Broad-spectrum bacteriostatic lactic acid bacteria metagen preparation, preparation method and application to laying hens

Abstract

The invention relates to a broad-spectrum bacteriostatic lactic acid bacteria postbiotic preparation, a preparation method and application to laying hens, the lactic acid bacteria postbiotic preparation comprises lactic acid bacteria thalli and fermentation products of the lactic acid bacteria thalli, and lactic acid bacteria comprise lactobacillus plantarum LP45 and bifidobacterium animalis BAL531. The feed additive is prepared from lactobacillus plantarum LP45 and bifidobacterium animalis BAL531 through mixed fermentation and low-temperature vacuum drying, pathogenic bacteria such as clostridium perfringens, escherichia coli and salmonella can be inhibited, the abundance of beneficial bacteria in the intestinal tracts of chicken flocks is improved, the state of excrement is improved, chicken necrotic enteritis and diarrhea are prevented and treated, the intestinal health of laying hens is improved, and the laying hens are prevented and cured. The digestion and absorption efficiency of the feed is improved, and the laying rate is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of application of probiotic products, and specifically relates to a broad-spectrum antibacterial lactic acid bacteria postbiotic preparation, a preparation method and application on laying hens. Background Art

[0002] Necrotic enteritis in laying hens is an acute intestinal infectious disease of laying hens caused by Clostridium perfringens. It is one of the most harmful intestinal diseases in the laying hen farming industry. It is usually characterized by severe intestinal bleeding, intestinal mucosal damage, ulcers, necrosis, etc. The disease can occur all year round, especially in the hot and rainy summer, and often occurs in broilers aged 2 to 6 weeks and commercial laying hens aged 3 to 6 months. The occurrence of acute necrotic enteritis in chickens can lead to sudden and large-scale deaths in the flock, while chronic necrotic enteritis in chickens will not lead to sudden deaths in the flock, but will lead to decreased appetite, decreased intestinal digestion and absorption function, decreased feed conversion efficiency, and weight loss, thereby hindering the growth of the flock. In addition, during the breeding process of laying hens, the indigestible protein in the feed and the anti-nutritional factors in it will damage the intestinal epithelial cells of the poultry, causing the feed to be excreted without being digested, resulting in overfeeding, which in turn causes bacterial and nutritional diarrhea in the chickens;

[0003] At present, antibiotics are usually used to prevent and treat the above diseases, but they are prone to drug resistance, accumulation and transmission in the food chain, and seriously affect human health.

[0004] Lactic acid bacteria are a type of probiotic that can grow using sugars to produce lactic acid. Because they have the function of improving intestinal microecology, postbiotics are a general term for the beneficial components of probiotics after processing, including bacteria and metabolites. Their health functions are based on their bacteria themselves, metabolites, or lysis products. These factors are officially named "postbiotics" by the International Probiotics Organization. Postbiotics are all inactivated / broken bacteria or contain a small amount of live bacteria. Their processing resistance is greatly improved compared to live bacteria preparations. Their application range and application forms are also wider than live bacteria preparations. It is a microecological preparation with a very broad application prospect.

[0005] Therefore, it is crucial to develop a lactic acid bacteria postbiotic preparation, preparation method and application that is easy to process and apply, has broad-spectrum antibacterial properties, and can effectively prevent and treat necrotic enteritis and overfeed diarrhea in chickens, in order to replace traditional antibiotics and solve a series of problems caused by the use of antibiotics. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects in the prior art and provide a broad-spectrum antibacterial lactic acid bacteria postbiotic preparation, a preparation method and application on laying hens, which can inhibit pathogenic bacteria such as Clostridium perfringens, Escherichia coli and Salmonella, prevent and treat necrotic enteritis and overfeed diarrhea in chickens, improve the intestinal health of laying hens, improve the digestion and absorption efficiency of feed, and increase egg production rate.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A broad-spectrum antibacterial lactic acid bacteria postbiotic comprises inactivated lactic acid bacteria and fermentation products thereof, wherein the lactic acid bacteria comprises Lactobacillus plantarum LP45 and Bifidobacterium animalis BAL531.

[0009] As a further technical solution, the fermentation product includes organic acids and antimicrobial peptides.

[0010] As a further technical solution, the following steps are included: inoculating Lactobacillus plantarum LP45 and Bifidobacterium animalis BAL531 into a fermentation medium, and after mixed fermentation and culture, low-temperature vacuum drying the fermentation liquid (during the drying process, part of the live bacteria are lysed and broken to release peptidoglycan, nucleotides and other components in the bacteria) to obtain lactic acid bacteria postbiotics.

