Lactobacillus plantarum NHE-LpB6401 and application thereof

文档序号：30142 发布日期：2021-09-24 浏览：36次中文

阅读说明：本技术 一株植物乳杆菌NHE-LpB6401及应用 (Lactobacillus plantarum NHE-LpB6401 and application thereof ) 是由张遨然尹望刘武蔡煕姮王红梅郭建强周航杨颜铱周桂莲李巍于 2021-07-16 设计创作，主要内容包括：本发明提供了一株植物乳杆菌(Lactobacillus plantarum)NHE-LpB6401,该菌分离自樱桃谷肉鸭肠道,保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮政编码：100101),保藏编号为CGMCC NO.22645,保藏日期为2021年5月31日。该菌株具有繁殖速度快、耐人工胃肠液、发酵性能强、抑菌性能强、产酸性和发酵性能强。能提高动物健康状态,改善生长性能,降低生产成本,提高经济效益,具有良好的应用前景。(The invention provides Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpB6401, which is separated from the intestinal tract of cherry valley meat ducks and is preserved in the general microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, with the address of the microbiological research institute of China academy of sciences No. 3, West Lu No.1, North Cheng, the area of the south of Beijing city, the postal code of 100101), the preservation number of CGMCC NO.22645 and the preservation date of 2021 year, 5 months and 31 days. The strain has the advantages of high propagation speed, artificial gastrointestinal fluid resistance, strong fermentation performance, strong bacteriostatic performance, strong acidity production and strong fermentation performance. Can improve the health state of animals, improve the growth performance, reduce the production cost, improve the economic benefit and have good application prospect.)

1. The Lactobacillus plantarum NHE-LpB6401 is classified and named as Lactobacillus plantarum NHE-LpB6401, and is preserved in China general microbiological culture Collection center (CGMCC) at 31/5/2021 with the preservation number of CGMCC NO.22645 and the preservation address of Beijing, China.

2. A liquid microbial inoculum containing lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No. 22645.

3. A microecological preparation and animal feed prepared from Lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No. 22645.

4. Use of lactobacillus plantarum NHE-LpB6401 according to any one of claims 1 to 3, having a accession number CGMCC No.22645, as a feed additive for animal drinking water.

5. The use of Lactobacillus plantarum NHE-LpB6401 having a accession number CGMCC No.22645 as claimed in claim 4 in a feed additive for animal drinking water, wherein the number of viable Lactobacillus plantarum NHE-LpB6401 in the feed additive is 1.0X 10⁶-1.0×10¹⁰CFU/g, preferably the viable count of Lactobacillus plantarum NHE-LpB6401 in the feed additive is 1X 10⁶CFU/g。

6. Use of lactobacillus plantarum NHE-LpB6401 according to any one of claims 1 to 3, having a accession number CGMCC No.22645, or a microbial inoculum containing same, for increasing feed conversion ratio and animal productivity.

7. Use of lactobacillus plantarum NHE-LpB6401 according to any one of claims 1 to 3, having a accession number CGMCC No.22645, or a bacterial agent comprising same, for the preparation of a medicament for the prevention or treatment of diarrhea in an animal.

8. Use of lactobacillus plantarum NHE-LpB6401 according to any one of claims 1 to 3, having a accession number CGMCC No.22645, or a microbial inoculum comprising same, for the preparation of an antimicrobial microbial preparation.

9. The use of lactobacillus plantarum NHE-LpB6401 having accession number CGMCC No.22645 or an agent comprising same as claimed in claim 8 for the preparation of an antibacterial microbial preparation comprising the following bacteria: enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, shigella, and clostridium perfringens.

10. Use of lactobacillus plantarum NHE-LpB6401 containing the accession number CGMCC No.22645 or a microbial inoculum containing the same as in any one of claims 1 to 3 in the production of cherry valley duck.

Technical Field

The invention relates to probiotics for livestock feeding, in particular to a probiotic Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpB6401 strain and application thereof.

Background

Lactobacillus plantarum (Lactobacillus plantarum) is one of lactic acid bacteria, the optimal growth temperature is 30-35, the Lactobacillus plantarum is anaerobic or facultative anaerobic, the strain is a straight or bent rod, the strain is single or sometimes paired or chained, the optimal pH value is about 6.5, and the Lactobacillus plantarum belongs to homofermentation lactic acid bacteria. The biological characteristics are as follows: the amount of the Bacillus circulans is usually 0.9-1.2 vtm × 3.0-8.0 μm, single, paired or short chain. Usually lacking flagella, but able to move. Gram positive, no sporulation. Facultative anaerobe, surface colony diameter about 3mm, convex, round, smooth surface, fine, white, occasionally light yellow or dark yellow. Belongs to chemoheterotrophic bacteria, needs a nutrient-rich culture medium for growth, needs calcium pantothenate and nicotinic acid, but does not need thiamine, pyridoxal or pyridoxamine, folic acid and vitamin B12. Can ferment pentose or gluconate, and the final product contains lactic acid more than 85%. Nitrate is generally not reduced, gelatin is not liquefied, and both catalase and oxidase are negative. Can produce DL-lactic acid, has the activity of fructose-1, 6-diphosphate aldolase and hexose monophosphate pathways, can grow in gluconate, and produces C02. Fermentation of 1 molecule of ribose or other pentose sugar produces 1 molecule of lactic acid and 1 molecule of acetic acid. Can grow at 15 ℃, and the optimal growth temperature is 30-35 ℃ generally.

The industrial application of lactobacillus plantarum reported at present is commonly applied to food fermentation, such as fermented milk production, and also can be applied to heavy metal degradation, and the lactobacillus produced in the propagation process can be used as a natural preservative, so the lactobacillus plantarum can also be used in the field of production and cultivation.

Disclosure of Invention

The invention aims to provide Lactobacillus plantarum (NHE-LpB 6401) with a probiotic effect and application thereof.

