Lactobacillus reuteri RZ-D15L14 and application of postbiotics in reduction of diarrhea rate and death and culling rate of weaned piglets

Abstract

The invention relates to the technical field of biology, in particular to application of lactobacillus reuteri RZ-D15L14 and a metagen to reduction of the diarrhea rate and the death and culling rate of weaned piglets, the lactobacillus reuteri is lactobacillus reuteri, the strain number of the lactobacillus reuteri is RZ-D15L14, the lactobacillus reuteri is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20231503. After 500mg / kg of lactobacillus reuteri RZ-D15L14 is added into daily ration, the antioxidant function of the body and the intestinal health can be enhanced by improving the activity of antioxidant enzyme, reducing the content of oxidation products and improving the content of short-chain fatty acid in the intestinal tract, so that the effect of reducing the diarrhea rate and the death and culling rate of the piglets is achieved, and the positive effect is particularly outstanding in weak piglets.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to the application of Limosilactobacillus reuteri RZ-D15L14 and postbiotics in reducing the diarrhea rate and mortality rate of weaned piglets. Background Art

[0002] The digestive system of weaned piglets is underdeveloped, the immune function is not perfect, the anti-stress and disease resistance are weak, and careful feeding management is required. Transferring and grouping is one of the important measures. Separating and feeding weaned piglets with different growth and development conditions and different health levels can effectively reduce the fighting and food grabbing among the pig groups and provide a healthy growth environment for weak piglets. However, due to their weak constitution and slow growth, the production performance of weak piglets is generally poor; and because they belong to the disease-susceptible group, the mortality rate is often higher than the normal level. The formation of weak piglets is mainly caused by adverse factors during pregnancy, lactation and nursery periods. In particular, intrauterine growth restriction (IUGR) will lead to low birth weight of piglets and subsequent high morbidity, high mortality and low growth performance throughout the period, which is one of the prominent problems existing in the pig industry. Therefore, improving the immunity of weak pigs and reducing the mortality rate are of great significance for improving the breeding efficiency.

[0003] Limosilactobacillus reuteri is famous for its excellent intestinal health care function and is widely used in the fields of food, health products, pharmaceuticals, etc. In the field of livestock and poultry breeding, there are more and more studies and reports on the improvement of intestinal health by Limosilactobacillus reuteri. However, there are few reports on using postbiotics prepared from Limosilactobacillus reuteri to specifically solve problems such as high diarrhea rate and high mortality rate of weak pigs.

[0004] The prior art CN101974463A discloses Limosilactobacillus reuteri L-HD5 and its compound live bacteria preparation, and its preservation number is CGMCC No. 4050. The first experimental group is the freeze-dried powder preparation group of Limosilactobacillus reuteri L-HD5. The diarrhea rate of the first experimental group is 12.5%, and that of the control group is 32.2%. The diarrhea rate of the experimental group is reduced by 19.7% compared with the control group.

[0005] The prior art CN105746921A discloses a novel feed additive for effectively preventing and treating diarrhea in piglets, which is composed of the following components in parts by weight: 3-5 parts of Bacillus subtilis powder, 1-3 parts of Enterococcus lactis powder, 2-4 parts of Pediococcus acidilactici powder, 1-5 parts of Lactobacillus reuteri powder, 4-8 parts of fermented perilla leaves, 1-3 parts of plant polysaccharides, 2-6 parts of locust tree flowers, 1-4 parts of fresh Imperata cylindrica roots, 1-3 parts of Magnolia officinalis flowers, 1-3 parts of garlic powder, and the rest is a carrier, and the carrier is composed of defatted rice bran, and the defatted rice bran is pre-crushed through a 40-mesh sieve. Compared with the control group, the diarrhea rates of piglets in Examples 1-4 were 32.28%, 6.64%, 4.15%, 5.37%, and 10.35% respectively, which were reduced by 79.42%, 87.14%, 83.36%, and 67.93%.

[0006] The prior art CN118787670A discloses the application of a Lactobacillus reuteri strain from Rongchang pigs in piglet breeding. The taxonomic name of the Lactobacillus reuteri strain from Rongchang pigs is Lactobacillus reuteri RS-X-811. The diarrhea rates of the animal experiments using the compound probiotic solution prepared in Examples 4-6 to replace the RS-X-811 probiotic solution in Example 3 were 11.75% ± 3.65, 9.21% ± 2.37, 10.79% ± 3.08, and 10.16% ± 2.55.

[0007] However, the above prior art does not disclose other indicators such as the mortality rate. Therefore, there is an urgent need to provide the application of Lactobacillus reuteri RZ-D15L14 and postbiotics in reducing the diarrhea rate and mortality rate of weaned piglets. Summary of the Invention

[0008] The present invention first provides a Lactobacillus reuteri, which is Lactobacillus reuteri, and its strain number is RZ-D15L14, and it is preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC NO: M 20231503.

[0009] The present invention also provides a compound probiotic, which contains the above-mentioned Lactobacillus reuteri and other probiotics.

[0010] The present invention also provides a probiotic metabolite, which is obtained by fermenting the above-mentioned Lactobacillus reuteri or the above-mentioned compound probiotic.

