CN108265019A - Leuconostoc mesenteroides subsp mesenteroides, preparation method and application - Google Patents

Abstract

The invention discloses a Leuconostoc mesenteroides subsp. Mesenteroides, a preparation method and an application thereof. The Leuconostoc mesenteroides subsp. mesenteroides is typical in China. The deposit number of the Culture Collection Center is CCTCC NO: M 2018129. The Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129 has a high ability to produce acid and degrade nitrite, and is resistant to salt and acid and alkali. It can improve the nutritional quality of the product and at the same time can The storage period of the product is extended, and the lactic acid bacteria resource is developed, which is beneficial to the development of the kimchi industry and has a broad market prospect.

Description

Leuconostoc intestinalis subspecies, preparation method and application

technical field

The invention relates to the field of microorganisms, in particular to a Leuconostoc enterococci subspecies, a preparation method and an application thereof.

Background technique

Leuconostoc is a genus of lactic acid bacteria, a type of gram-positive bacteria that ferment heterozygous lactic acid, exists on the surface and roots of plants, and is the dominant bacteria in naturally fermented foods of milk or plant materials. Fermentation can metabolize organic acids, alcohols, diacetyl and other aromatic compounds to improve product flavor; dextran can be used as a blood thickener, stretching agent and stabilizer; mannitol can be used as a low-calorie sweetener; The production of bacteriocin can be used as a biological preservative; the production of oligosaccharides can maintain the ecological balance of the normal flora in the human intestinal tract, and has a probiotic effect. Leuconostoc is expected to become a new microecological agent.

Leuconostoc enterica is an important strain of the genus Leuconostoc. It is a general term for a class of non-spore-forming Gram-positive bacteria. Leuconostoc subspecies has been listed as one of the edible fungi by the Ministry of Health of my country (Announcement No. 8 in 2012) and the US Food and Drug Administration (FDA). Leuconostoc enterica subspecies can regulate the balance of intestinal flora, promote the absorption of nutrients, improve the flavor of products, and also have the ability to resist oxidation and antagonize pathogenic bacteria. Because of its ability to produce special flavor substances, it is widely used in food fields such as milk and pickles.

Kimchi is a traditional food with a long history. At present, the popular kimchi in the world is mainly divided into Chinese kimchi, Korean kimchi and Japanese kimchi according to the place of origin and taste. Japanese kimchi is non-fermented, while Chinese kimchi and Korean kimchi are fermented. Korean kimchi is one of the Chinese kimchi with great ethnic characteristics. It has a wide variety of materials and a wide range of materials. It has different tastes depending on the raw materials, preparation methods, usage and time. It has a unique and strong flavor and is rich in lactic acid bacteria activity and lactic acid. Sakura is a characteristic vegetable grown by the Korean people. It has a short growth period and is seasonal. It is rich in nutrition and tastes suitable for the public. At present, Leuconostoc is mostly isolated from Henan Linzhou kimchi, Sichuan kimchi, and spicy cabbage, but Leuconostoc enterica has not been isolated from Korean traditional fermented cherry cabbage. Due to the different sources, isolation and cultivation methods of the same microorganism, there are certain differences in its characteristics and functions. In traditional fermented foods, such as kimchi, during the fermentation process, due to the slow growth of lactic acid bacteria or the weak ability to degrade nitrite, many bacteria are generated in some fermented foods, which affects the shelf life of kimchi, and kimchi will produce Raw nitrite, nitrite is a very harmful substance to the human body, excessive or long-term consumption is likely to cause cancer. Therefore need a kind of bacterium that can solve this kind of problem.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, provide a kind of Leuconostoc enterococci subspecies and its preparation method and application, the Leuconostoc enterocida have high acid production ability and degrade nitrite ability, and salt and acid-alkali resistance, through the leuconostoc enterococcus subsp. enterica can degrade nitrite in the fermentation of kimchi, and the high acid-producing ability can inhibit the reproduction of harmful bacteria and prolong the shelf life of kimchi.