In order to achieve the purpose of the invention, the Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpB6401 provided by the invention is a Lactobacillus plantarum strain separated from intestinal contents of healthy cherry valley meat ducks, is determined to be Lactobacillus plantarum through colony morphology observation, physiological and biochemical characteristics, molecular biology identification and the like, and has a 16S rDNA sequence shown in SEQ ID No. 1. The strain has the advantages of high propagation speed, artificial gastrointestinal fluid resistance, strong fermentation performance, strong bacteriostatic performance, strong acidity production and strong fermentation performance.

The Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpB6401 provided by the invention has been deposited in China general microbiological culture Collection center (CGMCC for short, address: Beijing West Lu No.1 of the sunward Chen, Ministry of microbiology, China academy of sciences, zip code 100101) in 2021, 5 and 31 days, and is classified and named as Lactobacillus plantarum (CGMCC No.22645) with the preservation number of CGMCC No. 22645.

The Lactobacillus plantarum NHE-LpB6401 provided by the invention has the following microbiological characteristics: lactobacillus plantarum NHE-LpB6401 is gram-positive bacillus, grows well on MRS agar medium, is cultured for 18h, has a colony diameter of 3-4mm, is round, milky white, smooth in surface, convex in the middle, and neat in edge, has a colony morphology shown in figure 1, has a linear rod shape under a microscope, grows in a facultative anaerobic manner, and has a strain morphology shown in figure 2 after gram staining; the lactobacillus plantarum NHE-LpB6401 has a suitable growth temperature range: 10-60 ℃, optimum growth temperature: 25-38 ℃; the growth is suitable for pH3.5-8, and the optimum pH value is 5-7. Some physiological and biochemical properties are shown in Table 1.

The invention provides a liquid microbial inoculum containing lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No. 22645.

The invention provides a microecological preparation and animal feed prepared from lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No. 22645.

The invention provides a feed additive for animal drinking water, which contains lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No. 22645.

The viable count of Lactobacillus plantarum NHE-LpB6401 in the feed additive is 1.0 × 10⁶-1.0× 10¹⁰CFU/g. Preferably, the viable count of the lactobacillus plantarum NHE-LpB6401 in the feed additive is 1 x 10⁶CFU/g。

The invention provides application of lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No.22645 or a microbial inoculum containing the lactobacillus plantarum to the improvement of feed conversion rate and animal production performance.

The invention provides application of lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No.22645 or a microbial inoculum containing the lactobacillus plantarum in preparing a medicament for preventing or treating animal diarrhea.

The invention provides application of lactobacillus plantarum NHE-LpB6401 with the preservation number of CGMCC No.22645 or a microbial inoculum containing the lactobacillus plantarum in preparing an antibacterial microbial preparation.

The method mainly comprises the following steps of resisting the following bacteria: enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, shigella, and clostridium perfringens.

In addition, the invention further provides application of the lactobacillus plantarum NHE-LpB6401 in production of cherry valley meat ducks.

The culture method of the lactobacillus plantarum NHE-LpB6401 comprises the following steps:

taking lactobacillus plantarum NHE-LpB6401 (preservation number is CGMCC No.22645) seed liquid (viable bacteria concentration is 10)⁹CFU/mL)3mL, inoculating the mixture into 300mL of shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation is finished, fermentation culture is carried out in a fermentation tank, 300mL of shake flask fermentation seed liquid is inoculated into a fermentation culture medium in a 50L fermentation tank for fermentation culture, and the liquid loading capacity of the 50L fermentation tank is 35L of the fermentation culture medium. After the fermentation is finished, detecting that the number of viable bacteria in the fermentation liquor is 3.4 multiplied by 10¹⁰CFU/mL。

The shake flask fermentation medium consists of the following components: 0.5-4% of cane sugar, 0.5-2.5% of glucose, 0.5-3.0% of yeast extract powder, 0.5-2.5% of soybean peptone, 0.01-0.5% of magnesium chloride, 0.01-1.0% of calcium carbonate, 0.01-0.5% of manganese sulfate and the balance of water.

Preferably: 1.2% of sucrose, 0.8% of glucose, 0.5% of yeast extract powder, 1.2% of soybean peptone, 0.1% of magnesium chloride, 0.1% of calcium carbonate, 0.04% of manganese sulfate and the balance of water.

The shake flask fermentation conditions are as follows: the inoculation amount is 1 percent (volume ratio), the fermentation temperature is 35 ℃, the pH value is 5.8, 200r/min, and the fermentation time is 6 hours.

The culture medium components and the fermentation conditions of the 50L fermentation tank are as follows: 1.0-4.0% of soft sugar, 0.2-2.0% of glucose, 0.5-3.5% of yeast extract, 0.5-2.5% of soybean peptone, 0.5-2.5% of corn dry powder, 0.01-0.5% of magnesium chloride, 0.01-1.0% of calcium carbonate, 0.01-0.5% of manganese sulfate, 0.78-0.2% of tween-800.05 and the balance of water.

Preferably: 1.0% of soft sugar, 0.5% of glucose, 0.5% of yeast extract, 0.8% of soybean peptone, 1% of corn dry powder, 0.1% of magnesium chloride, 0.1% of calcium carbonate, 0.04% of manganese sulfate, 800.1% of tween-800 and the balance of water.

The shake flask fermentation conditions are as follows: the liquid loading amount of a 50L fermentation tank is 35L of culture medium, the tank pressure is controlled to be 0.05-0.06MPa, the inoculation amount is 300mL, the fermentation temperature is 35 ℃, the fermentation time is 11h, the pH value is 5.8, and the stirring speed is 200 r/min.

The detection method for the lactobacillus plantarum NHE-LpB6401 in breeding generations comprises the following steps:

inoculating lactobacillus plantarum NHE-LpB6401 suspension into a sterile MRS broth culture medium according to the inoculation amount of 1% (v/v), loading the suspension into a liquid volume of 300mL/1000mL, carrying out shaking culture at 37 ℃ and 180r/min, measuring the viable count of lactobacillus in the MRS broth culture medium every half hour, drawing a growth curve to obtain the viable count change of the lactobacillus in the logarithmic growth phase, and then calculating the reproduction generation according to a formula, wherein the reproduction generation time of lactobacillus plantarum NHE-LpB6401 is 18 min/generation.

