Lactobacillus plantarum for preventing piglet diarrhea and promoting growth and application thereof

Abstract

The invention belongs to the technical field of animal husbandry disease prevention and microorganisms, and particularly relates to a lactobacillus plantarum for preventing piglet diarrhea and promoting growth and application thereof, the strain is lactobacillus plantarum SC-24 with the preservation number of CGMCC No.30256, and is preserved in China General Microbiological Culture Collection Center on April 3, 2024. When the lactobacillus plantarum SC-24 fermentation product is used for preparing a feed additive, the diarrhea rate of weaned piglets can be remarkably reduced, and the lactobacillus plantarum SC-24 fermentation product has a relatively good promoting effect on the growth performance and the health condition of the weaned piglets; therefore, the strain can be used as a new choice for micro-ecological treatment of piglet diarrhea.

Description

Technical Field

[0001] The invention belongs to the field of animal husbandry disease prevention and microbial technology, and particularly relates to a plant lactobacillus strain for preventing piglet diarrhea and promoting growth and an application thereof. Background Art

[0002] Because piglets have not yet established a stable micro-ecological system in their intestines, they have low resistance and are sensitive to external stimuli. They are easily invaded by various pathogenic microorganisms and various stress factors. Among them, piglet diarrhea is a common disease in current pig production. Diarrhea will interrupt the normal physiological development process of piglets, causing gastrointestinal dysfunction and insufficient nutrient intake, which will lead to growth retardation or even death of piglets, causing huge economic losses.

[0003] Traditionally, diarrhea in piglets is prevented by adding drug ingredients (antibiotics or synthetic antimicrobial drugs) to feed. However, feed antibiotics or synthetic antimicrobial drugs will form drug residues in the piglets' bodies, which may cause food safety problems and threaten human health.

[0004] Therefore, how to prevent piglet diarrhea safely and without toxic side effects is an urgent problem that needs to be solved. Summary of the invention

[0005] In order to overcome the problems of the prior art, the present invention provides a strain of Lactobacillus plantarum for preventing piglet diarrhea and promoting growth and application thereof.

[0006] The object of the present invention is achieved in that:

[0007] The first aspect of the present invention provides a strain of Lactobacillus plantarum SC-24, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 3, 2024, with a deposit number of CGMCC No.30256.

[0008] The second aspect of the present invention provides a method for culturing the above-mentioned Lactobacillus plantarum SC-24, comprising the following steps:

[0009] (1) transferring Lactobacillus plantarum SC-24 into MRS liquid culture medium and culturing at 37° C. for 24 h to obtain an activated bacterial solution;

[0010] (2) The activated bacterial solution in step (1) is inoculated into a liquid MRS culture medium at an inoculum amount of 1% to 3%, and the culture is continued to obtain a bacterial solution of Lactobacillus plantarum SC-24.

[0011] The third aspect of the present invention provides a probiotic preparation prepared by the above-mentioned Lactobacillus plantarum SC-24.

[0012] The fourth aspect of the present invention provides a probiotic fermentation product prepared by the above-mentioned Lactobacillus plantarum SC-24.

[0013] The fifth aspect of the present invention provides a product prepared from the above-mentioned Lactobacillus plantarum SC-24, probiotic agent or probiotic fermentation product.

[0014] Preferably, the product is feed or feed additive.

[0015] The sixth aspect of the present invention provides the use of the Lactobacillus plantarum SC-24, probiotic agents or probiotic fermented products in the preparation of feed or feed additives for preventing piglet diarrhea and promoting growth.

[0016] The seventh aspect of the present invention provides a feed additive for preventing diarrhea and promoting growth in piglets, comprising the following components in mass percentage: 15% to 25% of the probiotic fermentation product prepared by the plant lactobacillus SC-24, 2% to 8% of mannan, 2% to 8% of oligofructose, and the balance is water; and the following raw material components in concentration: 50 to 150 mg / L of vitamin A, 50 to 150 mg / L of vitamin D3, and 1 to 5 mg / L of vitamin E;

[0017] The probiotic fermentation product prepared by Lactobacillus plantarum SC-24 comprises the following steps:

[0018] A. Take Lactobacillus plantarum and culture it in MRS culture medium at 30-39°C for 8-16 hours to obtain Lactobacillus plantarum fermentation liquid I;

[0019] B. inoculating Lactobacillus plantarum fermentation liquid I into MRS culture liquid, adjusting the initial pH value to 4.5-8.5, and culturing at 30-39° C. for 48-72 hours to obtain Lactobacillus plantarum fermentation liquid II;

[0020] C. Resuspending the Lactobacillus plantarum fermentation liquid II into a 20-30% mixture using a NaCl solution, heating to 90-100° C. and maintaining for 5-10 minutes, then breaking the Lactobacillus plantarum cells to release the contents, and centrifuging at a speed of 3500-5000 rpm for 10-15 minutes, and taking the supernatant I after centrifugation;

[0021] D. Add composite protease to the supernatant I obtained in step C for enzymolysis at 35-45° C. for 8-15 hours. After enzymolysis, heat the system to 90-100° C. and keep it for 5-10 minutes, and take supernatant II;

[0022] E. Separate and recover the substances with a relative molecular mass less than 5000 in the supernatant II obtained in step D, and freeze-dry them to obtain the Lactobacillus plantarum SC-24 polypeptide complex.

