CN111944729A

CN111944729A - High-temperature-resistant lactobacillus plantarum microbial inoculum and preparation method and application thereof

Info

Publication number: CN111944729A
Application number: CN202010877058.XA
Authority: CN
Inventors: 李军超; 黄钦耿; 梁玲; 翁雪清; 吴松刚
Original assignee: Songgang Fujian Bioengineering Co Ltd
Current assignee: Songgang Fujian Bioengineering Co Ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-11-17
Anticipated expiration: 2040-08-27
Also published as: CN111944729B

Abstract

The invention discloses a lactobacillus plantarum and a preparation method and application of a lactobacillus plantarum product. The Lactobacillus plantarum is Lactobacillus plantarum (bacterial plantarum), the strain number of the Lactobacillus plantarum is SG-03, and the registration number of the Lactobacillus plantarum in Guangdong province microbial strain collection center is GDMCC No: 61035. the strain has high lactic acid yield, good antibacterial activity, and high temperature resistance. The live bacteria agent product prepared by high-temperature spray drying after lactobacillus plantarum SG-03 fermentation can promote the growth of broiler chickens and improve the productivity of the broiler chickens. The lactobacillus plantarum SG-03 is safe to people and livestock, does not have the problem of environmental pollution, is simple in culture condition, easy to store, resistant to high temperature and convenient to process, and is suitable for development and application.

Description

High-temperature-resistant lactobacillus plantarum microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to a lactobacillus plantarum microbial inoculum and a preparation method and application thereof in the technical field of biology.

Background

In recent years, the disadvantages of the widespread use of antibiotics in humans and animals, and even the abuse thereof, have been highlighted. The long-term use of antibiotics can cause the normal flora in intestinal tracts of human beings and animals to be disordered, influence the formation of the immune function of organisms, and even generate 'super bacteria' with multiple drug resistance due to the long-term abuse of the antibiotics. Therefore, the search for alternatives to antibiotics is an urgent task for the future survival and development of humans.

Protoplast fusion is a very useful breeding technique, and has the following advantages compared with other breeding techniques: (1) the incompatibility of sexual hybridization can be overcome, the boundary of species can be broken, the fusion among species, genus and even boundary can be achieved, and distant hybridization can be realized; (2) high recombination frequency, little restriction by conjugation or fertility, and complete transmission of genetic material without the need for complete understanding of the mechanism of action; (3) avoids the gene operations of separation, purification, shearing, splicing and the like, has simpler requirements on technology and instruments and equipment than the gene engineering, and has less investment; (4) the obstruction of cell walls is removed, and the whole set of genomes are contacted with each other after the amphiphilic strains are fused, so that a fusant integrating the excellent genetic characters of the parents is constructed.

Lactobacillus plantarum (lactobacillus plantarum) is a lactic acid bacterium that has been reported to occur naturally in almost all vertebrate and mammalian intestines. The lactobacillus plantarum has strong adhesion capacity to intestinal mucosa and excellent gastrointestinal digestive juice tolerance resistance, can be planted and propagated in human intestinal tracts, can improve the distribution of intestinal flora, antagonize harmful bacteria for planting, and avoids intestinal diseases; lactobacillus plantarum produces a broad-spectrum antibacterial substance called "lactobacillus plantarum", which is a polypeptide, and widely inhibits gram-positive and gram-negative bacteria, thus facilitating colonization in the gastrointestinal tract of animals. The lactobacillus plantarum used as a probiotic preparation can obviously increase the IgA content of intestinal mucosa, improve the immunity of organisms so as to maintain the ecological balance of intestinal tracts and promote the health of animals. The lactobacillus plantarum has high theoretical research and production application values as a novel internationally recognized probiotic lactic acid bacterium. However, lactobacillus plantarum is unable to form dormant bodies with strong stress resistance, such as spores, and has poor temperature tolerance, which also greatly limits the application of lactobacillus plantarum in feed processing.

The bacillus licheniformis has the characteristics of good enzyme production and spore production, excellent high temperature resistance, acid and alkali resistance, easy storage and the like, and can be widely used as a competitive inhibitor for probiotics or pathogenic bacteria in livestock and poultry breeding industry. In recent years, the application of bacillus licheniformis in various aspects is increasingly reported at home and abroad. The method obtains better research results in the industries of medicine, feed processing, pesticide and the like. Researches show that the bacillus licheniformis can further improve the digestion and absorption of nutrient substances by promoting the growth of intestinal dominant flora, and has obvious growth promoting effect and the function of reducing the material-to-weight ratio.

The lactobacillus plantarum and the bacillus licheniformis are beneficial probiotics for the health of animal intestinal tracts, the two bacteria are fused, the fusant has the strong capacities of producing protease and amylase of the bacillus licheniformis and forming spores capable of resisting high temperature, the lactobacillus has the capacities of resisting the low-acid environment of the stomach and the high-bile-salt environment of the front sections of the intestinal tracts, the colonization capacity of the intestinal tracts is greatly enhanced, the defects of difficult culture and preparation of the fusant can be overcome, and the fusant can grow well in anaerobic and aerobic environments and has the capacity of high stress resistance. The research on the fusion and breeding of the lactobacillus plantarum and the bacillus licheniformis is developed to obtain the fusion strain with the excellent properties of the two parent strains, realize multiple effects of one strain, is a brand new topic for obtaining the advantage complementation of the lactobacillus and the bacillus, and has extremely high practical significance and application value.

Disclosure of Invention

The invention aims to solve the technical problem of how to utilize a bacterial agent prepared by a fusion bacterial strain which can produce lactic acid and has the functions of enzyme production, stress resistance and high temperature resistance of spore bacteria and apply the bacterial agent.

In order to solve the above technical problems, the present invention provides a microbial inoculum containing Lactobacillus plantarum SG-03, which is Lactobacillus plantarum SG-03 having accession number GDMCCNo: 61035. the strain is preserved in Guangdong province microorganism culture collection center (GDMCC for short) in 27.5.2020, and the preservation address is microbial research institute of Guangdong province, 5 th floor, of Miehu 100 th building, 59 th floor, 5 th floor, in Guangzhou city, zip code 510070. Hereinafter, the strain is abbreviated as lactobacillus plantarum SG-03.

The culture of Lactobacillus plantarum SG-03 also belongs to the scope of protection of the present invention. The culture of Lactobacillus plantarum SG-03 is a substance obtained by culturing Lactobacillus plantarum SG-03 in a microbial culture medium (e.g., a fermentation broth containing Lactobacillus plantarum SG-03 and a substance secreted into a liquid medium, or a solid medium).

The culture of the lactobacillus plantarum SG-03 has at least one of the following functions W1-W3:

w1, production of short chain fatty acids (e.g. lactic acid);

w2, inhibiting the activity of gram-positive bacteria;

w3, inhibiting the activity of gram-negative bacteria;

w4, promoting the growth of broiler chickens;

w5, improving the meat-to-feed ratio;

w6, improving apparent digestibility of protein;

w7, increasing the weight of the broiler chicken;

w8, improving the immunity of the broiler chicken.

The invention also provides a product containing Lactobacillus plantarum SG-03 and/or a metabolite of said Lactobacillus plantarum SG-03 and/or a culture of said Lactobacillus plantarum SG-03.

