CN112011481A

CN112011481A - Lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof

Info

Publication number: CN112011481A
Application number: CN202010801032.7A
Authority: CN
Inventors: 刘滢; 刘雪连; 沈红霞; 邓莉萍; 孙晓杰; 李晟硕; 韩绍良; 刘英俊; 王玉华; 王丽英
Original assignee: Jiangxi Dabeinong Farming Technology Co ltd; Zhengzhou Dabeinong Feed Technology Co ltd; Beijing Dabeinong Technology Group Co Ltd
Current assignee: Jiangxi Dabeinong Farming Technology Co ltd; Zhengzhou Dabeinong Feed Technology Co ltd; Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2020-12-01
Anticipated expiration: 2040-08-11
Also published as: CN112011481B

Abstract

The invention discloses lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof. The lactobacillus reuteri is DBN-JP, and the preservation number of the lactobacillus reuteri is CGMCC No. 19493. The lactobacillus reuteri thallus, the fermentation liquid and the fermentation supernatant can be used as additives for preparing animal feed or added into animal drinking water or fed separately, and have the effects of stimulating the gastrointestinal development of young animals, improving the immunity of the animals and reducing diarrhea, wherein the animals include but are not limited to various animals such as pigs, chickens, ducks and the like. The feed has similar function to antibiotic colistin sulfate in preventing and treating bacterial diarrhea of livestock and poultry, but has no antibiotic side effect.

Description

Lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof

Technical Field

The invention belongs to the field of micro-ecology, and particularly relates to lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof.

Background

The newborn period and the weaning period are two key links for limiting the intensive large-scale pig raising production efficiency in China. The health condition of the intestinal tract is a key factor influencing the production performance of piglets and the overall survival rate of swinery. Improving the intestinal health of newborn and weaned piglets and avoiding or reducing the infection of pathogenic microorganisms in the intestinal tract are important problems to be solved urgently in pig raising production (Yiyunlong et al, 2010). Factors such as incomplete development of intestinal immune system of newborn piglets, no establishment of intestinal microecological environment balance, and susceptibility of weaned piglets to infection of pathogenic microorganisms due to weaning stress are major factors causing growth retardation and frequent diseases of piglets (Pacific et al, 2015). Recent researches show that the establishment and balance of intestinal microbial flora structure can promote the development and maturation of intestinal immune system of piglets, improve the intestinal functions of newborn piglets and weaned piglets, and play an important role in the establishment and the function development of normal stable intestinal state of piglets (Foushe et al, 2016).

The bacterial diarrhea of chickens, which is one of chicken diseases, affects the growth and development speed of chicken flocks and seriously causes death to bring huge loss to farmers. Pathogens inducing the chicken bacterial diarrhea mainly comprise escherichia coli, salmonella, pasteurella and the like, the pathogenic bacteria are sensitive to antibiotics, and the livestock farm usually prevents the bacterial diarrhea by adding the antibiotics into feed, but the escherichia coli and the salmonella are easy to generate drug resistance (Wachengwei, 2020).

Conventionally, antibiotics are added into feed to play roles in preventing and treating diseases and promoting growth. However, the problems of livestock and poultry intestinal flora imbalance, pathogenic bacteria drug resistance enhancement, livestock product safety, environmental pollution and the like caused by long-term use of a large amount of antibiotics can threaten animal health and even human health. In recent years, with the advancement of the feed "banning" mode in various countries around the world, the No. 2428 bulletin of the ministry of agriculture in China prohibited the use of colistin sulfate as a growth promoter in 2016, 11 and 1, and the publication of the No. 2638 bulletin is determined by stopping the production of olaquindox, arsanilic acid, rocarsone and the like for food animal production in 2018, 5. The european commission made a decision in 2017 that "the european union banned the use of high dose zinc oxide for piglets within five years". With the implementation of the national plan for suppressing animal-derived bacteria drug resistance (2017 and 2020), the development of safe and effective novel antibiotic substitutes is urgent.

Lactobacillus reuteri is a beneficial lactobacillus (Hsueh et al, 2010) which is commonly existing in intestinal tracts of human and animals, and lactobacillus reuteri and metabolites thereof have a plurality of excellent physiological functions and have positive significance for maintaining the health of animal organisms and promoting the growth and development of animals. Reuterin, one of the major metabolites of lactobacillus reuteri, inhibits the growth of harmful bacteria such as escherichia coli, salmonella, etc., protects the microecological balance of the intestinal tract (navy, et al, 2017), and orally administered lactobacillus reuteri significantly reduces the incidence and severity of diarrhea (Shornikova, et al, 1997).

Disclosure of Invention

In order to solve the problems, the invention provides a Lactobacillus reuteri DBN-JP for preventing and treating bacterial diarrhea of livestock and poultry and application thereof.

The Lactobacillus reuteri DBN-JP provided by the invention is preserved in China general microbiological culture Collection center at 3-19.2020, with the address: the collection number of the microbial research institute of the Chinese academy of sciences, No. 3 Xilu-Beijing province, Chaoyang, and the collection number is: CGMCC No. 19493. The Lactobacillus reuteri has strong stress resistance and bacteriostatic ability, and has probiotic performance for preventing and treating bacterial diarrhea of livestock and poultry.

The lactobacillus reuteri provided by the invention has the following biological properties:

acid resistance: the survival rate is more than 100 percent after being treated for 6 hours under the condition of simulated gastric juice with the pH value of 2.0.

