Biological feed leavening agent for preventing and treating African swine fever as well as preparation method and application of biological feed leavening agent

Abstract

The invention provides a biological feed leavening agent for preventing and treating African swine fever as well as a preparation method and application of the biological feed leavening agent, and belongs to the technical field of feeds. The biological feed leavening agent disclosed by the invention is prepared from the following components in parts by weight: 5 to 25 parts of micrococcus luteus, 10 to 50 parts of Weissella mesenteroides SL7, 10 to 50 parts of lactic acid bacteria, 10 to 20 parts of bacillus licheniformis and 40 to 80 parts of clostridium butyricum. The biological feed leavening agent for preventing and treating the African swine fever, provided by the invention, is added into feed, so that the completeness of mucosal barriers of organisms can be maintained, the non-specific immunity of the organisms can be improved, and the threshold value of infectious diseases, especially the threshold value of infection of the African swine fever can be increased.

Description

Technical Field

[0001] The present invention relates to the field of feed technology, and in particular to a biological feed fermentation agent for preventing and treating African swine fever, and a preparation method and application thereof. Background Art

[0002] African swine fever is an acute, febrile, highly contagious animal disease of domestic pigs and wild boars caused by the African swine fever virus. Pigs of all breeds and ages can be infected, with morbidity and mortality rates as high as 100%.

[0003] African swine fever can enter the pig's body through the upper respiratory system and mouth, and infect the nasopharynx or tonsils. The virus quickly spreads to the lymph nodes of the mandible, and then spreads throughout the body through the lymph and blood. The natural infection incubation period is about 5 to 9 days, and the clinical trial infection is about 2 to 5 days. The course of onset is relatively short, and the clinical manifestations are fever (body temperature up to 40℃~42℃), difficulty breathing, rapid heartbeat, some pigs accompanied by coughing symptoms, serous or mucopurulent secretions in the nose and eyes, obvious bleeding symptoms in the gastrointestinal mucosa, kidneys, and lymph nodes, and cyanosis of the skin.

[0004] At present, there is no vaccine that can effectively prevent African swine fever at home and abroad, nor is there a specific medicine to treat African swine fever. Therefore, it is very important to find a way to effectively prevent and control African swine fever. Summary of the invention

[0005] In view of this, the present invention provides a biological feed fermentation agent for preventing and treating African swine fever, and a preparation method and application thereof. After feeding pigs with feed containing the biological feed fermentation agent for preventing and treating African swine fever provided by the present invention, it can maintain the integrity of the body's mucosal barrier, improve the body's nonspecific immune ability, and increase the threshold of pigs' infection with diseases, especially African swine fever.

[0006] The first object of the present invention is to provide a biological feed fermentation agent for preventing and treating African swine fever, which comprises the following components in parts by weight: 5-25 parts of Micrococcus luteus, 10-50 parts of lactic acid bacteria, 10-50 parts of Weissella mesenteroides SL7, 10-20 parts of Bacillus licheniformis, and 40-80 parts of Clostridium butyricum.

[0007] The biological feed fermentation agent for preventing and treating African swine fever provided by the present invention contains Micrococcus luteus, lactic acid bacteria, Weissella mesenteroides SL7, Bacillus licheniformis, Clostridium butyricum and their metabolites at the same time, and its application effect is the result of the combined action of the above-mentioned probiotics and their metabolites.

[0008] Furthermore, the lactic acid bacteria are specifically at least one of Lactobacillus acidophilus, Enterococcus faecalis, Lactobacillus casei, and Pediococcus acidilactici.

[0009] The second object of the present invention is to provide a method for preparing a biological feed fermentation agent for preventing and treating African swine fever, comprising the following steps:

[0010] (1) weighing Micrococcus luteus, lactic acid bacteria, Weissella mesenteroides SL7, Bacillus licheniformis, and Clostridium butyricum according to weight, mixing to obtain a mixture for later use;

[0011] (2) Adding auxiliary materials to the mixture in step (1) to obtain a biological feed fermentation agent.

[0012] Furthermore, the auxiliary material in step (2) includes at least one of maltodextrin, glucose and corn starch.

[0013] Furthermore, the dosage form of the biological feed fermentation agent includes powder or liquid.

[0014] The third object of the present invention is to provide a method for preparing a fermented feed containing a biological feed starter, comprising the following steps:

[0015] (1) mixing the complete material and water in a ratio of 1:2-3 to obtain a liquid material;

[0016] (2) adding the bio-feed fermentation agent powder or the bio-feed fermentation liquid to the liquid material described in step (1), mixing them evenly, and then fermenting them to obtain fermented feed.