The generation time is calculated according to the following formula:wherein X1 is the number of bacteria at the beginning of log phase (T1); x2 is the number of late log phase (T2) bacteria; g is generation time, namely the reproduction generation time.

The preparation method of the lactobacillus plantarum NHE-LpB6401 microecological preparation comprises the following steps:

and (2) centrifuging the lactobacillus plantarum NHE-LpB6401 (with the preservation number of CGMCC No.22645) fermentation liquor at 13000r/min for 10min to obtain lactobacillus plantarum NHE-LpB6401 active bacterial sludge, and then uniformly mixing the lactobacillus plantarum active bacterial sludge and water according to the weight ratio of 1:2 to obtain high-concentration bacterial suspension. Uniformly mixing the lactobacillus plantarum NHE-LpB6401 thallus suspension and the protective agent according to the weight ratio of 1:2.2 to prepare microcapsule wet powder, preparing wet granules by a granulator, uniformly mixing the wet granules with the coating agent solution, putting the mixture into a fluidized drying bed, and drying and coating to obtain the NHE-LpB6401 microecological preparation, wherein the effective viable count reaches 5.0 multiplied by 10¹⁰CFU/g。

The protective agent is: 10-70% of corn starch, 1-5% of sodium carboxymethylcellulose, 1-8% of glucan, 0.5-8% of inulin, 5-20% of skimmed milk powder, 5-20% of gelatinized modified starch, 0.5-5% of sucrose, 0.5-5% of peptone, 0.1-2.5% of trehalose, 1-10% of glycerol, 1-10% of microcrystalline cellulose and the balance of water.

Preferably: 40% of corn starch, 2% of sodium carboxymethylcellulose, 2% of glucan, 2.4% of inulin, 15% of skimmed milk powder, 10% of gelatinized modified starch, 5% of sucrose, 2% of peptone, 2% of trehalose, 5% of glycerol, 5% of microcrystalline cellulose and the balance of water.

The coating agent solution is as follows: 2-20% of sodium alginate, 10-25% of hydroxymethyl cellulose, 10-30% of chitosan, 1-10% of mannan and the balance of water.

Preferably: 10% of sodium alginate, 6.5% of hydroxymethyl cellulose, 8.5% of chitosan, 4% of mannan and the balance of water.

The probiotic test method of lactobacillus plantarum NHE-LpB6401 of the invention is as follows:

on a sterile operating table, the concentration is 10⁹Adding bacterial suspension of CFU/mL pathogenic bacteria (enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Shigella, and Clostridium perfringens) into nutrient agar (the culture medium is changed into tryptone-sulfite-cycloserine agar by Clostridium perfringens, and strict anaerobic condition is required for culture condition) cooled to 45 deg.C, mixing, and making into pathogenic bacteria agar plate with thickness of about 4mm, wherein the concentration of pathogenic bacteria in the agar plate is 10⁹CFU/mL. The sterilized Oxford cup was placed on the medium, and lightly pressurized to make it contact with the medium without any gap, after 10 minutes, 200. mu.L of the fermentation broth prepared in example 2 was dropped into each vial, respectively, without overflowing, and cultured at 37 ℃ for 36-96 hours, and then the diameter of the zone of inhibition was measured. Each experiment was repeated three times, and the average was taken,

the nutrient agar culture medium comprises the following components in percentage by weight: 1% of peptone; 0.3% of beef extract; 0.5 parts of NaCl; the balance being water, pH 7.2 + -0.2.

The tryptone-sulfite-cycloserine agar culture medium comprises the following components in percentage by weight: tryptone 1.5%, soytone 0.5%, yeast powder 0.5%, sodium pyrosulfite 0.1%, ferric ammonium citrate 0.1%, agar 2%, and water in balance, pH 7.6 + -0.2, and filtering sterilized 0.5% D-cycloserine solution 20mL/250mL when cooled to 50 ℃ in use.

The stress resistance test method of the lactobacillus plantarum NHE-LpB6401 comprises the following steps:

1. measuring the artificial gastric juice resistance:

putting 10mL of lactobacillus plantarum NHE-LpB6401 bacterial suspension into 90mL of artificial gastric juice (250mL of triangular flask) and shaking at constant temperature of 200r/min at 37 ℃ for 180 min; after shaking, 10mL of sample solution is taken to adjust the pH value to 7.0, 90mL of normal saline is added, shaking is carried out at the constant temperature of 37 ℃ and 200r/min for 30min, and then dilution plate colony culture counting is carried out.

The preparation method of the artificial gastric juice comprises the following steps: according to the preparation method in the pharmacopoeia of the people's republic of China 2010, 16.4mL of dilute hydrochloric acid is taken, about 800mL of water and 10g of pepsin are added, the mixture is uniformly shaken, then the diluted mixture is diluted into 1000mL of water, the pH values are adjusted to be 1.5, 2.0 and 2.5 respectively, and a microporous filter membrane is sterilized (0.22 mu m) for later use.

2. Determination of resistance to artificial intestinal juice

Putting 1mL of lactobacillus plantarum NHE-LpB6401 bacterial suspension into 99mL (250mL triangular flask) of artificial intestinal juice, shaking at constant temperature of 37 ℃ and 200r/min for 5h, adding 99mL of physiological saline into 1mL of sample solution after shaking is finished, shaking at constant temperature of 37 ℃ and 200r/min for 30min, and then performing dilution plate colony culture counting.

The preparation of the artificial intestinal juice comprises the following steps: the preparation of the artificial intestinal juice refers to a preparation method in pharmacopoeia of the people's republic of China 2010, phosphate buffer (containing pancreatin) (pH6.8), 6.8g of monopotassium phosphate is taken, 500mL of water is added for dissolving, and the pH value is adjusted to 6.8 by 0.1mol/L of sodium hydroxide solution; dissolving pancreatin 10g in water, mixing the two solutions, diluting with water to 1000mL, and filtering with 0.22 μm microfiltration membrane for sterilization.

