CN110878266B

CN110878266B - Lactobacillus johnsonii and application thereof

Info

Publication number: CN110878266B
Application number: CN201911151651.XA
Authority: CN
Inventors: 李有全; 贾丹; 刘爱红; 王锦明; 孟林明; 王佳慧; 李贺海; 关贵全; 刘军龙; 殷宏; 罗建勋; 石红梅; 赵索南; 任巧云; 杨吉飞; 刘志杰; 独军政; 高闪电; 刘光远; 李东平
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2021-04-20
Anticipated expiration: 2039-11-21
Also published as: CN110878266A

Abstract

The invention discloses lactobacillus johnsonii and application thereof in diarrhea of piglets, lambs and calves. The strain is separated from the excrement of healthy pigs, and has good in-vitro probiotic potential and safety. The lactobacillus johnsonii and other strains are jointly applied to diarrhea young animals, and the strain has obvious prevention and treatment effects on diarrhea cases with different degrees.

Description

Lactobacillus johnsonii and application thereof

Technical Field

The invention relates to lactobacillus johnsonii and application thereof.

Background

China is a big animal husbandry country, and the healthy development of livestock and poultry is the key of the economic development of the breeding industry. In the pig industry, piglet diarrhea is the most serious problem faced by large-scale farms, and has become one of the main problems affecting the healthy and stable development and economic benefits of the pig industry. Many pathogenic microorganisms can cause diarrhea in young animals such as piglets, lambs and calves, and Escherichia coli, salmonella, streptococcus and the like are the main pathogenic microorganisms. In large-scale farms, the solution to such problems is the use of antibiotics. Although antibiotics play a certain positive role in preventing and resisting diseases and the like, the harm brought to human health and environment by long-term use of the antibiotics is self-evident. With the restriction and disablement of antibiotics in most countries, the search for alternatives to new antibiotics has received considerable attention. Research proves that supplementing a certain amount of probiotics in the livestock and poultry feed has good effects of preventing and curing gastrointestinal diseases of livestock, promoting nutrient absorption of livestock and the like.

Probiotics (probiotics) are a general term for active beneficial microorganisms that usually colonize the intestinal tract and reproductive system of animals and produce a definite health effect. The concept was first derived from 1965, which Lilly et al defined as a "growth-promoting-factor-producing microorganism". In 2002, the food probiotic evaluation guideline formally defined probiotics by the food probiotic organization (FAO) and the World Health Organization (WHO) as follows: when ingested in sufficient quantities, they can confer a healthy effect on the host. Common probiotics include lactobacillus, bifidobacterium, streptococcus, bacillus, yeast and the like. The probiotics which are currently most widely studied and used are lactobacilli and bifidobacteria.

Lactic acid bacteria are a general term for a group of gram-positive bacteria capable of fermenting carbohydrates and producing large amounts of lactic acid, and include various genera, mainly Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), coccus (Streptococcus), Enterococcus (Enterococcus), Lactococcus (Lactococcus), and the like. Lactic acid bacteria play an important physiological role in the body as microorganisms normally present in the intestinal tracts of humans and animals, and are internationally recognized as food-grade safe microorganisms (GRAS). At present, lactic acid bacteria as green, safe and effective probiotics are widely applied to food, medicines and feed additives.

The probiotic preparation is mainly made of living microorganisms, needs to have certain tolerance to the gastrointestinal tract environment of a host, and can only play a probiotic role after bacterial strains are adhered, colonized and propagated in intestinal tracts. Related researches show that the lactobacillus can secrete various enzymes to promote absorption of various nutrient substances and reduce loss of trace elements in vivo in an adsorption mode. In addition to producing bacteriocin, lactic acid bacteria can stimulate the body to produce defensins, and can promote humoral immunity and cellular immunity of the host by enhancing the activity of peripheral blood T lymphocytes and B lymphocytes. The direct feeding of lactobacillus screened in vitro has been proved to increase the weight gain of livestock and poultry, increase the number of beneficial bacteria in intestinal tract, reduce the number of pathogenic bacteria, enhance immunity, improve antioxidant function, increase feed utilization rate, reduce mortality and prevent diseases caused by gastrointestinal flora. Therefore, the lactobacillus has wide application prospect in livestock production and disease control, and has important significance in developing novel probiotic lactobacillus strains.