[0011] As a further technical solution, the inoculation amounts of Lactobacillus plantarum LP45 and Bifidobacterium animalis BAL531 are both 1-5%.

[0012] As a further technical solution, the fermentation medium includes: 2% glucose, 1% soy peptone, 1% yeast extract powder, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.05% manganese sulfate, 0.05% L-cysteine ​​salt, 0.1% Tween-80, and 1000L purified water.

[0013] As a further technical solution, the temperature of the mixed fermentation culture is 37°C.

[0014] As a further technical solution, the fermentation is carried out for 30-40 hours until the total acid content in the fermentation broth remains stable.

[0015] As a further technical solution, the low-temperature vacuum drying parameters are: temperature of 40-100°C and flow rate of 3L / min.

[0016] As a further technical solution, during the low-temperature vacuum drying process, the temperature is reduced gradually from 100° C. to 40° C. in a gradient manner.

[0017] As a further technical solution, during the low-temperature vacuum drying process, the temperature decreases gradually from 100°C to 40°C in the order of 100°C, 80°C, 70°C, 50°C, and 40°C.

[0018] A preparation containing the lactic acid bacteria postbiotics, the preparation comprising one or more of powder, capsule and tablet.

[0019] A broad-spectrum antibacterial lactic acid bacteria postbiotic powder comprises the lactic acid bacteria postbiotic and an acceptable carrier or auxiliary material in the powder.

[0020] As a further technical solution, the lactic acid bacteria postbiotic powder is obtained by physically mixing the lactic acid bacteria postbiotic and an acceptable carrier or auxiliary material in the powder in a ratio of 9 to 1:1.

[0021] As a further technical solution, the acceptable carrier or auxiliary material in the powder includes glucose.

[0022] The lactic acid bacteria postbiotics, the lactic acid bacteria postbiotics prepared by the preparation method, and the lactic acid bacteria postbiotic powder are used in the preparation of drugs for preventing and treating necrotic enteritis and overfeed diarrhea in laying hens or feed additives for improving the egg-laying performance of laying hens.

[0023] As a further technical solution, the lactic acid bacteria postbiotics or the lactic acid bacteria postbiotic powder has an antibacterial effect on Clostridium perfringens, Escherichia coli and Salmonella.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) Lactobacillus plantarum LP45 has excellent acid production ability. It can metabolize and produce a large amount of organic acids, including lactic acid, phenyllactic acid, acetic acid, butyric acid, etc., which can effectively inhibit intestinal pathogens such as Clostridium perfringens, Escherichia coli, and Salmonella, and can prevent necrotizing enteritis and diarrhea. It can increase the expression of tight junction proteins in the small intestine, enhance the mucosal barrier function, and prevent and treat ulcerative colitis. It can also regulate the balance of intestinal flora, increase the number of probiotics, reduce the number of harmful bacteria, and enhance the digestion and absorption function of the body and promote growth by increasing the small intestine chorionic gland ratio. Animal Bifidobacterium BAL531 metabolizes a variety of short-chain fatty acids, such as acetic acid, butyric acid, etc., which can provide energy for intestinal epithelial cells and promote intestinal development. The components of the cell membrane in its bacterial components: peptidoglycan, have the ability to activate the intestinal immune system, promote the proliferation of macrophages in the body and improve the activity of macrophages. The present invention uses the two in combination, utilizes their synergistic effect, and mixes and ferments them to make a product preparation that effectively prevents and treats necrotic enteritis and overfeed diarrhea in laying hens.

[0026] 2) The present invention fully mixes and ferments Lactobacillus plantarum LP45 and Bifidobacterium animalis BAL531 for a long time to produce more abundant metabolites, then dries the fermentation liquid into powder by low-temperature vacuum drying equipment while retaining more active substances, and finally physically mixes the postbiotic powder with a carrier to prepare a lactic acid bacteria postbiotic preparation, which includes organic acids, antimicrobial peptides, amino acids, extracellular polysaccharides, teichoic acid, peptidoglycan, etc., and can inhibit common pathogenic bacteria such as Clostridium perfringens, Escherichia coli and Salmonella in the breeding process, so that the effect on laying hen breeding is more obvious and no drug residue is left.

[0027] In summary, the lactic acid bacteria postbiotic preparation prepared by mixed fermentation of Lactobacillus plantarum LP45 and Bifidobacterium animalis BAL531 of the present invention can inhibit pathogenic bacteria such as Clostridium perfringens, Escherichia coli and Salmonella, increase the abundance of beneficial bacteria in the intestines of chickens, improve fecal status, prevent and treat necrotic enteritis and overfeed diarrhea in chickens, improve the intestinal health of laying hens, improve the digestion and absorption efficiency of feed, and increase egg production rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the antibacterial test diagram of the present invention;

[0029] exist Figure 1 In the figure, a: enterotoxigenic Escherichia coli (ETEC); b: enteroaggregative Escherichia coli (EAEC); c: Salmonella pullorum; d: Clostridium perfringens type C from chicken.