In order to achieve the above object, the present invention proposes the technical scheme of the first aspect: a kind of Leuconostoc mesenteroides subsp. mesenteroides, which is preserved in China Type Culture Collection Center, and the preservation number is: CCTCC NO : M 2018129.

It is isolated from cherry pickles of the Korean nationality in Yanbian, Jilin, and the Leuconostoc mesenteroides subsp. Mesenteroides is obtained. The strain was identified and the result was Leuconostoc mesenteroides subsp. mesenteroides. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on March 14, 2018, and was registered in the collection center, number CCTCC NO: M 2018129, and on March 28, 2018 Day to determine the test result as survival.

The present invention proposes a technical solution of the second aspect: a preparation method of Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129, comprising the following steps : Take kimchi as the fungus source, preferably, the kimchi is sakura. Culture of Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M2018129 in medium under aerobic conditions.

The culture medium of the present invention is a conventional culture medium in the field, and it only needs to be able to grow the Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129. Preferably, the medium includes MRS liquid medium, LMM solid medium and MRS solid medium containing vancomycin, preferably, the concentration of vancomycin is 30ug/ml. When vancomycin is used at this concentration, it can ensure the best Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129, and other bacteria cannot grow at this concentration, which is more conducive to obtaining Pure Leuconostoc enteritidis subsp. enterica JNZB003. The specific cultivation and isolation steps are as follows, firstly, proliferate and cultivate Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129 in MRS liquid medium, and secondly, separate and cultivate on LMM solid medium Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129, again, isolation and purification of Leuconostoc mesenteroides subsp. Mesenteroides in MRS solid medium containing vancomycin subsp. Mesenteroides) CCTCCNO: M 2018129, wherein the plate streaking method is used for isolation and culture on solid medium.

The temperature of the cultivation is the conventional temperature and conventional time in the art, so as to be able to grow Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129, preferably, cultured in MRS liquid medium The temperature is 25-36° C., and the culture time is 24-72 hours. Further, the culture temperature in MRS liquid medium is 30° C., and the culture condition is 120-160 r/min constant temperature shaking culture for 24-72 hours. Optimally, the culture temperature in the MRS liquid medium is 30°C, and the culture condition is 140 r/min constant temperature shaking culture for 48 hours. Under this condition, Leuconostoc mesenteroides subsp. Mesenteroides) CCTCC NO: M 2018129.

Preferably, the culture temperature in the LMM solid medium and the vancomycin-containing MRS solid medium is 25-36°C, and the culture time is 24-72h. Further, in the LMM solid medium and in the MRS solid medium containing vancomycin The culture temperature of vancomycin in the MRS solid medium is 30°C, and the culture conditions are inverted constant temperature and static culture to obtain Leuconostoc mesenteroides subsp. Mesenteroides with the best performance CCTCC NO: M 2018129 .

The present invention proposes a third technical solution: application of Leuconostocmesenteroides subsp. Mesenteroides CCTCC NO: M 2018129 in pickle production.

The beneficial effect of the invention is that the leuconostoc enterococci subspecies enterococcus isolated from the Korean traditional fermented sakura has high acid production ability and nitrite degradation ability, and is resistant to salt and acid and alkali. It can improve the nutritional quality of the product and at the same time extend the storage period of the product, and has developed lactic acid bacteria resources. It is beneficial to the development of the kimchi industry and has broad market prospects.

Description of drawings

Fig. 1 is the bacterium colony shape figure that bacterial strain JNJB003 of the present invention is isolated on LMM medium;

Fig. 2 is the bacterium colony morphology figure that bacterial strain JNJB003 of the present invention purifies on MRS medium;

Fig. 3 is the Gram stain microscopic examination photo of some bacterial strains;

Fig. 4 is the bacterial strain morphological diagram after the strain in Fig. 3 through catalase experiment;

Figure 5a is a picture of the identification of the growth test of the strain at 37°C;

Figure 5b is a growth test identification picture of the strain in a medium containing 30 μg/mL vancomycin;

Fig. 5c is the identification picture of the salt tolerance test of bacterial strain;

Fig. 5d is the identification picture of the arginine hydrolysis test of the bacterial strain;

Fig. 5e is the identification picture of the escin hydrolysis test of the strain;