Lactic acid bacteria are the earliest and most widespread probiotics used in animal husbandry. Research shows that after lactobacillus is added into broiler feed, the oxidation resistance and growth performance of broilers can be improved, the occurrence of osteoporosis can be reduced, and the weight gain of broilers can be promoted; after the lactobacillus is used for feeding weaned piglets, the diarrhea occurrence frequency and the death rate of the piglets are obviously reduced, and the weight gain of the livestock body is promoted; after the lactobacillus is applied in the pig production, the growth development and the production performance of the pig can be obviously promoted; the lactobacillus feed is applied to the milk cow breeding, and has the effects of improving the nutrient utilization rate of the feed, increasing the milk yield, improving the milk quality and preventing mastitis. In a word, after the probiotic lactic acid bacteria are added into the livestock and poultry feed, the growth of animals can be effectively promoted, the immunity of organisms can be improved, the death rate can be reduced, and animal diseases can be prevented and treated. Naturally, the probiotics as a new safe, green and pollution-free antibiotic substitute plays an important role in reducing the problems of bacterial drug resistance increase, antibiotic residue, environmental pollution and the like caused by long-term use of antibiotics in the breeding industry, and provides important guarantee for the safe production of livestock and poultry and the human health.

Therefore, the method has important significance for meeting the requirements of the breeding industry in China, promoting the healthy growth of livestock and poultry and developing the probiotic strains which have the characteristics of probiotics and can prevent and treat or slow diarrhea of young livestock.

Disclosure of Invention

The invention provides a probiotic strain which can be used in livestock breeding, enriches the strains of the feed probiotics and provides strain resources for the development of a compound probiotic preparation.

The invention provides a strain of lactobacillus johnsonii GHZ10a (separated from healthy pig manure)Lactobacillus johnsoniiGHZ10a), which is deposited in China general microbiological culture Collection center (CGMCC for short) of China Committee for culture Collection of microorganisms in Beijing, and is classified as Lactobacillus johnsonii (Lactobacillus johnsonii, preservation number CGMCC No. 18472, preservation date of 2019, 6 months and preservation unit address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

The lactobacillus johnsonii can be applied to the preparation of feeds for treating piglets, lambs, calves and the like, and can also be applied to the preparation of medicaments for treating diarrhea of the piglets, the lambs, the calves and the like.

The strain sieve provided by the invention is selected from 325 newly separated lactic acid bacteria, and the specific method comprises the following steps: firstly, inoculating 325 newly separated lactic acid bacteria into sterile PBS (phosphate buffer solution) with pH of 2.5 and containing 0.3% pig bile salt, respectively tolerating for 2h and 4h at 37 ℃, calculating the number of viable bacteria, and screening 64 separated strains with better tolerance; secondly, determining the bacteriostatic activity of the screened 64 strains of tolerant lactic acid bacteria by an oxford cup diffusion method, wherein the strain with the number of GHZ10a has the widest bacteriostatic spectrum and the highest bacteriostatic activity; finally, the strain is identified as Lactobacillus johnsonii GHZ10a through morphological observation, physiological and biochemical identification and 16S rDNA amplification.

The lactobacillus johnsonii has good in-vitro probiotic potential, can rapidly enter logarithmic phase and has strong acid production capacity; survival rate was 83.93% after 3h tolerance in simulated gastric fluid and 78.55% after 6h tolerance in simulated intestinal fluid; has good hydrophobicity and copolymerization ability; the adhesion rate to Caco-2 cells is 12.37 +/-0.32%, and the adhesion of Escherichia coli to Caco-2 cells can be inhibited.

The lactobacillus johnsonii adopts a K-B method to determine the antibiotic sensitivity of the strain, and the test shows that the GHZ10a strain is sensitive to 7 of 10 antibiotics used in the test and resistant to kanamycin, naproxen acid and cefotaxime; the strain GHZ10a showed no hemolytic activity after streaking on a blood plate; GHZ10a high concentration (1X 10)⁹One mouse per day), the mouse is normal in mental state, normal in stool color and shape, and free of abnormal secretion and death condition of mouth and nose; after the mice are dissected, the indexes of all organs are normal and have no obvious difference with those of a control group.

The lactobacillus johnsonii and other probiotic strains are compounded into a composite probiotic preparation to feed 1511 diarrheal piglets, except that some sick animals die due to serious diarrhea and other factors, the illness state of other sick animals is improved in the next day of feeding, and the sick animals are basically cured after 3-5 days. The recovered piglets have no diarrhea recurrence phenomenon.

