South medicine probiotic compound preparation for preventing and treating porcine diarrhea as well as preparation method and application of south medicine probiotic compound preparation

Abstract

The invention discloses a probiotic composition, which is prepared from the following components in parts by mass: 1 to 2 parts of lactobacillus rhamnosus, 1 to 2 parts of saccharomycetes and 1 to 2 parts of bacillus subtilis. The concentration of the lactobacillus rhamnosus is 1 * 10 < 10 >-5 * 10 < 10 > CFU / mL, the concentration of the saccharomycetes is 1 * 10 < 11 >-5 * 10 < 11 > CFU / mL, and the concentration of the bacillus subtilis is 5 * 10 < 8 >-1 * 10 < 9 > CFU / mL. When the probiotic composition is mixed with feed to feed pigs, the diarrhea index of the pigs in the feeding process can be effectively reduced, the feed conversion ratio of the pigs is increased, the growth performance of the pigs is improved, and the diarrhea of the pigs is effectively prevented and treated. In addition, pithecellobium clypearia, jasminum elongatum, Chinese knotweed herb, ovateleaf holly bark, cape jasmine, radix sophorae flavescentis, citrus chachiensis hortorum and poria cocos are combined with the probiotic composition for fermentation to obtain the south medicine probiotic compound preparation, and the diarrhea index of pigs in the feeding process can be more effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry and veterinary technology, specifically to a compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea, its preparation method, and its application. Background Technology

[0002] In recent years, with the development of modern technology and animal husbandry, large-scale and intensive farming models have been vigorously promoted. However, high-density farming increases the possibility of disease occurrence and spread, leading to problems such as antibiotic abuse, increased drug-resistant bacteria, and drug residues, which seriously endanger the development of the livestock and poultry industry and human health.

[0003] During weaning, piglets are prone to intestinal microecology imbalance due to factors such as dietary changes and environmental stress, which can lead to atrophy, shedding, and vacuolation of intestinal villi. This results in thinning of the intestinal wall, a reduction in the absorption surface area, and interference with the balance of gut microbiota (GM), reducing the number of probiotics. Consequently, the small intestine's absorption and digestion functions are reduced, making piglets more susceptible to pathogens and diseases such as diarrhea.

[0004] Piglet diarrhea is a highly dangerous and common infectious disease in pigs, primarily affecting newborn, weaned piglets. It ranks first in morbidity and mortality among diarrheal diseases in pig herds, causing significant losses to pig farmers. To prevent and control pig diarrhea and reduce the overuse of antibiotics, current efforts are focused on finding non-antibiotic alternatives to improve animal health.

[0005] Probiotics are live microorganisms that can provide health benefits to the host. They are a type of non-antibiotic alternative. Ingesting appropriate amounts of probiotics can help maintain or restore the normal gut microbiota composition, thereby enhancing the host's immunity and promoting the digestion and absorption of nutrients. For piglets, certain types of lactic acid bacteria, such as Lactobacillus johnsonii, can attach to the intestinal surface by forming a biofilm, providing a stable interface to resist external threats.

[0006] However, in the process of using probiotics to treat diarrhea in piglets, many probiotics cannot survive long enough in the host's body, and many probiotics cannot effectively resist stomach acid, bile salts, or other adverse conditions, resulting in poor colonization ability of probiotics in the piglet's intestines, thus leading to poor prevention and treatment effects for piglet diarrhea. Furthermore, currently, the prevention and treatment of diarrhea in piglets often involves feeding a single probiotic, which is quite sensitive to changes in the environment, especially in the complex intestinal environment. A single strain may not be able to effectively colonize or maintain a sufficient number to exert a sustained effect.

[0007] Therefore, there is an urgent need for a compound probiotic that can effectively prevent and treat diarrhea in piglets. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned deficiencies of the prior art and provide a compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea, its preparation method, and its application.

[0009] The first objective of this invention is to provide a probiotic composition.

[0010] A second objective of this invention is to provide the use of the above-described probiotic composition in the preparation of a medicament for the prevention and treatment of swine diarrhea.

[0011] A third objective of this invention is to provide the use of the above-described probiotic composition in the preparation of a medicament for enhancing the immunity of pigs.

[0012] The fourth objective of this invention is to provide a compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea.

[0013] The fifth objective of this invention is to provide the application of the above-mentioned probiotic composition and / or the above-mentioned herbal probiotic compound preparation in reducing the feed conversion ratio of pigs.

[0014] The sixth objective of this invention is to provide a method for preparing a compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea.

[0015] To achieve the above objectives, the present invention is implemented through the following solution:

[0016] A probiotic composition, by weight, contains 1-2 parts of Lactobacillus rhamnosus (ATCC53103), 1-2 parts of yeast (ATCC204508) and 1-2 parts of Bacillus subtilis (ATCC23857).