Fig. 5f is the identification picture of the citric acid metabolism test of bacterial strain;

Figure 5g is a picture of the glucan production and identification of the strain;

Figure 5h is a carbohydrate fermentation test identification picture of the strain;

Fig. 6 is the result figure of the strain bacterial PCR amplification product that screens out;

Figure 7 is a bar graph showing the growth of Leuconostoc enterococci subspecies JNZB003 at different temperatures;

Fig. 8 is a bar graph of the growth of Leuconostoc enteritidis subspecies JNZB003 under different salt concentration conditions;

Fig. 9 is a graph showing the change of OD value of Leuconostoc enterococci subspecies JNZB003 with culture time;

Fig. 10 is a graph showing the acid production of Leuconostoc enterococci subspecies JNZB003 over time;

Fig. 11 is a graph showing the optical density growth of Leuconostoc enterolis subspecies JNZB003 at different pH values;

Fig. 12 is a graph of the degrading ability of Leuconostoc enterica subspecies JNZB003 to different concentrations of nitrite;

Fig. 13 is a picture of an identification test for the ability of Leuconostoc enterococci subsp. enterica JNZB003 to degrade nitrite.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The control bacterial strain of the present invention is selected as Leuconostoc enterica bacterial strain (BNCC195309), obtains Leuconostoc enterococci strain by comparison of the bacterial strain, and this bacterial strain (BNCC195309) is purchased from North Nachuanglian Biotechnology Co., Ltd.; MRS solid Culture medium, MRS liquid medium, 1% vancomycin were purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Park; ; L-rhamose was purchased from Shanghai Reagent No. 2 Factory; galactose, sorbitol, cellobiose, lactose, maltose, sucrose, fructose, L-arabinose, trehalose, D-xylose, raffinose, L -Sorbose and melibiose were purchased from SIGMA; Ezup Column Bacterial Genomic DNA Extraction Kit, SanPrep Column DNA Gel Recovery Kit were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

Instruments and equipment of the present invention: PRESOCLAVE-II high-pressure sterilizer Spain SELECTA; 2720 thermalcycler PCR instrument Applied Biosystems; HC-2518R refrigerated high-speed centrifuge BBI; CX41-RF fluorescence microscope Japan OLYMPUS.

The reagents and raw materials used in the present invention are all commercially available.

The methods not mentioned in the preparation method of the present invention are conventional methods.

Embodiment 1, isolation and purification of bacterial strain JNJB003

The source of the strain: the traditional fermented sakura was taken from the Jingang Mountain pickle store, farmer’s market and supermarket in Yanbian Korean Autonomous Prefecture, and a single strain of Leuconostoc enterica was screened out by means of separation and purification. The specific method is: aseptic Under certain conditions, take 1 mL of Yanbian Korean traditional fermented cherry juice, put it in a sterilized test tube, and use the plate streaking method to draw a line in the petri dish containing LMM solid medium. Under aerobic conditions at 30°C, culture at a constant temperature for 48 hours. Pick the colonies that produce drop-shaped viscous polysaccharides from the petri dish containing LMM solid medium, streak and purify them three times in the culture dish containing MRS solid medium containing 30ug/mL vancomycin, and pick a single colony The colony was examined under a microscope and compared with the Leuconostoc enterococcus strain (BNCC195309), and it was confirmed that a single pure culture was obtained, which was named JNJB003, as shown in Figures 1 and 2 .

Embodiment 2, identification of bacterial strain JNJB003

1. Preliminary identification of strain JNJB003

Gram staining is a widely used identification method in bacteriology. Catalase test is an important test to identify Gram-positive bacteria, to further identify strains, and to isolate and purify 20 Gram staining and catalase experiments were carried out on bacterial strains suspected of Leuconostoc, as shown in Figure 3, Gram staining microscope photos of some bacterial strains, wherein, a is bacterial strain JNZB003; b is bacterial strain JNZB005; c is bacterial strain JNZB011, d is JNZB020. As shown in Figure 4, the photo of the catalase test. Microscopically, 20 strains of bacteria were Gram-positive, and 17 strains were negative for catalase. The 17 strains of catalase-negative bacteria were further identified.