The product can be a microbial inoculum, a microecological preparation containing the microbial inoculum, or a feed containing the microbial inoculum or the microecological preparation.

The active ingredient of the product can be Lactobacillus plantarum SG-03 or/and a metabolite of Lactobacillus plantarum SG-03 or/and a culture of the Lactobacillus plantarum SG-03, and the active ingredient of the product can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the product can be determined by a person skilled in the art according to the effect of the product.

The product can be any one of the following products:

v1, a product for producing lactic acid;

v2, a product inhibiting gram-positive bacteria;

v3, a product inhibiting gram negative bacteria;

v4, animal growth promoting products;

v5, product for improving the meat-to-feed ratio of animals;

v6, a product that increases the apparent digestibility of proteins;

v7, products for increasing animal weight;

v8, and a product for improving animal immunity.

In the product, the product can improve the immune function of animals and is embodied by all or part of the following Z1-Z7:

z1, improving the antioxidant capacity of the liver;

z2, improving the antioxidant capacity of pectoral muscles;

z3, improving the oxidation resistance of jejunum;

z4, improving the antioxidant capacity of serum;

z5, increasing serum IgG content;

z6, increasing the content of serum interleukin I;

z7, increasing the content of SIGA in jejunal mucosa.

The animal may be poultry, in particular broiler chicken.

The product may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier can be at least one of various solid culture media, calcium carbonate, defatted rice bran and the like; the liquid carrier may be water or at least one of various liquid media and the like.

In the product, the lactobacillus plantarum SG-03 or/and a metabolite of lactobacillus plantarum SG-03 or/and a culture of said lactobacillus plantarum SG-03 may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture or a mixture of cells and filtrate.

The preparation formulation of the product can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the product can also be added with surfactant (such as Tween 20, Tween 80, etc.), binder, stabilizer (such as antioxidant), pH regulator, etc.

The following applications of the microbial inoculum or/and the lactobacillus plantarum SG-03 metabolite or/and the lactobacillus plantarum SG-03 culture or/and the product also belong to the protection scope of the invention:

u1, use in lactic acid production;

u2, application in bacteriostasis;

u3, application in promoting animal growth;

u4, application in animal breeding;

u5, and its application in improving animal immunity.

As mentioned above, the metabolite of Lactobacillus plantarum SG-03 may be a fermentation broth of Lactobacillus plantarum SG-03. The fermentation liquor of lactobacillus plantarum SG-03 can be prepared by the following method: culturing lactobacillus plantarum SG-03 in a liquid fermentation culture medium, and collecting a fermentation liquid (containing the lactobacillus plantarum SG-03 and substances secreted into the liquid culture medium), wherein the fermentation liquid is a metabolite of the lactobacillus plantarum SG-03.

The method for culturing the lactobacillus plantarum SG-03 also belongs to the protection scope of the invention.

The method for culturing the lactobacillus plantarum SG-03 comprises the step of culturing the lactobacillus plantarum SG-03 in a culture medium.

The method for preparing the product also belongs to the protection scope of the invention.

The method for preparing the product comprises the step of taking the lactobacillus plantarum SG-03 and/or the metabolite of the lactobacillus plantarum SG-03 and/or the culture of the lactobacillus plantarum SG-03 as a component of the product to obtain the product, wherein the product is a liquid microbial inoculum or a solid microbial inoculum.

In the method, the product can be a liquid microbial inoculum or a solid microbial inoculum.

Experiments prove that the lactobacillus plantarum SG-03 has high lactic acid yield, good bacteriostatic activity and high temperature resistance. The live bacteria agent product prepared by high-temperature spray drying after lactobacillus plantarum SG-03 fermentation can promote the growth of broiler chickens and improve the productivity of the broiler chickens. The lactobacillus plantarum SG-03 is safe to people and livestock, does not have the problem of environmental pollution, is simple in culture condition, easy to store, resistant to high temperature and convenient to process, and is suitable for development and application.

Biological material preservation instructions

Taxonomic nomenclature of biological materials: lactobacillus plantarum

Latin literature name of biomaterial: lactobacillus plantarum

Strain number of biological material: SG-03

The preservation unit is called as follows: guangdong province microbial strain preservation center

The preservation unit is abbreviated as: GDMCC

Address: microbiological research institute of Guangzhou city, first furcellaran No. 100, No. 59, No. 5, Guangdong province; and E, postcode: 510070 preservation date: year 2020, 5 and 27

The preservation number is: GDMCCNo: 61035

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

In the quantitative tests in the following examples, three replicates were set up and the results averaged, unless otherwise specified.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

1. Bacterial strains

Lactobacillus plantarum SG-03 in the following examples is lactobacillus plantarum (lactobacillus plantarum), belonging to the genus lactobacillus (lactobacillus sp.) with accession number GDMCCNo at the guangdong provincial collection of microorganisms: 61035. the strain is preserved in Guangdong province microorganism culture collection center (GDMCC for short) in 27.5.2020, and the preservation address is microbial research institute of Guangdong province, 5 th floor, of Miehu 100 th building, 59 th floor, 5 th floor, in Guangzhou city, zip code 510070.

Lactobacillus plantarum (Lactobacillus plantarum) CICC20265 in the following examples was collected in the China Center for Industrial Culture Collection (CICC) at 03/31 2006, and was publicly available from CICC since the date of Collection. Hereinafter referred to as Lactobacillus plantarum CICC 20265.

Bacillus licheniformis (Bacillus licheniformis) CICC10092 in the following examples was collected at the China Center for Industrial Culture Collection (CICC) at 08.1979, and was publicly available from CICC since the date of Collection. Hereinafter referred to as Bacillus licheniformis CICC 10092.

2. Enzyme

In the following examples, lysozyme and helicase are both products of the company of bioengineering (Shanghai).

In the following examples, the muramidase is a product of institute of microorganisms of Guangdong province.

3. Culture medium and reagent

In the following examples, MRS agar medium and MRS broth medium are all products of Kyork Biotechnology, Inc. of Guangdong.

In the following examples, the MRS broth containing glycine at a concentration of 10g/L was a liquid medium obtained by adding glycine to the MRS broth; the glycine concentration in the liquid medium was 10 g/L.

In the following examples, calcium carbonate-containing MRS agar medium is a solid medium obtained by adding calcium carbonate to MRS agar medium; the calcium carbonate concentration in the solid medium was 3.0% (w/v).

In the following examples, DY solid medium was prepared as follows: adding yeast extract 20g, sodium chloride 5g, glucose 5g, and agar 15-20g into 1000ml tap water, adjusting pH to 7.0, and sterilizing with high pressure steam at 121 deg.C for 22min to obtain solid culture medium.

In the following examples, DY liquid medium was prepared as follows: adding yeast extract 20g, sodium chloride 5g, and glucose 5g into 1000ml tap water, adjusting pH to 7.0, and sterilizing with high pressure steam at 121 deg.C for 22min to obtain solid culture medium.

In the following examples, DY liquid culture medium containing glycine at a concentration of 10g/L was a liquid culture medium obtained by adding glycine to DY liquid culture medium; the glycine concentration in the liquid medium was 10 g/L.