The lactobacillus johnsonii and other probiotic strains are combined into a compound probiotic preparation which is primarily applied to diarrhea lambs and calves, and the compound probiotic preparation has the functions of slowing down and curing diarrhea diseases.

The lactobacillus johnsonii GHZ10a has high safety, good in-vitro probiotic potential and certain antibiotic resistance, and can effectively inhibit porcine diarrhea diseases.

Drawings

FIG. 1, graph of GHZ10a inhibiting Pasteurella 393.

Figure 2, a plot of GHZ10a inhibiting staphylococcus haemolyticus ZSY 2.

FIG. 3, graph of GHZ10a inhibiting pathogenic E.coli ATCC 43888.

FIG. 4, colony morphology of GHZ10 a. The lactobacillus johnsonii GHZ10a is flat on MRS plate, and has irregular edge, smooth surface and approximately transparent shape.

FIG. 5, gram stain of GHZ10a (100X). The gram staining of lactobacillus johnsonii GHZ10a is positive, the thallus under the microscope is in a long rod shape, the two ends are round, and the thallus appears in a single, paired, chain or cluster shape.

FIG. 6, PCR amplified fragment map of GHZ10 a. The first lane is a DNAmarker with a DNA molecular mass of 2000. 1. Lane 2 shows the fragment length of the strain GHZ10a, which is about 1500 bp.

Figure 7, growth curve-pH curve of GHZ10 a.

FIG. 8, a graph of hemolysis assay of GHZ10 a. The upper area of the plate is the strain GHZ10a, and hemolysis does not occur; the area under the plate was positive control (E.coli), with hemolytic ring and hemolytic activity.

Detailed Description

The following is a detailed description of aspects of the invention with reference to the drawings.

Example isolation, screening and identification of lactic acid bacteria

1.1 isolation of lactic acid bacteria

50 parts of fresh excrement of healthy pigs in a plurality of pig farms in Qinghai province is collected. Weighing 10g of a fecal sample, uniformly mixing the fecal sample with 90mL of sterile PBS, filtering, taking 1mL of filtrate, performing gradient dilution, and mixing 10g of the filtrate with the sterile PBS^-3、10^-4、10^-5The diluted solutions were spread on MRS plates containing 0.75% calcium carbonate, respectively, and cultured at 37 ℃ for 24 hours under anaerobic conditions. About 4-7 single colonies with different forms and colors and generating calcium-dissolving rings in each sample are respectively streaked and purified on an MRS (methicillin resistant Staphylococcus aureus) plateAfter three times, single colonies are selected and inoculated in MRS liquid culture medium, anaerobic culture is carried out at 37 ℃ for 24 hours for later use, and 325 isolates are obtained through preliminary separation in total.

The formula of the MRS liquid culture medium used in the invention is as follows:

10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 801.0mL of Tween and K₂HPO₄·3H₂O2.0 g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, MgSO₄·7H₂O 0.29g、MnSO₄·H₂O0.058 g, adding the above components into distilled water, diluting to 1000mL, adjusting pH to 6.3, and autoclaving at 121 deg.C for 20 min.

The formula of the calcium-added MRS solid culture medium used in the invention is as follows:

adding 7.5g of calcium carbonate and 15.0g of agar into the liquid MRS culture medium, adjusting pH to 6.3, and autoclaving at 121 ℃ for 20 min.

The formulation of the sterile PBS used in the invention is as follows:

weighing 8.0g of NaCl, 0.2g of KCl and Na₂HPO₄1.42 g，KH₂PO₄Adding 0.27 g of the extract into 1L of deionized water, fully stirring and dissolving, dropwise adding concentrated hydrochloric acid to adjust the pH to 7.4, carrying out autoclaving at 121 ℃ for 20min, and storing at room temperature for later use.

1.2 tolerant Primary Screen for lactic acid bacteria

Acid resistance measurement: 1mL of fresh culture medium of each of the 325 new isolates was inoculated into sterile PBS (pH 2.5), and after 3 hours of tolerance at 37 ℃, samples were taken and serially diluted to 10^-4、10^-50.1mL of diluent is absorbed and coated on an MRS plate, viable count is counted and determined after anaerobic culture is carried out for 24h at 37 ℃, and the survival rate is calculated by taking the viable count of 0h as a control.