[0011] The present invention also provides a postbiotic of Lactobacillus reuteri, which is prepared by treating the above-mentioned Lactobacillus reuteri through heat treatment, physical treatment, high hydrostatic pressure treatment, freeze-drying or ultrasonic oscillation processing methods.

[0012] The present invention also provides a postbiogenic Limosilactobacillus reuteri, which is prepared by processing the above-mentioned Limosilactobacillus reuteri through heat treatment, physical treatment, high hydrostatic pressure treatment, freeze-drying or ultrasonic oscillation processing methods.

[0013] The present invention also provides the use of the above-mentioned Limosilactobacillus reuteri or the above-mentioned Limosilactobacillus reuteri postbiogenic or the above-mentioned probiotic metabolites or the above-mentioned Limosilactobacillus reuteri postbiogenic or the above-mentioned Limosilactobacillus reuteri postbiogenic in the preparation of animal feed or the preparation of animal feed additives or the preparation of drugs for treating diarrhea.

[0014] In certain embodiments, the animal is a non-human mammal.

[0015] In certain embodiments, the animal is a pig.

[0016] In certain embodiments, the pig is a piglet.

[0017] In certain embodiments, the pig is a weaned piglet.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects:

[0019] The present invention first isolated Limosilactobacillus reuteri RZ-D15L14. Adding 500 mg / kg of the postbiogenic Limosilactobacillus reuteri RZ-D15L14 to the diet may enhance the body's antioxidant function and intestinal health by increasing the activity of antioxidant enzymes, reducing the content of oxidation products, and increasing the content of intestinal short-chain fatty acids, thereby achieving the effect of reducing the diarrhea rate and mortality rate of piglets, and this positive effect is particularly prominent in weak piglets.

[0020] Preservation description

[0021] The Limosilactobacillus reuteri RZ-D15L14 was isolated from the intestinal mucus of rural free-range annual pigs and was sent to the China Center for Type Culture Collection for preservation on August 17, 2023. The taxonomic name is: Limosilactobacillus reuteri RZ-D15L14, and the preservation number is: CCTCC NO: M20231503, address: Wuhan University, Wuhan, Hubei, China. Detailed implementation manners

[0022] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments.

[0023] Example 1: Preparation of Postbiotics from Lactobacillus reuteri RZ-D15L14

[0024] (1) Medium preparation:

[0025] MRS medium: 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder (added when preparing solid medium, not added to liquid medium), 1000 ml distilled water, pH 6.2 ± 0.2, sterilized at 121 °C for 20 minutes.

[0026] (2) Preparation of postbiotics:

[0027] Pick a loop of purified single colonies and inoculate them into MRS liquid medium, statically culture at 37 °C for 24 h to make a seed solution; inoculate the seed solution at 2% into a 50 L fermenter and ferment in MRS liquid medium for 48 h (temperature 37 °C, control pH at a constant 6.0, stirring speed 80 rpm); add 10% - 20% maltodextrin to the fermentation broth and mix evenly; spray-dry the fermentation broth (Shanghai Yacheng YC-015) to obtain postbiotics of Lactobacillus reuteri RZ-D15L14.

[0028] Example 2: Effect of Postbiotics from Lactobacillus reuteri RZ-D15L14 on the Growth Performance of Intrauterine Growth Retarded Piglets

[0029] Select 48 male piglets with intrauterine growth retardation at 20 days old (4.5 ± 0.5 kg), randomly divide them into two groups, namely the control group and the experimental group, with 4 replicates in each group and 6 pigs in each replicate. Piglets in the control group were fed a basal diet, and the diet formula was prepared according to the NRC-2012 piglet nutritional requirement standard. Piglets in the experimental group were fed a basal diet and supplemented with 500 mg / kg of RZ-D15L14 postbiotics. During the animal experiment, all piglets were allowed free access to food and water and were immunized according to the regular immunization program as required. The diarrhea situation of piglets was recorded during the breeding period, and all piglets were weighed and the feed consumption was calculated at the end of the experiment. The experiment lasted for 45 days.

[0030] The results showed (Table 1) that compared with the control group, adding RZ-D15L14 postbiotics to the diet could significantly reduce the diarrhea rate of piglets (P < 0.05), and could reduce the feed-to-gain ratio by 5.36%.

[0031] Table 1 Effect of Postbiotics of RZ-D15L14 on the Growth Performance of Intrauterine Growth Retarded Piglets

[0032]

[0033] Example 3: Effects of Postbiotics of Lactobacillus reuteri RZ-D15L14 on Weak Piglets

[0034] A total of 544 healthy male weaned piglets at 24 days of age with uniform body weight were randomly divided into two treatment groups, namely the control group and the experimental group. There were 8 replicates in each treatment, with 34 pigs in each replicate. The two groups were divided into two pens according to the initial body weight of the piglets: the weak pig pen (6.0 ± 0.5 kg) and the strong pig pen (8.0 ± 0.5 kg). Piglets in the control group were fed a basal diet, and the diet formula was prepared according to the NRC-2012 piglet nutrient requirement standard. Piglets in the experimental group were fed a basal diet supplemented with 500 mg / kg of RZ-D15L14 postbiotics. The experiment lasted for 48 days. During the animal experiment, all piglets had free access to food and water and received routine immunization procedures as required. The number of deaths and culls was recorded during the experiment, and all piglets were weighed and feed consumption was calculated at the end of the experiment. At the same time, two pigs were selected from each replicate, that is, 16 pigs in each group, for anterior vena cava blood collection to detect antioxidant-related indicators. Fresh feces were collected to detect short-chain fatty acid levels.