[0017] Furthermore, the amount of the biological feed starter powder added in step (2) is 0.1-1wt%.

[0018] Furthermore, the amount of the biological feed fermentation agent liquid added in step (2) is 0.1-1wt%.

[0019] Furthermore, in step (2), the fermentation temperature is 35°C ± 5°C, and the fermentation time is 6-10 hours.

[0020] The fourth object of the present invention is to provide the application of the fermented feed in preventing and treating African swine fever.

[0021] Compared with the prior art, the present invention has the following beneficial effects: by feeding pigs with the feed containing the biological feed fermentation agent for preventing and treating African swine fever prepared by the present invention, it can maintain the integrity of the body's mucosal barrier, improve the body's nonspecific immune ability and the level of antiviral antibodies in the body, increase the threshold of pigs infected with diseases, especially African swine fever, and assist in curing or preventing the infection of African swine fever virus. At the same time, the biological feed fermentation agent containing the biological feed fermentation agent for preventing and treating African swine fever prepared by the present invention can also increase the activity of antioxidant enzymes in the pig body, reduce the damage to the body caused by excessive free radicals generated by relieving weaning stress, help promote the health of the pig herd, and thus effectively reduce the probability of the pig herd being infected with African swine fever. In addition, the biological feed fermentation agent containing the biological feed fermentation agent for preventing and treating African swine fever prepared by the present invention also has the effects of improving the digestibility of nutrients, reducing intestinal inflammation, improving the balance of intestinal flora, and improving daily weight gain and feed conversion efficiency. DETAILED DESCRIPTION

[0022] In some embodiments, a biological feed fermentation agent for preventing and treating African swine fever includes the following components, measured by weight: 5-25 parts of Micrococcus luteus, 10-50 parts of lactic acid bacteria, 10-50 parts of Weissella mesenteroides SL7, 10-20 parts of Bacillus licheniformis, and 40-80 parts of Clostridium butyricum.

[0023] In some embodiments, the strain code is as follows:

[0024] Micrococcus luteus: CCTCC No: M2017514;

[0025] Weissella mesenteroides SL7: GDMCC No:62429;

[0026] Bacillus licheniformis: CICC 10037;

[0027] Clostridium butyricum JBH-BD1: CCTCC NO:M 2022151;

[0028] Lactobacillus acidophilus: CICC 6095;

[0029] Enterococcus faecalis: CICC 21869;

[0030] Pediococcus acidilactici: CICC 24367;

[0031] Lactobacillus casei: CICC 6114.

[0032] In some embodiments, the lactic acid bacteria are specifically at least one of Lactobacillus acidophilus, Enterococcus faecalis, Lactobacillus casei, and Pediococcus acidilactici.

[0033] In some embodiments, the present invention also provides a method for preparing a biological feed starter for preventing and treating African swine fever, comprising the following steps:

[0034] (1) weighing Micrococcus luteus, lactic acid bacteria, Weissella mesenteroides SL7, Bacillus licheniformis, and Clostridium butyricum according to weight, mixing to obtain a mixture for later use;

[0035] (2) Adding auxiliary materials to the mixture in step (1) to obtain a biological feed fermentation agent.

[0036] In some embodiments, 10-hydroxy-2-decenoic acid and Rosa laevigata extract can also be added to the biological feed fermentation agent for preventing and treating African swine fever. The amount of 10-hydroxy-2-decenoic acid added is 0.05% of the mass of the biological feed fermentation agent; the amount of Rosa laevigata extract added is 5% of the mass of the biological feed fermentation agent. The present invention has no special restrictions on the method of adding Rosa laevigata extract.

[0037] In some embodiments, the extraction method of the Rosa laevigata extract is as follows: weigh 120 g of Rosa laevigata powder, add 8 times the amount of distilled water, stir evenly, then extract at 80°C for 2.5 hours, repeat the extraction twice, combine the filtered extracts, and concentrate to obtain 150 ml of Rosa laevigata water extract.

[0038] After a lot of research, it was found that biological feed fermentation agents with the addition of 10-hydroxy-2-decenoic acid and Rosa laevigata extract have a better effect in reducing the probability of pig herds being infected with African swine fever. The reason may be that 10-hydroxy-2-decenoic acid, Rosa laevigata extract and a variety of probiotics can synergistically enhance the matrix immune function and improve resistance to viruses.

