CN113046280A - Salt-tolerant low-temperature-resistant lactobacillus plantarum for inhibiting pathogenic bacteria and application thereof - Google Patents

Salt-tolerant low-temperature-resistant lactobacillus plantarum for inhibiting pathogenic bacteria and application thereof Download PDF

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CN113046280A
CN113046280A CN202110515656.7A CN202110515656A CN113046280A CN 113046280 A CN113046280 A CN 113046280A CN 202110515656 A CN202110515656 A CN 202110515656A CN 113046280 A CN113046280 A CN 113046280A
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谈重芳
张淼
刘媛
陈骏
庞会利
王敏
关毅飞
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Abstract

The invention discloses a salt-tolerant and low-temperature-resistant lactobacillus plantarum for inhibiting pathogenic bacteria and application thereof. The invention provides Lactobacillus (Lactobacillus plantarum) QZW5 with the preservation number of CCTCC NO: m2021323, the strain has the following functions: 1) inhibiting pathogenic bacteria at low temperature; 2) inhibiting pathogenic bacteria under repeated freeze thawing conditions; 3) extending the shelf life of frozen products under refrigerated, partially frozen or frozen conditions; 4) the preservation period of the frozen product under the condition of repeated freeze thawing is prolonged. The invention finds out a lactic acid bacterium which is low temperature resistant and high salt resistant, can be used for inhibiting various low temperature resistant or high salt resistant pathogenic bacteria, and can be used for refrigeration preservation, micro-freezing preservation or repeated freezing and thawing of frozen and preserved products.

Description

Salt-tolerant low-temperature-resistant lactobacillus plantarum for inhibiting pathogenic bacteria and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a salt-tolerant and low-temperature-resistant lactobacillus plantarum for inhibiting pathogenic bacteria and application thereof.
Background
Low temperature is one of the most important means for ensuring the quality of food raw materials, processing and finished products. The production and sale of low-temperature prefabricates, slightly frozen aquatic products and frozen foods in China are in the forefront all over the world, but the foods cannot avoid repeated freeze-thaw states in the production and sale processes, because low-temperature-resistant microorganisms form the capability of resisting various stresses in the long-term evolution process, the foods can slowly grow under the low-temperature condition, even under the freezing condition, the metabolic activity is still kept in a sublethal state for a long time, even under the repeated freeze-thaw, the pathogenic capability can be recovered, and the cold-resistant microorganisms can secrete low-temperature-resistant enzymes to decompose proteins or lipids in the foods under the low-temperature and freezing conditions, so that the quality of the foods is slowly reduced, and serious potential safety hazards are brought to the refrigerated and frozen foods, and therefore the control of low-temperature-resistant pathogenic bacteria is always a difficult problem of the safety of refrigerated and frozen food.
Disclosure of Invention
One object of the present invention is to provide a lactic acid bacterium.
The preservation number of the Lactobacillus (Lactobacillus plantarum) QZW5 is CCTCC NO: m2021323.
The application of the lactobacillus or the bacterial suspension thereof or the culture solution thereof or the supernatant of the culture solution thereof in at least one of the following 1) to 4) or at least one functional product with the following 1) to 4) is also within the protection scope of the invention:
1) inhibiting pathogenic bacteria;
2) inhibiting pathogenic bacteria under low temperature or repeated freeze thawing condition;
3) extending the shelf life of frozen products under refrigerated, partially frozen or frozen conditions;
4) the storage period of the frozen product under the condition of repeated freeze thawing is prolonged.
In the above application, the frozen product is a food frozen product.
In the application, the pathogenic bacteria are low temperature resistant bacteria;
or, the low temperature is less than or equal to 4 ℃;
or the repeated freezing and thawing conditions are repeated freezing and thawing at-7 ℃ and 7 ℃;
or, the refrigeration conditions are-4 ℃ to 0 ℃;
or, the partial freezing condition is-2 ℃ to-4 ℃;
alternatively, the freezing conditions are below-4 ℃.
It is another object of the invention to provide a product.
The product provided by the invention comprises the lactic acid bacteria or the bacterial suspension thereof, the culture solution thereof or the supernatant of the culture solution thereof.