[0030] Figure 2 The photographs of chicken droppings of the chicken flock before and after taking the present invention;

[0031] exist Figure 2 In the figure, a: before the experiment, experimental group; b: before the experiment, control group; c: the 7th day, experimental group; d: the 7th day, control group. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0034] In the present invention, Lactobacillus plantarum LP45, with a deposit number of CGMCC NO.8072, has been disclosed in the Chinese invention patent with a publication number of CN103642716B;

[0035] Bifidobacterium animalis BAL531, with a deposit number of CGMCC NO.17329, which has been disclosed in the Chinese invention patent with a publication number of CN114250182A;

[0037] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0038] Example 1

[0039] A method for preparing lactic acid bacteria postbiotics comprises the following steps:

[0040] Inoculate Lactobacillus plantarum LP45 at an inoculation rate of 1% and Bifidobacterium animalis BAL531 at an inoculation rate of 1% in a fermentation medium, respectively, and mix and ferment at 37° C. for 35 hours until the total acid content in the fermentation broth remains stable, and then low-temperature vacuum dry the fermentation broth to obtain lactic acid bacteria postbiotics;

[0041] The fermentation medium comprises: 2% glucose, 1% soy peptone, 1% yeast extract powder, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.05% manganese sulfate, 0.05% L-cysteine ​​salt, 0.1% Tween-80, and 1000L purified water.

[0042] The low-temperature vacuum drying parameters are: temperature decreasing from 100°C to 80°C to 70°C to 50°C to 40°C, and flow rate 3L / min.

[0043] A method for preparing a lactic acid bacteria postbiotic powder comprises the following steps:

[0044] The lactic acid bacteria postbiotics and the carrier glucose are physically mixed in a ratio of 1:1 and then packaged to obtain the lactic acid bacteria postbiotic powder.

[0045] Example 2

[0046] A method for preparing lactic acid bacteria postbiotics comprises the following steps:

[0047] Inoculate Lactobacillus plantarum LP45 at an inoculation rate of 3% and Bifidobacterium animalis BAL531 at an inoculation rate of 3% in a fermentation medium, respectively, and mix and ferment at 37° C. for 42 hours until the total acid content in the fermentation broth remains stable, and then low-temperature vacuum dry the fermentation broth to obtain lactic acid bacteria postbiotics;

[0048] The fermentation medium comprises: 2% glucose, 1% soy peptone, 1% yeast extract powder, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.05% manganese sulfate, 0.05% L-cysteine ​​salt, 0.1% Tween-80, and 1000L purified water.

[0049] The low-temperature vacuum drying parameters are: temperature decreasing from 100°C to 80°C to 70°C to 50°C to 40°C, and flow rate 3L / min.

[0050] A method for preparing a lactic acid bacteria postbiotic powder comprises the following steps:

[0051] The lactic acid bacteria postbiotics and carrier glucose are physically mixed in a ratio of 5:1 and then packaged to obtain lactic acid bacteria postbiotic powder.

[0052] Example 3

[0053] Inoculate Lactobacillus plantarum LP45 at an inoculation rate of 5% and Bifidobacterium animalis BAL531 at an inoculation rate of 5% in a fermentation medium, respectively, and mix and ferment at 37° C. for 37 hours until the total acid content in the fermentation liquid remains stable, and then low-temperature vacuum dry the fermentation liquid to obtain lactic acid bacteria postbiotics;

[0054] The fermentation medium comprises: 2% glucose, 1% soy peptone, 1% yeast extract powder, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.05% manganese sulfate, 0.05% L-cysteine ​​salt, 0.1% Tween-80, and 1000L purified water.

[0055] The low-temperature vacuum drying parameters are: temperature decreasing from 100°C to 80°C to 70°C to 50°C to 40°C, and flow rate 3L / min.

[0056] A method for preparing a lactic acid bacteria postbiotic powder comprises the following steps:

[0057] The lactic acid bacteria postbiotics and the carrier glucose are physically mixed in a ratio of 9:1 and then packaged to obtain the lactic acid bacteria postbiotic powder.