In the following examples, the fusion regeneration medium RM was prepared as follows: adding glucose, peptone, beef extract, yeast extract, diammonium hydrogen citrate, Tween 80, sodium acetate and K into distilled water₂HPO₄、MgSO₄、MnSO₄Sucrose, MgCl₂·6H₂O and maleic acid, wherein the concentrations of the O and the maleic acid are respectively 20g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L yeast extract, 2g/L diammonium hydrogen citrate, 801ml/L tween and 5g/L, K sodium acetate₂HPO₄2g/L、MgSO₄0.6g/L、MnSO₄0.25g/L, 171g/L, MgCl of sucrose₂·6H₂O4.066g/L, maleic acid 2.32g/L, autoclave sterilization at 121 ℃ for 20min to obtain the fusant regeneration medium RM.

In the following examples, the seed culture medium was prepared as follows: adding corn steep liquor, glucose, yeast extract, peptone, sodium acetate, diammonium hydrogen citrate, ammonium sulfate, light calcium carbonate, magnesium sulfate, manganese sulfate, tween 80 and soybean oil into distilled water to ensure that the mass volume percentage (g/100mL) of the corn steep liquor, the glucose is 3.0%, the yeast extract is 1%, the peptone is 1%, the sodium acetate is 0.5%, the diammonium hydrogen citrate is 0.2%, the ammonium sulfate is 0.1%, the light calcium carbonate is 0.6%, the magnesium sulfate is 0.02%, the manganese sulfate is 0.005%, the tween is 800.1% and the soybean oil is 0.1%, adjusting the pH value to 6.0-6.5, and carrying out high-pressure sterilization at 121 ℃ for 20min to obtain the seed culture medium.

In the following examples, the liquid fermentation medium was prepared as follows: adding 3% of soybean cake powder, 2% of corn starch, 2% of sucrose, 2.0% of corn steep liquor, 0.5% of glucose, 1% of peptone, 0.5% of yeast extract, 0.2% of diammonium hydrogen citrate, 800.1% of tween, 0.5% of sodium acetate, 0.2% of ammonium sulfate, 0.01% of magnesium sulfate, 0.01% of manganese sulfate, 2% of agar powder and the balance of tap water into tap water, wherein the added mass volume percentage (g/100mL) of the soybean cake powder, the added corn starch, the added sucrose, the added sodium acetate, the added ammonium sulfate, the added magnesium sulfate, the added manganese sulfate and the added agar powder are soybean cake powder, corn starch, sucrose and the added corn steep liquor are mixed.

In the following examples, LB solid medium (cat. L1015) was manufactured by Beijing Soilebao Tech Co.

In the following examples, the SMM hypertonic solution was formulated as follows: adding sucrose and MgCl into distilled water₂·6H₂O and maleic acid in an amount of 0.5mol/L, MgCl mol based on sucrose₂·6H₂O0.02mol/L and maleic acid 0.02mol/L, adjusting pH to 7.0, sterilizing at 121 deg.C for 22min to obtain SMM hypertonic solution, and storing at 4 deg.C for use.

In the following examples, the melting promoters were formulated as follows: and adding polyethylene glycol (PEG-6000) into the SMM hypertonic solution to ensure that the mass percentage of the polyethylene glycol is 40 percent to obtain the melting promoter.

In the following examples, enramycin (specifically 4% enramycin premix) is a product of jinyue animal health products, ltd, hangzhou.

Example 1 fermentation application of Lactobacillus plantarum SG-03

Lactobacillus plantarum SG-03 used in this example was lactobacillus plantarum (lactobacillus plantarum), which was registered with GDMCCNo: 61035. the strain is preserved in Guangdong province microorganism culture collection center (GDMCC for short) in 27.5.2020, and the preservation address is microbial research institute of Guangdong province, 5 th floor, of Miehu 100 th building, 59 th floor, 5 th floor, in Guangzhou city, zip code 510070. . Hereinafter, the strain is abbreviated as lactobacillus plantarum SG-03.

The Lactobacillus plantarum SG-03 thalli are short rods, the average size of the thalli is 0.9-1.2 microns multiplied by 4.0-9.0 microns, gram staining is positive, spores are not produced, flagella are not produced, but movement is possible, and the lactobacillus plantarum has no fluorescence characteristics. The bacterial colony of the lactobacillus plantarum SG-03 on the MRS culture medium is milk white, viscous and smooth in surface, and opaque circular bulges are usually displayed on a flat plate. Can ferment pentose or gluconate, and produce acid and no gas. Nitrate is generally not reduced, gelatin is not liquefied, and both catalase and oxidase are negative. The growth temperature range of Lactobacillus plantarum SG-03 is 15-45 deg.C, the optimum growth temperature is 30-35 deg.C, the growth pH value is 3.5-8.5, and the optimum pH value is 4.5-6.5. Lactobacillus plantarum SG-03 has 16SrDNA shown in a sequence 1 in a sequence table.

Fermentation of Lactobacillus plantarum SG-03

1. Activation culture of seeds

1.1, inoculating Lactobacillus plantarum SG-03 glycerol strain to an MRS agar culture medium slant (18 multiplied by 180mm), and culturing for 48h at 30 ℃ in an incubator.

1.2, washing the conidia with 5ml of sterile physiological saline, transferring the conidia to an MRS eggplant flask culture medium (an eggplant flask filled with an MRS agar culture medium), and culturing for 24 hours at 30 ℃ in an incubator, namely, activating and culturing the strain to obtain an activated eggplant flask strain, namely activated lactobacillus plantarum SG-03 strain.

2. Seed culture in shake flasks

And (2) adding 20mL of sterile water into the activated lactobacillus plantarum SG-03 strain obtained in the step (1) for elution to prepare a bacterial suspension, transferring the bacterial suspension into a 2L shake flask filled with 500mL of MRS broth culture medium, and culturing for 24 hours at the constant temperature of 120r/min and 30 ℃ to further improve the total number of bacterial colonies and simultaneously strengthen the activity of the strain to obtain shake flask seeds.

3. Seed scale-up culture

And (3) carrying out enlarged culture on the shake flask seeds by adopting a seeding tank, transferring the shake flask seeds obtained in the step (2) into the seeding tank containing a seed culture medium according to the inoculation amount of 2 percent (the inoculation amount can be properly adjusted within the range of 2-10 percent), and culturing to obtain the seeds. The control process comprises the following steps: the pressure in the tank is 0.06Mpa, the temperature in the tank is 30 DEG CThe initial stirring is carried out for 150r/min, the dissolved oxygen is naturally reduced, the bottom sugar (reducing sugar) is 3 percent, the seed transferring amount is 20 percent, the period is 18h, and the colony count reaches 10⁹cfu/mL。

4. Cultivation in fermenter

And (3) transplanting the seeds cultured in the seeding tank in the step (3) to a fermentation tank (filled with a liquid fermentation culture medium), wherein the transplanting amount is 20%. In the initial stage of fermentation culture, the aeration rate of the fermentation tank is controlled to be 150-200ml/min, the stirring is controlled to be 100-200rpm, the temperature is controlled to be 30 ℃, the initial reduction concentration is controlled to be 20-30g/l, and the dissolved oxygen is naturally controlled. The whole process is controlled to feed 70% glucose aqueous solution, so as to maintain the glucose content in the fermentation tank to be 0.5-1.0g/100 mL. And (3) after the aerobic fermentation period is 24h and 24h, closing stirring, maintaining the positive pressure in the fermentation tank, performing anaerobic fermentation for 8h, and stopping fermentation to obtain the lactobacillus plantarum SG-03 fermentation liquor.

