Bacterial preparation for preventing and treating diarrhea of weaned piglets and promoting growth performance as well as preparation method and application of bacterial preparation

Abstract

The invention discloses a bacterial preparation for preventing and treating diarrhea of weaned piglets and promoting growth performance as well as a preparation method and application of the bacterial preparation, and provides application of Rhodococcus opacus PD630 in preparation of the bacterial preparation for preventing and treating diarrhea of weaned piglets and promoting growth performance. The Rhodococcus opacus PD630 capable of producing oil, disclosed by the invention, has the advantages that the immunocompetence of an immune system of a piglet is enhanced, the weaning stress of the piglet is relieved, and the Rhodococcus opacus PD630 plays a role in preventing and treating diarrhea of the weaned piglet and plays a positive role in promoting the growth performance.

Description

Technical Field

[0001] The present invention belongs to the field of prevention and treatment of livestock and poultry diseases, and particularly relates to a bacterial preparation for preventing and treating diarrhea of weaned piglets and promoting growth performance, a preparation method thereof, and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to increase some understanding of the overall background of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] China is a major country in pig farming. The total output value of pigs is nearly 1.3 trillion yuan, and the pig inventory ranks first in the world. The average annual number of pigs slaughtered in the past 5 years is about 700 million. Implementing early weaning measures for piglets is one of the key technologies in intensive modern pig farming production. However, during the early weaning period, piglets will be stimulated in terms of nutrition, environment, and psychology, which will exacerbate the occurrence of weaning stress, causing symptoms such as decreased appetite, indigestion, intestinal inflammation, and intestinal flora disorder. Subsequently, it will lead to diarrhea or even death of piglets. If the diarrhea of piglets cannot be controlled early, it will induce infectious diarrhea caused by various pathogenic bacteria, resulting in the aggravation of piglet diarrhea. Even if cured, it will have an impact on the production performance of piglets, and thus bring huge losses to the pig farming industry.

[0004] Currently, in the measures for preventing and treating piglet diarrhea, usually means such as strengthening feeding management, adding probiotics, and fecal microbiota transplantation are adopted, but the effect is very small; secondly, in Western medicine clinics, mainly antibiotic drugs such as enrofloxacin, florfenicol, oxytetracycline, kanamycin, gentamicin, and sulfonamides are used for control. However, with the continuous emergence of drug-resistant strains and the poor resistance of piglets themselves, some pig farms have poor control effects of diarrhea using existing drugs, and it causes high drug residues and high toxicity in livestock products, which is seriously harmful to the human body. Banning antibiotics has become an inevitable trend.

[0005] In July 2019, the Ministry of Agriculture and Rural Affairs issued a notice, deciding to stop the production, import, operation, and use of some drug feed additives, and since January 1, 2020, to withdraw all growth-promoting drug feed additive varieties except traditional Chinese medicine. Since July 1 of the same year, feed production enterprises have stopped producing commercial feeds containing growth-promoting drug feed additives (except traditional Chinese medicine).

[0006] Therefore, it is necessary to explore safer, green, and pollution-free preparations to prevent and treat diarrhea of weaned piglets and play a positive role in promoting their growth performance. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention aims to provide a bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance, its preparation method and application, so as to solve the problem of diarrhea in weaned piglets at the present stage, achieve the prevention and treatment of diarrhea in weaned piglets and have a positive promoting effect on the growth performance of piglets, thereby improving economic benefits.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In the first aspect of the present invention, there is provided an application of Rhodococcus opacus PD630 in the preparation of a bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance.

[0010] Through the research of the present invention, it is found that Rhodococcus opacus PD630 can reshape the intestinal microbiota, and further alleviate the inflammatory response caused by the disorder of intestinal lipid metabolism induced by weaning stress.

[0011] In the second aspect of the present invention, there is provided a bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance, and the bacterial preparation uses Rhodococcus opacus PD630 as an active ingredient.

[0012] In the third aspect of the present invention, there is provided a preparation method of the bacterial preparation described in the second aspect, and the method includes the following steps:

[0013] Rhodococcus opacus PD630 is first cultured in a proliferation medium. When the OD600 of the bacteria growth is greater than or equal to 0.7, the sample is collected, the precipitate is collected by centrifugation, the bacterial cells are washed 2-3 times with sterilized water, and then added to an activation medium for activation. When the nitrogen source is exhausted, the sample is collected, and the precipitate is collected by centrifugation;

[0014] The collected bacterial cells are added with a cryoprotectant and then freeze-dried to obtain the Rhodococcus opacus PD630 bacterial preparation.