[0023] Further, in step E, the supernatant II is separated using a dialysis bag with a molecular weight cut-off of 5000 to recover substances with a relative molecular mass of less than 5000; and / or

[0024] The supernatant II was concentrated by ultrafiltration using a cellulose acetate membrane with a molecular weight cutoff of 5000 to recover substances with a relative molecular mass of less than 5000.

[0025] Furthermore, in step E, a gel chromatography column is used to separate and recover substances with a relative molecular mass of less than 5000 in the supernatant II obtained in step D.

[0026] Information on deposited strains:

[0027] Lactobacillus plantarum SC-24, depository institution: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101. Deposit date: April 3, 2024, deposit number: CGMCC No.30256, classification name: Lactobacillus plantarum.

[0028] The advantages and beneficial effects of the present invention are:

[0029] 1. The present invention provides a new strain of Lactobacillus plantarum SC-24, which has an excellent ability to prevent diarrhea in piglets;

[0030] 2. The use of the plant lactobacillus SC-24 fermentation product of the present invention to prepare feed additives can significantly reduce the diarrhea rate of weaned piglets and has a good promoting effect on the growth performance and health status of weaned piglets; this shows that the strain described in this application can be used as a new choice for microecological treatment of piglet diarrhea. DETAILED DESCRIPTION

[0031] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0032] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention.

[0034] The culture medium formula involved in the embodiment of the present invention includes:

[0035] MRS culture medium: carbon source 1-2.5%, nitrogen source 1-2.5%, dipotassium hydrogen phosphate 0.1-0.8%, beef extract 0.5-1%, yeast powder 0.5-1%, diamine citric acid 0.2%, sodium acetate 0.5%, magnesium sulfate 0.058%, manganese sulfate 0.025%, and the balance is distilled water.

[0036] Embodiment 1:

[0037] The present embodiment provides a feed additive for preventing diarrhea and promoting growth of piglets, comprising the following components in percentage by mass: 20% of the probiotic fermentation product prepared by Lactobacillus plantarum SC-24 described in Example 1, 5% of mannan, 5% of fructooligosaccharides (FOS), and the remainder is distilled water; and the following concentrations of raw material components: vitamin A 100 mg / L, vitamin D3 100 mg / L, and vitamin E 2 mg / L.

[0038] The probiotic fermentation product prepared by Lactobacillus plantarum SC-24 comprises the following steps:

[0039] A. Take Lactobacillus plantarum, activate it, and culture it in MRS culture medium at 30-39°C and 180 rpm for 8-16 hours to obtain Lactobacillus plantarum fermentation liquid I.

[0040] B. Inoculate Lactobacillus plantarum fermentation liquid I into MRS culture medium at an inoculation amount of 1-5%, adjust the initial pH value to 4.5-8.5, and culture at 30-39° C. for 48-72 hours to obtain Lactobacillus plantarum fermentation liquid II.

[0041] C. Use 2% mass concentration NaCl solution to resuspend Lactobacillus plantarum fermentation liquid II into a 20-30% mixture, heat to 90-100°C, and keep for 5-10 minutes, then break the Lactobacillus plantarum cells to release the contents, and centrifuge at a speed of 3500-5000 rpm for 10-15 minutes, and take the supernatant I after centrifugation.

[0042] D. Add composite protease to the supernatant I obtained in step C for enzymolysis at 35-45°C for 8-15 hours. After enzymolysis, heat the system to 90-100°C and maintain for 5-10 minutes, and take supernatant II.

[0043] E. Separate the supernatant II using a dialysis bag with a molecular weight cutoff of 5000 to recover substances with a relative molecular mass of less than 5000; then perform gel chromatography on the substances with a relative molecular mass of less than 5000 and freeze-dry them to obtain the Lactobacillus plantarum SC-24 polypeptide complex.

[0044] Test Example 1

[0045] This test example uses animal experiments to study the feed additive prepared in Example 2 and its efficacy in preventing and treating diarrhea in weaned piglets.