[0035] (1) Effects of Postbiotics of RZ-D15L14 on the Growth Performance of Weak and Strong Piglets

[0036] As shown in the results (Table 2), the postbiotics of Lactobacillus reuteri RZ-D15L14 had no significant effects on the average daily gain, average daily feed intake, and feed-to-gain ratio of piglets. Compared with the indicators of weak piglets in the two groups, the postbiotics of Lactobacillus reuteri RZ-D15L14 could significantly reduce their diarrhea rate and death and cull rate (P < 0.005), from 0.96% and 25% to 0.77% and 11.76% respectively; at the same time, for strong piglets, the postbiotics of Lactobacillus reuteri RZ-D15L14 had a tendency to reduce the diarrhea rate (P = 0.068) and significantly reduced the death and cull rate (P < 0.05).

[0037] Table 2 Effects of Postbiotics of RZ-D15L14 on the Growth Performance of Weak and Strong Piglets

[0038]

[0039] (2) Effects of Postbiotics of RZ-D15L14 on the Serum Biochemical Indexes of Weak and Strong Piglets

[0040] Superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and glutathione peroxidase (GSH-Px) in serum are key indicators reflecting the body's oxidation level and antioxidant capacity. As shown in Table 3, when comparing the indicators of the two groups of weak piglets, the postbiotics of Lactobacillus reuteri RZ-D15L14 can significantly reduce the content of MDA in their serum and significantly increase the content of SOD (P < 0.05); when comparing the indicators of the two groups of strong piglets, the postbiotics of Lactobacillus reuteri RZ-D15L14 have a tendency to reduce MDA (P = 0.081).

[0041] Table 3 Effects of postbiotics of RZ-D15L14 on serum biochemical indicators of weak and strong piglets

[0042]

[0043] (3) Effects of postbiotics of RZ-D15L14 on short-chain fatty acids in feces of weak and strong piglets

[0044] An increase in the content of short-chain fatty acids in the intestine is generally considered to be beneficial to the intestinal health of piglets. By measuring the content of short-chain fatty acids in feces, it was found that for both weak and strong piglets, the postbiotics of Lactobacillus reuteri RZ-D15L14 have a tendency to increase the content of acetic acid in their feces (P = 0.065, P = 0.052), and significantly increase the content of propionic acid and butyric acid (Table 4).

[0045] Table 4 Effects of postbiotics of RZ-D15L14 on short-chain fatty acids in feces of weak and strong piglets

[0046]

[0047] In summary: Adding 500 mg / kg of postbiotics of Lactobacillus reuteri RZ-D15L14 to the diet may enhance the body's antioxidant function and intestinal health by increasing the activity of antioxidant enzymes, reducing the content of oxidation products, and increasing the content of intestinal short-chain fatty acids, thereby achieving the effect of reducing the diarrhea rate and mortality rate of piglets, and this positive effect is particularly prominent in weak piglets.

[0048] The above is the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Lactobacillus reuteri, characterized in that, The Limosilactobacillus reuteri is Limosilactobacillus reuteri with the strain number RZ-D15L14, which is preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO:M 20231503.

2. A compound probiotic, characterized in that, The compound probiotic contains the Limosilactobacillus reuteri described in claim 1 and other probiotics.

3. A probiotic metabolite, characterized in that, The probiotic metabolite is obtained by fermenting the Limosilactobacillus reuteri described in claim 1 or the compound probiotic described in claim 2.

4. A postbiotic of Lactobacillus reuteri, characterized in that, The postbiotics of the Limosilactobacillus reuteri is prepared by processing the Limosilactobacillus reuteri described in claim 1 through heat treatment, physical treatment, high hydrostatic pressure treatment, freeze-drying or ultrasonic oscillation processing methods.

5. A compound probiotic postbiotic element, characterized in that, The postbiotics of the compound probiotic is prepared by processing the compound probiotic described in claim 2 through heat treatment, physical treatment, high hydrostatic pressure treatment, freeze-drying or ultrasonic oscillation processing methods.

6. Use of the Limosilactobacillus reuteri described in claim 1 or the compound probiotic described in claim 2 or the probiotic metabolite described in claim 3 or the postbiotics of the Limosilactobacillus reuteri described in claim 4 or the postbiotics of the compound probiotic described in claim 5 in the preparation of animal feed or the preparation of animal feed additives or the preparation of drugs for treating diarrhea.

7. The application according to claim 6, wherein The animal is a non-human mammal.

8. The application according to claim 6, characterized in that, The animal is a pig.

9. The application according to claim 8, characterized in that, The pig is a piglet.

10. The application according to claim 9, wherein The pig is a weaned piglet.

Images