The performance of resisting bile salt: the survival rate is more than 95 percent after being treated for 8 hours under the condition of 0.3 percent simulated gastric juice.

Heat resistance: the heating temperature is less than or equal to 80 ℃, and the bacteriostatic ability of the Lactobacillus reuteri DBN-JP is not influenced after 15 min.

The active ingredients of the lactobacillus reuteri product provided by the invention are the lactobacillus reuteri DBN-JP, fermentation liquor and fermentation supernatant thereof.

The lactobacillus reuteri DBN-JP, the fermentation liquor and the fermentation supernatant thereof have at least one of the following uses:

antibacterial property: minimum inhibitory concentration for inhibiting escherichia coli k 88: the minimum inhibitory concentration of the Lactobacillus reuteri DBN-JP fermentation liquid to Escherichia coli k88 is 32 mu l/ml; minimum inhibitory concentration for inhibiting salmonella: the minimum inhibitory concentration of the lactobacillus reuteri DBN-JP fermentation liquid to the salmonella is 64 mu l/ml; minimum inhibitory concentration for inhibiting staphylococcus aureus: the minimum inhibitory concentration of the lactobacillus reuteri DBN-JP fermentation liquor to staphylococcus aureus is 32 mu l/ml.

The function of preventing and treating the bacterial diarrhea of the livestock and poultry is as follows: the results of the piglet animal experiments show that: compared with a blank group, the addition of the active ingredient of the Lactobacillus reuteri DBN-JP into the feed can prevent and treat piglet bacterial diarrhea (P is less than 0.05) by remarkably reducing the number of Escherichia coli and salmonella in caecum and increasing the number of lactobacillus; the broiler test result shows that compared with the blank group, the addition of the lactobacillus reuteri DBN-JP active ingredient in the feed can prevent and treat broiler bacterial diarrhea (P is less than 0.05) by remarkably reducing the number of escherichia coli and salmonella in the cecum of broiler and increasing the number of lactobacillus.

Improving the immunity function of animals: the results of the piglet animal experiments show that: compared with a blank group, the addition of the active ingredient of the lactobacillus reuteri DBN-JP into the feed can improve the immunity of piglets by obviously improving the content of SIgA in jejunal mucous membranes (P is less than 0.05); the broiler test result shows that compared with a blank group, the feed added with the lactobacillus reuteri DBN-JP active ingredient can improve the broiler immunity (P is less than 0.05) by obviously improving the content of IgA and IgM in broiler blood.

The lactobacillus reuteri thallus, the fermentation liquor and the fermentation supernatant can be used as additives for preparing animal feed or adding the additive into animal drinking water or feeding the additive alone, and have the effects of improving the immunity of animals and reducing diarrhea for young animals, wherein the animals include but are not limited to various animals such as pigs, chickens, ducks and the like. The feed has similar function to antibiotic colistin sulfate in preventing and treating bacterial diarrhea of livestock and poultry.

Drawings

FIG. 1 shows the growth curve of Lactobacillus reuteri DBN-JP.

FIG. 2 shows the microscopic observation of Lactobacillus reuteri DBN-JP.

FIG. 3 shows the colony morphology of Lactobacillus reuteri DBN-JP plate.

FIG. 4 shows the acid tolerance of Lactobacillus reuteri DBN-JP.

FIG. 5 shows the bile salt resistance of Lactobacillus reuteri DBN-JP.

FIG. 6 shows the acid production curve of Lactobacillus reuteri DBN-JP.

FIG. 7 shows a circle diagram of the genome of Lactobacillus reuteri DBN-JP.

FIG. 8 shows the functional distribution diagram of Lactobacillus reuteri DBN-JP GOC annotation.

FIG. 9 shows the minimum inhibitory concentration of Lactobacillus reuteri DBN-JP fermentation broth.

FIG. 10 shows the Lactobacillus reuteri DBN-JP bacteriostasis electron microscope photograph.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 Lactobacillus reuteri DBN-JP culture method of the present invention

1. Strain Lactobacillus reuteri DBN-JP (isolated from piglet intestinal contents in the national focus laboratory of microbiological engineering for feed by Beijing Dabei agricultural technology group GmbH); lactobacillus reuteri CICC6118, lactobacillus reuteri CICC6119 and lactobacillus reuteri CICC6121 (the three strains are purchased from China center for culture Collection of industrial microorganisms); lactobacillus reuteri DSM17938 (isolated from Bigaia products).

2. Culture medium

Wherein the culture medium is any culture medium suitable for culturing lactobacillus reuteri (such as MRS medium, Rogosa SL medium, TPY medium or other modified medium, etc.), and those skilled in the art know which culture medium is suitable for culturing lactobacillus reuteri. The following take MRS medium as an example:

MRS culture medium: casein peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 5.0g, sodium acetate 5.0g, diammonium citrate 2.0g, Tween 801.0 g, and K₂HPO₄ 2.0g，MgSO₄.7H₂O 0.2g，MnSO₄.H₂O 0.05g，CaCO₃20.0g, agar 15.0g, distilled water 1.0L, pH6.8, 115 deg.C sterilization for 20 min.

MRS solid culture medium 15g/L agar was added to MRS liquid culture medium for activation of Lactobacillus reuteri and counting of viable flat cells.