[0039] In some embodiments, the auxiliary material in step (2) includes at least one of maltodextrin, glucose, and corn starch.

[0040] In some embodiments, the dosage form of the biological feed fermentation agent includes powder or liquid.

[0041] In some embodiments, the present invention provides a method for preparing a fermented feed containing a biological feed starter, comprising the following steps:

[0042] (1) mixing the complete material and water in a ratio of 1:2-3 to obtain a liquid material;

[0043] (2) adding the bio-feed fermentation agent powder or the bio-feed fermentation liquid to the liquid material described in step (1), mixing them evenly, and then fermenting them to obtain fermented feed.

[0044] In some embodiments, the amount of the biological feed starter powder added in step (2) is 0.1-1 wt%, preferably 0.1 wt%.

[0045] In some embodiments, the amount of the biological feed fermentation agent liquid added in step (2) is 0.1-1wt%, preferably 1wt%.

[0046] In some embodiments, the fermentation temperature in step (2) is 35°C±5°C, preferably 35°C, and the fermentation time is 6-10 hours, preferably 8 hours.

[0047] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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 belong to the scope of protection of the present invention.

[0048] The experimental methods or test methods described in the following examples are conventional methods unless otherwise specified; the raw materials and auxiliary agents are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0049] The formula composition and nutritional index of the complete feed used in the following Examples 1 and 2 are shown in Tables 1 and 2:

[0050] Table 1 Complete feed formula

[0051]

[0052]

[0053] Table 2 Nutritional indicators of complete feed formula

[0054] Nutritional indicators content(%) Dry matter content 86.40 Crude Protein 15.50 calcium 0.60 Total Phosphorus 0.47 Salt 0.37 Lysine 1.00 Methionine + Cysteine 0.60 Threonine 0.65 Tryptophan 0.20

[0055] Example 1

[0056] A method for preparing a fermented feed comprises the following steps:

[0057] (1) Preparing a biological feed fermentation agent: weighing 10 parts of Micrococcus luteus, 10 parts of Weissella mesenteroides SL7, 20 parts of lactic acid bacteria, 10 parts of Bacillus licheniformis, and 40 parts of Clostridium butyricum by weight, crushing and sieving, and then mixing to obtain a mixture, and then adding auxiliary material maltodextrin to the mixture to obtain a biological feed fermentation agent for standby use;

[0058] (2) preparing a liquid material: mixing the complete material and water in a ratio of 1:3 to obtain a liquid material for later use;

[0059] (3) Preparing fermented feed: Weighing a biological feed fermentation agent and adding it to the liquid material described in step (2), wherein the mass ratio of the biological feed fermentation agent to the complete feed is 1:1000, mixing evenly, and fermenting at a temperature of 35° C. for 8 hours to obtain a fermented feed.

[0060] Example 2

[0061] A method for preparing a fermented feed comprises the following steps:

[0062] (1) Preparing biological feed fermentation liquid: weighing 10 parts of Micrococcus luteus fermentation liquid, 10 parts of Weissella mesenteroides SL7 fermentation liquid, 20 parts of lactic acid bacteria fermentation liquid, 10 parts of Bacillus licheniformis fermentation liquid, and 40 parts of Clostridium butyricum fermentation liquid by weight, mixing them well to obtain a mixture, and then adding corn starch to the mixture to obtain biological feed fermentation liquid for later use;

[0063] (2) preparing a liquid material: mixing the complete material and water in a ratio of 1:3 to obtain a liquid material for later use;

[0064] (3) Preparing fermented feed: Weigh the biological feed fermentation liquid and add it to the liquid material described in step (2), wherein the mass ratio of the biological feed fermentation liquid to the complete feed is 1:100. After mixing evenly, ferment at a temperature of 35° C. for 8 hours to obtain fermented feed.

[0065] The content of Micrococcus luteus in the Micrococcus luteus fermentation broth in step (1) is 2.0*10 8 CFU / mL;

[0066] The content of Weissella mesenteroides SL7 in the Weissella mesenteroides SL7 fermentation liquid is 1.0*10 9 CFU / mL;

[0067] The content of lactic acid bacteria in the lactic acid bacteria fermentation liquid is 1.0*10 9 CFU / mL;

[0068] The content of Bacillus licheniformis in the Bacillus licheniformis fermentation liquid is 1.0*10 9 CFU / mL;

[0069] The content of Clostridium butyricum in the Clostridium butyricum fermentation broth is 2.0*10 8 CFU / mL.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 1 is that Micrococcus luteus was not added, and the remaining steps were the same as Example 1.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 1 is that Weissella mesenteroides SL7 is not added, and the remaining steps are the same as Example 1.