2. Physiological and biochemical identification of strain JNJB003

Physiological Characterization of Strains

The 17 screened strains were identified for physiological characteristics, including 37°C growth test, 1°C growth test, catalase test, vancomycin resistance test, 3% sodium chloride growth test and 6.5% sodium chloride growth test The test results are shown in Table 1. As can be seen from Table 1, the 17 strains screened all have tolerance to 30ug/ml vancomycin, and can grow in 3% sodium chloride and 6.5% sodium chloride solution, but each bacterial strain There are great differences in the growth degree of JNZB003, JNZB009, JNZB011, JNZB012, JNZB015 and NZB019 in 3% sodium chloride and 6.5% sodium chloride solutions, and the growth rate is close to that of JNZB005 and JNZB017 in 3% Sodium chloride grows well, but grows weakly in 6.5% sodium chloride solution; 17 strains do not grow at 1°C, 11 strains grow at 37°C, and 6 strains do not grow.

Wherein, the tolerance test method to 30ug/ml vancomycin is as follows:

(1) Take the activated culture, streak it on the MRS solid medium containing 30ug/mL vancomycin, and culture it at 37°C for 24-72h. If it grows, repeat the inoculation and culture of the grown colonies again. If there are still colonies , indicating that it can grow at 37°C.

(2) Take the activated culture, streak it on the MRS solid medium containing 30ug/mL vancomycin, and culture it at 1°C for 48 hours. If it grows, repeat the inoculation and culture of the grown colonies again. If there are still colonies, it means Can grow at 1°C.

The salt tolerance test method is as follows:

Bacterial colonies were picked on a solid medium containing 30ug/mL vancomycin, inserted into MRS liquid medium containing 3.0% and 6.5% NaCl, cultured at 30°C for 24h, shaken well, and the OD ₆₀₀ value was determined to MRS liquid medium was used as blank control.

The catalase test method is as follows:

Drop 3% hydrogen peroxide on a sterilized and clean glass slide, pick the colony on the MRS solid medium, mix it with 3% hydrogen peroxide, if there are bubbles, it means a positive reaction, and if there are no bubbles, it means a negative reaction reaction.

Table 1 Identification results of physiological characteristics of strains

"+" stands for growth; "-" stands for no growth

Biochemical identification results of strain JNJB003

On the basis of the identification of the above physiological characteristics, basic biochemical identification was carried out on the 17 screened strains, including arginine hydrolysis test, citric acid utilization test, dextran production test, and escin hydrolysis test. The identification results are shown in Table 2 . As can be seen from Table 2, the arginine hydrolysis test of 17 strains was all positive reactions, which was inconsistent with the characteristics of Leuconostoc described in the "Bergey's Bacteria Identification Manual"; 7 of the 17 strains screened The strains could metabolize citric acid, 10 strains could not metabolize citric acid; 6 strains could hydrolyze aescin, 11 strains could not hydrolyze escin; 3 strains could use sucrose to produce dextran. In the Leuconostoc genus, only Leuconostoc enterica subspecies can use sucrose to produce glucan and form a viscous lawn on the medium. Other species do not have this feature. "Handbook of Bacteria Identification" and "Lactic Acid Bacteria Classification and Identification and Experimental Methods", combined with the identification of physiological characteristics, the six strains JNZB003, JNZB005, JNZB009, JNZB011, JNZB015 and JNZB018 were further tested for carbohydrate fermentation and 16S rDNA molecular identification.

All 6 strains can use cellobiose, melibiose, sucrose, fructose, L-arabinose, arrutose, trehalose, mannose, D-xylose, salicin, amygdalin; none of the 6 strains can utilize Erythritol, sorbitol, L-sorbose and L-rhamnose; in addition, strain JNZB003 can utilize galactose, maltose and ribose, the results are shown in Table 3. According to the description of Leuconostoc in "Bergey's Bacteria Identification Handbook" and "Lactic Acid Bacteria Classification and Identification and Experimental Methods", combined with morphological characteristics, physiological characteristics identification, basic biochemical identification and carbohydrate fermentation identification. It was preliminarily judged that the strain JNZB003 was Leuconostoc enterolis subspecies, JNZB005, JNZB009 and JNZB011 were Leuconostoc colder, and JNZB015 and JNZB018 were Leuconostoc sarcoides.