3. Culture method

The culture is carried out using the above-mentioned medium, and the specific culture process is generally known to those skilled in the art.

Seed culture: culturing MRS agar in Hungates rolling tube culture medium at 37 deg.C for 16-18 h;

and (3) amplification culture: culturing in MRS broth at 37 deg.C for 16-24 hr with the growth curve of Lactobacillus reuteri DBN-JP shown in figure 1. In the case of the scale-up culture, whether the obtained culture was pure bacteria was observed by a microscope at 4 hours, 8 hours and 24 hours, as shown in FIG. 2.

4. Viable count determination

Shaking up the cultured fermentation liquor, accurately sucking 1ml of bacterial liquid, uniformly mixing in 9ml of sterile normal saline, sequentially carrying out gradient dilution to a proper gradient according to the method, taking 1ml of diluted bacterial liquid, inoculating the diluted bacterial liquid into an MRS solid culture medium, uniformly coating, putting the MRS solid culture medium into a constant-temperature incubator at 37 ℃ for culturing for 48 hours, carrying out viable bacteria counting, and determining that the number of bacterial colonies on a flat plate is 30-300 cfu/plate as effective. The morphology of the Lactobacillus reuteri DBN-JP colony plate is shown in FIG. 3.

5. Lactobacillus reuteri active ingredient

The lactobacillus reuteri fermentation liquid is liquid obtained by primary fermentation, secondary fermentation or multiple fermentation in any culture medium suitable for the growth of lactobacillus reuteri, and comprises lactobacillus reuteri thalli and metabolites. The lactobacillus reuteri fermentation supernatant is liquid obtained by centrifuging, filtering or other methods of the lactobacillus reuteri fermentation liquid and removing part or all of lactobacillus reuteri thallus.

The lactobacillus reuteri thallus, the fermentation liquor and the fermentation supernatant can be prepared into powder, granules or paste by the modes of freeze drying, spray drying, fluidized bed drying and the like generally known by the technical personnel in the field, and the powder, the granules or the paste is added into animal feed and drinking water for feeding or is fed separately.

Example 2 probiotic Properties of Lactobacillus reuteri DBN-JP of the invention

1. Acid resistance test

The strain is anaerobically cultured in a liquid culture medium at 37 ℃ overnight, and thalli are collected by centrifugation and washed for 2 times by the liquid culture medium. Resuspending in simulated gastric juice with pH of 2.0 and 4.0, standing at 37 deg.C for culture, sampling at 4h and 6h, diluting with 10 times of sterile water, inoculating MRS plate at 100 μ l at appropriate dilution, inoculating 2 parallel samples at each dilution, culturing at 37 deg.C for 36-48h, and counting.

As can be seen from FIG. 4, Lactobacillus reuteri DBN-JP has good acid resistance, and acid treatment only slows down its growth rate but does not cause death compared to normal pH.

2. Bile salt resistance test

The strain is statically cultured in a liquid culture medium at 37 ℃ overnight, thalli are collected by centrifugation, and the liquid culture medium is washed for 2 times. Resuspending in simulated bile with bile salt concentration of 0.3% and 0.5%, standing at 37 deg.C for culture, sampling at 6 hr and 8 hr, diluting with 10 times of sterile water, inoculating solid plate at 100 μ l at appropriate dilution, inoculating 2 parallel samples at each dilution, culturing at 37 deg.C for 36-48 hr, and counting.

As can be seen from FIG. 5, Lactobacillus reuteri DBN-JP is well tolerant to 0.3% of bile salts, with a survival rate > 95%; the survival rate of the treated mixture can be more than 80% after being treated for 6 hours by 0.5% of the concentration of the bile salt, and the survival rate of the treated mixture can be more than 70% after being treated for 8 hours.

3. Acid production capacity

The strain is statically cultured in a liquid culture medium at 37 ℃ overnight, centrifuged to collect supernatant, and the content of lactic acid, acetic acid and propionic acid in the supernatant is determined by an HPLC method. HPLC method: mobile phase: 0.05% sulfuric acid solution, column: c18 column, flow rate: 1ml/min, ultraviolet detector, 210 nm. And (3) passing the sample through a filter membrane of 0.45 mu m, and then detecting on a machine.

TABLE 1 acid productivity of different samples fermented for different times

Unit: g/L

Item	Lactic acid	Acetic acid
			Blank of culture Medium	/	/
DBN-JP(16h)	8.77±0.10^a	9.60±0.02^a
			DBN-JP(24h)	10.76±0.25^b	9.80±0.04^b
DSM17938(16h)	9.24±0.05^c	4.38±0.03^c
			DSM17938(24h)	9.69±0.04^d	4.47±0.05^c

As can be seen from Table 1, in 16h of fermentation, the lactic acid production amount of the Lactobacillus reuteri DBN-JP is lower than that of DSM17938(P < 0.05), but the acetic acid production amount is higher than that of DSM17938(P < 0.05), and the total acid production amount (lactic acid + acetic acid) is combined, so that the Lactobacillus reuteri DBN-JP has obvious acid production advantages; after fermentation is carried out for 24 hours, the lactic acid and acetic acid production amount of the Lactobacillus reuteri DBN-JP is obviously higher than that of DSM17938(P is less than 0.05), the total acid production amount (lactic acid + acetic acid) is comprehensively obtained, and the Lactobacillus reuteri DBN-JP has obvious acid production advantages.