And (3) testing the bile salt resistance: respectively taking 1mL of fresh culture solution of the 325 isolated bacteria, inoculating the fresh culture solution into sterile PBS containing 0.3% of pig bile salt, counting and determining the number of viable bacteria after tolerating for 3h at 37 ℃, and calculating the survival rate by taking the number of viable bacteria of 0h as a control.

The tolerance result shows that 168 lactic acid bacteria in 325 suspected lactic acid bacteria strains have higher acid tolerance and the survival rate is higher than 80 percent; 64 test strains in the acid-tolerant lactic acid bacteria have high tolerance to bile salts, the survival rate is over 80 percent, and the rest strains show different degrees of sensitivity to acids and bile salts.

1.3 bacteriostatic lactic acid bacteria rescreening

Preparation of fermentation supernatant: centrifuging fresh bacterial liquid of the 64 acid and bile salt tolerant isolates at 4 ℃ and 6000r/min for 10min, filtering supernatant with a 0.22 mu m microporous filter membrane, and storing at 4 ℃ for later use.

The antibacterial activity of the strain is measured by adopting a diffusion method, and 16 pathogenic bacteria such as haemophilus parasuis, pasteurella, mycoplasma bovis, escherichia coli and the like are selected as indicator bacteria. The activated pathogenic bacteria are regulated by sterile PBS to the concentration of about 1 × 10⁶CFU/mL, uniformly coating 100 mu L of pathogenic bacteria on corresponding agar plates, and standing for 10min at room temperature; after the surface bacteria liquid of the flat plate is slightly dried, uniformly punching the flat plate by using a puncher with the aperture of 7mm, and adding 150 mu L of the cell-free fermentation supernatant into the holes; the plate is horizontally placed at room temperature for pre-diffusion for 2h, and cultured at 37 ℃ for 18 h; after the test is finished, the diameter of the antibacterial ring is measured by using a vernier caliper, and the test is repeated for 3 times to obtain an average value.

The results of bacteriostatic tests show that the strain with the number of GHZ10a shows inhibitory action of different degrees on 15 strains of 16 strains of pathogenic bacteria (figure 1-figure 3), has the widest bacteriostatic spectrum and the largest bacteriostatic diameter compared with other strains, has the strongest bacteriostatic action on salmonella, but has no inhibitory action on Clostridium perfringens MQ 5. The bacteriostatic effect of the strain GHZ10a is shown in Table 1.

TABLE 1 determination of the bacteriostatic ability of the Strain GHZ10a (mm)

Based on the above experimental results, the strain GHZ10a was finally screened as a candidate strain for probiotics.

1.4 identification of the Strain

And (3) morphology observation: strain GHZ10a was flat on MRS agar plates with irregular edges, smooth surface, and near transparency (fig. 4). Gram staining is purple, is gram positive bacteria, and the microscopical thallus is long rod-shaped, with two round ends, appearing in single, paired, chain-forming or cluster-like shapes (fig. 5).

Physiological and biochemical identification: the physiological and biochemical characteristics of strain GHZ10a are shown in Table 2. The gelatin test, the indole test, the hydrogen peroxide test and the starch hydrolysis test are all negative. In the carbohydrate fermentation test, the strain GHZ10a can ferment glucose and fructose, and can not utilize arabinose and lactose.

TABLE 2 physiological and biochemical characteristics of Strain GHZ10a

Test strains	Gelatin test	Indole test	Hydrogen peroxide	Starch hydrolysis
					GHZ10a	-	-	-	-
Test strains	Glucose	Fructose	Ara-laPrimary sugar	Lactose
					GHZ10a	+	+	-	-

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

Molecular biological identification: extracting genome of the strain GHZ10a by using a DNA extraction kit, and performing PCR amplification by using 16S rDNA universal primers 27F and 1492R for bacterial identification, wherein the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, and performing 35 cycles; finally, extending for 8min at 72 ℃; the amplified product is detected by 1% agarose gel electrophoresis to obtain a target fragment with the length of about 1500bp (figure 6), the sequence is submitted to NCBI for BLAST comparison to obtain the sequence with the homology of 99.99% with Lactobacillus johnsonii, and the accession numbers are obtained: MK 208491.

Combining the morphology, physiological and biochemical characteristics and 16S R DNA sequence of the strain GHZ10a, the strain is finally determined to be Lactobacillus johnsonii (Lactobacillus johnsonii). The strain is submitted in 2019, 9 and 6 days and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO: 18472.