In the above product, the supernatant of the lactic acid bacteria culture solution is prepared as follows: culturing the lactobacillus to obtain a culture solution, and collecting the supernatant of the culture solution.
The product has at least one function of 1) to 4) as follows:
1) inhibiting pathogenic bacteria;
2) inhibiting pathogenic bacteria under low temperature or repeated freeze thawing condition;
3) extending the shelf life of frozen products under refrigerated, partially frozen or frozen conditions;
4) the storage period of the frozen product under the condition of repeated freeze thawing is prolonged.
It is a further object of the present invention to provide a method of:
the invention provides a method for prolonging the preservation period of a frozen product, which comprises the following steps: the frozen product is soaked in the supernatant of the culture solution of the lactic acid bacteria, and then is refrigerated or frozen, so that the storage period of the frozen product is prolonged.
Or, the invention provides a method for prolonging the storage period of a frozen product under the condition of repeated freeze thawing, which comprises the following steps: the frozen product is soaked in the supernatant of the culture solution of the lactic acid bacteria, and then repeated freezing and thawing are carried out, so that the storage period of the frozen product under the repeated freezing and thawing condition is prolonged.
In the method, the refrigeration condition is-4 ℃ to 0 ℃;
or, the freezing conditions are below-4 ℃;
or the repeated freezing and thawing conditions are repeated freezing and thawing at-7 ℃ and 7 ℃.
The invention provides a lactic acid bacterium which is low temperature resistant and high salt resistant, can be used for inhibiting various low temperature resistant or high salt resistant pathogenic bacteria, and can be used for cold storage, micro-freezing storage or repeated freezing and thawing storage of frozen products.
Deposit description
The strain name is as follows: lactobacillus plantarum
Latin name: lactobacillus plantarum
The strain number is as follows: QZW5
The preservation organization: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (China center for cell communication)
Address: university of Wuhan, China
The preservation date is as follows: 2021 year, 4 months and 2 days
Registration number of the preservation center: CCTCC NO: m2021323
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The pathogen indicator strains in the following examples are as follows:
micrococcus luteus (Micrococcus luteus)28001 (purchased from institute of biologics, Henan province), Staphylococcus aureus (Staphylococcus aureus)12600 (purchased from institute of biologics, Henan province), Staphylococcus aureus 26003 (purchased from institute of biologics, Henan province), Staphylococcus aureus 29213 (purchased from institute of biologics, Henan province), Bacillus subtilis 63501 (purchased from institute of biologics, Henan province) and listeria BAA (purchased from institute of biologics, Henan province), Escherichia coli (Escherichia coli)30105 (purchased from institute of biologics, Henan province) and Salmonella (Salmonella enterica) ATCC14028, Salmonella ATTC13076, pseudomonas aeruginosa 10104 (purchased from institute of biologics, Henan province), and Salmonella 50094 (purchased from institute of biologics, Henan province).
The media in the following examples are as follows:
medium for lactobacillus strain culture (MRS solid medium): 10g of peptone, 5g of yeast extract, 10g of beef extract, 20g of glucose, 2g of dipotassium phosphate, 2g of ammonium citrate, 5g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 80 g of tween 80lmL, 15g of agar, 1.0L of distilled water and 6.5 of pH value.
The MRS liquid culture medium is different from the MRS solid culture medium in that agar is not added.
NA culture medium (for culturing Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Salmonella): 3g of beef extract, 10g of peptone, 5g of sodium chloride, 15g of agar, 1.0L of distilled water and pH 7.0-7.2.
LB medium (for e.coli culture): 10g of peptone, 5g of yeast extract, 10g of sodium chloride, l.0L of distilled water, pH 7.0.
Example 1 isolation and identification of Lactobacillus plantarum QZW5 Strain
Firstly, separation and purification
A strain QZW5 is obtained by separating fresh wheat material from Qinghai, and the specific method is as follows:
weighing 5g of a fresh sample, adding the fresh sample into sterilized 45ml of distilled water, oscillating for 30s by using a vortex oscillator, sequentially diluting for 5 times according to a 10-fold gradient, then respectively coating 20 mul of 10, 1000 and 100000-fold diluted sample liquid on an MRS culture medium, placing the mixture on an anaerobic culture box, culturing for 24 hours, separating and further purifying to obtain QZW5, wherein the bacteria are gram-positive bacilli, catalase-negative and spore-non-producing bacteria.