3. Determination of the bile salt resistance

Putting 1mL of lactobacillus plantarum NHE-LpB6401 bacterial suspension into 99mL (250mL of triangular flask) of solutions with different cholate concentrations, wherein the cholate concentrations are 0.15%, 0.3%, 1% and 1.5%, shaking at the constant temperature of 200r/min at 37 ℃ for 120min, adding 99mL of physiological saline into 1mL of sample solution after shaking is finished, shaking at the constant temperature of 200r/min at 37 ℃ for 30min, and then performing dilution plate colony culture counting.

The preparation method of the bile salt solution with different concentrations comprises the following steps: adding 9mL, 18mL, 60mL and 90mL of 5% bile salt solution into PBS solution with pH7.4, diluting to 300mL, and mixing to obtain PBS solution containing 0.15%, 0.30%, 1% and 1.5% bile salt.

The preparation method of the 5% bile salt solution comprises the following steps: accurately weighing 5.0g of bile salt, dissolving with 100ml of LPBS solution to constant volume, and sterilizing at 121 deg.C for 20 min.

The preparation method of the PBS solution comprises the following steps: 0.8% of sodium chloride, 0.02% of potassium chloride, 0.363% of disodium hydrogen phosphate, 0.024% of potassium dihydrogen phosphate and the balance of water. Adjusting pH to 7.4 with 6mol/L HCl, and sterilizing at 121 deg.C for 20 min.

The invention discloses a method for detecting lactobacillus plantarum NHE-LpB6401 for preventing and treating diarrhea, which comprises the following steps:

selecting 60 common Kunming mice, female, 11-13g, and conventionally feeding. The mice are randomly divided into three groups, and each group is fed with basic ration for 5 days, so that the mice can adapt as soon as possible. The formal test is started and divided into two stages. The first stage of growth performance observation test, experimental group added with lactobacillus plantarum fermentation liquor with the concentration of 1 × 10⁶CFU/mL. The control group and the negative control group were drinking purified water without lactobacillus plantarum for two weeks. The mice were weighed 1 time before and after the test, the growth of the mice was observed during the test, and the average growth rate of the body weight of each group was calculated, and the results are shown in table 5. Performing salmonella challenge test in the second stage, performing salmonella challenge on the negative control group and the test group, wherein the challenge mode adopts intragastric administration, specifically intragastric administration of 0.3 mL/0.2% sodium bicarbonate solution, and the intragastric administration concentration is 10 after 5min⁹The order of magnitude of Salmonella is 0.8 mL/mouse, and the control group is intragastrically administered with 0.8 mL/mouse of physiological saline. Observing the mental state and death condition of mice after challenge, and continuing drinking 1 × 10⁶The lactobacillus plantarum NHE-LpB6401 fermentation liquor with the concentration of CFU/mL, and the control group and the negative control group normally drink tap water. During the test period, mice are raised in cages in the same room, naturally illuminated and freely fed. The environmental temperature is controlled to be 25 +/-2 ℃, the humidity is 60 percent,

the invention has the beneficial effects that:

(1) the lactobacillus plantarum NHE-LpB6401 has the advantages of high propagation speed, strong acid production capacity, strong antibacterial performance and strong stress resistance; through the optimization of a fermentation culture medium and a process,fermenting at 35 deg.C for 11 hr to obtain fermentation liquid with viable count of 3.4 × 10¹⁰CFU/mL is more suitable for the development of feed industry and livestock and poultry breeding industry, can produce microecologics, is applied to animal production, can greatly reduce production cost and improve economic benefit due to high propagation speed, and lays a solid strain foundation for future development.

(2) The lactobacillus plantarum NHE-LpB6401 can be used for preparing animal microecological preparations, greatly reduces the fermentation cost, can effectively reduce the use cost, and lays a foundation for large-scale use.

(3) The lactobacillus plantarum NHE-LpB6401 can effectively maintain the balance of animal intestinal flora, improve the intestinal performance and improve the animal production performance. Through a mouse challenge test, the average weight increase rate of a test mouse is obviously higher than that of a control group (P is less than 0.05); after salmonella is attacked, the mental state and the survival rate of the mice in the test group are obviously higher than those of the mice in the negative control group. The lactobacillus plantarum NHE-LpB6401 bacterial liquid fed by the strain can effectively prevent and treat the diarrhea of the mice, improve the survival rate of the mice attacked by pathogenic bacteria, and can promote the growth of the mice.

(4) The lactobacillus plantarum NHE-LpB6401 microecological preparation is applied to the production of cherry valley meat ducks, after the lactobacillus plantarum NHE-LpB6401 microecological preparation is added into daily ration, the growth performance is obviously improved, the feed-meat ratio can be obviously reduced (P is less than 0.05), the daily gain and the feed intake are improved, the death and culling rate is improved, the production cost is reduced, and the culture benefit is improved.

Drawings

FIG. 1 is a colony morphology of Lactobacillus plantarum NHE-LpB6401 on MRS medium.

FIG. 2 is a gram stain of Lactobacillus plantarum NHE-LpB6401 strain.

Detailed Description

The invention is described below by means of specific embodiments. The embodiments are to be considered as illustrative and not restrictive in character, the spirit and scope of the invention being limited only by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.