5. Adding adjuvant and spray drying

Adding calcium carbonate and defatted rice bran into Lactobacillus plantarum SG-03 fermentation broth until the content of calcium carbonate is 5.0g/100mL and the content of defatted rice bran is 10.0g/100mL, and directly spray drying (outlet temperature is about 125 deg.C) to obtain high temperature resistant lactobacillus product containing Lactobacillus plantarum SG-03, wherein the water content in the product is less than 10%, and the viable count of Lactobacillus plantarum SG-03 is 1.0 × 10⁹cfu/g is named as Lactobacillus plantarum SG-03 viable bacteria agent.

6. Preparation of parent bacterial agent

According to the steps 1-5, replacing SG-03 with CICC20265 and CICC10092 respectively, preparing a microbial inoculum, wherein the viable count of the lactobacillus plantarum CICC20265 is 110cfu/g (the high temperature resistance is poor, and the influence of high-temperature spraying on the viable count is great), and the microbial inoculum is named as a CICC20265 lactobacillus plantarum viable dose; the viable count of the bacillus licheniformis CICC10092 is 1.0 multiplied by 10¹⁰cfu/g is named as CICC10092 bacillus licheniformis viable bacteria agent.

Secondly, the application of the viable bacteria agent of the lactobacillus plantarum SG-03 in the AA broiler chicken (the contrast of the single bacteria agent of the lactobacillus plantarum CICC20265 and the single bacteria agent of the bacillus licheniformis CICC10092 is preferably added in the following animal experiments, and the certain performance of the viable bacteria agent of the lactobacillus plantarum SG-03 is better than that of the parent strain)

1. Influence of high temperature resistant lactobacillus plantarum SG-03 on production performance of AA broiler chickens

The basic ration is corn-soybean meal-corn protein powder-DDGS type feed, the granular feed used in the whole process is a product of animal science and technology college of Shandong agricultural university, and the specific basic ration and the nutrition level are shown in Table 1.

Table 1 basal ration composition and nutritional level (air-dried basal) units: is based on

¹⁾The nutrient water is calculated on average. Nutrientlevelswellealculatestvalues.

The viable bacteria agent of the lactobacillus plantarum SG-03 is the viable bacteria agent of the lactobacillus plantarum SG-03 prepared in the step one, and the viable bacteria number of the lactobacillus plantarum SG-03 is 1.0 multiplied by 10⁹cfu/g。

The enramycin product (specifically 4% of enramycin premix) is a Hangzhou Jinyue animal health product, namely a product of Limited company, and the content of enramycin is 4%.

432 healthy AA broiler chickens with 1 day age and consistent body conditions are selected, the weight of each chicken is 44.1g +/-5.5 g, the number of the chickens is randomly divided into 6 treatments, each treatment is 6 times of repetition, and each repetition is 12 chickens. The 6 process settings were as follows:

a0 group is blank control group without addition of microbial inoculum, and is fed with basal diet;

the A1 group is 100g/TCICC20265 group, and is fed with 100g/TCICC202653 daily ration. The 100g/TCICC20265 daily ration is prepared by adding viable bacteria agent of Lactobacillus plantarum CICC20265 into basal daily ration. The content of the lactobacillus plantarum SG-03 viable bacteria agent in 100g/T CICC20265 daily ration is 100g/1000kg, namely the content of the lactobacillus plantarum CICC20265 is 1.1cfu/kg (the temperature resistance is poor, and the feed is basically free of viable bacteria after being granulated at high temperature).

The A2 group is 100g/TCICC10092 group, and is fed with 100g/TCICC10092 and daily food. The 100g/TCICC10092 daily ration is obtained by adding bacillus licheniformis CICC10092 viable bacteria agent into basic daily ration. 100g/T CICC10092 daily ration contains 100g/1000kg of viable bacteria agent of Bacillus licheniformis CICC10092, namely the content of the Bacillus licheniformis CICC10092 is 1.0 multiplied by 10⁹cfu/kg。

The group B is 100g/TSG-03 group, and is fed with 100g/TSG-03 daily ration. The 100g/TSG-03 daily ration is obtained by adding viable bacteria agent of Lactobacillus plantarum SG-03 into basic daily ration. The content of viable bacteria agent of Lactobacillus plantarum SG-03 in 100g/TSG-03 daily ration is 100g/1000kg, i.e. the content of Lactobacillus plantarum SG-03 is 1.0 × 10⁸cfu/kg。

The group C is 500g/TSG-03 group, and is fed with 500g/TSG-03 daily ration. The 500g/TSG-03 daily ration is obtained by adding viable bacteria agent of lactobacillus plantarum SG-03 into basic daily ration. The content of viable bacteria agent of Lactobacillus plantarum SG-03 in 500g/TSG-03 daily ration is 500g/1000kg, i.e. the content of Lactobacillus plantarum SG-03 is 5.0 × 10⁸cfu/kg。

The group D is 200g/T enramycin group, and is fed with 200g/T enramycin daily ration. The 200g/T enramycin daily ration is obtained by adding enramycin into basic daily ration. The enramycin commodity content in 200g/T enramycin daily ration is 200g/1000kg, namely the enramycin content is 0.008%.

During the test period, the feed intake, the number of dead-panned chickens and the weight of dead-panned chickens were counted for each treatment. And (4) forbidding feeding for 12 hours at the age of 21 days and the age of 35 days, only ensuring normal drinking water and illumination in the period, recording the weight of the broiler chickens, carefully pouring out the residual materials and weighing. Recording the body weight gain and the material consumption of the two stages of 1-21 days old and 21-35 days old. The broiler chickens with the statistical production performance are fasting for 12 hours before sampling, and only normal drinking water and illumination are provided. The average Body Weight (BW), average Body Weight Gain (BWG), feed-to-weight ratio (F/G), and Average Feed Intake (AFI) were counted.

The test data are expressed by mean value plus or minus standard deviation, and single factor variance analysis is carried out by using ANOVA process in SAS9.1 statistical software, wherein the difference is obvious when P is less than 0.05, and the difference is not obvious when P is more than or equal to 0.05. The results are shown in Table 2.

Table 2. influence of different addition amounts of live lactobacillus plantarum SG-03 on production performance of AA broiler chickens

The results show that: 100-500g of lactobacillus plantarum SG-03 viable bacteria agent is added into each chicken every day, the production performance of broiler chickens is remarkably improved, the daily gain is remarkably increased, the feed conversion ratio is remarkably reduced, the substitution effect of growth promotion by antibiotics (enramycin) is effectively achieved, and the addition effect of 100g is better than 500 g.

Compared with the parental strain control, the growth promoting effect of the same addition of 100g of SG-03 microbial inoculum is obviously better than that of the live bacterial agents of lactobacillus plantarum CICC20265 and bacillus licheniformis CICC 10092.