[0046] The experiment selected 30 healthy, similar-weight, weaned Sego pigs at 28 days of age and randomly divided them into three treatment groups (one control group and two experimental groups), with two replicates in each group, five pigs in each replicate, and one replicate as one pen. After weighing the initial weight, SAS software was used for analysis to confirm that there was no difference in initial weight among the treatments. Pens fed with different feeds were arranged crosswise to eliminate environmental differences.

[0047] The experiment adopts single factor experimental design, experimental treatment:

[0048] Control group: only fed with the basic diet for piglets;

[0049] Experimental group 1: fed with basic diet + 3% "feed additive prepared in Example 2";

[0050] Experimental group 2: Feeding: basic diet + 5% of “feed additive prepared in Example 2”.

[0051] After 3 days of pre-feeding, a four-week feeding test was conducted. Piglets were fed with milk replacer and piglet treasure in the two stages before weaning (weeks 1-2) and after weaning (weeks 3-4), respectively. The basic diet is based on the complete feed ("Sano" premix + energy feed) fed in the pig farm. The experimental diet is divided into three types: ① Basic diet (see Table 1, without any antibiotics) ② Basic diet + 3% "Feed additive prepared in Example 2" ③ Basic diet + 5% "Feed additive prepared in Example 2". During the test, the powder was added to the "feed additive prepared in Example 2" at a ratio of 1:100 and stirred to mix evenly, and then fed to the experimental weaned piglets.

[0052] Table 1 Basic diet formula for the experiment

[0053]

[0054] Note: ①4% premix contains: Vitamin A 300 KIU, Vitamin D 356 KIU, Vitamin E 800 IU, Vitamin K 365 mg, Vitamin B1 71 mg, Vitamin B2 331 mg, Vitamin B6 81 mg, Vitamin B12 1 mg, Niacin 1175 mg, Pantothenic acid 900 mg, Folic acid 32 mg, Biotin 1.3 mg, Choline chloride 6 g, Iron 5 g, Copper 6.3 g, Zinc 5.5 g, Manganese 1.9 g, Iodine 20 mg, Selenium 15 mg, Cobalt 25 mg, Calcium 135-225 g, Phosphorus 57 g. The premix also contains phytase, 10% aminophenylarsonic acid 25000 mg, 10% colistin sulfate premix 5000 mg, 5% quinolone 25000 mg; ②Nutritional ingredients are all calculated values.

[0055] The nursery house uses slatted floor nursery equipment, the house temperature is controlled at 20-25℃, and the relative humidity is 50%-70%. The experimental piglets are fed ad libitum, 4-6 times a day, and the amount of each addition should be slightly left in the feed trough.

[0056] The pre-test period is 5 days, and vaccination, deworming and health care are carried out at the same time. After entering the main test period, each group is fed with corresponding diets. Free food and water are available, and immunization is carried out according to the normal procedures of the pig farm. The test period is 28 days.

[0057] At the beginning and end of the experiment, each piglet was weighed on an empty stomach. The feed amount, residual feed amount and loss were recorded, and the feed-to-weight ratio was calculated based on the average daily weight gain and average daily feed intake.

[0058] 1. Average daily weight gain

[0059] The piglets were weighed on an empty stomach in the morning on the first and last day of the experiment, and the average daily weight gain of weaned piglets was calculated based on the initial and final weights.

[0060]

[0061] 2. Average daily feed intake

[0062] The feed intake of each group was recorded separately, and the average daily feed intake was calculated based on the number of experimental days.

[0063]

[0064] 3. Material-to-weight ratio

[0065] The feed-to-weight ratio was calculated based on the average daily feed intake and average daily weight gain.

[0066]

[0067] 4. Diarrhea rate

[0068] Observe the feces of piglets regularly at 8 am every day, record the number of diarrhea pigs, and calculate the diarrhea rate.

[0069]

[0070] Data processing: Data were recorded daily according to the experimental design, and the experimental data were preliminarily sorted using Excel. After the feeding was completed, the data were processed using SPSS14.0 software for variance analysis.

[0071] Results and Analysis

[0072] 1. Effect of “Feed Additive Prepared in Example 2” on Diarrhea Rate in Weaned Piglets

[0073] Table 2 Effect of "Feed additive prepared in Example 2" on diarrhea in weaned piglets

[0074]

[0075] In the early stage of the experiment, all groups had diarrhea. In the middle stage of the experiment, the number of diarrhea in the experimental group decreased, and there was no diarrhea in the later stage. During the experiment, one pig in the control group died of diarrhea, and the rest of the piglets had fever. The physical condition and health level of the piglets in the experimental group were better. The diarrhea rate of pigs in each group is shown in Table 2. From the statistical results in Table 2, it can be seen that the diarrhea rate of the experimental group was 6.65 and 6.79 percentage points lower than that of the control group, and the health level was significantly better than that of the control group.