Example 1 isolation, screening and characterization of Lactobacillus plantarum NHE-LpB6401

1. And (3) separating and purifying lactic acid bacteria:

slaughtering healthy cherry valley meat ducks to take intestinal tracts, quickly collecting 2.5 g of intestinal tract contents under an aseptic condition, placing the intestinal tract contents in a triangular flask containing 22.5mL of sterilized normal saline, shaking for 1h at a constant temperature of 37 ℃, sequentially diluting to 10 ten thousand times by adopting a 10-time-to-dilution method, selecting three dilution degrees of 1000 times, 1 ten thousand times and 10 ten thousand times, sucking 0.1mL of the dilution to coat on an improved MRS agar plate, carrying out inverted culture for 48h at 37 ℃ after coating the plate, picking bacterial colonies of suspected lactic acid bacteria with obvious calcium-soluble rings and larger than 5mm on the MRS agar plate by using an inoculating ring, carrying out streaking separation culture, picking bacterial colonies with good separation effects after 48h culture, transferring the bacterial colonies to an MRS agar inclined plane by using the inoculating ring for pure culture, repeatedly carrying out passage pure culture for 3 times, suspending the bacterial cells in a 20% glycerol solution, and storing the bacterial cells in a refrigerator at-80 ℃ for later use.

2. Observation of colony morphology:

according to the size of the calcium dissolving ring in the step 1, the strains with strong acid production capacity are preliminarily screened out and can be preliminarily determined as the lactic acid bacteria, and the total number of the strains is 256. Activating the glycerol tube strain preserved in the step 1 for 2-3 times by using an MRS agar plate, then inoculating the glycerol tube strain into an MRS broth culture medium, carrying out shake culture at the constant temperature of 37 ℃ for 18-20 h at constant temperature of 180r/min, taking a clean glass slide for gram staining and microscopic examination, observing the microscopic morphology of the strain, and selecting bacillus-producing-free bacteria with gram staining as positive for later use.

3. Preparation of lactic acid bacteria suspension and fermentation liquor

Streaking the lactic acid bacteria obtained in the step 1 on an MRS agar plate, culturing for 48h at 37 ℃, picking single bacterial colony from the plate, performing shake culture for 24h at 37 ℃ and 180r/min in 100mL of MRS liquid culture medium, and obtaining lactic acid bacteria suspension for later use; and continuing to culture at 37 ℃ and shaking at 180r/min for 96h to obtain lactobacillus fermentation liquor for later use.

4. Screening acid production capacity of lactic acid bacteria:

inoculating the lactobacillus suspension obtained in the step 3 into an MRS liquid culture medium according to the inoculation amount of 1% (volume ratio), culturing at 37 ℃ for 12h, screening out lactobacillus which is 12h and enables the pH of the fermentation liquor to be lower than 4.2, and obtaining 56 strains of lactobacillus in total.

5. Screening of bacteriostatic lactic acid bacteria:

taking pathogenic bacteria (enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Shigella, and Clostridium perfringens) with concentration of 10⁹2mL of CFU/mL of bacterial suspension is added into 200mL of nutrient agar culture medium which is sterilized and then cooled to about 45 ℃, 10mL of unset bacteria-carrying culture medium is sucked and transferred onto the nutrient agar plate which is poured with 10mL of bottom plate, and a plurality of pathogen plates are prepared (the culture medium of pathogen clostridium perfringens is changed into tryptone-sulfite-cycloserine agar, and the culture condition needs strict anaerobic condition). Clamping 1 sterilized Oxford cup (a round small tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10 mm) on each pathogen nutrition agar plate on an ultraclean workbench by using sterile tweezers, putting the sterilized Oxford cups on a plate, enabling the sterilized Oxford cups to be in contact with a culture medium without gaps, after several minutes, respectively sucking 200 mu L of suspected lactobacillus strain fermentation liquor (obtained in the step 3) into the Oxford cups, and culturing at the constant temperature of 37 ℃ for 24 hours. At least 3 times of each strain are repeated, and the size of the inhibition zone is observed and measured, wherein the number of the strains with the large inhibition zone is 9, and the strains are respectively marked as B802, B3601, B4401, B5202, B6401, B6802, B7201, B7202 and B8003.

6. Screening of the reproductive performance of the lactic acid bacteria:

inoculating the lactic acid bacteria suspension prepared in the step 3 into a sterile MRS broth culture medium according to the inoculation amount of 1% (v/v), loading the liquid in the MRS broth culture medium for 300mL/1000mL, carrying out shaking culture at 37 ℃ for 180r/min, measuring the number of live bacteria in the MRS broth culture medium every half hour, drawing a growth curve to obtain the change of the number of live bacteria in the logarithmic growth phase of the lactic acid bacteria, and then calculating the breeding generations according to a formula to obtain 4 lactic acid bacteria with strong breeding performance, namely B3601, B6401, B7202 and B8003 respectively, wherein the breeding generations are less than 25 minutes.

7. And (3) screening the temperature resistance of lactic acid bacteria:

and (3) putting 10mL of the lactic acid bacteria suspension obtained in the step (3) into a sterile test tube, then putting the test tube into a water bath kettle at 55, 65 and 75 ℃ for treatment for 30min, then measuring the number of the viable bacteria of the treated lactic acid bacteria, and calculating the survival rate according to the number of the viable bacteria before treatment. Obtaining the lactic acid bacteria B6401 with the best temperature resistance, treating the lactic acid bacteria B6401 in a water bath kettle at the temperature of 75 ℃ for 30min, wherein the survival rate reaches 92.23%, and taking the lactic acid bacteria B as the strains to be researched next step.

8. Identification of the strain genus:

the strain B6401 is subjected to morphological, physiological and biochemical identification, after the strain B6401 grows on an MRS agar plate for 24 hours, a circular colony with the diameter of 2-3mm is formed, the colony is milky, smooth and opaque in surface, and is raised, neat in edge and easy to pick up, the colony is in a shape shown in a figure 1, the microscopic shape is in a figure 2, the colony is gram-positive, rod-shaped, free of spores, motionless and facultative anaerobic, the growth suitable temperature range is 4-65 ℃, the optimal growth temperature is 15-45 ℃, the growth pH is 3.5-8.5, and the optimal pH is 4.5-7.5. The strain species are identified by performing physiological and biochemical tests on the strain B6401 according to a common bacteria system identification manual. The results showed that strain B6401 was lactobacillus.