2. Influence of high-temperature-resistant lactobacillus plantarum SG-03 on immunity of AA broiler chickens

2.1, anti-oxidation index

Repeatedly selecting 1 chicken wing vein for blood sampling at 21 and 35 days of age, obliquely standing, centrifuging at 3000r/min for 10min to separate serum, transferring separated serum into 1.5ml centrifuge tube with pipette, and storing in refrigerator at-20 deg.C. After dissection, the liver, pectoral muscle and jejunum were placed in a 2ml cryopreservation tube and stored in a refrigerator at-20 ℃ for testing.

The total antioxidant capacity (T-AOC) of serum, liver, pectoralis muscle and jejunum is measured according to the method of the kit (the kit is purchased from Nanjing institute of bioengineering research, Ltd., product number A015-1-2) please indicate a specific detection method or reference), the results are shown in Table 3, the data are expressed as the mean value +/-standard deviation, the single-factor variance analysis is carried out by using the ANOVA process in SAS9.1 statistical software, the difference is obvious when P is less than 0.05, and the difference is not obvious when P is more than 0.05.

Table 3. unit of influence of live lactobacillus plantarum SG-03 on broiler oxidation resistance: u/g

The results show that 100g and 500g of the lactobacillus plantarum SG-03 viable bacteria agent can be added to obviously improve the oxidation resistance of the liver, the pectoralis muscle and the jejunum and improve the oxidation resistance of serum, and the effect of adding 100g is better than 500 g.

Compared with parent strains, the lactobacillus plantarum SG-03 is superior to lactobacillus plantarum CICC20265 viable bacteria agent and bacillus licheniformis CICC10092 viable bacteria agent under the same addition condition of remarkably improving the oxidation resistance of the liver, the pectoral muscle and the jejunum.

2.2 serum immune index

Blood sampling and serum separation and detection of antioxidant indexes.

Jejunal mucosa: the jejunum was cut off, the contents were separated, rinsed with normal saline, and blotted dry with filter paper. The mucosa was scraped off with a glass slide into a 2ml cryopreservation tube. Immediately placing the sample in a liquid nitrogen tank for storage, and transferring the sample to a refrigerator at the temperature of minus 80 ℃ for testing after sampling.

Serum immunoglobulin G (IgG) (purchased from Nanjing Biotech institute of Biotechnology, Inc., product No. E026-1-1), interleukin-I (IL-1) (purchased from Nanjing Biotechnology institute of Biotechnology, Inc., product No. H001), and secretory immunoglobulin A (SIGA) of jejunal mucosa (purchased from Nanjing Biotechnology institute of Biotechnology, product No. H108-2) were measured by ELISA.

The results are shown in Table 4.

Table 4. influence unit of live lactobacillus plantarum SG-03 on immune index of broiler chicken serum: mg/ml

The results show that the lactobacillus plantarum SG-03 viable bacteria agent can obviously improve the humoral immunity and the cellular immunity of the broiler chicken, increase the content of IgG and the content of the intestinal mucosa SIgA and improve the immune function.

Compared with parent strains, the lactobacillus plantarum SG-03 has the same obvious effects of improving humoral immunity and cellular immunity, and is obviously superior to lactobacillus plantarum CICC20265 viable bacteria agent and bacillus licheniformis CICC10092 viable bacteria agent under the same addition condition.

Example 2 Breeding of novel high temperature resistant lactic acid bacterium, Lactobacillus plantarum SG-03

Preparation of Lactobacillus plantarum protoplast suspension

1. Preparation of Lactobacillus plantarum activation and bacterial suspension

The lactobacillus plantarum (lactobacillus plantarum) used in this example was lactobacillus plantarum cic 20265, which was purchased from china industrial collection of microorganisms management center.

Inoculating glycerol strain of Lactobacillus plantarum CICC20265 to a slant (18 x 180mm test tube slant) containing MRS agar culture medium, culturing at 30 deg.C for 24h, which is F1 substitute slant, taking one ring from F1 substitute slant to fresh empty slant, and culturing at 30 deg.C for 16h to obtain activated Lactobacillus plantarum CICC20265 strain.

Then, the cells in the activated slant were washed with sterile water to prepare a bacterial suspension, and the number of colonies was controlled to 10⁶CFU/mL. Inoculating the bacterial suspension into MRS broth containing glycine with concentration of 10g/L according to the inoculation amount of 2% (V/V), wherein the liquid content in a 250mL triangular flask is 50mL, culturing for 12h at 30 ℃ and 200r/min in a constant temperature oscillator to obtain logarithmic phase cell culture solution, centrifuging at 8000rpm for 10min, and collecting thalli.

Then, the thalli obtained by the centrifugation is washed twice by adopting an SMM hypertonic solution, the supernatant is removed, and finally 10ml of SMM solution is taken for re-suspending the cells, so that the lactobacillus plantarum CICC20265 bacterial suspension is obtained, wherein the bacterial colony number of the lactobacillus plantarum CICC20265 is about 10⁷CFU/mL。

2. Preparation of lactobacillus plantarum protoplast suspension by enzymolysis wall breaking

Carrying out combined digestion by adopting lysozyme and helicase, and respectively adding enzyme mother liquor after filtration sterilization into the lactobacillus plantarum CICC20265 bacterial suspension obtained in the step 1 to prepare the bacterial suspension containing the enzyme, wherein the optimal using concentration of the lysozyme is 0.7% (w/v), and the optimal using concentration of the helicase is 0.5% (w/v). Placing the bacterial suspension containing the enzyme in a shaking table, carrying out enzymolysis at 80r/min, carrying out microscopic examination once every 10min, and observing the formation condition of a protoplast, wherein the optimal enzymolysis temperature is 30 ℃; the optimal enzymolysis time is 1.5 h; the optimum pH value for enzymolysis is 6.5. When about 90% of cells are converted into protoplasts, centrifuging at 3000rpm to collect cells, washing and centrifuging twice with SMM hypertonic solution, and resuspending with 5mL of SMM hypertonic solution to obtain Lactobacillus plantarum protoplast suspension with Lactobacillus plantarum protoplast concentration of 2.0 × 10⁷The protoplast formation rate reaches 91.3 percent per mL.

3. Inactivated lactobacillus plantarum protoplast suspension

And (3) carrying out 60 ℃ heat inactivation treatment on the lactobacillus plantarum protoplast suspension obtained in the step (2), setting the time to be 5min, 10min, 15min and 20min respectively, slightly oscillating for 1 time every 5min to uniformly heat the protoplasts, coating regeneration flat plates (flat plates made of a fusant regeneration culture medium RM) respectively after the treatment is finished, culturing for 72h at the temperature of 30 ℃, and calculating the number of viable bacteria. The result shows that the heat inactivation is carried out for 15min, the inactivation rate reaches 100 percent, and the requirement of parent inactivation is met.

And (3) inactivating the lactobacillus plantarum protoplast suspension by adopting a condition of heat inactivation at 60 ℃ for 15 min. Secondly, preparation of a suspension of Bacillus licheniformis protoplasts

1. Preparation of Bacillus licheniformis activation and bacterial suspension

The bacillus licheniformis (bacillus licheniformis) used in this example is bacillus licheniformis cic 10092, which is purchased from china industrial microorganism culture collection management center.