[0015] In one or some embodiments of the present invention, the formula of 1L of the proliferation medium is: 3g of beef extract, 10g of peptone, 5g of sodium chloride, and the rest is made up with water. The formula of 1L of the activation medium is: 8-10g of Na2HPO4·12H20, 1-2g of KH2PO4, 0.1-0.3g of MgS04·7H20, 1.0-1.5mg of FeNa-EDTA, 15-25mg of CaCl2, 1.5-2.5ml of Hoagland solution, 0.4-0.6g of NaHCO3, and 1-5g of NH4C1, and the rest is made up with water.

[0016] In one or some embodiments of the present invention, the volume ratio of the bacterial cells to the cryoprotectant is 1:5 to 10. 750 g of skim milk powder, 100 g of sucrose, 100 g of trehalose and 50 g of antioxidant are added to 1 L of the cryoprotectant. The antioxidant can be selected from vitamin C, sodium thiosulfate, etc.

[0017] In one or some embodiments of the present invention, after freeze-drying, it is packaged. The packaging is carried out using vials and stored in a low-temperature environment to maintain the activity and stability of the bacterial preparation.

[0018] In the fourth aspect of the present invention, there is provided the use of the bacterial preparation described in the second aspect in the preparation of additives for livestock and poultry breeding feeds or drugs.

[0019] Compared with the related technologies known to the inventors of the present invention, one of the technical solutions of the present invention has the following beneficial effects:

[0020] The Rhodococcus opacus PD630 of the present invention that can produce oil can enhance the immune ability of the immune system of piglets, reduce the weaning stress of piglets, play a role in preventing and treating diarrhea in weaned piglets and have a positive promoting effect on growth performance.

[0021] The bacterial preparation in the present invention can not only reduce the diarrhea rate and mortality of weaned piglets, but also improve the growth performance of animals, enhance the resistance of the body to pathogenic bacteria, stimulate the generation of the body's immune response, and maintain a relatively high level of immune response in the body. Feeding the bacterial preparation in the present invention to weaned piglets can significantly improve the intestinal microecological environment of the body and enhance the resistance of the body to pathogenic bacteria in the case of pathogenic Escherichia coli infection.

[0022] The bacterial preparation described in the present invention can inhibit the colonization and reproduction of harmful pathogenic bacteria, reduce the long-term sub-therapeutic dose and ultra-high dose addition of antibiotics as a role in preventing and treating animal diseases in feeds, and avoid the harms in aspects such as bacterial drug resistance, antibiotic drug residues and food safety caused by the abuse of feed antibiotics. The bacterial preparation in the present invention can significantly improve the intestinal mucosal immune level, reduce or avoid the occurrence of intestinal diseases, and maintain the microecological balance of the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0024] Figure 1 It is a process flow chart for the preparation of the Rhodococcus opacus PD630 bacterial preparation.

[0025] Figure 2 It is a test flow chart for Example 2.

[0026] Figure 3 It is the result graph of average daily gain, average daily feed intake, feed to gain ratio and diarrhea rate of piglets in each group in Example 2.

[0027] Figure 4 It is the test flow chart of Example 3.

[0028] Figure 5 It is the result graph of average daily feed intake and average daily gain of piglets in each group in Example 3.

[0029] Figure 6 It is the intestinal length of piglets in each group in Example 3.

[0030] Figure 7 It is the HE staining graph of piglet colon tissue in Example 3.

[0031] Figure 8 It is the expression of inflammatory factors of piglets in each group in Example 3.

[0032] Figure 9 It is the expression of ZO-1, MUC2, OCLN, and CLDN-1 proteins of piglets in each group in Example 3. Detailed implementation manners

[0033] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0035] The core technology of the present invention is that Rhodococcus opacus PD630 is made into a Rhodococcus opacus PD630 bacterial preparation after activation, which enhances the immune ability of the piglet immune system, reduces the weaning stress of piglets, plays a role in preventing and treating diarrhea in weaned piglets and has a positive promoting effect on growth performance. Specifically, the key points of the present invention include the following aspects:

[0036] (1) Strain screening: Compared with traditional endogenous probiotic preparations on the market, the present invention innovatively selects the exogenous strain Rhodococcus opacus PD630. (2) The formulation of the Rhodococcus opacus PD630 bacterial preparation, the main component of which is the activated live bacteria.