TABLE 1 part of the physio-biochemical characteristics of Strain B6401

Item	B6401	Item	B6401
				Aerobic growth	+	Mannitol	+
Anaerobic growth	+	Mannose	+
				Contact enzyme	-	Sorbose	+
Production of ammonia from arginine	-	Sucrose	+
				Shushu sugar	-	Lactose	+
Galactose	+	Maltose	+
				Melibiose	+	Cellobiose	+
Glucose	+	Trehalose	+
				Fructose	+	Arabinose	+

Note: "+" indicates positive reaction; "-" indicates negative reaction.

9. 16S rDNA sequencing assay:

and extracting the B6401 strain genome DNA by using a kit for extracting bacterial DNA. Sequencing the 16S rDNA gene fragment of the B6401 strain through primers F and R to obtain a sequence shown as SEQ ID NO.1, and comparing the determined sequence with the 16S rDNA sequence in GenBank by BLAST analysis, the homology of the B6401 strain and the lactobacillus plantarum can reach 100%. The strain B6401 is determined to be Lactobacillus plantarum (Lactobacillus plantarum) through morphological characteristics, physiological and biochemical characteristics and 16S rDNA characteristics of the strain B6401, and is formally marked as NHE-LpB 6401.

10. Strain preservation:

the Lactobacillus plantarum NHE-LpB6401 obtained by separation, purification and screening is preserved in China general microbiological culture Collection center (CGMCC, the address is the institute of microbiology of China academy of sciences 3, North road 1 institute of North Cheng, south China, Beijing City, postal code: 100101), the preservation number is CGMCC NO.22645, and the Lactobacillus plantarum is classified and named as Lactobacillus plantarum (Lactobacillus plantarum) in 2021, 31.5.31.1.

The improved MRS agar culture medium comprises the following components: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, Tween 800.1%, and K₂HPO₄ 0.2％，MgSO₄0.058 percent of ammonium citrate, 0.2 percent of diammonium citrate and MnSO₄0.025 percent, agar 1.8 percent, calcium carbonate 1 percent and the balance of water, and the pH value is 7.0 +/-0.2.

The MRS agar culture medium comprises the following components: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, Tween 800.1%, and K₂HPO₄ 0.2％，MgSO₄0.058 percent of ammonium citrate, 0.2 percent of diammonium citrate and MnSO₄0.025 percent, agar 1.8 percent and the balance of water, and the pH value is 7.0 +/-0.2.

The MRS broth culture medium comprises the following components: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, Tween 800.1%, and K₂HPO₄ 0.2％，MgSO₄0.058 percent of ammonium citrate, 0.2 percent of diammonium citrate and MnSO₄0.025%, and the balance of water, and the pH value is 7.0 +/-0.2.

The nutrient agar culture medium comprises the following components: peptone 1%, beef extract 0.3%, agar 2%, NaCl 0.5%, and water in balance, and pH 7.2 + -0.2.

The tryptone-sulfite-cycloserine agar culture medium comprises the following components: tryptone 1.5%, soytone 0.5%, yeast powder 0.5%, sodium pyrosulfite 0.1%, ferric ammonium citrate 0.1%, agar 2%, and water in balance, pH 7.6 + -0.2, and filtering sterilized 0.5% D-cycloserine solution 20mL/250mL when cooled to 50 ℃ in use.

EXAMPLE 2 preparation of Lactobacillus plantarum NHE-LpB6401 fermentation broth

Taking lactobacillus plantarum NHE-LpB6401 (preservation number is CGMCC No.22645) seed liquid (viable bacteria concentration is 10)⁹CFU/mL)3mL, inoculating the mixture into 300mL of shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation is finished, fermentation culture is carried out in a fermentation tank, 300mL of shake flask fermentation seed liquid is inoculated into a fermentation culture medium in a 50L fermentation tank for fermentation culture, and the liquid loading capacity of the 50L fermentation tank is 35L of the fermentation culture medium. The fermentation is finishedThen, the number of viable bacteria in the fermentation liquor is detected to be 3.4 multiplied by 10¹⁰CFU/mL。

EXAMPLE 3 preparation of Lactobacillus plantarum NHE-LpB6401 Microecological preparation

The lactobacillus plantarum NHE-LpB6401 (preservation number is CGMCC No.22645) is prepared into NHE-LpB6401 fermentation broth by the method of the embodiment 2, the fermentation broth is centrifuged at 13000r/min for 10min to obtain lactobacillus plantarum NHE-LpB6401 active bacterial sludge, and then the lactobacillus plantarum active bacterial sludge and water are uniformly mixed according to the weight ratio of 1:2 to obtain high-concentration bacterial suspension. Uniformly mixing the lactobacillus plantarum NHE-LpB6401 thallus suspension and the protective agent according to the weight ratio of 1:2.2 to prepare microcapsule wet powder, preparing wet granules by a granulator, and then preparing the granulesMixing with coating agent solution, adding into fluidized drying bed, drying and coating to obtain NHE-LpB6401 microecological preparation with effective viable count of 5.0 × 10¹⁰CFU/g。

The protective agent is: 40% of corn starch, 2% of sodium carboxymethylcellulose, 2% of glucan, 2.4% of inulin, 15% of skimmed milk powder, 10% of gelatinized modified starch, 5% of sucrose, 2% of peptone, 2% of trehalose, 5% of glycerol, 5% of microcrystalline cellulose and the balance of water.

The coating agent solution is as follows: 10% of sodium alginate, 6.5% of hydroxymethyl cellulose, 8.5% of chitosan, 4% of mannan and the balance of water.