Among them, the arginine hydrolysis test method is as follows:

Take the activated culture, inoculate it into the arginine hydrolysis medium, drop 1 drop of sterile paraffin mineral oil on it, and incubate at 30°C for 24-72h. If the medium is still purple, it means that arginine is hydrolyzed to produce alkaline substances, a positive reaction; if it turns yellow, it means acid production, a negative reaction.

The test method for dextran production is as follows:

The colonies on the MRS solid medium were picked, streaked on the dextran production medium, and cultured at 30°C for 24-72h. Observe the colony, if a drop-shaped, sticky lawn is formed around the colony, it means that dextran is produced, and it is a positive reaction; no water drop-shaped, sticky lawn is formed, which is a negative reaction.

The citric acid utilization test method is as follows:

Take the activated culture, streak it on the citric acid utilization medium, and culture it at 30°C for 24-72 hours. If blue colonies are formed, it means that it has the ability to metabolize citric acid; if no color develops, it means that it does not metabolize citric acid.

Aescin hydrolysis test method is as follows:

Take the activated culture, inoculate it in the aescin medium, cultivate it at 30°C for 72 hours, take the aescin culture solution and place it in a colorimetric plate, add a little iron citrate dropwise, if it turns black, it means that the aescin is hydrolyzed, A positive reaction; no color development is a negative reaction. The uninoculated culture medium was used as a control.

The carbohydrate fermentation test method is as follows:

Take activated cultures, inoculate them in fermentation media containing different carbohydrates, and culture them at 30°C for 24-72 hours. If the culture medium turns yellow, it means that carbohydrates are metabolized to produce acid, which is a positive reaction; otherwise, it is a negative reaction.

Table 2 Identification results of the basic biochemical characteristics of the strains

Arginine hydrolysis: "+" means that arginine is hydrolyzed, and a purple color is a positive reaction; "-" means that arginine is not hydrolyzed, and a yellow color is a negative reaction.

Utilization of citric acid: "+" means metabolizing citric acid, forming blue colonies; "-" means not metabolizing citric acid, forming white colonies.

Dextran formation: "+" represents the formation of viscous lawns; "-" represents no formation of viscous lawns.

Aescin hydrolysis: "+" means aescin hydrolysis, black color is a positive reaction; "-" means aescin is not hydrolyzed, no color is a negative reaction.

Table 3 Biochemical identification results of carbohydrate fermentation of strains

"+" represents the use of carbohydrates to produce acid, and the yellow color is a positive reaction; "-" represents a negative reaction.

3. Molecular identification of 16S rDNA of Leuconostoc enteritidis subsp.

In order to identify strain JNJB003 more accurately and effectively, on the basis of traditional physiological and biochemical identification, molecular biology 16S rDNA sequence analysis technology was used for identification. The DNA of the 6 strains screened was extracted with the Ezup Column Bacterial Genomic DNA Extraction Kit, and a 1400±100bp band was obtained by PCR amplification. The band of the PCR amplification product was recovered and connected to Takara pMD*18- T Vector vector clones were transformed for blue and white screening, and then sequenced using M13+/- primers. The amplified product is shown in Figure 6, and the sequence of the 16S rDNA gene is shown as sequence 1 in the sequence listing.

Sequence homology comparisons were performed on the sequencing results with the BLAST program in the NCBI database, and the comparison results are shown in Table 4. As can be seen from Table 4, bacterial strain JNZB003 and Leuconostoc mesenteroides subsp. The similarity of the 16s rDNA gene sequence of LC260034.1 reached 99%, and the strain JNZB003 was identified as Leuconostoc enteritidis subsp. CCTCC NO: M 2018129, and the test result was determined to be alive on March 28, 2018.