Inoculating glycerol strain of Bacillus licheniformis CICC10092 to 18 × 180mm test tube slant made of DY solid culture medium, activating twice at 30 deg.C for 24 hr, washing with sterile water to obtain activated slant with viable count of 10⁸CFU/mL. Inoculating with an inoculum size of 2%Culturing in DY liquid culture medium containing glycine 10g/L with constant temperature oscillator at 30 deg.C and 200r/min for 6 hr, adding ampicillin to make final concentration 5U/mL, continuously oscillating and culturing for 2 hr to obtain culture solution in logarithmic growth phase, centrifuging at 8000rpm for 10min, washing twice with SMM hypertonic solution, and re-suspending in 10mL of SMM hypertonic solution to obtain Bacillus licheniformis CICC10092 suspension with colony count of 10⁸CFU/mL。

2. Preparation of bacillus licheniformis protoplast suspension by enzymolysis wall breaking

Carrying out combined digestion by adopting lysozyme and muramidase, and respectively adding enzyme mother liquor after filtration and sterilization into the bacillus licheniformis CICC10092 bacterial suspension obtained in the step 1 to prepare bacterial suspension containing the enzyme, wherein the optimal enzymolysis concentration of the lysozyme is 1.0 percent, and the optimal enzymolysis concentration of the muramidase is 0.3 percent; the optimal enzymolysis temperature is 30 ℃; the optimal enzymolysis time is 1.5 h; the optimum pH for enzymatic hydrolysis was 7.0. Placing the bacterial suspension containing enzyme in a shaking table, performing enzymolysis at 80r/min, performing microscopic examination every 10min, observing the protoplast formation condition, centrifuging at 3000rpm to collect cells when about 90% of the cells are converted into protoplasts, washing and centrifuging twice by using an SMM (SMM) hypertonic solution, re-suspending by using 5mL of the SMM hypertonic solution to obtain the bacillus licheniformis protoplast suspension, and finally obtaining the bacillus licheniformis protoplast concentration of 1.1 multiplied by 10⁸The protoplast formation rate reaches 93.7 percent per mL.

3. Inactivated bacillus licheniformis protoplast suspension

And (3) performing ultraviolet inactivation on the bacillus licheniformis protoplast suspension obtained in the step (2): and (3) adopting an ultraviolet lamp with the power of 30W, wherein the irradiation distance is 20cm, carrying out ultraviolet irradiation inactivation in an environment avoiding white light interference, respectively and vertically irradiating for 30s, 60s, 90s, 120s and 150s, respectively coating regeneration flat plates after treatment, culturing in a dark place for 72h, and calculating the number of viable bacteria. The result shows that the inactivation rate of the bacillus licheniformis CICC20265 reaches 100 percent by 120s of ultraviolet irradiation, and the requirement of parent inactivation is met.

And inactivating the bacillus licheniformis by adopting the condition of ultraviolet irradiation for 120s to obtain the inactivated bacillus licheniformis protoplast suspension.

Cell fusion of inactivated parents

And (3) mixing the suspension of the lactobacillus plantarum protoplast inactivated in the step one and the suspension of the bacillus licheniformis protoplast inactivated in the step two respectively, centrifuging at 3000r/min for 15min, adding 3mL of a melting promoting agent for heavy suspension, and placing in a water bath at 35 ℃ for heat preservation and fusion for 30 min.

After the cell fusion is finished, centrifuging for 15min at 2500r/min, discarding the supernatant, washing and centrifuging three times by using an SMM hypertonic solution, and removing polyethylene glycol. Diluting the obtained protoplast precipitate to 10 with SMM hypertonic solution⁵And (4) taking the diluted solution with the concentration per mL, coating the diluted solution on a fusant regeneration culture medium RM, placing the culture medium in a constant temperature incubator at 30 ℃, and culturing in a dark place until a regenerated fusion single colony grows out. The number of recovered single colonies obtained was 1652 in total, each single colony serving as a fusion.

Screening of fusions

1. Primary screen

And (3) respectively putting the 1652 fusions obtained in the third step into an MRS agar medium containing calcium carbonate, culturing for 48h at 30 ℃, performing one-round primary screening according to whether the fusions have the acid-producing capacity, selecting a single colony with an obvious transparent ring, and obtaining 190 strains in total, namely obtaining 190 fusions.

2. Secondary screening

And (3) performing fermentation culture on 190 fusants with acid production capacity subjected to primary screening for secondary screening by using a 96-well plate (with the aperture of 6.0mm), wherein the culture medium is MRS broth culture medium, and the culture conditions comprise that the initial pH value is 6.5, the temperature is 30 ℃, the speed is 220r/min, and the fermentation time is 48 h. After the fermentation is finished, the following detection is carried out:

2.1, respectively taking Lactobacillus plantarum CICC20265 and Bacillus licheniformis CICC10092 as control strains, detecting the acid production capacity of fermentation of different fusants, and measuring the lactic acid content of fermentation liquor by adopting a biosensor analyzer SBA-40 (product of Jinan Byelsen biotechnology, Co., Ltd.).

2.2, respectively detecting the antibacterial activity of each fusant fermentation broth on gram-positive bacteria (the detected bacteria are staphylococcus aureus ATCC25923) and gram-negative bacteria (the detected bacteria are escherichia coli ATCC25922) by adopting an agar diffusion method, wherein the specific method comprises the following steps:

1) and (3) gram-positive bacteria bacteriostatic activity determination: an LB solid culture medium is used, the mixture is heated, placed at room temperature and cooled to 45-55 ℃, a proper amount of staphylococcus aureus ATCC25923 is added, the mixture is poured into a flat plate after being mixed evenly, a sterile puncher with the diameter of 6.0mm is used for punching on the flat plate, agar blocks are removed, 50ul of fermentation liquor of different fusants are respectively added into sample application holes for sample application, the mixture is placed into an incubator with the temperature of 4 ℃ for refrigeration and absorption for 2 hours and then is cultured for 24 hours at the temperature of 30 ℃, the flat plate is taken out, and the diameters of inhibition zones of the different fusants are.

2) And (3) gram-negative bacteria bacteriostatic activity determination: an LB solid culture medium is used, the mixture is heated, placed at room temperature and cooled to 45-55 ℃, an appropriate amount of Escherichia coli ATCC25922 is added, the mixture is poured onto a flat plate after being mixed evenly, a sterile puncher with the diameter of 6.0mm is used for punching holes on the flat plate, agar blocks are removed, 50ul of fermentation liquor of different fusants is respectively added into sample application holes for sample application, the mixture is placed into an incubator with the temperature of 4 ℃ for refrigeration and absorption for 2 hours and then cultured for 24 hours at the temperature of 30 ℃, the flat plate is taken out, and the diameters of inhibition zones of the different fusa.

2.3, detecting different fusion casein hydrolysis loops, wherein the determination method comprises the following steps: 1% of casein and 1.5% of agar powder, sterilizing at 121 ℃ for 20min, cooling, pouring the plate, punching the plate by using an aseptic puncher with the diameter of 6.0mm, removing agar blocks, adding 50ul of fermentation liquor of the fusant into sample application holes respectively for sample application, putting the mixture into an incubator at 4 ℃ for cold storage and absorption for 2 hours, then culturing in an incubator at 30 ℃ for 24 hours, taking out the plate, and measuring the diameters of casein hydrolysis rings of different fusants.