II, physiological, biochemical and molecular identification
1. Physiological and biochemical identification
The above-mentioned strain QZW5 was grown under different conditions and tested for growth characteristics under different carbon sources by API50, the results are shown in Table 1.
Table 1 shows the physiological and biochemical properties of QZW5
Figure BDA0003061895640000031
Figure BDA0003061895640000041
In the above table, + growth, w weakly growth, and-no growth.
3. Molecular identification
A single colony of the strain QZW5 is extracted for colony PCR, and the primers are universal primers for 16S rRNA gene amplification of bacteria: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') to obtain PCR amplification products.
And (3) sequencing the PCR amplification product to obtain a 16S rRNA gene sequence, wherein the nucleotide sequence is sequence 1.
And performing BLAST comparison on the sequencing result, and determining that the separated strain is Lactobacillus plantarum by combining morphological analysis comparison.
The strain QZW5 is preserved in China center for type culture Collection (CCTCC for short, address: China, Wuhan university) at 4/2/2021, with the preservation number of CCTCC NO: m2021323, classified and named Lactobacillus plantarum.
4. Bacteriostatic experiment of Lactobacillus plantarum QZW5
Oxford cup double-layer plate method: cooling the indicator bacterium culture medium containing 1.5% agar to about 50 ℃, pouring the indicator bacterium culture medium into a sterile culture dish according to 15mL per plate, and cooling the indicator bacterium culture medium in an ultra-clean workbench; preparing an indicator bacterium culture medium containing 0.8% of agar, cooling to about 50 ℃, adding an indicator bacterium suspension as an upper layer culture medium according to the inoculation amount of 1%, pouring 5mL of the upper layer culture medium into a flat plate containing the 1.5% of agar culture medium for cooling, then gently placing an oxford cup on the flat plate by using a sterile forceps, adding a cell-free fermentation supernatant into the oxford cup, spreading the cell-free fermentation supernatant on an ultraclean workbench for 5 hours, culturing the cell-free fermentation supernatant for 24 hours under a proper culture condition, and observing the appearance of a bacteriostatic circle.
The indicator bacteria are strains in columns 1 and 3 in table 2, the indicator bacteria culture medium is a culture medium corresponding to the strains, and the indicator bacteria suspension is a culture solution obtained by culturing the indicator bacteria in the culture medium corresponding to the indicator bacteria.
The results are shown in Table 2.
Table 2 shows the results of the experiments (zone of inhibition in mm)
Figure BDA0003061895640000051
As can be seen from the above, QZW5 inhibited the growth of the indicator bacteria shown in Table 2, which were all inhibitory greater than 7.8 mm.
Example 2 application of Lactobacillus plantarum QZW5 in Cold chain repeated Freeze-thaw preservation of food
High salt tolerance experiment of Lactobacillus plantarum QZW5
QZW5 in example 1 was cultured in MRS liquid medium at 30 ℃ to a viable count of 108Centrifugation at CFU/mL or more was carried out, the supernatant was removed, and 1mL of a sterile NaCl solution (solvent water) having a concentration shown in Table 3 was added for 3 hours, followed by anaerobic culture at 28 ℃ for 24 hours on MRS solid plates.
As shown in Table 3, the survival rate of Lactobacillus plantarum QZW5 after 3 hours treatment with 10% to 20% NaCl solution was more than 90%, indicating that it is high salt resistant.
Table 3 shows the activity of the strains at different salt concentrations
Figure BDA0003061895640000052
Second, drug sensitivity test of Lactobacillus plantarum QZW5
QZW5 in example 1 was cultured in MRS liquid medium (not containing agar) at 30 ℃ for 24 hours to obtain QZW5 activated bacterial suspension.