The acid production capability of the lactobacillus is related to the bacteriostatic performance and the capability of adjusting the pH value of the intestinal tract, the lactobacillus reuteri DBN-JP has better lactic acid and acetic acid production capability, and the fermentation time can be adjusted according to the acid production curve (figure 6) according to the production requirement in specific implementation.

Example 4 Lactobacillus reuteri DBN-JP Whole genome sequencing analysis of the invention

The whole genome of the Lactobacillus reuteri DBN-JP is obtained through a third generation sequencing platform, the genome size is 2243904bp, the gene number is 2160, the total gene length is 1923786bp, the average gene length is 890.64, and the GC content is 39.65 percent, which is shown in figure 7; 3239 CDS were analyzed, of which 2160 CDS with COG functional annotation were obtained, and the gene length accounted for 85.73% of the total genome length. Comparing COG database to complete homologous gene annotation classification, and obtaining coding genes (shown as figure 8) with unknown functions, including information storage and processing, cell biological process and signal transduction, basic metabolism, wherein translation, ribosome structure and biogenesis; a moving body: prophage, transposon; amino acid transport and metabolism; a general function prediction gene; the proteins that replicate, recombine and repair account for a large proportion, 8.15%, 7.5%, 5.97%, 5.69% and 5.05%, respectively. The results of carbohydrate-associated enzyme (CAZY) database annotation indicate that the strain contains 32 glycoside hydrolase genes, 5 carbohydrate-associated modules genes and 17 glycosyltransferase genes, and does not contain a polysaccharide lyase gene, a carbohydrate esterase gene and auxiliary functions.

Through whole genome sequencing and comparative genomics analysis, the strain comprises specific genes including but not limited to SEQ ID No. 1-3.

Example 5 bacteriostatic ability of Lactobacillus reuteri DBN-JP of the present invention

Bacteriostatic property

Oxford cup method: the sterilized LB agar medium was heated to completely melt, poured into petri dishes at 15ml per dish (lower layer), and allowed to solidify. In addition, the thawed medium is cooled to about 50 deg.C and mixed with a concentration of pathogenic bacteria (Escherichia coli, Salmonella, Staphylococcus aureus) of about 1 × 10⁸cfu/ml, 5ml of a medium mixed with pathogenic bacteria was added to the coagulated medium to be coagulated (upper layer). An Oxford cup is vertically placed on the surface of the culture medium, 150 mu l of a sample to be detected (culture medium blank, Lactobacillus reuteri DBN-JP, CICC6118, CICC6119, CICC6121, DSM17938 fermentation liquor (fermentation for 16h) and 0.02% colistin sulfate) is added into the cup, and the culture is carried out for 16-18 hours at 37 ℃ after the cup is filled. And measuring the size of the bacteriostatic zone by using a vernier caliper.

TABLE 2 bacteriostatic ability of different samples against pathogenic bacteria

Unit: mm is

Item	Escherichia coli k88	Staphylococcus aureus	Salmonella
				Blank of culture Medium	6.00±0.1	6.00±0.1	6.00±0.1
DBN-JP	18.73±0.23^a	19.68±0.26^a	18.84±0.04^a
				CICC6118	14.55±0.22^b	15.48±0.21^b	14.47±0.20^b
CICC6119	14.85±0.08^b	15.30±0.17^b	15.28±0.19^c
				CICC6121	14.99±0.10^b	15.81±0.09^b	15.97±0.09^d
DSM17938	17.32±0.13^c	18.60±0.31^c	16.82±0.11^e
				0.02% colistin sulfate	20.08±0.05^d	19.92±0.03^a	19.34±0.30^a

As can be seen from Table 2, the bacteriostatic ability of Lactobacillus reuteri DBN-JP on Escherichia coli k88 is lower than 0.02% colistin sulfate (P < 0.05), but significantly higher than that of Lactobacillus reuteri CICC6118, CICC6119, CICC6121 and DSM17938(P < 0.05); the bacteriostatic ability to staphylococcus aureus is not different significantly (P is more than 0.05) compared with 0.02% colistin sulfate, but is significantly higher than that to lactobacillus reuteri CICC6118, CICC6119, CICC6121 and DSM17938(P is less than 0.05); the bacteriostatic ability to salmonella is not significantly different (P is more than 0.05) compared with 0.02% colistin sulfate, but is significantly higher than that of lactobacillus reuteri CICC6118, CICC6119, CICC6121 and DSM17938(P is less than 0.05).

In conclusion, the bacteriostatic ability of lactobacillus reuteri DBN-JP on staphylococcus aureus and salmonella is equivalent to 0.02% colistin sulfate, the bacteriostatic ability on escherichia coli is slightly lower than 0.02% colistin sulfate, and the lactobacillus reuteri DBN-JP has better bacteriostatic ability compared with a control strain.

Effect of Heat on the bacteriostatic ability of Lactobacillus reuteri DBN-JP

Generally, the temperature for preparing the granules is generally 75-80 ℃ for 15min, and according to the feed addition requirement, the fermentation liquor of lactobacillus reuteri DBN-JP and DSM17938 is subjected to heat treatment under different conditions, and the fermentation liquor is heated at 80 ℃ for 15min to obtain a sample, and the diameter of the inhibition zone is measured by an Oxford cup method.