[0058] Comparative Example 1

[0059] A method for preparing lactic acid bacteria postbiotics comprises the following steps: the same as Example 1, except that only Lactobacillus plantarum L45 is inoculated in the fermentation medium, and Bifidobacterium animalis BAL531 is not inoculated, and the inoculation amount is 2%.

[0060] A method for preparing lactic acid bacteria postbiotic powder comprises the following steps: same as Example 1.

[0061] Comparative Example 2

[0062] A method for preparing lactic acid bacteria postbiotics comprises the following steps: the same as Example 1, except that only animal Bifidobacterium BAL531 is inoculated in the fermentation medium, and Lactobacillus plantarum L45 is not inoculated, and the inoculation amount is 2%.

[0063] Application Example 1: In vitro antibacterial test

[0064] 1. Experimental Methods

[0065] A variety of common intestinal pathogens were selected as indicator bacteria, including Clostridium perfringens type C, toxigenic Escherichia coli, Salmonella pullorum, and Salmonella enteritidis. The size of the inhibition zone of the lactic acid bacteria postbiotic preparation against various indicator bacteria was measured by the Oxford cup method to evaluate its antibacterial performance. Figure 1 and Table 1;

[0066] Table 1 Inhibitory effect of lactic acid bacteria postbiotics on different pathogenic bacteria (inner circle diameter 5mm)

[0067]

[0068]

[0069] From the results in Table 1, it can be seen that the postbiotic preparation of Lactobacillus plantarum alone has a certain antibacterial effect on the above-mentioned common pathogens, and the postbiotic preparation of Bifidobacterium animalis alone only has an inhibitory effect on Clostridium perfringens. However, the postbiotic preparation prepared by mixing the two strains has a significantly increased inhibitory effect on several common pathogens, indicating that the present invention has a broad-spectrum antibacterial property, and the two strains have a synergistic effect, which can enrich each other's metabolites more, and the inhibitory effect on pathogenic bacteria is greatly enhanced.

[0070] Application Example 2: Application in laying hen farming

[0071] 1. Experimental Methods

[0072] Lactobacillus postbiotic preparations can be added by mixing with feed or dissolved in water and used as drinking water, which is more convenient for farmers to use. In this experiment, the preparations were added by mixing with feed.

[0073] 1. The composition of the basic diet is based on the Chinese laying hen feeding standards, with corn and soybean meal as the main raw materials. The diet is prepared according to the laying period of laying hens, and the nutritional components meet the laying needs of laying hens. The formula and nutritional level are detailed in Table 2;

[0074] Table 2 Basic diet composition and nutritional level (air-dry basis %)

[0075]

[0076] 2. Feeding and management

[0077] The observation period before the test was 15 days, and the formal feeding period was 30 days, for a total of 45 days. The surrounding environment, chicken houses, and utensils were disinfected before the test. The experimental chickens were raised in three-layer cages of type A, and 6 fixed points were selected for feces sampling in the experimental group and the control group. The chicken cages were all automatically temperature-controlled with infrared heating devices. During the test period, a constant light and mechanical ventilation system of 16h / d was maintained, and 24℃ was constant. Manure was removed manually once a day during the test period. The mental state and feces status of the chickens were observed every day.

[0078] 3. Trial Grouping and Plan

[0079] This study selected 280-day-old Jingfen laying hens, all of which had been fed, and randomly selected 2 groups, 3 replicates in each group, 30 hens in each replicate, 90 hens in each group, a total of 180 hens. The three replicates of the control group were marked as group 1, group 2, and group 3, and were fed a corn-soybean meal-based basal diet; the three replicates of the experimental group were marked as group 4, group 5, and group 6, and lactic acid bacteria postbiotic preparations were added to the basal diet at a dosage of 500g / t.

[0080] The test period was 30 days. The test group and the control group took daily diet and feces samples 15 days before the test, 7 days before the test, and on the 0th, 7th, 14th, 21st, and 30th days of the test, and took photos and recorded them. The test chicken feed (collected according to GB / T14699.1-2005) and excrement (no feed or feathers and other debris in the feces) were collected once in the morning and evening for each replicate group. 100g of fresh feces were taken from each group each time, 50g of which was fixed with 10% 4molHCL, and 50g was refrigerated. The feces sample fixed with 10% 4molHCL for 3 days in each replicate group was mixed for the determination of crude protein content; the fresh sample without HCl was mixed for the detection of other indicators. The feed samples collected from each group were mixed separately and tested together with the feces samples.