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0038] Example 1. Preparation of the Rhodococcus opacus PD630 bacterial preparation (as Figure 1 ):

[0039] (1) Bacterial strain purchase: Rhodococcus opacus PD630 was purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains (BNCC).

[0040] (2) Medium preparation: The formula of 1L of proliferation medium is: 3g of beef extract, 10g of peptone, 5g of sodium chloride, and the rest is made up with water; the formula of 1L of activation medium is: 8 - 10g of Na2HPO4·12H20, 1 - 2g of KHPO, 0.1 - 0.3g of MgS04·7H20, 1.0 - 1.5mg of FeNH-citrate, 15 - 25mg of CaCl2, 1.5 - 2.5ml of Hoagland solution, 0.4 - 0.6g of NaHCO3, and 1 - 5g of NH4C1, and the rest is made up with water.

[0041] (3) Cultivation: Rhodococcus opacus PD630 is first cultured in the proliferation medium. When the OD600 of the bacteria growth reaches 0.7, the sample is collected, the precipitate is collected by centrifugation, the bacterial cells are washed 2 - 3 times with sterilized water, and then added to the activation medium for activation. When the nitrogen source is exhausted, the sample is collected and the precipitate is collected by centrifugation.

[0042] (4) Drying: Add the cryoprotectant to the bacterial cells at a volume ratio of 1:9 (the formula of the cryoprotectant is: by mass percentage, 20% skim milk powder, 10% sucrose, 10% trehalose, and 5% antioxidant, 55% deionized water. The antioxidant is vitamin C), place it in a -80°C freezer and then freeze-dry it in a freeze dryer to obtain the Rhodococcus opacus PD630 bacterial preparation. The viable bacteria count in this bacterial preparation is 1.5×10 9 CFU / g.

[0043] (5) Packaging: Use vials for packaging and store in a low-temperature environment to maintain the activity and stability of the bacterial preparation.

[0044] Effect of Different Doses of Rhodococcus opacus PD630 Bacterial Preparation on Diarrhea and Growth Performance of Early-Weaned Piglets

[0045] Healthy 7-day-old unweaned piglets with similar body weights were selected as experimental animals. After inspection, the body weights of the experimental animals did not differ significantly (±1 kg) (P > 0.05). They were randomly divided into four groups, with 30 pigs in each group, namely the control group (CON), the high-dose bacterial preparation group (H-R), the medium-dose bacterial preparation group (M-R), and the low-dose bacterial preparation group (L-R).

[0046] In each treatment group, 10 g of Rhodococcus opacus PD630 bacterial preparation with different doses was added to the creep feed for piglets at 7 days of age. The low-dose bacterial preparation group had 2×10 8 CFU / g, the medium-dose bacterial preparation group had 2×10 9 CFU / g, and the high-dose bacterial preparation group had 2×10 10 CFU / g. During the experiment, routine daily management was carried out, and the average daily gain, average daily feed intake, feed-to-gain ratio, and diarrhea rate of each group were statistically analyzed (as Figure 3 ). The daily feed consumption of each pig was recorded. On the 1st day and the 16th day of the experiment, all experimental pigs were weighed on an empty stomach before eating the feed, and the ADG and ADFI of the piglets during the experiment were calculated. The bacterial preparation was added every day.

[0047] The results showed that supplementing the medium-dose bacterial preparation (with viable bacteria count of 2×10 9 CFU / g) in the creep feed for piglets significantly increased the average daily gain and average daily feed intake compared with other groups, and had a relieving effect on growth retardation and diarrhea caused by weaning stress. It indicated that Rhodococcus opacus PD630 bacterial preparation could significantly improve the daily gain of piglets, promote animal growth, relieve piglet diarrhea, and thus improve animal survival rate.

[0048] After statistics, the preventive effect of Rhodococcus opacus PD630 bacterial preparation on diarrhea of 14-day-old unweaned piglets reached 100%, the therapeutic effect on diarrhea of 21-day-old weaned piglets reached 90%, and the weaning survival rate of piglets increased by more than 80%. After weaning, the feed utilization rate of piglets increased by 20%, the average daily gain increased by 20%, and the economic benefit per pig increased by 2.5%.

