represented by the degradation rate of sodium nitrite, and the specific calculation formula is as follows:

the test results are shown in Table 1.

Table 1: nitrite degradation capability test result of partial screening bacteria

The degradation capability of each degradation bacterium on sodium nitrite is greatly different, and the degradation rate of only 27 strains of the 81 strains of degradation bacterium screened on sodium nitrite is more than 90 percent. As shown in Table 1, the strain with the strain number G9 has the strongest degradation capacity on sodium nitrite, and the degradation rate of sodium nitrite reaches 99.72%, so that the strain can be used as a potential strain for controlling nitrite in pickle.

(2) Test of degradation ability of biogenic amine

According to the results of the nitrite degradation capability test in the above examples, the bacterial strain with the sodium nitrite degradation rate higher than 90% is selected to further test the biogenic amine degradation capability.

After activating the strain with the degradation rate of sodium nitrite higher than 90%, respectively centrifuging at 4 ℃ and 5000rpm for 10min, discarding the supernatant, and leaving a precipitate (namely thalli). The cells were resuspended in 0.05mol/L PBS buffer (pH=6.5), centrifuged at 5000rpm at 4℃for 10min, the supernatant was discarded, the cells were left, and the cells were washed repeatedly. Resuspension of bacterial cells with 0.05mol/L PBS buffer solution (containing tryptamine, beta-phenethylamine, tyramine, histamine, putrescine, cadaverine, spermine and spermidine, and the final concentration is 100 mg/L), and regulating bacterial liquid OD _600nm To 0.8 to obtain bacterial suspension. The bacteria suspension is used as an experimental group by taking PBS buffer solution containing biogenic amine and having the concentration of 0.05mol/L as a control group. After the control group and the experimental group were cultured in a constant temperature incubator at 37℃for 48 hours, they were centrifuged at 12000rpm for 10 minutes, respectively, and the supernatants were collected. The biogenic amine concentration in the culture solutions of the experimental and control groups was determined according to liquid chromatography of GB 5009.208-2016 "determination of biogenic amine in food". The biological amine degradation capacity of each strain is expressed by the biological amine degradation rate, and is specifically calculatedThe calculation formula is as follows:

the test results are shown in Table 2.

Table 2: results of test of the biodegradability of partially screened bacteria

The degradation capability of 27 strains (the degradation rate of sodium nitrite is higher than 90%) on biogenic amine is greatly different, wherein only 7 strains with the degradation rate of biogenic amine above 10% are provided. As is clear from Table 2, the degradation rate of biogenic amine of the strain with the strain number G9 was as high as 14.05%.

From the above, the strain with the strain number G9 had the ability to efficiently degrade nitrite and biogenic amine, the degradation rate of nitrite was 99.72%, and the degradation rate of biogenic amine was 14.05%, so that G9 was used as a strain for subsequent study.

3. Nitrite and biogenic amine production ability test of Strain No. G9

After activating the strain with the strain number G9, the strain was centrifuged at 5000rpm at 4℃for 10min, and the supernatant was discarded to leave a precipitate (i.e., cell). The cells were resuspended in 0.05mol/L PBS buffer (pH=6.5), centrifuged at 5000rpm at 4℃for 10min, the supernatant was discarded, the cells were left, and the cells were washed repeatedly. Re-suspending thallus with MMRS-B culture medium and MMRS-C culture medium, and regulating bacterial liquid OD _600nm To 0.8, MMRS-B culture medium bacterial suspension and MMRS-C culture medium bacterial suspension are respectively obtained. MMRS-B culture medium and MMRS-C culture medium are respectively used as a nitrite control group and a biogenic amine control group, wherein MMRS-B culture medium bacterial suspension is a nitrite experimental group, and MMRS-C culture medium bacterial suspension is a biogenic amine experimental group. After culturing the nitrite control group, the biogenic amine control group and the experimental group in a constant temperature incubator at 37 ℃ for 48 hours, the nitrite control group, the biogenic amine control group and the experimental group are respectively centrifuged at 12000rpm for 10 minutes, and the supernatant is collected. Test of nitrite control group and nitrite test with reference to GB5009.33-2016 determination of nitrite and nitrate in foodNitrite concentration in the group, and biogenic amine concentration in biogenic amine control group and biogenic amine experimental group culture solution were measured by referring to GB 5009.208-2016 "determination of biogenic amine in food", and nitrite and biogenic amine production of strain with strain number G9 was obtained.

The detection results show that the concentrations of nitrite and biogenic amine in the culture solution of the experimental group are lower than the detection limit, which indicates that the strain with the strain number G9 cannot metabolize to produce nitrite and biogenic amine.