Example in vitro probiotic Studies of Lactobacillus johnsonii GHZ10a

2.1 preparation of Lactobacillus johnsonii GHZ10a seed solution

Selecting a single colony from a purified MRS plate, inoculating the single colony in 10mL of MRS culture medium, culturing at the constant temperature of 37 ℃ for 24h, and continuously carrying out passage twice to prepare separated strain liquid for later use.

2.2 measurement of growth Curve and pH Curve of Lactobacillus johnsonii GHZ10a

Inoculating 2.1 Lactobacillus johnsonii GHZ10a liquid in MRS culture medium at 2%, standing at 37 deg.C for culture,taking liquid culture medium without inoculated bacteria as control, sampling every 2h within 0-24h after inoculation culture to determine bacterial liquid OD_(600nm)Value and pH value. The results show (FIG. 7) that the Lactobacillus johnsonii GHZ10a entered logarithmic growth phase 4h after inoculation, and OD was within 4-8h_(600nm)The value shows a straight line increase, the bacteria enter a stationary phase after 10h of culture, and the OD of the thallus is at the moment_(600nm)The value was 3.551, and the cell count reached the highest value at this time. The pH value is slowly reduced within the first 4h after the strain GHZ10a is inoculated, and the acid production capability is weak; after the logarithmic growth phase, the pH of the bacterial liquid is obviously reduced, and when the bacterial liquid is grown for 10 hours, the bacterial liquid is slowly reduced in a stable phase. Along with the extension of the inoculation time, the increase of the number of the thalli and the decrease of the pH value of the fermentation liquor are in a certain relation, and when the thalli are in a logarithmic growth phase, the pH value of the bacterial liquid is decreased rapidly.

2.3 study of the gastrointestinal tolerance of the GHZ10a Strain

Preparing simulated gastric juice: pepsin (1:15000) was weighed and dissolved in sterilized normal saline (0.9% w/v, hydrochloric acid to adjust pH to 3.0) to prepare a solution with a concentration of 3 g/L. After filtration through a 0.22 μm sterile filter membrane, the cells were stored at 4 ℃.

Preparing simulated intestinal juice: trypsin (1:250) was weighed out and dissolved in sterilized normal saline (0.9% w/v, pH adjusted to 8.0 with NaOH) to prepare a solution with a concentration of 1g/L, and 0.3% bile salt was added. After filtration through a 0.22 μm sterile filter membrane, the cells were stored at 4 ℃.

Centrifuging the strain liquid separated from 2.1, collecting the strain, resuspending with sterile PBS, and adjusting the concentration to 1 × 10⁹And CFU/mL, adding 1mL of bacterial liquid into 9mL of simulated gastric juice, standing and culturing at 37 ℃ for 1h, 2h and 3h, and sampling and counting respectively. After 3h, 1mL of the above culture was placed in 9mL of simulated intestinal fluid, cultured at 37 ℃ for 2h, 4h, and 6h, and then sampled and counted respectively to determine the total number of viable bacteria. The results of gastrointestinal tolerance of the strain GHZ10a are shown in Table 3, the survival rate of the strain GHZ10a after 3 hours of simulated gastric fluid treatment is 83.93%, and the survival rate of the simulated intestinal fluid treatment is 78.55%, so that the strain can well tolerate the gastrointestinal environment.