Pouring 15mL of MRS containing 1.0% agar into a 9cm culture dish, adding 200 μ L of the above QZW5 activated bacteria liquid into 5mL of MRS containing 0.8% agar at 50 deg.C, mixing, and controlling the concentration of QZW5 living cells at 1.5 × 107CFU/mL, QZW 5-containing medium was spread evenly on the bottom medium in the chamberStanding at room temperature for about 15min, cooling and solidifying the upper agar layer, drying, placing antibiotic drug sensitive test strip (from OXOID) on the surface of agar by aseptic technique, culturing at 37 deg.C for 24h, measuring the diameter of inhibition zone with vernier caliper, reading to millimeter, and taking the average of 3 measurements as the result.
As a result, as shown in Table 4, it can be seen that Lactobacillus plantarum QZW5 is sensitive to most antibiotics and suitable for safe preservation of food, since antibiotic-sensitive strains do not transfer resistance genes to pathogenic microorganisms easily.
Table 4 shows the results of the drug sensitivity test of Lactobacillus plantarum QZW5
Figure BDA0003061895640000061
In the above table, -means not sensitive.
Thirdly, bacteriostatic detection of Lactobacillus plantarum QZW5 under low temperature condition
1. Bacteriostatic detection at 4 ℃ under refrigeration
Culturing Lactobacillus plantarum QZW5 in MRS liquid medium at 28 deg.C for 24 hr, centrifuging at 5000 rpm, collecting supernatant, and adding 10 per ml of supernatant6CFU the following table 5 pathogenic bacteria, 4 degrees C under bacteriostatic culture, get the culture solution, in the following table 5 different bacteriostatic culture time respectively: the number of pathogenic bacteria in the culture solution was measured at 4, 8, 12, 24, and 72 hours. An isolated strain of the same origin, QZ50, was used as a control.
Culturing the culture solution obtained in different antibacterial culture time at 28 deg.C for 24 hr, counting and detecting pathogenic bacteria number, 10%4Above CFU/mL is ++, 102-104CFU/mL plus or minus 0-102CFU/mL is + -, and does not grow as-.
QZW5 at 4 ℃ for different periods of time, the results are shown in Table 5, which shows that QZW5 inhibits a wide variety of pathogens at 4 ℃.
TABLE 5 bacteriostasis time of QZW5 to different pathogenic bacteria at 4 deg.C
Figure BDA0003061895640000062
Figure BDA0003061895640000071
2. Inhibition of pathogenic bacteria by Lactobacillus plantarum QZW5 under the condition of partial freezing at-2 ℃ to-4 DEG C
Culturing Lactobacillus plantarum QZW5 in MRS liquid culture medium at 28 deg.C for 24 hr, centrifuging at 5000 rpm, collecting supernatant, adding 10 to the supernatant6CFU/mL of the following pathogenic bacteria in Table 6 were subjected to bacteriostatic culture at-4 ℃ to obtain culture solutions, and the culture solutions were cultured for the following different bacteriostatic culture times (culture times after adding the pathogenic bacteria): and detecting the number of pathogenic bacteria in the mixed bacteria liquid at 4, 8, 12, 24 and 72 hours. An isolated strain of the same origin, QZ50, was used as a control.
Culturing the above different culture solutions on NA solid plate at 28 deg.C for 24 hr, and detecting pathogenic bacteria amount, 10%4Above CFU/mL is ++, 102-104CFU/mL plus or minus 0-102CFU/mL is + -, and does not grow as-.
QZW5 the results of inhibiting pathogenic bacteria at-4 ℃ for various periods of time are shown in Table 6, and it can be seen that QZW5 inhibits a wide variety of pathogenic bacteria at-4 ℃ in the case of partial freezing.
TABLE 6 bacteriostasis time of QZW5 to different pathogenic bacteria at-4 deg.C
Figure BDA0003061895640000072
In the above table, + growth, -no growth.
3. Inhibition result of Lactobacillus plantarum QZW5 on pathogenic bacteria under repeated freeze-thawing condition of-7 ℃ to 7 ℃ for 72 hours
Culturing Lactobacillus plantarum QZW5 in MRS liquid culture medium at 28 deg.C for 24 hr, centrifuging at 5000 rpm, collecting supernatant, adding 10 to the supernatant6CFU/mL of the pathogens listed in Table 7 below, were left at-7 ℃ for 12 hours and thenStanding at 7 deg.C for 12 hr, and repeating for 5 times (-7 deg.C/7 deg.C). The isolated strain QZ50 from the same source was used as a control.