2.4, evaluating the acid production, bacteriostasis and casein hydrolysis performances of different fusions according to the detection results of the steps 2.1, 2.2 and 2.3, wherein the determination results of part of fusions are shown in Table 5, and accordingly selecting 4 strains with better acid production and bacteriostasis activity capacities: SG-03, SG116, SG169 and SG 333.

TABLE 5 fermentation results (partial) for different fusions in 96 well plates

3. Shake flask rescreen

Performing shake flask fermentation and re-screening on 4 strains (SG-03, SG116, SG169 and SG333) with better acid production and bacteriostatic activity selected in the step 2, wherein the used culture medium is a liquid fermentation culture medium; the liquid loading of a 250mL shake flask is 50mL, and fermentation is carried out for 48h at 30 ℃. After the fermentation is finished, centrifuging at 8000r/min and collecting fermentation supernatant.

And (3) determining the bacteriostatic activity of the supernatant on gram-positive bacteria and gram-negative bacteria and the content of lactic acid, wherein the determination method is the same as the step 2, and the experiment is repeated for 3 times. The test data were analyzed for one-way anova using SPSS13.0 statistical software in combination with duncan's multiple comparisons. The difference is very significant when P is less than 0.01, the difference is significant when P is less than 0.05, the difference is not significant when P is more than 0.05, and the result is expressed by Mean +/-standard deviation (Mean +/-SD).

The results are shown in Table 6.

TABLE 6 shake flask fermentation double-screen verification

The results show that the SG-03 strain is better than other fusions in both lactic acid metabolism and bacteriostatic and protease metabolic activities. The lactic acid content of the SG-03 fusion is 6.7 percent (P is less than 0.05), 17.3 percent (P is less than 0.01) and 14.5 percent (P is less than 0.01) higher than that of the SG116, SG169 and SG333 fusions respectively, and the differences reach significant levels; has obvious difference in bacteriostatic activity compared with SG116 (G)^-，35.1％，P＜0.05；G⁺，17.1％，P＜0.05)、SG169(G^-，42.9％，P＜0.05；G⁺11.4%, P < 0.05) and SG333 (G)^-，22.0％，P＜0.05；G⁺22.5%, P < 0.05) are all preferred; the casein hydrolysis ability was also better than other fusions, but the difference was not significant (P > 0.05).

G of SG-03 Strain compared with the original Strain⁺The antibacterial activity is better than that of two female parents, is improved by 76.2 percent (P is less than 0.01) compared with the CICC20265 parent, has obvious difference, is improved by 12.1 percent (P is more than 0.05) compared with the CICC20265 parent, and has no obvious difference; SG-03 fusions also exhibit better parent fusion properties in the metabolic capacity of lactic acid and proteases, fusing the parentsIs excellent in the characteristics of (1).

Meanwhile, the collected fermentation supernatant is treated for 10min at 60 ℃, 80 ℃ and boiling water bath, the untreated fermentation supernatant is used as a control, the number of viable bacteria is detected, and the high temperature resistance of the fusion strain is inspected, wherein the detection method comprises the following steps: sucking 1mL fermentation liquid and diluting the fermentation liquid to 10 times by serial dilution⁴And (3) taking 0.1mL of the bacterial liquid treated at different temperatures respectively, coating an MRS agar plate (a plate made of an MRS agar culture medium), culturing for 48h in a 30 ℃ incubator, calculating the number of bacterial colonies on the plate, taking three plates for each temperature treatment, repeating for three times, observing and recording the number of bacterial colonies on the plates, and calculating the temperature tolerance of the bacterial strains. The test data were analyzed for one-way anova using SPSS13.0 statistical software in combination with duncan's multiple comparisons. The difference is very significant when P is less than 0.01, the difference is significant when P is less than 0.05, the difference is not significant when P is more than 0.05, and the result is expressed by Mean +/-standard deviation (Mean +/-SD).

The results are shown in Table 7.

TABLE 7 temperature tolerance of re-screened strains

Strain numbering	Untreated	60℃	80℃	100℃
					CICC20265	3.3±0.3×10⁹cfu/ml	4.2×10⁵cfu/ml	0	0
CICC10092	2.5±0.2×10¹⁰cfu/ml	2.1±0.2×10¹⁰cfu/ml	1.9±0.2×10¹⁰cfu/ml	1.6±0.2×10¹⁰cfu/ml
					SG-03	1.8±0.2×10⁹cfu/ml	1.7±0.2×10⁹cfu/ml	1.1±0.5×10⁹cfu/ml	0.9±0.2×10⁹cfu/ml
SG116	1.9±0.2×10⁹cfu/ml	0.3±0.05×10⁸cfu/ml	2.1±0.5×10⁶cfu/ml	3.2±1.0×10²cfu/ml
					SG169	1.4±0.2×10⁹cfu/ml	0.5±0.05×10⁸cfu/ml	4.0±0.7×10⁵cfu/ml	3.0±1.1×10²cfu/ml
SG333	2.1±0.3×10⁹cfu/ml	0.1±0.01×10⁸cfu/ml	3.0±0.8×10⁵cfu/ml	2.2±1.0×10²cfu/ml

The result shows that the SG-03 fusant has very good temperature tolerance, under the condition that the initial viable counts are basically consistent, the viable counts are maintained at 94.4 percent after being treated at 60 ℃ for 10min, while the viable counts of the SG116, SG169 and SG333 fusants are respectively 1.6 percent, 3.6 percent and 0.5 percent, and the difference is very obvious (P is less than 0.01); after 10min of treatment at 80 ℃, the SG-03 fusions still maintained 61.1% of the survival rate, while the survival rates of other fusions were all below 0.1%; however, after 10min of treatment at 100 ℃, the survival rate of the SG-03 fusion is still 50.0%, and the survival rates of other fusions are all below 0.001%, and the difference is very significant (P < 0.01).

According to the table 5, the table 6 and the table 7, the high temperature resistant strain SG-03 which has good acid production capacity and can metabolize to generate high content of antibacterial active substances is finally obtained by screening.

SG-03 has better lactobacillus fermentation characteristic and better protease production capability, has better bacteriostatic activity to staphylococcus aureus ATCC25923 than the bacillus licheniformis CICC1009 and the lactobacillus plantarum CICC20265 of the initial strain, has greatly improved bacteriostatic activity to escherichia coli ATCC25922 than the bacillus licheniformis CICC1009 of one of the initial strains, and is slightly lower than the lactobacillus plantarum CICC20265 of the other initial strain; the hydrolysis capacity of the casein is also between two parent strains, and more remarkably, the fusion strain SG-03 shows excellent temperature tolerance, the viable count still keeps 50% after treatment at 100 ℃, shows good high-temperature tolerance, is very suitable for the high-temperature pelleting process of the feed, and ensures the high-temperature viable count of the feed pelleting.