TABLE 3 Effect of heating on the bacteriostatic ability of different Lactobacillus reuteri fermentation broths

Unit: mm is

Species of	Escherichia coli k88	Staphylococcus aureus	Salmonella
				DBN-JP	18.46±0.30^a	19.27±0.36^a	18.35±0.43^a
DBN-JP (80 ℃ for 15min)	18.35±0.34^a	19.23±0.26^a	18.06±0.11^a
				DSM17938	18.08±0.13^a	19.74±0.24^a	17.23±0.24^b
DSM17938(80 deg.C for 15min)	17.21±0.56^b	16.87±1.21^b	15.60±0.34^c

As can be seen from Table 3, the bacteriostatic ability of Lactobacillus reuteri DBN-JP was not significantly affected by heating at 80 ℃ for 15min (P > 0.05); heating at 80 deg.C for 15min has significant effect on the bacteriostatic ability of Lactobacillus reuteri DSM17938(P < 0.05), but still retains a part of the bacteriostatic ability. The heat resistance of the lactobacillus is directly related to industrialization, the good heat resistance is beneficial to the addition and application of the lactobacillus in the feed, and the data show that the lactobacillus reuteri DBN-JP has the advantage of being added in the feed compared with the lactobacillus reuteri DSM 17938.

Lactobacillus reuteri DBN-JP minimum inhibitory concentration

Inoculum: inoculating Escherichia coli k88, Staphylococcus aureus, and Salmonella into nutrient broth culture medium, culturing at 37 deg.C and 180r/min for 12 h.

Taking a sterile 48-well plate, adding 1ml of nutrient broth into each tube except 1.6ml of nutrient broth into the 1 st hole, adding 0.4ml of lactobacillus reuteri DBN-JP fermentation broth solution (with the concentration of 1280 mu l/ml) into the 1 st hole, uniformly mixing, sucking 1ml into the 2 nd hole, uniformly mixing, sucking 1ml into the 3 rd hole, continuously diluting to the 11 th hole in a multiple ratio manner, sucking 1ml from the 11 th hole, discarding, and taking the 12 th hole as a growth control without a medicament. The concentration of the fermentation liquor of Lactobacillus reuteri DBN-JP is 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 mul/ml in sequence. Then 1ml of each of the prepared inocula was added to each well to give a final bacterial liquid concentration of about 5X 10 per well⁶CFU/ml. The drug concentrations of the 1 st to 11 th wells are 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25 and 0.125. mu.l/ml respectively. And (4) standing and culturing for 8h at 37 ℃, and observing by naked eyes, wherein the hole with the lowest concentration of the medicament has no bacteria growth, namely the MIC of the tested bacteria.

As can be seen from FIG. 9, the minimum inhibitory concentration of the Lactobacillus reuteri DBN-JP fermentation broth to Staphylococcus aureus is 32. mu.l/ml, the minimum inhibitory concentration to Escherichia coli k88 is 32. mu.l/ml, and the minimum inhibitory concentration to Salmonella is 64. mu.l/ml.

Lactobacillus reuteri DBN-JP antibacterial electron microscope picture

Escherichia coli k88 was inoculated into nutrient broth, cultured at 37 ℃ at 180r/min for 12 h. Taking 1 sterile 50ml centrifuge tube with a cover, adding 30ml of escherichia coli k8812h culture solution and 9.6mg of lactobacillus reuteri DBN-JP16h supernatant (1000r/min, 5min), sealing, placing in a 37 ℃ incubator, standing and culturing for 8h, centrifuging for 10000r/min and 5min, washing with sterile ultrapure water twice, scraping off bacterial sludge lightly, collecting in a 1.5ml centrifuge tube, adding 1ml of stationary liquid (provided by China institute of agricultural science atomic energy utilizing microscope room), resuspending, keeping out of the sun at room temperature for 48h, and taking samples to prepare electron microscope samples.

As can be seen from FIG. 10, the active ingredient of Lactobacillus reuteri DBN-JP has the ability to disrupt the cell membrane of pathogenic bacteria.

Example 5: application of lactobacillus reuteri DBN-JP in prevention and treatment of piglet diarrhea

1. Test materials

Lactobacillus reuteri fermentation broth: the viable count of Lactobacillus reuteri is about 5-6 × 10⁸cfu/g (if viable count is too high, dilute with lactobacillus reuteri fermentation supernatant); lactobacillus reuteri powder: centrifuging the fermentation broth of Lactobacillus reuteri at 10000r/min for 3min, adding protective agent, and lyophilizing to obtain Lactobacillus reuteri with viable count of about 5-6 × 10⁸cfu/g; lactobacillus reuteri supernatant: centrifuging the lactobacillus reuteri fermentation liquor at 10000r/min for 3min to obtain supernatant; prepared by a national key laboratory of microbiological engineering for feed of Beijing Dabei agricultural technology group GmbH.

2. Test animals and test design

Randomly selecting 90 weaned piglets of 21-day-old Du grown-up piglets, and randomly dividing the piglets into 5 groups and blank groups according to the principles of similar body weight and same sex proportion: feeding basic ration; antibiotic group: feeding basal diet +300mg/kg aureomycin (15%); bacterium powder group: feeding basic ration and lactobacillus reuteri powder by 1 kg/t; supernatant group: feeding basic ration and lactobacillus reuteri supernatant at 1L/t; and (3) fermentation liquor group: feeding basal diet and lactobacillus reuteri fermentation liquor 1L/t. Each group had 6 replicates, 3 pigs each, with a test period of 49 days.