Example 4 Lactobacillus plantarum NHE-LpB6401 probiotic validation

On a sterile operating table, the concentration is 10⁹Adding bacterial suspension of CFU/mL pathogenic bacteria (enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Shigella, and Clostridium perfringens) into nutrient agar (the culture medium is changed into tryptone-sulfite-cycloserine agar by Clostridium perfringens, and strict anaerobic condition is required for culture condition) cooled to 45 deg.C, mixing, and making into pathogenic bacteria agar plate with thickness of about 4mm, wherein the concentration of pathogenic bacteria in the agar plate is 10⁹CFU/mL. The sterilized Oxford cup was placed on the medium, and lightly pressurized to make it contact with the medium without any gap, after 10 minutes, 200. mu.L of the fermentation broth prepared in example 2 was dropped into each vial, respectively, without overflowing, and cultured at 37 ℃ for 36-96 hours, and then the diameter of the zone of inhibition was measured. Three replicates of each experiment were averaged and the results are shown in table 2.

The nutrient agar culture medium comprises the following components in percentage by weight: 1% of peptone; 0.3% of beef extract; 0.5 parts of NaCl; the balance being water, pH 7.2 + -0.2.

TABLE 2 bacteriostatic effect of Lactobacillus plantarum NHE-LpB6401 on pathogenic bacteria

Pathogenic bacteria	Bacteriostatic diameter (mm)
		Intestinal pathogenic Escherichia coli	18.22
Staphylococcus aureus StBphylococcus Bureus	31.23
		Salmonella typhimurium	21.15
Salmonella	25.67
		Shigella Shigella Castellani	14.23
Clostridium perfringens Clostridium perfringens perfringen	16.75

Example 5 Lactobacillus plantarum NHE-LpB6401 stress resistance validation

1. Measuring the artificial gastric juice resistance:

10mL of lactobacillus plantarum NHE-LpB6401 bacterial suspension (prepared by the method described in example 1) is placed in 90mL of artificial gastric juice (250mL of triangular flask) and shaken at constant temperature of 200r/min and 37 ℃ for 180 min; after shaking, 10mL of sample solution is taken to adjust the pH value to 7.0, 90mL of normal saline is added, shaking is carried out at the constant temperature of 37 ℃ and 200r/min for 30min, and then dilution plate colony culture counting is carried out. The results are shown in Table 3. As can be seen from Table 3, the survival rate of Lactobacillus plantarum NHE-LpB6401 in artificial gastric juice (containing enzymes) with pH1.5, pH2.0 and pH2.5 after being treated for 3 hours is more than 99%, which indicates that the strain NHE-LpB6401 has high acid resistance, can resist gastric acid and can successfully reach the intestinal tract to play a role.

TABLE 3 survival of Lactobacillus plantarum NHE-LpB6401 after 3h treatment in artificial gastric juice

Treatment of	pH1.5	pH2.0	pH2.5
				Initial Activity CFU/mL	8.17×10⁹	8.17×10⁹	8.17×10⁹
Activity CFU/mL after treatment	8.09×10⁹	8.17×10⁹	8.17×10⁹
				Survival rate after treatment%	99.02	100	100

2. Determination of resistance to artificial intestinal juice

1mL of lactobacillus plantarum NHE-LpB6401 bacterial suspension (prepared according to the method described in example 1) is placed in 99mL (250mL triangular flask) of artificial intestinal fluid, the mixture is shaken at the constant temperature of 37 ℃ and 200r/min for 5 hours, 1mL of sample solution is added with 99mL of physiological saline after shaking is finished, the mixture is shaken at the constant temperature of 37 ℃ and 200r/min for 30 minutes, and then dilution plate colony culture counting is carried out. The results show that the activity of the lactobacillus plantarum NHE-LpB6401 in the artificial intestinal juice is not reduced, the survival rate is 100 percent, and the bacterial strain can well survive and preserve the activity in the intestinal juice, thereby exerting the probiotic effect.

3. Determination of the bile salt resistance

1mL of lactobacillus plantarum NHE-LpB6401 bacterial suspension (prepared according to the method described in example 1) is placed in 99mL (250mL triangular flask) of solutions with different concentrations of bile salts, wherein the concentrations of the bile salts are 0.15%, 0.3%, 1% and 1.5%, and then shaking is carried out at constant temperature of 200r/min at 37 ℃ for 120min, 1mL of sample solution is added with 99mL of normal saline after shaking is finished, shaking is carried out at constant temperature of 200r/min at 37 ℃ for 30min, and then dilution plate colony culture counting is carried out. The results are shown in Table 4. The survival rate of lactobacillus plantarum NHE-LpB6401 treated in 0.3% bile salt solubility solution for 2 hours is 95.23%, which shows that the strain has higher bile salt resistance, can resist bile salt in duodenal juice and can reach intestinal tracts to exert the function.

TABLE 4 survival of Lactobacillus plantarum NHE-LpB6401 after treatment for 2h in bile salt solutions of different concentrations

Treatment of	0.15％	0.3％	1％	1.5％
					Initial Activity CFU/mL	8.17×10⁹	8.17×10⁹	8.17×10⁹	8.17×10⁹
Activity CFU/mL after treatment	8.17×10⁹	7.78×10⁹	7.01×10⁹	6.92×10⁹
					Survival rate after treatment%	100	95.23	85.80	84.70

Example 6 mouse challenge test

Selecting 60 common Kunming mice, female, 11-13g, and conventionally feeding. The mice are randomly divided into three groups, and each group is fed with basic ration for 5 days, so that the mice can adapt as soon as possible. The formal test is started and divided into two stages. The first stage of growth performance observation test, experimental group added with lactobacillus plantarum fermentation liquor with the concentration of 1 × 10⁶CFU/mL. The control group and the negative control group were drinking purified water without lactobacillus plantarum for two weeks. The mice were weighed 1 time before and after the test, the growth of the mice was observed during the test, and the average growth rate of the body weight of each group was calculated, and the results are shown in table 5. Performing salmonella challenge test in the second stage, performing salmonella challenge on the negative control group and the test group, wherein the challenge mode adopts intragastric administration, specifically intragastric administration of 0.3 mL/0.2% sodium bicarbonate solution, and the intragastric administration concentration is 10 after 5min⁹The order of magnitude of Salmonella is 0.8 mL/mouse, and the control group is intragastrically administered with 0.8 mL/mouse of physiological saline. Observing the mental state and death condition of mice after challenge, and continuing drinking 1 × 10⁶In CFU/mL concentrationLactobacillus plantarum NHE-LpB6401 fermentation broth, and normal tap water for the control group and the negative control group. During the test period, mice are raised in cages in the same room, naturally illuminated and freely fed. The environmental temperature is controlled to be 25 +/-2 ℃, the humidity is 60 percent,

the lactobacillus plantarum fermentation broth is the fermentation broth prepared in example 2, and then the thalli are collected by centrifugation, and the collected thalli are washed with physiological saline for 3-5 times to remove the fermentation broth.