Table 4 Alignment of 16S rDNA gene sequences of strains

Example 3, the characteristics of Leuconostoc enterococci subspecies JNZB003

Due to different sources of the same microorganism, there are certain differences in some characteristics. In order to master the characteristics of the Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 of the present invention, after the screened Leuconostoc enterocida enterica JNJB003CCTCC NO: M 2018129 was activated, the volume ratio was 2% and 3 % The culture solution was inoculated in the MRS liquid medium.

The optimal growth temperature of the strain was investigated by culturing under different temperature conditions. The results are shown in Figure 7. The growth temperature range of Leuconostoc enterococci subsp. The optimum growth temperature is 30°C.

Salt tolerance, observe the effect of different salt concentrations on the growth of Leuconostoc enterica subsp. The growth ability of JNJB003CCTCCNO: M 2018129 gradually weakened. When the salt concentration was 4% and 6%, it could grow well. When the salt concentration increased to 8%, the growth of Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 was slow. When the salt concentration reached 10%, Leuconostoc enterolis subsp. enterica JNJB003CCTCC NO: M 2018129 grew weakly.

The growth status and acid production ability were evaluated by measuring the dynamic changes of OD600 value and pH value during the growth process of the strain in MRS liquid medium. It can be seen from Figure 9 that Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 has good After 14 hours of culture, it entered a stable period, at which time the OD ₆₀₀ value reached 1.52; at 48 hours of culture, the OD ₆₀₀ value was the highest, as high as 1.909, and then the OD ₆₀₀ value began to decrease slowly, entering the decay period; The subspecies JNJB003CCTCCNO: M 2018129 produced acid quickly. After 12 hours of cultivation, the pH value dropped to 4.60. After 24 hours of cultivation, the pH value dropped to 4.16. After 28 hours of cultivation, the pH value dropped to about 4.0 and then no longer decreased. See Figure 10 for details.

Observe the growth status of Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 under different pH conditions, and investigate the acid and alkali resistance of Leuconostoc enterocida subspecies JNJB003CCTCC NO: M 2018129. The results are shown in Figure 11. The optimal growth pH value of Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 is 6. When the pH value is <4 and the pH value is >10, the growth of Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 is slow; When the value is 2 and 11, Leuconostoc enteritidis subsp. enterica JNJB003CCTCC NO: M 2018129 can also grow weakly.

Screening of the nitrite degradation ability of Leuconostoc enterica subspecies JNJB003CCTCC NO: M 2018129, see Figures 12 and 13, from which it can be seen that with the increase of nitrite concentration, Species JNJB003CCTCC NO: M 2018129 first increased and then decreased the degradation rate of nitrite. Leuconostoc enterococci subspecies JNJB003CCTCC NO: M 2018129 had the highest degradation rate of 150ug/ml nitrite for 24 hours, reaching 95.92%, followed by 75ug/ml, reaching 94.18%.

Embodiment 4, the application of Leuconostoc enterococci subspecies JNJB003 in kimchi

The pickle of the present invention can choose conventional production method, and the raw material of pickle can be selected conventional raw material, is not as the limitation of the present invention, preferably, pickle raw material is ripe cherry cherry, whole tree, and preparation method can adopt the following steps.

(1) Preparation of bacterial suspension of Leuconostoc enteritidis subsp. Leuconostoc membranaceus subsp. JNJB003CCTCC NO: M 2018129 was inoculated in MRS liquid medium and cultured at 30°C for 48 hours to obtain bacterial suspension of Leuconostoc membranaceus subsp. JNJB003CCTCC NO: M 2018129. The content of Leuconostoc enterica subspecies JNJB003CCTCC NO: M 2018129 was 118 cfu/ml.

Wherein, the solvent of MRS liquid medium is water, and the solute and its concentration are as follows: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate 2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L; pH6.8.

(2) Soak the whole cherry tree at the mature stage with 6% salt water for 12 hours, take it out, control the moisture in the cherry tree, take 500g and put it in a jar, and put the leuconostoc intestinal membrane obtained in step (1) Subspecies JNJB003CCTCC NO: M 2018129 put 300 μl of each bag of bacterial suspension into jars, mix well, seal, and ferment in a 4°C freezer to obtain traditional sakura kimchi.