TABLE 3 detection of gastrointestinal tolerance by Strain GHZ10a

2.4 determination of surface Properties of Strain GHZ10a

The overnight cultured Lactobacillus johnsonii GHZ10a and Escherichia coli ATCC43888 were centrifuged at 5000r/min and 4 ℃ for 10min, and the cells were collected. Determination of the self-agglutination rate of the strain (%): adjusting the absorbance of the concentration of Lactobacillus gasseri GHZ10a at 600nm to 0.5 + -0.02 (A)₀) Standing for 24h, and measuring light absorption value A₂₄Calculating the self-agglutination rate of the strain GHZ10a, and the formula is (A)₀-A₂₄)/A₀X is 100%; determination of the co-aggregation rate (%) of the strain and E.coli: adjusting the light absorption value of the mixed suspension liquid of the Lactobacillus gasseri GHZ10a and the Escherichia coli ATCC43888 at the wavelength of 600nm to be 0.5 +/-0.02 (A)₀) Standing for 24h, and measuring light absorption value A₂₄Calculating the co-aggregation rate of the strain GHZ10a, wherein the formula is (A)₀-A₂₄)/A₀X is 100%; measurement of the hydrophobicity of the strain (%): the absorbance of Lactobacillus johnsonii GHZ10a at a wavelength of 600nm was made 0.5. + -. 0.02 with sterile PBS (A)₀) Adding 1mL of dimethylbenzene into 3mL of bacterial suspension, pre-culturing for 10min at room temperature, then fully mixing the two-phase system by vortex oscillation for 2min, standing at 37 ℃ for co-culturing for 1h, removing the dimethylbenzene phase, and measuring the light absorption value A of the water phase at 600nm₁Calculating the hydrophobic rate of the strain GHZ10a, wherein the formula is (A)₀-A₁)/A₀X 100%. The measurement results are shown in Table 4, and it is understood that the strain GHZ10a has a self-aggregation rate of 69.53%, a co-aggregation rate with Escherichia coli of 69.28% and a hydrophobicity of 51.79%, indicating that Lactobacillus johnsonii GHZ10a can aggregate with Escherichia coli and has good hydrophobicity.

TABLE 4 determination of surface Properties of Lactobacillus johnsonii GHZ10a

Bacterial strains	Self-aggregation rate	Co-aggregation ratio	Rate of water repellency
				Lactobacillus johnsonii GHZ10a	69.53％	69.28％	51.79％

2.5GHZ10a Strain adhesion Capo-2 cell assay

In the test, Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) is selected as a control strain.

Fluorescence labeling of test strains: selecting the activated test strains, centrifuging at 4 ℃ and 6000r/min to collect thalli, washing for 3 times by using sterile PBS, adding the thalli into Fluorescein Isothiocyanate (FITC) solution with the working concentration of 500 mu g/mL, and performing dark treatment at 37 ℃ for 2 hours; centrifuging the marked bacteria liquid at 6000r/min, and washing for 3 times by PBS to remove unbound FITC; the bacterial cells were resuspended in RPMI-1640 cell culture medium, and the concentration of the bacterial liquid was adjusted to 2X 10⁸And (3) absorbing 100 mu L of the fluorescence-labeled bacterial liquid into a 96-well plate, and measuring the fluorescence intensity of the bacteria when the wavelength of absorbed light is 485nm and the wavelength of emitted light is 530nm by using a microplate reader, wherein the fluorescence intensity is the initial relative fluorescence value.

Adhesion test: to a 12-well plate cultured to a monolayer of cells, 0.5mL of FITC-labeled bacteria was added at 37 ℃ with 5% CO₂Sealing and incubating in the incubator for 2h, rinsing with sterile PBS for 3-5 times to remove non-adhered bacteria; adding 300 μ L pancreatin, digesting cells for 5min, adding cell culture solution to stop reaction when cells are snowflake-shaped and scattered under microscope, and blowing and mixing cell suspension. Absorbing 100 mu L of cell suspension into a 96-well plate and measuring the fluorescence intensity, namely the cell suspension after adhesionRelative fluorescence values. The adhesion rate of the strain is calculated by the formula:

the adhesion rate (relative fluorescence value after adhesion/initial relative fluorescence value) × 100%

The adhesion rates of strain GHZ10a and control strain GG are shown in Table 5. The lactobacillus johnsonii GHZ10a shows that the adhesion rate is higher and has no obvious difference with the adhesion rate of lactobacillus rhamnosus GG in probiotic products.

TABLE 5 adhesion rates of Strain GHZ10a

Bacterial strains	Adhesion Rate (%)
		Lactobacillus johnsonii GHZ10a	12.37±0.32
Lactobacillus rhamnosus GG	12.15±0.58

2.6 determination of the ability of the Strain GHZ10a to inhibit the adhesion of E.coli to Caco-2 cells by different means

Fluorescence labeling of E.coli: the labeling method was the same as that of the strain GHZ10a in 2.5.

And (3) competitive test: test and control groups were set. Test groups test strains (GHZ10a, LGG; 2X 10) were added to 12-well culture plates cultured in monolayer cells, respectively⁸CFU/mL) and 0.25mL of marked Escherichia coli respectively and mixing uniformly; adding 0.5mL of FITC-labeled Escherichia coli into a control group; 37 ℃ and 5% CO₂After incubation in the incubator for 2h, the cells were washed 3-5 times with sterile PBS.