Fifth, identification and preservation of SG-03

SG-03 cells are short rods, the average size of the cells is 0.9-1.2 μm multiplied by 4.0-9.0 μm, gram staining is positive, no spore is produced, no flagellum exists, but movement is possible, and no fluorescence characteristic exists. SG-03 is milk white, viscous and smooth in surface on MRS medium, and opaque circular bulges are often displayed on the plate. Can ferment pentose or gluconate, and produce acid and no gas. Nitrate is generally not reduced, gelatin is not liquefied, and both catalase and oxidase are negative. The growth temperature range of SG-03 is 15-45 deg.C, the optimum growth temperature is 30-35 deg.C, the growth pH value is 3.5-8.5, and the optimum pH value is 4.5-6.5. SG-03 has 16SrDNA shown in a sequence 1 in a sequence table.

The 16SrDNA sequence of SG-03 is shown as sequence 1 in the sequence table by 16SrDNA sequencing. According to the 16SrDNA sequence and BLAST analysis, the identity of the 16SrDNA sequence of SG-03 and the 16SrDNA sequence of Lactobacillus plantarum CICC20265 is 99.97%, and the identity of the 16SrDNA sequence of Bacillus licheniformis CICC10092 is 84.16%. The obtained high-temperature resistant fusion strain SG-03 is identified as lactobacillus plantarum (Lactobacillus plantarum), the lactobacillus plantarum SG-03 is preserved in Guangdong provincial microorganism culture collection (GDMCC for short; address: institute for microorganisms in Guangdong province, 5 th floor, Miehrlo, 100 th building, 59 th building, Miehleya, Guangzhou city; zip code: 510070) in 27 th month in 2020, and the preservation number is GDMCCNo: 61035.

sixthly, the genetic stability of the lactobacillus plantarum SG-03

The lactobacillus plantarum SG-03 is subjected to subculture to examine the genetic stability, the subculture is carried out once every 3 days for 15 generations, and shake flask fermentation (adopting a liquid fermentation medium) is carried out every other generation to check the bacteriostatic activity, the lactic acid yield and the temperature tolerance. The result shows that the content of each index of the lactobacillus plantarum SG-03 in the passage process has no obvious change, and the lactobacillus plantarum SG-03 has good genetic stability.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Songgang (Fujian) bioengineering Co.Ltd

<120> high-temperature-resistant lactobacillus plantarum microbial inoculum as well as preparation method and application thereof

<130> GNCSY201616

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 1438

<212> DNA

<213> Lactobacillus plantarum (Lactobacillus plantarum)

<400> 1

acatgcagtc gaacgaactc tggtattgat tggtgcttgc atcatgattt acatttgagt 60

gagtggcgaa ctggtgagta acacgtggga aacctgccca gaagcggggg ataacacctg 120

gaaacagatg ctaataccgc ataacaactt ggaccgcatg gtccgagttt gaaagatggc 180

ttcggctatc acttttggat ggtcccgcgg cgtattagct agatggtggg gtaacggctc 240

accatggcaa tgatacgtag ccgacctgag agggtaatcg gccacattgg gactgagaca 300

cggcccaaac tcctacggga ggcagcagta gggaatcttc cacaatggac gaaagtctga 360

tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt aaaactctgt tgttaaagaa 420

gaacatatct gagagtaact gttcaggtat tgacggtatt taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga tttattgggc 540

gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa gccttcggct caaccgaaga 600

agtgcatcgg aaactgggaa acttgagtgc agaagaggac agtggaactc catgtgtagc 660

ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctgtc tggtctgtaa 720

ctgacgctga ggctcgaaag tatgggtagc aaacaggatt agataccctg gtagtccata 780

ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc ttcagtgctg cagctaacgc 840

attaagcatt ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgaagct acgcgaagaa ccttaccagg 960

tcttgacata ctatgcaaat ctaagagatt agacgttccc ttcggggaca tggatacagg 1020

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080

caacccttat tatcagttgc cagcattaag ttgggcactc tggtgagact gccggtgaca 1140

aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200

cgtgctacaa tggatggtac aacgagttgc gaactcgcga gagtaagcta atctcttaaa 1260

gccattctca gttcggattg taggctgcaa ctcgcctaca tgaagtcgga atcgctagta 1320

atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380

accatgagag tttgtaacac ccaaagtcgg tggggtaacc tttaggaacc agccgcct 1438

Claims

1. A microbial inoculum, which is characterized in that: the microbial inoculum contains Lactobacillus plantarum SG-03 and/or a metabolite of Lactobacillus plantarum SG-03, wherein the Lactobacillus plantarum SG-03 is Lactobacillus plantarum (Lactobacillus plantarum), and the accession number of the Lactobacillus plantarum SG-03 in Guangdong province microbial strain collection center is GDMCC No: 61035.

2. a culture of lactobacillus plantarum SG-03 characterized by: the lactobacillus plantarum SG-03 is obtained by culturing lactobacillus plantarum SG-03 in a microbial culture medium.

3. Culture of lactobacillus plantarum SG-03 according to claim 2, characterized in that: has at least one function of W1-W8:

w1, producing lactic acid;

w2, inhibiting the activity of gram-positive bacteria;

w3, inhibiting the activity of gram-negative bacteria;

w4, promoting the growth of broiler chickens;

w5, improving the meat-to-feed ratio;

w6, improving apparent digestibility of protein;

w7, increasing the weight of the broiler chicken;

w8, improving the immunity of the broiler chicken.

4. A product characterized by: a culture comprising Lactobacillus plantarum SG-03 according to claim 1 or/and a metabolite of Lactobacillus plantarum SG-03 and/or Lactobacillus plantarum SG-03 according to claim 2 or 3.

5. The product of claim 4, wherein: is any one of the following products:

v1, a product for producing lactic acid;

v2, a product inhibiting gram-positive bacteria;

v3, a product inhibiting gram negative bacteria;

v4, animal growth promoting products;

v5, product for improving the meat-to-feed ratio of animals;

v6, a product that increases the apparent digestibility of proteins;

v7, products for increasing animal weight;

v8, and a product for improving animal immunity.

6. The product of claim 5, wherein: the animal immune function improving agent is represented by all or part of the following Z1-Z7:

z1, improving the antioxidant capacity of the liver;

z2, improving the antioxidant capacity of pectoral muscles;

z3, improving the oxidation resistance of jejunum;

z4, improving the antioxidant capacity of serum;

z5, increasing serum IgG content;

z6, increasing the content of serum interleukin I;

z7, increasing the content of SIGA in jejunal mucosa.

7. Any of the following uses of the inoculant of claim 1 or/and said Lactobacillus plantarum SG-03 and/or said Lactobacillus plantarum SG-03 metabolite, and/or said Lactobacillus plantarum SG-03 culture of claim 2 or 3, or/and the product of any of claims 4-6:

u1, use in lactic acid production;

u2, application in bacteriostasis;

u3, application in promoting animal growth;

u4, application in animal breeding;

u5, and its application in improving animal immunity.

8. The method for culturing lactobacillus plantarum SG-03 of claim 1, which comprises: comprising the step of culturing said Lactobacillus plantarum in a culture medium.

9. A process for preparing the product of any of claims 4-6, characterized in that: comprising the step of obtaining a product by using the Lactobacillus plantarum SG-03 of claim 1 and/or a metabolite of said Lactobacillus plantarum SG-03 and/or a culture of said Lactobacillus plantarum SG-03 of claim 2 or 3 as an ingredient of said product.