Growth performance of the first stage shows that the weight of mice fed with lactobacillus plantarum NHE-LpB6401 is increased obviously, and the lactobacillus plantarum NHE-LpB6401 is shown to promote digestion and absorption and improve feed efficiency.

After salmonella challenge, the mice were observed daily for the onset of symptoms: listlessness and inactivity; dull and shaky manifestations; the hair is not glossy and the back hair is messy when the hair is curled together; poor appetite, prominent back bow and serious attack, blood in the eyes of the mice with serious disease and incapability of opening the eyes. After the challenge, on day 1, the negative control group began to have symptoms of morbidity, died 4, and other survivors had symptoms of morbidity to different extents, fleeed all around, and was mentally impatient. The test group died 1 mouse each on day 1 and 2, and other mice had poor spirit, no food, and accumulated in the pile within 3 days, and then gradually returned to normal.

The death time, death number and survival rate of each group after challenge are shown in table 6, and the poisoning phenomenon of the mice in the test group after challenge is not serious, which shows that the lactobacillus plantarum NHE-LpB6401 has a certain immunoprophylaxis effect on salmonella infection, effectively reduces the morbidity of the mice infected by salmonella, delays the morbidity time after challenge, and effectively improves the survival rate.

TABLE 5 weight gain in mice

Grouping	Average initial weight (g/only)	Average final weight (g/piece)	Average growth rate (%)
				Control group	15.14±0.22	29.23±0.56	93.06^b
Negative control group	15.56±0.38	29.12±0.51	87.15^c
				Test group	15.66±0.32	39.74±0.56	153.77^a

TABLE 6 mice mortality before and after challenge

Example 7 Effect of Lactobacillus plantarum NHE-LpB6401 Microecological preparation on the Productivity of cherry valley meat Duck

4200 cherry valley meat ducks of 1 day old are selected for the test and randomly divided into two groups, wherein each group is 20 in number of repetitions, and each repetition is 105 in number of repetitions. Taking basic daily ration as a control group, without adding lactobacillus plantarum NHE-LpB6401 microecological preparation,the experimental group was prepared by adding the Lactobacillus plantarum NHE-LpB6401 microecological preparation prepared in example 3 of the present invention (1X 10 per kg feed) to the basal diet⁶CFU Lactobacillus plantarum NHE-LpB 6401). The meat ducks for the test are fed twice a day (07:00 and 16:00), fed with free food and free drinking water, and raised until the meat ducks are 38 days old and slaughtered. The feeding amount, the remaining amount, the death and elimination number and the death and elimination weight are recorded every day, and other procedures of feeding management such as immunization and the like are executed according to the conventional regulations. .

The test results show that, as compared with the control group, the feed-meat ratio of the meat ducks of the lactobacillus plantarum group in the later period is remarkably reduced (P is less than 0.05), and the average daily gain and the average daily feed intake tend to be improved. In the whole period, the slaughtering weight of the meat ducks in the lactobacillus plantarum group is remarkably higher than that of a control group (P <0.05), the feed-meat ratio (1.74) is lower than that of the control group (1.77), and the survival rate (97.8%) is also higher than that of the control group (96.6). From the meat production cost, the lactobacillus plantarum group (4.38) was 0.24 yuan/kg lower than the competitive group (4.62). . The experiment shows that the growth performance of the meat ducks can be improved by adding the plant lactobacillus micro-ecological preparation into the cherry valley meat duck feed, the survival rate of the meat ducks can be improved, and the meat production cost is reduced.

TABLE 7 influence of Lactobacillus plantarum microecologics on growth performance of cherry valley ducks

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> New hope six and shares Limited

SICHUAN NEW HOPE ANIMAL HUSBANDRY TECHNOLOGY Co.,Ltd.

CHENGDU FENGLAN SCIENCE AND TECHNOLOGY Co.,Ltd.

<120> lactobacillus plantarum NHE-LpB6401 and application

<130> 2029

<141> 2021-05-31

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1434

<212> DNA

<213> Lactobacillus plantarum

<400> 1

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tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata acacctggaa 120

acagatgcta ataccgcata acaacttgga ccgcatggtc cgagcttgaa agatggcttc 180

ggctatcact tttggatggt cccgcggcgt attagctaga tggtggggta acggctcacc 240

atggcaatga tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg 300

cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg 360

agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt taaagaagaa 420

catatctgag agtaactgtt caggtattga cggtatttaa ccagaaagcc acggctaact 480

acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt attgggcgta 540

aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggctcaa ccgaagaagt 600

gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 660

gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg 720

acgctgaggc tcgaaagtat gggtagcaaa caggattaga taccctggta gtccataccg 780

taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 840

aagcattccg cctggggagt acggccgcaa ggctgaaact caaaggaatt gacgggggcc 900

cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct 960

tgacatacta tgcaaatcta agagattaga cgttcccttc ggggacatgg atacaggtgg 1020

tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080

cccttattat cagttgccag cattaagttg ggcactctgg tgagactgcc ggtgacaaac 1140

cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200

gctacaatgg atggtacaac gagttgcgaa ctcgcgagag taagctaatc tcttaaagcc 1260

attctcagtt cggattgtag gctgcaactc gcctacatga agtcggaatc gctagtaatc 1320

gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380

atgagagttt gtaacaccca aagtcggtgg ggtaaccttt taggaaccag ccgc 1434

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