[0074] Application effect test:

[0075] Test Example 1

[0076] A pig farm in Jun County, Henan Province, with 80 sows, has been using the fermented feed prepared in Example 1 and Example 2 for free feeding since 2018. From 2019 to 2022, when African swine fever was rampant, people have been visiting the farm for study, with a total of 20,000 visitors. Visitors do not need to take any protective measures, and the pig farm has not had an outbreak of African swine fever. It can be seen that the fermented feed prepared in Example 1 and Example 2 of the present invention is very effective in preventing and controlling African swine fever.

[0077] Test Example 2

[0078] From August 2021 to December 2022, 10 farms in northern Henan were counted, with a total of 25,000 pigs, which were fed with the fermented feed provided in Example 1 and Example 2, respectively. The experiment was carried out for 3 months, with free feeding, and the incidence of African swine fever was recorded. The results show that since the fermented feed prepared in Example 1 and Example 2 was used, the incidence of African swine fever during the test was 0, while the incidence of African swine fever in pigs in the surrounding farms that did not add the fermented feed prepared by the present invention was as high as 50% (the breeding conditions of the surrounding farms were consistent with those in Test Example 2, the only difference being that the fermented feed prepared by the present invention was not added to the surrounding farms). It can be concluded that the fermented feed prepared by the present invention can be used to feed pigs, which can effectively prevent and treat African swine fever.

[0079] Test Example 3

[0080] A total of 240 21-day-old "Du Changda" weaned piglets with the same genetic background and similar weight were randomly divided into 4 groups, namely the control group, the experimental group, the comparative group 1 and the comparative group 2. There were 3 replicates in each group, with 20 piglets in each replicate. Each treatment was fed with the corresponding feed, and the piglets were allowed to eat and drink freely throughout the experiment. The feeding management procedures and immunization procedures during the experiment were carried out in accordance with the conventional commercial pig feeding management manual.

[0081] The control group was fed a basal diet, the formula composition and nutritional indicators of the basal diet are shown in Tables 3 and 4;

[0082] The experimental group was fed with a basal diet and the fermented feed prepared in Example 1, wherein the fermented feed was added in an amount of 5% of the basal diet.

[0083] The control group 1 was fed with a basic diet and the fermented feed prepared in control group 1, and the fermented feed was added in an amount of 5% of the basic diet.

[0084] The control group 2 was fed with a basic diet and the fermented feed prepared in control group 2, wherein the fermented feed was added in an amount of 5% of the basic diet.

[0085] Table 3 Basic diet formula

[0086]

[0087]

[0088] Table 4 Nutritional index content of basic diet

[0089] Nutritional indicators content(%) Crude Protein 18.00 calcium 0.55 Total Phosphorus 0.62 Salt 0.98 Lysine 1.40 Methionine + Cysteine 0.81 Threonine 0.88 Tryptophan 0.27 Moisture 10.20

[0090] Determination of immune and antioxidant indicators in serum: At 8:00 am on the 14th day of the experiment, two piglets of intermediate weight were selected from each treatment group for fasting blood collection from the anterior vena cava using a blood collection tube without anticoagulant, and the serum was obtained by centrifugation at 3000×rpm for 15 minutes, which was packaged and frozen at -80℃ for testing. The activities of catalase (CAT), glutathione peroxidase (GSH-px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in serum samples were detected; at the same time, the levels of serum immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) were detected by enzyme linked immunosorbent assay (ELISA). The kit of Nanjing Jiancheng Biological Research Institute was used for determination according to the instructions.