3. Experimental diet and feeding management

The test basic ration is a corn-soybean meal type ration, and is prepared according to the nutritional needs of pigs in the NCR (2012) stage of 7-11kg and the NCR stage of 11-25 kg. The basic feed composition and the nutrition level that this embodiment provided are reference scheme, can add according to the actual use daily ration of plant during specific application.

The basal diet composition and nutritional levels are shown in table 3. The test is carried out in a Yutian farm of the great northern agricultural group, the temperature of the colony house is controlled to be 26 +/-1 ℃, the relative humidity is controlled to be 50-80%, all pigs can freely eat and drink water, the colony house is cleaned regularly and disinfected by alternately using disinfectant, and the ventilation and sanitation of the colony house are kept.

TABLE 4 basic diet composition and nutritional levels (air-dried basis)

％

1) The vitamin premix is provided for each kilogram of feed: VA8000IU, VD₃2000IU，VE25.0IU，VK1.2mg，VB₁2.5 mg，VB₂6.5 mg，VB₆10.0mg，VB₁₂50mg, biotin 0.15mg, folic acid 1.0mg, D-pantothenic acid 20.0mg, nicotinic acid 45 mg.

2) The mineral premix is provided for each kilogram of diet: fe 100mg, Cu100mg, Zn100mg, Mn4mg and Se 0.35 mg.

3) The nutrient levels are calculated values.

4. Sample collection and processing

Diets were settled on trial day 49 and all pigs fasted for 12 h. All test pigs were weighed and recorded on day 50 of the trial, and 1 piglet with a weight closest to the average weight of the group was selected and labeled in each replicate and slaughtered in order. Collecting duodenal mucosa samples and jejunum mucosa samples and colon and caecum chyme samples, quickly freezing by using liquid nitrogen, and storing in a refrigerator at the temperature of minus 80 ℃ to be tested.

5. Intestinal microflora

And respectively establishing a standard curve of each flora by taking the plasmid containing the target fragment as a standard plasmid, determining the copy number of all samples by adopting a real-time fluorescence quantitative method, and calculating by using the standard curve to obtain the number of escherichia coli, lactobacillus and salmonella in the colon and the caecum of the weaned pig.

6. Immune function

The content of SigA in the mucous membranes of duodenum and jejunum is determined by adopting a pig secretory immunoglobulin A (SigA) enzyme-linked immunosorbent assay kit.

7. Data analysis

All test data are counted by using Excel2010, single-factor variance analysis is carried out by using span 20.0 statistical software, all data are expressed by 'mean value plus or minus standard error', the difference is obvious when P is less than 0.05, and the significant difference trend is that P is more than or equal to 0.05 and less than 0.10.

8. Data analysis and conclusions

TABLE 5 Effect on the cecal and colonic flora of 70 day-old piglets

Lg(CFU/g)

Item	Blank group	Antibiotic group	Fungus powder group	Supernatant group	Fermentation liquor group
						Cecum
Escherichia coli	8.53±0.15^a	8.51±0.17^a	8.25±0.51^b	8.35±0.14^b	8.15±0.69^b
						Lactobacillus strain	10.21±0.11^a	10.23±0.27^a	10.71±0.36^b	10.56±0.24^b	11.04±0.16^c
Salmonella	3.45±0.32^a	2.56±0.41^b	2.73±0.46^b	2.77±0.66^b	2.48±0.42^b
						Colon
Escherichia coli	8.59±0.23^ab	8.79±0.05^ab	8.48±0.29^a	8.76±0.30^ab	8.47±0.74^a
						Lactobacillus strain	10.86±0.10^a	10.93±0.21^a	11.25±0.31^b	11.05±0.19^ab	11.23±0.22^b
Salmonella	3.44±0.33^a	2.60±0.47^b	2.91±0.28^b	2.84±0.58^b	2.74±0.25^b

As can be seen from Table 5, compared with the blank group, the numbers of Escherichia coli and Salmonella in the cecum of the bacterial powder group, the supernatant group and the fermentation liquid group are remarkably reduced, the number of Lactobacillus is remarkably increased (P is less than 0.05), and the numbers of Escherichia coli, Salmonella and Lactobacillus among the groups have a remarkable difference trend (P is more than or equal to 0.05 and less than 0.1); compared with the antibiotic group, the differences of the amounts of the Escherichia coli, the salmonella and the lactobacillus in the cecum of the bacterial powder group, the supernatant group and the fermentation liquor group are not significant (P is more than or equal to 0.05).

Compared with the blank group, the number of salmonella in the colon of the bacterium powder group, the supernatant group and the fermentation liquid group is obviously reduced (P is less than 0.05), the number of lactobacillus is obviously improved (P is more than or equal to 0.05 and less than 0.1), the number of escherichia coli has no obvious difference (P is more than 0.1), the number of lactobacillus and salmonella among all groups has obvious difference (P is less than 0.05), and the number of escherichia coli has no obvious difference (P is more than 0.1); compared with the antibiotic group, the colon bacillus number of the bacterium powder group and the fermentation liquid group is obviously reduced (P is less than 0.05), and the lactobacillus number is obviously increased (P is less than 0.05); the number of salmonella in the bacterial powder group, the fermentation liquid group and the supernatant group is obviously reduced (P is less than 0.05).