Because Leuconostoc enterococci enterica subspecies is mainly enriched in the early stage of kimchi fermentation, it rapidly ferments and produces a large amount of lactic acid, which reduces the pH value of the fermentation environment, inhibits the growth of harmful microorganisms and inhibits the formation of nitrite, thereby achieving long-term Preservation, improving nutritional quality and ensuring food safety purposes. In addition, Leuconostoc enterococci subspecies JNJB003CCTCC NO: M2018129 has a higher ability to produce acid and degrade nitrite, making the quality and consumption of pickles healthier and safer.

The technical contents and technical characteristics of the present invention have been disclosed above, but those skilled in the art may still make various replacements and modifications based on the teachings and disclosures of the present invention without departing from the spirit of the present invention. Therefore, the protection scope of the present invention should not be limited to The content disclosed in the embodiment should include various replacements and modifications that do not depart from the present invention, and are covered by the claims of this patent application.

<110> Jilin Academy of Agricultural Sciences

<120> Leuconostoc enterolis subspecies, preparation method and application

<160> 1

<210> 1

<211> 1385

<212>DNA

<213> Leuconostoc mesenteroides subsp. Mesenteroides

<400> 1

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gatccgcggt gcattagtta gttggtgggg taaaggccta ccaagacaat gatgcatagc 240

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gctgcagtag ggaatcttcc acaatgggcg aaagcctgat ggagcaacgc cgcgtgtgtg 360

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cttgagtgca gtagaggtaa gtggaactcc atgtgtagcg gtggaatgcg tagatatatg 660

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gtgggtagca aacaggatta gataccctgg tagtccaacac cgtaaacgat gaacactagg 780

tgttaggagg tttccgcctc ttagtgccga agctaacgca ttaagtgttc cgcctgggga 840

gtacgaccgc aaggttgaaa ctcaaaggaa ttgacgggga cccgcacaag cggtggagca 900

tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tttgaagctt 960

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ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac accatgggag tttgtaatgc 1380

Tccaaag 1385

Claims (10)

1. A Leuconostoc mesenteroides subsp. Mesenteroides, characterized in that it is preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCCNO: M 2018129. 2. a preparation method of Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129, characterized in that it comprises the following steps: taking pickles as bacterial source, under aerobic conditions and CCTCC NO: M 2018129 was cultivated in the medium for Leuconostoc mesenteroides subsp. Mesenteroides. 3. The preparation method according to claim 2, characterized in that, said pickles are cherry blossoms. 4. The preparation method according to claim 2, characterized in that Leuconostoc mesenteroides subsp. Mesenteroides (Leuconostoc mesenteroides subsp. Mesenteroides) CCTCC NO: M 2018129 is included in the MRS liquid medium in the culture medium Proliferation and cultivation of Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129, isolation and cultivation of Leuconostoc mesenteroides subsp. Mesenteroides on LMM solid medium CCTCC NO: M 2018129 and CCTCC NO: M 2018129 for isolation and purification of Leuconostocmesenteroides subsp. Mesenteroides in MRS solid medium containing vancomycin. 5. The preparation method according to claim 4, characterized in that the vancomycin concentration is 30ug/ml. 6 . The preparation method according to claim 4 , wherein the culture temperature in the MRS liquid medium is 25-36° C., and the culture time is 24-72 hours. 7 . The preparation method according to claim 6 , wherein the culture temperature in the MRS liquid medium is 30° C., and the culture condition is 120-160 r/min constant temperature shaking culture. 8. The preparation method according to claim 4, characterized in that, the culture temperature in the LMM solid medium and in the MRS solid medium containing vancomycin is 25-36°C, and the culture time is 24-72h . 9. The preparation method according to claim 8, characterized in that, the culture temperature in the LMM solid medium and in the MRS solid medium containing vancomycin is 30°C, and the culture condition is an inverted constant temperature static culture . 10. The application of Leuconostoc mesenteroides subsp. Mesenteroides CCTCC NO: M 2018129 according to claim 1 in the production of kimchi.