4. Nitrite and biogenic amine degrading ability test of Strain No. G9 in the same System (mock Pickle fermentation System)

After activating the strain with the strain number G9, the strain was centrifuged at 5000rpm at 4℃for 10min, and the supernatant was discarded to leave a precipitate (i.e., cell). The cells were resuspended in 0.05mol/L PBS buffer (pH=6.5), centrifuged at 5000rpm at 4℃for 10min, the supernatant was discarded, the cells were left, and the cells were washed repeatedly. Re-suspending thallus with MMRS-D culture medium and regulating bacterial liquid OD _600nm To 0.8 to obtain bacterial suspension. MMRS-D culture medium is used as control group, and the bacterial suspension is used as experimental group. After the control group and the experimental group were cultured in a constant temperature incubator at 37℃for 48 hours, they were centrifuged at 12000rpm for 10 minutes, respectively, and the supernatants were collected. Sodium nitrite degradation rate and biogenic amine degradation rate are calculated by referring to the calculation formulas in the above examples by referring to GB5009.33-2016 'determination of nitrite and nitrate in food' and GB 5009.208-2016 'determination of biogenic amine in food', respectively determining sodium nitrite and biogenic amine concentrations in culture solutions of a control group and an experimental group, so as to evaluate the ability of a strain with a strain number G9 to degrade nitrite and biogenic amine in the same system.

The test results show that the strain with the strain number G9 shows excellent nitrite and biogenic amine degrading ability, wherein the nitrite degrading rate is 61.42%, and the biogenic amine degrading rate is 13.69%. Therefore, the strain with the strain number G9 can be used as a potential superior fermentation strain of kimchi.

5. Molecular biological identification of G9

The strain with the strain number of G9 is identified by adopting a 16S rDNA method, the sequencing result is shown as SEQ ID NO.1, and the specific sequence is as follows:

5’-TTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTTAACACCCAAAGTCGGTGGGGAACCTTTAGGAGCCAGCC-3’(SEQ ID NO.1)。

the sequencing results were BLAST aligned with the nucleic acid database in NCBI for molecular biological identification, and the identification results are shown in table 3.

Table 3: molecular biological identification of strain with strain number G9

As is clear from Table 3, the strain with the strain number G9 selected in the above example has a homology of up to 99.93% with Lactobacillus fermentum (Lactobacillus fermentum) CAU5298 with accession No. MF582936.1, and the strain with the strain number G9 is designated Lactobacillus fermentum (Lactobacillus fermentum) G9.

Lactobacillus fermentum G9 was deposited with the collection of microorganisms and cell cultures (GDMCC) in the cantonese province at 11/2 of 2021, and the GDMCC was located on floor 5 of the institute 59, university, first middle road, guangzhou city, accession No. GDMCC No. 62025.

Growth characteristics of Lactobacillus fermentum G9

1. Growth curve

The growth capacity is an important parameter for evaluating the ferment, and the growth curve can intuitively understand the growth rule of the bacteria.

Inoculating activated lactobacillus fermentum G9 into MRS liquid culture medium at 0.5% (V/V), standing at 37deg.C for 24 hr, sampling every 2 hr, and measuring bacterial density (OD) _600nm )。

The detection results are shown in FIG. 2.

As can be seen from FIG. 2, 0-4 hours, the Lactobacillus fermentum G9 is in a growth delay period and grows slowly; after 6 hours, entering a logarithmic growth phase, and rapidly increasing the number of thalli; after 14h, growth was slow, starting to enter the stationary phase. Lactobacillus fermentum G9 has good growth ability.

2. Salt tolerance and acid tolerance test

After lactobacillus fermentum G9 was activated, inoculated in 0.5% (V/V) inoculum size to MRS liquid media having NaCl concentrations of 0%, 2%, 4%, 6%, 8% and 10% and pH values of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0, respectively, and after stationary culture at 37 ℃ for 24 hours, the bacterial density (OD) of each culture was measured _600nm ) To evaluate the salt tolerance and acid tolerance of lactobacillus fermentum G9.

The detection results are shown in FIG. 3.

As can be seen from fig. 3A, lactobacillus fermentum G9 was less inhibited at a salt concentration of 2% -4%; when the salt concentration was increased to 6%, OD _600nm Above 2.0; however, when the salt concentration was increased to 8% or more, lactobacillus fermentum G9 hardly grew normally. As can be seen from FIG. 3B, when the pH is less than 3.0, hair is developedLactobacillus fermentum G9 hardly grows normally; and when the pH was raised to 4.0, OD _600nm Above 2.0; when the pH is raised to 4.5, the lactobacillus fermentum G9 is less inhibited; when the pH is raised to above 5.0, the lactobacillus fermentum G9 can grow normally.