[0049] Example 3. Effect of Rhodococcus opacus PD630 Bacterial Preparation on Diarrhea and Growth Performance Complicated with Colitis Induced by DSS in Piglets (as Figure 4 )

[0050] Weaned piglets at 21 days of age with healthy and similar body weights were selected as experimental animals. After inspection, the body weights of the experimental animals did not differ significantly (±1.5 kg) (P > 0.05). They were randomly divided into four groups, with 5 pigs in each group, namely the (control group) Health group, Rhodococcus opacus group, DSS group, and DSS + Rhodococcus opacus group.

[0051] The experimental colitis-induced diarrhea model was induced by gavage with 4% DSS. The experimental group was supplemented with 7 - 10 g of Rhodococcus opacus PD630 bacterial preparation in the piglet diet. During the experiment, routine daily management was carried out, and growth performance such as average daily gain and average daily feed intake of each group was statistically analyzed. The experiment lasted for 16 days, and intestinal tissues were collected at the end of the experiment.

[0052] The results showed that in the piglet model induced by DSS, the addition of Rhodococcus opacus PD630 bacterial preparation could significantly reverse the effect of DSS on piglet growth performance, restoring the reduction in the average daily feed intake and average daily gain of the affected piglets (as Figure 5 ); at the same time, it improved the shortening of the intestinal length caused by DSS (as Figure 6 ); the results of histological evaluation showed that edema and infiltration of inflammatory cells appeared in the submucosa of the colon of piglets after DSS treatment, while the addition of Rhodococcus opacus PD630 reversed the colon inflammation (as Figure 7 ). By detecting the expression of immune factors and tight junction proteins in intestinal tissues using qPCR technology, it was found that the supplementation of Rhodococcus opacus PD630 significantly inhibited the inflammatory response caused by DSS (as Figure 8 ) and the damage of the intestinal barrier (as Figure 9 ). This indicates that Rhodococcus opacus PD630 may have the potential as an intervention measure to help maintain intestinal health and promote the repair of damaged intestinal barriers. The results of this study provide important clues for exploring the role of Rhodococcus opacus PD630 in regulating intestinal immunity and maintaining intestinal health.

[0053] After statistics, compared with the control group, the diarrhea rate of the compound bacterial preparation group decreased significantly by 87% in the 1st - 15th day stage after weaning (21 days of age), and the mortality rate decreased by 70%. The compound probiotic preparation effectively prevented piglet diarrhea.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An application of Rhodococcus opacus PD630 in the preparation of a bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance.

2. A bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance, characterized in that: The bacterial preparation uses Rhodococcus opacus PD630 as an active ingredient.

3. A method for preparing a bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance, characterized in that: The method comprises the following steps: Rhodococcus opacus PD630 was first cultured in a proliferation medium, and when the bacterial growth OD600 was greater than or equal to 0.7, the sample was collected, and the precipitate was collected by centrifugation. The bacteria were washed with sterile water for 2-3 times, and then activated by adding activation medium. When the nitrogen source was exhausted, the sample was collected, and the precipitate was collected by centrifugation. The collected bacterial cells were added with a cryoprotectant and then freeze-dried to obtain a Rhodococcus opacus PD630 bacterial preparation.

4. The method for preparing the bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance as claimed in claim 3, characterized in that: The formula for 1L proliferation medium is: 3g beef extract, 10g peptone, 5g sodium chloride, and the rest is made up with water.

5. The method for preparing the bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance as claimed in claim 3, characterized in that: The formula of 1L activation culture medium is: 8-10g Na2HPO4·12H20, 1-2g KH2PO4, 0.1-0.3g MgS04·7H20, 1.0-1.5mg FeNa-EDTA, 15-25mg CaCl2, 1.5-2.5ml Hoagland solution, 0.4-0.6g NaHCO3 and 1-5gNH4C1, and the rest is made up with water.

6. The method for preparing the bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance as claimed in claim 3, characterized in that: The volume ratio of bacteria to cryoprotectant is 1:5-10.

7. The method for preparing the bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance as claimed in claim 6, characterized in that: 750 g skim milk powder, 100 g sucrose, 100 g trehalose and 50 g antioxidant were added to 1 L cryoprotectant.

8. The method for preparing the bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance as claimed in claim 7, characterized in that: The oxidizing agent is selected from vitamin C or sodium thiosulfate.

9. The method for preparing the bacterial preparation for preventing and treating diarrhea in weaned piglets and promoting growth performance as claimed in claim 3, characterized in that: After freeze-drying, the product is packaged in vials and stored in a low-temperature environment.

10. Use of the bacterial preparation according to claim 2 in the preparation of livestock feed or drug additives.

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