Rejection test: test group test strains (GHZ10a, LGG; 2) were added to 12-well culture plates cultured in monolayer cells10⁸CFU/mL) 0.5 mL; adding 0.5mL of cell culture solution into a control group; at 37 ℃ with 5% CO₂And (3) incubating in an incubator for 1h, removing liquid in the holes, rinsing with sterile PBS for 3 times, adding 0.5 mLFITC-labeled escherichia coli into each hole, incubating at 37 ℃ for 1h in a sealed manner, and rinsing with sterile PBS for 3-5 times.

Replacement test: to a 12-well plate cultured to a monolayer of cells, 0.5 mM FITC-labeled E.coli was added at 37 ℃ with 5% CO₂Sealing and incubating in an incubator for 1h, discarding liquid in the hole, and rinsing with sterile PBS for 3 times; the test group was added with 0.5mL of the test strain (GHZ10a, LGG; 2X 10)⁸CFU/mL), adding 0.5mL of cell culture solution to a control group, carrying out closed incubation at 37 ℃ for 1h, and rinsing with sterile PBS for 3-5 times.

After the test is finished, 300 mu L of pancreatin is added into the hole, the cells are digested for 5min, when the cells are scattered like snowflake under a microscope, cell culture solution is added to stop the reaction, and cell suspension is mixed uniformly. The cell suspension was pipetted into a 96-well plate and the relative fluorescence intensity was measured with a microplate reader. The adhesion inhibition rate of the test strain on the Escherichia coli ATCC43888 in Caco-2 cells is calculated according to the following formula:

the adhesion inhibition (%) was 1- (test group relative fluorescence intensity/control group relative fluorescence intensity) × 100%

As shown in table 6, lactobacillus johnsonii GHZ10a can inhibit escherichia coli from adhering Caco-2 cells through competition, rejection and replacement, but mainly inhibits escherichia coli from adhering through competition and rejection, and the inhibition rate of the competition mode is more significant, while the inhibition rate of the rejection mode is lower.

TABLE 6 determination of the ability of Lactobacillus johnsonii GHZ10a to inhibit the adhesion of E.coli to Caco-2 cells

Bacterial strains	Competition test (%)	Rejection test (%)	Displacement test (%)
				Lactobacillus johnsonii GHZ10a	52.19±1.05	1.65±0.32	23.04±2.17

Example safety study of Lactobacillus johnsonii GHZ10a

3.1 susceptibility test of Strain GHZ10a (K-B method)

The activated strain GHZ10a was centrifuged and adjusted to a concentration of about 1X 10 with sterile PBS⁶CFU/mL, dipping the bacteria liquid with a sterilized cotton stick and uniformly coating the bacteria liquid on an MRS solid plate; uniformly placing the drug sensitive paper sheets on the surface of a flat plate by using sterile forceps, carrying out anaerobic culture at 37 ℃ for 24 hours, recording the diameter of a bacteriostatic circle of each drug sensitive paper sheet, and repeating 3 times of tests to obtain an average value. In the test, Escherichia coli (Escherichia coli) ATCC 25922 is selected as a quality control strain, and the sensitivity of the strain to antibiotics is judged by referring to the diameter of the minimum zone of inhibition in the international standards of CLSI (clinical and laboratory standards institute) of the United states of America to the sensitivity of antibacterial drugs. Of the 10 antibiotics used in this test, the strain GHZ10a was sensitive to 7 antibiotics such as tetracycline, ampicillin, vancomycin, and the like, and resistant to kanamycin, naproxen, and cefotaxime.

TABLE 7 results of drug susceptibility test of Strain GHZ10a to 10 antibiotics

Note: r, drug resistance; s, sensitivity; -, there is no drug sensitive ring.

3.2 hemolytic assay of Strain GHZ10a

The bacterial liquid of a freshly cultured strain GHZ10a is picked by an inoculating loop and streaked and inoculated on a blood agar plate, and after anaerobic culture at 37 ℃ for 20 hours, whether hemolytic rings are formed around colonies is observed. The results showed that no hemolytic ring was present around lactobacillus johnsonii GHZ10a (fig. 8), indicating that the strain is not hemolytic.

The blood agar plate used in the invention has the following formula:

10.0g of peptone, 5.0g of beef extract, 5.0g of sodium chloride and 15g of agar powder, adding the components into distilled water, fixing the volume to 1000mL, carrying out autoclaving at 121 ℃ for 20min, cooling to 60 ℃, adding 50mL of sterile defibrinated sheep blood, and mixing uniformly.