The lactobacillus fermentum G9 has certain salt tolerance and acid tolerance.

3. Tolerance test to nitrite and biogenic amine

Inoculating lactobacillus fermentum G9 after activation to MRS liquid culture medium with 0.5% (V/V) sodium nitrite concentration of 0, 50, 100, 150 and 200mg/L and biogenic amine concentration of 0, 50, 100, 200 and 300mg/L (biogenic amine including tryptamine, beta-phenethylamine, tyramine, histamine, putrescine, cadaverine, spermine and spermidine, total concentration of tryptamine, beta-phenethylamine, tyramine, histamine, putrescine, cadaverine, spermine and spermidine), standing at 37deg.C for 24 hr, and determining bacterial density (OD _600nm ) To evaluate the tolerance characteristics of lactobacillus fermentum G9 to nitrite and biogenic amines.

The detection results are shown in FIG. 4.

As is clear from FIG. 4A, when the concentration of sodium nitrite is 0-150mg/L, the growth of Lactobacillus fermentum G9 is hardly affected, and when the concentration of sodium nitrite is increased to 200mg/L, lactobacillus fermentum G9 is less inhibited, and the bacterial density is only 19.13% lower than that of the control group (sodium nitrite concentration is 0 mg/L). As can be seen from FIG. 4B, the growth of Lactobacillus fermentum G9 was hardly affected when the biogenic amine concentration was 0-200mg/L, and the growth was inhibited when the biogenic amine concentration was increased to 300mg/L, but OD _600nm Still remain above 5.0.

Lactobacillus fermentum G9 has better tolerance to nitrite and biogenic amine.

Degradation capability of lactobacillus fermentum G9 on nitrite and biogenic amine in pickle

(1) Preparation of activated seed liquid

The lactobacillus fermentum G9 preserved in a refrigerator at-80 ℃ is streaked in MRS solid culture medium, and then cultured at 37 ℃ for 48 hours. Picking single colony in MRS solid culture medium, inoculating in MRS liquid culture medium, and culturing at 37deg.C for 16 hr to activate lactobacillus fermentum G9; inoculating the seed liquid into MRS liquid culture medium according to the inoculation amount of 0.5% (V/V) to perform secondary activation, and obtaining the activated seed liquid.

(2) Raw material treatment

Cleaning fresh leaf mustard and Chinese cabbage, airing the water, and cutting the leaf mustard and Chinese cabbage for later use; removing shells of the moso bamboo shoots, cleaning, airing, and cutting into slices with a length of about 5cm, a width of about 3cm and a thickness of about 1cm for later use. Scalding the treated mustard, cabbage and bamboo shoots in boiling water for 1min, taking out, draining, and packaging into sterile fermentation jar.

(3) Fermentation

Sterile water containing 4% NaCl and 2% sucrose was boiled and cooled to room temperature to give solution A. Adding the solution A into the fermentation tank in the step (2) according to the solid-liquid mass ratio of 1:2, and ensuring that mustard, cabbage and bast shoots in the fermentation tank are completely immersed; inoculating the activated seed solution (final concentration of Lactobacillus fermentum G9 of 10) in step (1) at an inoculum size of 3% (V/W solution A mass) ⁷ CFU/mL), shaking up, and then taking the mixture as an experimental group (marked as lactobacillus fermentum G9); the control group was treated without starter culture. The experimental and control groups were fermented at 30 ℃. Samples were taken on days 1, 2, 3, 5, 7, 9, 11, 13 and 15, respectively, and the concentrations of sodium nitrite and biogenic amine in the fermentation broths of the control and experimental groups (lactobacillus fermentum G9) were determined, respectively, with reference to GB5009.33-2016 "determination of nitrite and nitrate in food" and GB 5009.208-2016 "determination of biogenic amine in food".

The detection results are shown in FIG. 5.

As can be seen from FIG. 5A, the nitrite content in the kimchi of the control group reached the peak value (83.88 mg/kg) at 2d of fermentation, far exceeding the national safety limit standard (20 mg/kg), and began to decrease with the prolongation of fermentation time, and after 9d of fermentation, the nitrite content tended to be substantially 0mg/kg. The change trend of the nitrite content in the kimchi of the experimental group is similar to that of the control group, but compared with the control group, the nitrite content in the kimchi of the experimental group is always lower than the national safety limit standard, and the nitrite content is basically about 0mg/kg after fermentation for 5 d. As can be seen from fig. 5B, the biogenic amine content in the kimchi of the control group is greatly increased, the biogenic amine content reaches 148.72mg/kg only after fermentation for 1d, then is reduced, and finally becomes gentle, the biogenic amine content is stabilized at about 100mg/kg at the later stage of fermentation, while the biogenic amine content in the kimchi of the experimental group can be remarkably reduced by lactobacillus fermentum G9 during the fermentation process, and the biogenic amine content in the kimchi of the experimental group is always lower than 30mg/kg during the whole fermentation process.