Culturing the mixed bacterial liquid with the freeze thawing times on an NA solid plate at 28 ℃, counting, detecting the number of pathogenic bacteria under different culture time, and 10 ℃4Above CFU/mL is ++, 102-104CFU/mL plus or minus 0-102CFU/mL is + -, and does not grow as-.
QZW5 at-7 deg.C/7 deg.C for different times of repeated freezing and thawing, the results of inhibiting pathogenic bacteria are shown in Table 7, and it can be seen that QZW5 can inhibit various pathogenic bacteria under the repeated freezing and thawing conditions at-7 deg.C/7 deg.C.
Table 7 shows the results of inhibiting pathogenic bacteria by QZW5 repeated freezing and thawing at-7 deg.C/7 deg.C
Figure BDA0003061895640000081
As can be seen from the above, Lactobacillus plantarum QZW5 can inhibit various pathogenic bacteria in low temperature and repeated freeze-thaw state, so it can be applied to fresh-keeping of frozen food.
Example 3 preservation of frozen food products by Lactobacillus plantarum QZW5 by repeated Freeze-thawing
Lactobacillus plantarum QZW5 was cultured in MRS liquid medium at 28 ℃ for 24 hours, centrifuged at 5000 rpm, and the supernatant was collected.
Various frozen food products shown in table 8 were soaked with the above supernatant for 10 minutes and then taken out, and stored for 15 days under the following conditions, respectively: repeated freeze thawing at 25 deg.C, 4 deg.C, 7 deg.C and 7 deg.C for 5 times, with no soaking treatment as control.
Observing the state of the frozen food, weighing 5g of the frozen food, adding the weighed frozen food into 45ml of sterilized distilled water, shaking the frozen food for 30s by using a vortex oscillator, sequentially diluting the frozen food for 5 times according to 10-fold gradient, and then respectively taking 20 mu l of each of 10-fold, 1000-fold and 100000-fold diluted sample liquid, culturing the diluted sample liquid on an eosin methylene blue solid culture medium for 28 ℃ for 24 hours to detect escherichia coli; aerobic bacteria were detected in an NA solid plate at 28 ℃ for 24 hours and anaerobic bacteria were detected in an anaerobic culture for 24 hours (the above-mentioned microorganism detection references: Pang H, Tan Z, Qin G, Wang Y, Li Z, Jin Q, Cai Y (2012) photosynthetic and photosynthetic analysis of lactic acid bacteria isolated from bacteria crystals and grains in the Tibeta Plateau.J. Microbiol 50(1): 63-71), and the results are shown in Table 8:
TABLE 8 frozen food status
Figure BDA0003061895640000091
Figure BDA0003061895640000101
Figure BDA0003061895640000111
The above results show that Lactobacillus plantarum QZW5 can prolong the shelf life of frozen food products under repeated freeze-thaw conditions.