TABLE 6 Effect on the content of SIgA in the duodenal and jejunal mucous membranes of 70-day-old piglets

μg/g

Item	Blank group	Antibiotic group	Fungus powder group	Supernatant group	Fermentation liquor group
						Duodenum	5.56±0.13^b	5.68±0.09^b	5.38±0.18^b	5.89±0.29^a	5.59±0.30^b
Jejunum	11.39±1.03^a	10.66±0.17^a	14.52±1.53^b	13.04±0.93^c	13.01±0.33^c

As can be seen from Table 6, compared with the blank group and the antibiotic group, the content difference of SIgA in the duodenal mucosa of the bacterial powder group and the fermentation liquid group is not significant (P is more than 0.05), the content difference of SIgA in the duodenal mucosa of the supernatant group is significant (P is less than 0.05), and the difference between the groups is significant (P is less than 0.05); compared with the blank group and the antibiotic group, the content difference of SIgA in jejunal mucosa of the bacteria powder group, the supernatant group and the fermentation liquid group is obvious (P is less than 0.05), the content difference of SIgA in duodenal mucosa of the supernatant group is obvious (P is less than 0.05), and the difference between the groups is obvious (P is less than 0.05).

The results show that the active ingredients (live bacteria, supernatant and fermentation liquor) of the lactobacillus reuteri DBN-JP can prevent and treat piglet diarrhea by reducing the number of harmful escherichia coli and salmonella in the caecum and colon of piglets and increasing the number of beneficial bacteria lactobacillus; and the intestinal immunity function can be improved by obviously improving the content of SIgA in the mucosa of duodenum and jejunum.

Example 6: application of lactobacillus reuteri DBN-JP in broiler chickens

1. Test materials

Lactobacillus reuteri fermentation broth: the viable count of the Lactobacillus reuteri is more than or equal to 10¹⁰cfu/g; lactobacillus reuteri powder: centrifuging the fermentation liquid of Lactobacillus reuteri at 10000r/min for 3min, adding protective agent, and lyophilizing to obtain the final product with viable count of Lactobacillus reuteri not less than 10⁸cfu/g; lactobacillus reuteri supernatant: centrifuging the lactobacillus reuteri fermentation liquor at 10000r/min for 3min to obtain supernatant; prepared by a national key laboratory of microbiological engineering for feed of Beijing Dabei agricultural technology group GmbH. Colistin sulfate, a commercially available product.

2. Test diet composition and nutritional level

The compound is prepared according to NRC broiler chicken nutrition standard. The composition and nutrient level of the diet at the early stage (age of 1-21 days) and the late stage (age of 22-42 days) are shown in tables 6-1 and 6-2. The material type is powder, only anticoccidial drug salinomycin (60 mug/ml) is added into basic daily ration, and other antibiotics are not added.

TABLE 7-1 composition of daily ration at later stage of experiment

Daily ration formula	Content%	Nutritional index	Nutrient content
				Corn (corn)	59.1	Metabolic energy	12.77
Bean pulp	30.6	Crude protein%	21.2
				Corn protein powder	3.8	Available phosphorus%	0.43
Oil and fat	1.7	Calcium content%	1.0
				Stone powder	1.31	Lysine%	1.08
Sodium chloride	0.42	Methionine%	0.5
				Calcium hydrogen phosphate	1.77	Methionine + cystine%	0.82
Lysine%	0.15
				Methionine%	0.15
Premix compound	1

TABLE 7-2 composition of daily ration at early stage of experiment

Daily ration formula	Content%	Nutritional index	Nutrient content
				Corn (corn)	64.3	Metabolic energy	12.77
Bean pulp	24.3	Crude protein%	19.3
				Corn protein powder	4.5	Available phosphorus%	0.43
Oil and fat	2.5	Calcium content%	0.91
				Stone powder	1.23	Lysine%	0.95
Sodium chloride	0.33	Methionine%	0.43
				Calcium hydrogen phosphate	1.58	Methionine + cystine%	0.73
Lysine%	0.16
				Methionine%	0.1
Premix compound	1

3. Test animals and test design

Test site: jilin honghai animal husbandry.

Randomly selecting 720 feathers of 1-day-old AA white feather broilers with basically consistent weight and health conditions, and randomly dividing the feathers into 5 groups and a blank group according to the principles of similar weight and same sex proportion: feeding basic ration; antibiotic group: feeding basic ration and 30g/t of colistin sulfate; bacterium powder group: feeding basic ration and lactobacillus reuteri powder by 1 kg/t; supernatant group: feeding basic ration and lactobacillus reuteri supernatant at 1L/t; and (3) fermentation liquor group: feeding basal diet and lactobacillus reuteri fermentation liquor 1L/t. Each group had 6 replicates, each replicate 24-feather chicken, the experimental period was 42 days, conventional rearing, free feeding, daily management and immunization programs were performed according to the chicken house conventional methods.

4. Sample collection and processing

Diets were settled on trial day 43 and all chickens were fasted for 8 h. Weighing and recording all test chickens on the 44 th day of the test, selecting and marking 3 feathers of broilers with the weight closest to the average weight of the group in each repetition, sequentially cutting off jugular veins to die, respectively tying the cecum at two ends by using a sanitary cotton thread, cutting off the cecum from the outer end, placing the cecum in a closed sample bag, freezing and storing the cecum in a laboratory, taking intestinal chyme in a sterilized centrifuge tube by a sterile method, quickly freezing by using liquid nitrogen, and storing the cecum in a refrigerator at the temperature of minus 80 ℃ to be tested.