[0076] 2. Effect of “Feed Additive Prepared in Example 2” on Growth Performance of Weaned Piglets

[0077] Table 3 Effect of "Feed additive prepared in Example 2" on growth performance of weaned piglets

[0078]

[0079] From the statistical results in Table 3 and the observations during the test, it can be seen that the piglets in the test group are better than those in the control group in terms of growth performance and health level. The piglets in the test group had less weaning stress, were able to enter the nursery state quickly and healthily, gained weight faster in the middle and late stages, and no disease occurred during the nursery; while the piglets in the control group had greater weaning stress, had severe diarrhea, and could only gradually turn to the nursery state after medication. During the test, one piglet died of diarrhea, and the meat loss was very obvious in the initial stage, and the overall health status was relatively poor. It shows that "the feed additive prepared in Example 2" has a good promoting effect on the growth performance and health status of weaned piglets.

[0080] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus plantarum SC-24, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 3, 2024, with the deposit number CGMCCNo.30256.

2. The method for cultivating Lactobacillus plantarum SC-24 according to claim 1, wherein The steps include: (1) transferring Lactobacillus plantarum SC-24 into MRS liquid culture medium and culturing at 37° C. for 24 h to obtain an activated bacterial solution; (2) The activated bacterial solution in step (1) is inoculated into a liquid MRS culture medium at an inoculum amount of 1% to 3%, and the culture is continued to obtain a bacterial solution of Lactobacillus plantarum SC-24.

3. A probiotic agent prepared from Lactobacillus plantarum SC-24 according to claim 1.

4. A probiotic fermentation product prepared by the plant lactobacillus SC-24 according to claim 1.

5. A product prepared from the Lactobacillus plantarum SC-24 according to claim 1, the probiotic agent according to claim 3 or the probiotic fermentation product according to claim 4.

6. The product according to claim 5, characterized in that The product is feed or feed additive.

7. Use of the Lactobacillus plantarum SC-24 according to claim 1, the probiotic agent according to claim 3 or the probiotic fermentation product according to claim 4 in the preparation of feed or feed additives for preventing diarrhea and promoting growth in piglets.

8. A feed additive for preventing diarrhea and promoting growth in piglets, characterized in that: The feed additive comprises the following components in mass percentage: 15% to 25% of the probiotic fermentation product prepared by the plant lactobacillus SC-24 of claim 1, 2% to 8% of mannan, 2% to 8% of oligofructose, and the balance is water; and the following raw material components in concentration: 50 to 150 mg / L of vitamin A, 50 to 150 mg / L of vitamin D3, and 1 to 5 mg / L of vitamin E; The probiotic fermentation product prepared by Lactobacillus plantarum SC-24 comprises the following steps: A. Take Lactobacillus plantarum and culture it in MRS culture medium at 30-39°C for 8-16 hours to obtain Lactobacillus plantarum fermentation liquid I; B. inoculating Lactobacillus plantarum fermentation liquid I into MRS culture liquid, adjusting the initial pH value to 4.5-8.5, and culturing at 30-39° C. for 48-72 hours to obtain Lactobacillus plantarum fermentation liquid II; C. Resuspending the Lactobacillus plantarum fermentation liquid II into a 20-30% mixture using a NaCl solution, heating to 90-100° C. and maintaining for 5-10 minutes, then breaking the Lactobacillus plantarum cells to release the contents, and centrifuging at a speed of 3500-5000 rpm for 10-15 minutes, and taking the supernatant I after centrifugation; D. Add composite protease to the supernatant I obtained in step C for enzymolysis at 35-45° C. for 8-15 hours. After enzymolysis, heat the system to 90-100° C. and keep it for 5-10 minutes, and take supernatant II; E. Separate and recover the substances with a relative molecular mass less than 5000 in the supernatant II obtained in step D, and freeze-dry them to obtain the Lactobacillus plantarum SC-24 polypeptide complex.

9. The feed additive for preventing diarrhea and promoting growth in piglets according to claim 8, characterized in that: In step E, the supernatant II is separated using a dialysis bag with a molecular weight cut-off of 5000 to recover substances with a relative molecular mass of less than 5000; and / or The supernatant II was concentrated by ultrafiltration using a cellulose acetate membrane with a molecular weight cutoff of 5000 to recover substances with a relative molecular mass of less than 5000.

10. The feed additive for preventing diarrhea and promoting growth in piglets according to claim 8 or 9, characterized in that: In step E, a gel chromatography column is used to separate and recover substances with a relative molecular mass of less than 5000 in the supernatant II obtained in step D.

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