The lactobacillus fermentum G9 can effectively reduce the accumulation of nitrite and biogenic amine in the pickle fermentation process, and can be used as a starter for fermenting pickle.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Sequence listing

<110> institute for processing silkworm industry and agricultural products at the national academy of agricultural sciences in Guangdong province

<120> Lactobacillus fermentum and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1415

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ttgattgatg gtgcttgcac ctgattgatt ttggtcgcca acgagtggcg gacgggtgag 60

taacacgtag gtaacctgcc cagaagcggg ggacaacatt tggaaacaga tgctaatacc 120

gcataacaac gttgttcgca tgaacaacgc ttaaaagatg gcttctcgct atcacttctg 180

gatggacctg cggtgcatta gcttgttggt ggggtaacgg cctaccaagg cgatgatgca 240

tagccgagtt gagagactga tcggccacaa tgggactgag acacggccca tactcctacg 300

ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca acaccgcgtg 360

agtgaagaag ggtttcggct cgtaaagctc tgttgttaaa gaagaacacg tatgagagta 420

actgttcata cgttgacggt atttaaccag aaagtcacgg ctaactacgt gccagcagcc 480

gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaaga gagtgcaggc 540

ggttttctaa gtctgatgtg aaagccttcg gcttaaccgg agaagtgcat cggaaactgg 600

ataacttgag tgcagaagag ggtagtggaa ctccatgtgt agcggtggaa tgcgtagata 660

tatggaagaa caccagtggc gaaggcggct acctggtctg caactgacgc tgagactcga 720

aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc 780

taggtgttgg agggtttccg cccttcagtg ccggagctaa cgcattaagc actccgcctg 840

gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900

agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atcttgcgcc 960

aaccctagag atagggcgtt tccttcggga acgcaatgac aggtggtgca tggtcgtcgt 1020

cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgttactagt 1080

tgccagcatt aagttgggca ctctagtgag actgccggtg acaaaccgga ggaaggtggg 1140

gacgacgtca gatcatcatg ccccttatga cctgggctac acacgtgcta caatggacgg 1200

tacaacgagt cgcgaactcg cgagggcaag caaatctctt aaaaccgttc tcagttcgga 1260

ctgcaggctg caactcgcct gcacgaagtc ggaatcgcta gtaatcgcgg atcagcatgc 1320

cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga gagttttaac 1380

acccaaagtc ggtggggaac ctttaggagc cagcc 1415

Claims

1. A strain of lactobacillus fermentum, characterized by the designation lactobacillus fermentum (Lactobacillus fermentum) G9, accession number: the collection date of the microorganism strain collection in Guangdong province: 2021, 11/2, deposit number: GDMCC No. 62025.

2. A microbial inoculum comprising the Lactobacillus fermentum G9 according to claim 1.

3. Use of lactobacillus fermentum G9 according to claim 1 or of the microbial agent according to claim 2 for degrading nitrite and/or biogenic amine.

4. Use according to claim 3, wherein the nitrite comprises sodium nitrite; the biogenic amine comprises at least one of tryptamine, beta-phenethylamine, tyramine, histamine, putrescine, cadaverine, spermine and spermidine.

5. Use of lactobacillus fermentum G9 according to claim 1 or a microbial inoculum according to claim 2 for the production of a fermentation product.

6. Use according to claim 5, characterized in that lactobacillus fermentum G9 according to claim 1 or the microbial inoculum according to claim 2 is inoculated onto a raw material for fermentation.

7. A fermented product obtained by inoculating lactobacillus fermentum G9 according to claim 1 or a microbial inoculum according to claim 2 and fermenting the raw material.

8. A method for degrading nitrite and/or biogenic amine, which is characterized in that lactobacillus fermentum G9 according to claim 1 or the microbial inoculum according to claim 2 is inoculated in raw materials for fermentation.

9. The method according to claim 8, wherein the viable bacterial inoculum size of the lactobacillus fermentum G9 according to claim 1 or the bacterial inoculum according to claim 2 is 10 ⁶ -10 ⁸ CFU/mL。

10. The method of claim 8, wherein the fermentation temperature is 25 ℃ to 33 ℃.