5. Intestinal microflora

And respectively establishing a standard curve of each flora by taking the plasmid containing the target fragment as a standard plasmid, determining the copy number of all samples by adopting a real-time fluorescence quantitative method, and calculating by using the standard curve to obtain the number of escherichia coli, lactobacillus and salmonella in the cecum of the broiler chicken.

6. Immune function

After the test is finished, 1.0ml of blood is collected from all chickens by the winged veins, serum is separated, and the contents of IgM, IgG and IgA are measured by adopting an ELISA kit.

7. Data analysis

8. Data analysis and conclusions

TABLE 8 Effect on cecal bacteria flora of broilers

Lg(CFU/g)

Item	Blank group	Antibiotic group	Fungus powder group	Supernatant group	Fermentation liquor group
						Escherichia coli	11.20±0.17^a	11.23±0.34^a	11.15±0.15^a	10.44±0.23^b	10.73±0.33^b
Lactobacillus strain	8.60±0.30^a	8.20±0.10^b	8.76±0.38^a	8.23±0.21^b	9.20±0.37^c
						Salmonella	8.56±0.19^a	8.23±0.11^b	8.52±0.23^a	8.45±0.43^a	8.14±0.78^b

TABLE 9 influence on broiler immunity index

mg/ml

Item	Blank group	Antibiotic group	Fungus powder group	Supernatant group	Fermentation liquor group
						IgA	1.15±0.08ab	1.03±0.07a	1.20±0.23ab	1.25±0.20ab	1.31±0.23b
IgG	1.88±0.19a	1.74±0.84a	1.96±0.13a	2.18±0.27b	2.14±0.12b
						IgM	0.89±0.11	0.85±0.74	0.82±0.13	0.85±0.09	0.94±0.05

As can be seen from table 8, compared with the blank group, the number of escherichia coli in the cecum of the supernatant group and the fermentation broth group was significantly reduced, the number of lactobacillus was significantly increased, and the number of salmonella in the fermentation broth group was significantly reduced (P < 0.05); compared with the antibiotic group, the number of escherichia coli in the supernatant group and the fermentation liquid group is obviously reduced, the number of lactobacillus in the bacterial powder group and the fermentation liquid group is obviously increased, and the number of salmonella in the bacterial powder group and the supernatant group is obviously reduced (P is less than 0.05); the number of colibacillus, salmonella and lactobacillus between groups is obviously different (P is less than 0.05).

As can be seen from Table 9, compared with the blank group, the IgA content of the bacterial powder group, the supernatant group and the fermentation liquid group is not significantly different (P is more than 0.05), and the difference between the groups is not significant; compared with the antibiotic group, the fermentation liquid group has the function of remarkably improving IgA in the blood of the broiler chicken (P is less than 0.05), and the supernatant group has the trend of remarkably improving IgA in the blood of the broiler chicken (P is more than or equal to 0.1 and less than 0.05). Compared with a blank group and an antibiotic group, the supernatant group and the fermentation liquid group have the effect of obviously improving the content of IgG (P is less than 0.05), and the difference between the groups is obvious (P is less than 0.05); compared with the antibiotic group, the bacterial powder group has the tendency of obviously improving the content of IgG (P is more than or equal to 0.1 and less than 0.05); compared with the blank group and the antibiotic group, the IgM content difference among the bacterial powder group, the supernatant group and the fermentation liquid group is not significant (P is more than 0.05), and the IgM content difference among the groups is not significant (P is more than 0.05).

In conclusion, the addition of the active ingredient of the lactobacillus reuteri DBN-JP in the feed can reduce the number of escherichia coli and salmonella in cecum of the broiler chicken, increase the number of lactobacillus, and improve the immunity of the broiler chicken by increasing the content of IgA and IgM in blood of the broiler chicken.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing Dabei agricultural technology group, GZHONG feed science and technology Co., Ltd, Jiangxi Dabei agricultural technology Co., Ltd

<120> lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof

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Claims

1. Lactobacillus reuteri DBN-JP for preventing and treating bacterial diarrhea of livestock and poultry, wherein the deposit number of the Lactobacillus reuteri DBN-JP is as follows: CGMCC No. 19493.

2. The lactobacillus reuteri product for preventing and treating bacterial diarrhea of livestock and poultry according to claim 1.

3. The lactobacillus reuteri product for preventing and treating bacterial diarrhea of livestock and poultry according to claim 2, wherein the product comprises but is not limited to one or more of lactobacillus reuteri thallus, lactobacillus reuteri fermentation liquor or lactobacillus reuteri fermentation supernatant.

4. Feed additive, premix, concentrate or batch containing the lactobacillus reuteri product for preventing and treating bacterial diarrhea of livestock and poultry according to claim 2 or 3.

5. The use of the lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry according to claim 1 in the preparation of medicaments for inhibiting intestinal pathogenic bacteria of livestock and poultry.

6. The use of lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry according to claim 1 in the preparation of medicaments for improving the immunity of animals.

7. The use according to claim 5 or 6, said livestock and poultry including but not limited to pigs or chickens.

8. The use according to claim 5, wherein the enteropathogenic bacteria include but are not limited to Escherichia coli k88, Salmonella, Staphylococcus aureus.