SEQUENCE LISTING
<110> Zhengzhou university
<120> salt-tolerant and low-temperature-resistant lactobacillus plantarum for inhibiting pathogenic bacteria and application thereof
<160> 1
<170> PatentIn version 3.5
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<213> Lactobacillus plantarum
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cgaactctgg tattgattgg tgcttgcatc atgatttaca tttgagtgag tggcgaactg 120
gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata acacctggaa acagatgcta 180
ataccgcata acaacttgga ccgcatggtc cgagtttgaa agatggcttc ggctatcact 240
tttggatggt cccgcggcgt attagctaga tggtgaggta acggctcacc atggcaatga 300
tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg gcccaaactc 360
ctacgggagg cagcagtagg gaatcttcca caatggacga aagtctgatg gagcaacgcc 420
gcgtgagtga agaagggttt cggctcgtaa aactctgttg ttaaagaaga acatatctga 480
gagtaactgt tcaggtatta gacggtattt aaccagaaag ccacggctaa ctacgtgcca 540
gcagccgcgg taatacgtag gtggcaagcg ttgtccggat ttattgggcg taaagcgagc 600
gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc aaccgaagaa gtgcatcgga 660
aactgggaaa cttgagtgca gaagaggaca gtaaggctcc atgtgtagcg gtgaaatgcg 720
tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac tgacgctgag 780
gctcgaaagt atgggtagca aacaggatta gataccctgg tagtccatac cgtaaacgat 840
gaatgctaag tgttggaggg tttccgccct tcagtgctgc agctaacgca ttaagcattc 900
cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag 960
cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac cttaccaggt cttgacatac 1020
tatgcaaatc taagagatta gacgttccct tcggggacat ggatacaggt ggtgcatggt 1080
tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt 1140
atcagttgcc agcattaagt tgggcactct ggtgagactg ccggtgacaa accggaggaa 1200
ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1260
ggatggtaca acgagttgcg aactcgcgag agtaagctaa tctcttaaag ccattctcag 1320
ttcggattgt aggctgcaac tcgcctacat gaagtcggaa tcgctagtaa tcgcggatca 1380
gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgagagt 1440
ttgtaacacc caaagtcggt ggggtaacct tttaggaacc agccgcctaa ggtgggacag 1500
atgattaggg tgaagtcgta acaaggtaac c 1531

Claims (10)

1. Lactobacillus (Lactobacillus plantarum) QZW5 with the preservation number of CCTCC NO: m2021323.
2. The use of the lactic acid bacteria or its bacterial suspension or its culture solution or its supernatant of the culture solution according to claim 1 in at least one of the following 1) to 4) or in a functional product having at least one of the following 1) to 4):
1) inhibiting pathogenic bacteria;
2) inhibiting pathogenic bacteria under low temperature or repeated freeze thawing condition;
3) extending the shelf life of frozen products under refrigerated, partially frozen or frozen conditions;
4) the storage period of the frozen product under the condition of repeated freeze thawing is prolonged.
3. Use according to claim 2, characterized in that: the frozen product is a food frozen product.
4. Use according to claim 2 or 3, characterized in that: the low temperature is less than or equal to 4 ℃;
or the repeated freezing and thawing conditions are repeated freezing and thawing at-7 ℃ and 7 ℃;
or, the refrigeration conditions are-4 ℃ to 0 ℃;
or, the partial freezing condition is-2 ℃ to-4 ℃;
alternatively, the freezing conditions are below-4 ℃.
5. A product comprising the lactic acid bacterium of claim 1, or a suspension thereof, or a culture thereof, or a supernatant thereof.
6. The product of claim 5, wherein: the supernatant of the lactic acid bacteria culture solution is prepared according to the following method: culturing the lactic acid bacterium according to claim 1 to obtain a culture solution, and collecting a supernatant of the culture solution.
7. The product according to claim 5 or 6, characterized in that:
the product has at least one function of 1) to 4) as follows:
1) inhibiting pathogenic bacteria;
2) inhibiting pathogenic bacteria under low temperature or repeated freeze thawing condition;
3) extending the shelf life of frozen products under refrigerated, partially frozen or frozen conditions;
4) the storage period of the frozen product under the condition of repeated freeze thawing is prolonged.
8. A method for extending the shelf life of a frozen product comprising the steps of: the frozen product is soaked in the supernatant of the culture solution of the lactic acid bacteria according to claim 1, and then refrigerated or frozen to prolong the storage period of the frozen product.
9. A method for extending the shelf life of a frozen product under repeated freeze-thaw conditions, comprising the steps of: the frozen product is soaked in the supernatant of the culture solution of the lactic acid bacteria according to claim 1, and then repeated freezing and thawing is carried out, so that the storage period of the frozen product under the repeated freezing and thawing condition is prolonged.
10. The method according to claim 8 or 9, characterized in that:
the refrigeration condition is-4 ℃ to 0 ℃;
or, the freezing conditions are below-4 ℃;
or the repeated freezing and thawing conditions are repeated freezing and thawing at-7 ℃ and 7 ℃.
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