[0081] 2. Test indicators and results

[0082] 1. Detection of apparent digestibility:

[0083] The moisture content was determined using the GB / T6435-2014 method. The feces were dried at 65°C for 48 hours, rehydrated at room temperature and made into air-dried samples, crushed and passed through a 40-mesh sieve. The total energy was determined using an oxygen bomb energy meter (IKA-C200) in accordance with ISO9831:1998, the crude protein content was determined using the Kjeldahl method in accordance with GB / T6432-1994, and the hydrochloric acid insoluble ash (AIA) in feed and feces was determined using the endogenous indicator method in accordance with GB / T6438-2007. The apparent digestibility of nutrients was calculated. The results are shown in Table 3

[0084] Nutrient apparent digestibility = 100% - nutrient content in feces × AIA content in feed / (nutrient content in feed × AIA content in feces) × 100%.

[0085] Table 3 Effects of lactic acid bacteria postbiotics on apparent digestibility of nutrients in laying hens

[0086]

[0087] 2. Changes in feces status: Before the experiment, take photos of the chicken feces of the chicken flock. During the experiment, take photos of the chicken feces at a fixed point every week to observe the changes in the diarrhea of ​​the chicken flock. The results are shown in Figure 2 , Figure 2 It showed that before the experiment, the feces of the chickens in both the experimental and control groups were unformed. However, after the experiment, the feces of the chickens in the experimental group improved on the 7th day and became formed, compared with the control group, while the feces of the chickens in the control group were still unformed.

[0088] 3. Changes in egg production rate: Results are shown in Table 4

[0089] Table 4 Effects of lactic acid bacteria postbiotics on egg production rate of laying hens

[0090]

[0091] From the results in Table 4, we can see that the egg production rates of the experimental group and the control group were the same at the beginning of the experiment. After using lactic acid bacteria postbiotics, the egg production rate of the experimental group increased and the feed-egg ratio decreased, indicating that lactic acid bacteria postbiotics improved the absorption and conversion efficiency of laying hens for feed and improved the production performance of laying hens.

[0092] In summary, lactic acid bacteria postbiotic preparations can effectively inhibit intestinal pathogens such as Clostridium perfringens, Escherichia coli and Salmonella, and can prevent and treat necrotic enteritis in laying hens caused by Clostridium perfringens, Escherichia coli and Salmonella. In addition, adding lactic acid bacteria postbiotic preparations during laying hen breeding can increase the abundance of beneficial bacteria in the intestines of the flock, improve the state of feces, prevent and treat overfeed diarrhea in the flock, improve feed digestibility, and increase egg production rate.

[0093] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A broad-spectrum antibacterial lactic acid bacteria postbiotic, characterized in that: The invention comprises lactic acid bacteria and fermentation products thereof, wherein the lactic acid bacteria comprises plant lactobacillus LP45 and animal bifidobacterium BAL531.

2. The lactic acid bacteria postbiotic according to claim 1, characterized in that The fermentation products include organic acids and antimicrobial peptides.

3. A method for preparing lactic acid bacteria postbiotics as claimed in claim 1, characterized in that: The method comprises the following steps: inoculating plant lactobacillus LP45 and animal bifidobacterium BAL531 into a fermentation medium, performing mixed fermentation and culturing, and then low-temperature vacuum drying the fermentation liquid to obtain lactic acid bacteria postbiotics.

4. The preparation method according to claim 3, characterized in that: The inoculation amounts of the plant lactobacillus LP45 and the animal bifidobacterium BAL531 are both 1-5%; and the temperature of the mixed fermentation culture is 37°C.

5. The preparation method according to claim 3, characterized in that: The fermentation is carried out for 30-40 hours until the total acid content in the fermentation broth remains stable.

6. The preparation method according to claim 3, characterized in that: The low-temperature vacuum drying parameters are: temperature of 40-100° C., flow rate of 3 L / min.

7. A preparation comprising the lactic acid bacteria postbiotic according to claim 1, characterized in that: The preparation includes one or more of powder, capsule and tablet.

8. A broad-spectrum antibacterial lactic acid bacteria postbiotic powder, characterized in that: The invention comprises the lactic acid bacteria postbiotic as claimed in claim 1, and an acceptable carrier or auxiliary material in the powder.

9. Use of the lactic acid bacteria postbiotic according to any one of claims 1-2, the lactic acid bacteria postbiotic prepared by the preparation method according to any one of claims 3-6, and the lactic acid bacteria postbiotic powder according to claim 8 in the preparation of drugs for preventing and treating necrotic enteritis and overfeed diarrhea in laying hens or feed additives for improving the egg-laying performance of laying hens.

10. The use according to claim 9, characterized in that: The lactic acid bacteria postbiotics or the lactic acid bacteria postbiotics powder has an antibacterial effect on Clostridium perfringens, Escherichia coli and Salmonella.

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