[0091] The experimental results are shown in Table 5 and Table 6:

[0092] Table 5 Effects of biological feed fermentation agents on the activity of antioxidant enzymes in the serum of weaned piglets

[0093]

[0094]

[0095] From the results of the determination of antioxidant related indicators in Table 5, it can be obtained that the biological feed fermentation agent obtained by the present invention can well alleviate weaning stress. Compared with the control group, the antioxidant enzyme activities in the experimental group were all improved, while the MDA content was reduced. When the experiment was carried out to the 14th day, the MDA in the serum of the weaned piglets in the experimental group decreased by 44.85%, and T-AOC increased by 24% compared with the control group. At the same time, the activities of antioxidant enzymes such as CAT, SOD, GSH-px in the comparative example 1 group and the comparative example 2 group without adding Micrococcus luteus or Weissella mesenteroides SL7 were improved to varying degrees compared with the control group, but were still lower than the experimental group. It shows that there is a synergistic effect between the Micrococcus luteus and Weissella mesenteroides SL7 in the biological feed fermentation agent provided by the present invention, which can synergistically improve the activity of antioxidant enzymes in the pig body, thereby reducing the damage to the body caused by excessive free radicals generated by relieving weaning stress.

[0096] From the effect of the weaning stress-alleviating biological feed fermentation agent on the growth performance of weaned piglets and the activity of antioxidant enzymes in serum, it can be concluded that the solution provided in Example 1 has a better effect in alleviating oxidative stress and helps to promote the health of the pig herd, thereby effectively reducing the probability of the pig herd being infected with African swine fever.

[0097] Table 6 Effects of biological feed fermentation agents on piglet mucosal immune factors

[0098] Antioxidant index (14 days) Control group Experimental Group Comparative Example 1 Comparative Example 2 IgA (ug / g) 4366.84±1152.31 4561.01±649.97 4458.04±746.36 44.247±347.91 IgM (mg / g) <![CDATA[115.22±22.01 b ]]> <![CDATA[131.24±15.50 b ]]> 118.64±46.58 <![CDATA[121.68±18.61 b ]]> IgG (mg / g) 135.33±17.49 150.70±21.48 138.90±28.39 140.35±69.23 SIgA (ug / mL) <![CDATA[507.30±39.43 b ]]> <![CDATA[794.58±56.16 a ]]> <![CDATA[637.44±36.87 a ]]> 687.57±47.38

[0099] From the results of the piglet mucosal immune factor determination in Table 6, it can be concluded that the IgA, IgM, IgG, and SIgA of the test group increased by 4.5%, 13.9%, 11.4%, and 56.6% respectively compared with the control group, while the increase in the comparative example 1 group and the comparative example 2 group was lower than that in the test group. It can be concluded that adding the biological feed fermentation agent of Example 1 to feed fermentation and then feeding can significantly enhance the non-specific antibody level of piglets.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biological feed fermentation agent for preventing and treating African swine fever, characterized in that: The invention comprises the following components in parts by weight: 5-25 parts of Micrococcus luteus, 10-50 parts of lactic acid bacteria, 10-50 parts of Weissella mesenteroides SL7, 10-20 parts of Bacillus licheniformis and 40-80 parts of Clostridium butyricum.

2. The method for preparing the biological feed fermentation agent for preventing and treating African swine fever according to claim 1, characterized in that: The following steps are involved: (1) weighing Micrococcus luteus, lactic acid bacteria, Weissella mesenteroides SL7, Bacillus licheniformis, and Clostridium butyricum according to weight, mixing to obtain a mixture for later use; (2) Adding auxiliary materials to the mixture in step (1) to obtain a biological feed fermentation agent.

3. The method for preparing the biological feed fermentation agent for preventing and treating African swine fever according to claim 2, characterized in that: The auxiliary material in step (2) includes at least one of maltodextrin, glucose and corn starch.

4. The biological feed fermentation agent for preventing and treating African swine fever according to claim 1, characterized in that: The dosage form of the biological feed fermentation agent includes powder or liquid.

5. A method for preparing a fermented feed containing the biological feed starter according to claim 1, characterized in that: The following steps are involved: (1) mixing the complete material and water in a ratio of 1:2-3 to obtain a liquid material; (2) adding the bio-feed fermentation agent powder or the bio-feed fermentation liquid to the liquid material described in step (1), mixing them evenly, and then fermenting them to obtain fermented feed.

6. The method for preparing fermented feed according to claim 5, characterized in that: The amount of the biological feed fermentation agent powder added in step (2) is 0.1-1wt%.

7. The method for preparing fermented feed according to claim 5, characterized in that: The amount of the biological feed fermentation agent liquid added in step (2) is 0.1-1wt%.

8. The method for preparing fermented feed according to claim 5, characterized in that: The fermentation temperature in step (2) is 35°C ± 5°C, and the fermentation time is 6-10h.

9. Use of the fermented feed according to any one of claims 5 to 8 in preventing and treating African swine fever.

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