3.3 high concentration feeding test of Strain GHZ10a

20 healthy Kunming mice were purchased and weighed approximately 15g, and the mice were randomly divided into two groups (10 each), a test group and a control group. Test group mice were fed basal diet + GHZ10a (1X 10)⁹/day), control mice were fed basal diet + MRS liquid medium. The test period was 30 days. During the test period, the two groups of mice have good mental state, bright hair color, no abnormal secretion of mouth and nose, normal diet and drinking water, normal color and shape of excrement and no death. After the test is finished, weighing the weight of the mouse, killing the mouse by a cervical dislocation method, dissecting and collecting organs such as heart, liver, spleen, lung, kidney, thymus and the like, washing with normal saline, sucking dry by filter paper, weighing and calculating the index of each organ. The results (table 8) showed that the difference in organ index between the test group and the control group was not significant, and it was found that lactobacillus johnsonii GHZ10a did not exert adverse effects on the body organs, and was judged as a safe strain and used as a candidate strain for a microecological preparation.

TABLE 8 calculation of organ index after feeding mice with high concentration of Lactobacillus johnsonii GHZ10a

Heart (%)

Liver (%)

Spleen (%)

Lung (%)

Kidney (%)

Thymus (%)

Control group

0.63±0.08

4.77±0.29

0.36±0.09

1.05±0.07

1.36±0.16

0.55±0.15

Test group

0.79±0.27

4.93±0.56

0.40±0.12

1.00±0.25

1.20±0.62

0.46±0.09

Example preliminary application study of Lactobacillus johnsonii GHZ10a in prevention and treatment of diarrhea in young livestock

The lactobacillus johnsonii GHZ10a is compounded with laboratory separated bacillus subtilis BSC16a and lactobacillus salivarius ZLp4b in a certain proportion to form a composite microecological preparation for preventing and treating piglet diarrhea. The results are shown in table 9, 1511 diarrhea piglets in 5 farms of Gansu Yongjing, Pingyang, folk music, Shanxi Yangling and Henan Minggu are fed respectively, diarrhea symptoms are obviously relieved in the next day of feeding, sick animals are basically cured after continuous feeding for 3-5 days, no animal death occurs in the Gansu Pingyang farms during feeding, the cure rate reaches 100%, and individual sick animals in the rest farms die due to severe diarrhea and other factors, wherein 5 pigs in the Gansu Mingyle farms die due to severe dehydration. Except that the cure rate of diarrhea of piglets in the Yangtze rabdosia Shaanxi farm is 87.60%, the cure rate of diarrhea cases in other farms is higher than 90.00%. In the feeding experiment, the probiotic preparation has obvious effect of curing diarrhea of piglets, and the disease relapse and other phenomena of recovered animals do not occur. In addition, the composite microecological preparation is primarily applied to diarrhea lambs and calves, and has the effects of relieving and curing diarrhea diseases. Therefore, the lactobacillus johnsonii GHZ10a can be used as a compound microecological preparation compound strain to be applied to prevention and treatment of diarrhea of young livestock.

TABLE 92019 statistical table of control results of diarrhea cases of piglets

Region of land

Animal(s) production

Cultivation scale

Number of onset of disease

Number of rehabilitation

Number of deaths

Cure rate

Mortality rate

Gansu yongjing

Piglet

2000

326

302

20

92.63％

6.67％

Gansu calm and cool

Piglet

600

56

0

100.00％

0.00％

Gansu folk music

Piglet

8000

532

496

36

93.23％

7.67％

Shaanxi Yangling

Piglet

4000

121

106+14*

3

87.60％

2.47％

Right of south river

Piglet

6000

476

452

24

94.96％

5.04％

Remarking: wherein 14 of the above drugs are drenched for 3 days to find no obvious improvement, and then the drugs are treated with antibiotics, wherein 11 of the drugs are recovered, and 3 of the drugs die. But in statistics, it was not calculated according to the number of convalescent pigs.

Claims

1. A strain of Lactobacillus johnsonii (Lactobacillus johnsonii) GHZ10a is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9 and 6 days, and the preservation number is CGMCC NO: 18472.

2. Use of lactobacillus johnsonii according to claim 1 for the preparation of a feed for piglets, lambs and calves.

3. Use of lactobacillus johnsonii according to claim 1 for the manufacture of a medicament for the treatment of diarrhoea in piglets, lambs and calves.