[0017] The concentration of the Lactobacillus rhamnosus is 1*10. 10 ~5*10 10 CFU / mL, yeast concentration 1*10 11 ~5*10 11 CFU / mL, the concentration of Bacillus subtilis is 5*10 8 ~1*10 9 CFU / mL.

[0018] Preferably, by weight, it contains 1 part Lactobacillus rhamnosus, 2 parts yeast and 2 parts Bacillus subtilis.

[0019] More preferably, the concentration of the Lactobacillus rhamnosus is 1.76 × 10⁻⁶. 10 CFU / mL, yeast concentration 2.7 × 10⁻⁶ 11 The concentration of Bacillus subtilis was 9.5 × 10 CFU / mL. 8 CFU / mL.

[0020] More preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus ATCC53103, the yeast is yeast ATCC204508, and the Bacillus subtilis is Bacillus subtilis ATCC23857.

[0021] When pigs are fed with the above-mentioned probiotic composition mixed with feed, the diarrhea index of pigs during feeding can be effectively reduced, the feed conversion ratio of pigs can be increased, and the growth performance of pigs can be improved.

[0022] Therefore, the present invention also claims protection for the use of any of the above-described probiotic compositions in the preparation of a medicament for the prevention and treatment of swine diarrhea.

[0023] Preferably, the pig is a weaned piglet.

[0024] This invention also claims protection for the use of any of the above-described probiotic compositions in the preparation of a medicament for enhancing the immunity of pigs.

[0025] This invention also claims protection for a compound preparation of southern Chinese medicinal herbs and probiotics for the prevention and treatment of swine diarrhea, comprising a southern Chinese medicinal herb composition and any of the above-described probiotic compositions;

[0026] The mass-volume ratio of the herbal composition to any of the above-mentioned probiotic compositions is 100:3-5; the herbal composition contains, by mass parts, 60-80 parts of *Eriocaulon buergerianum*, 4-11 parts of *Tinospora sinensis*, 4-11 parts of *Polygonum chinense*, 4-11 parts of *Impatiens balsamina*, 2-8 parts of *Gardenia jasminoides*, 1-6 parts of *Sophora flavescens*, 1-6 parts of *Citrus reticulata*, and 1-3 parts of *Poria cocos*.

[0027] Preferably, the southern medicinal herb composition contains, by weight parts:

[0028] 80 parts of monkey earring, 4.5 parts of twisted belly vine, 4.5 parts of fire charcoal mother, 4.5 parts of savior, 2.7 parts of gardenia, 1.4 parts of sophora flavescens, 1.4 parts of dried tangerine peel and 1 part of poria cocos.

[0029] Preferably, the mass-to-volume ratio of the herbal medicine composition and any of the above-described probiotic compositions is 100g:3mL.

[0030] This invention also claims protection for the use of any of the above-described probiotic compositions and any of the above-described herbal probiotic compound preparations in reducing the feed conversion ratio of pigs.

[0031] Preferably, the pig is a weaned piglet.

[0032] This invention also claims protection for a method for preparing a compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea, comprising the following steps, by weight:

[0033] S1. Mix the southern medicinal materials with water and boil thoroughly. Separate the solid and liquid, collect the liquid and concentrate it to a concentration of 0.5 g / mL to 2 g / mL to obtain the medicinal liquid.

[0034] The composition of the southern medicinal herbs, by mass parts, contains 60-80 parts of monkey ear ring, 4-11 parts of twisted belly vine, 4-11 parts of fire charcoal mother, 4-11 parts of savior, 2-8 parts of gardenia, 1-6 parts of sophora flavescens, 1-6 parts of tangerine peel and 1-3 parts of poria cocos.

[0035] S2. Add any of the above-mentioned probiotic compositions to the medicinal liquid obtained in step S1, and after full fermentation, a southern Chinese medicinal herb probiotic compound preparation is obtained; the mass-volume ratio of any of the above-mentioned probiotic compositions to the southern Chinese medicinal herb composition in step S1 is 3-5 mL: 100 g.

[0036] Preferably, the herbal composition in step S1 contains, by mass parts, 80 parts of *Eriocaulon buergerianum*, 4.5 parts of *Tricholoma matsutake*, 4.5 parts of *Polygonum chinense*, 4.5 parts of *Eupatorium fortunei*, 2.7 parts of *Gardenia jasminoides*, 1.4 parts of *Sophora flavescens*, 1.4 parts of *Citrus reticulata*, and 1 part of *Poria cocos*.

[0037] Preferably, in step S1, the concentration of the raw drug is concentrated to 1 g / mL.

[0038] Preferably, the full fermentation in step S2 specifically refers to fermentation at 25–37°C for 12–48 hours.

[0039] More preferably, fermentation is carried out at 37°C for 24 hours.

[0040] This invention also protects the probiotic compound preparation of southern Chinese medicinal herbs prepared by the above preparation method.

[0041] The present invention also claims protection for a method for reducing the feed conversion ratio of pigs, comprising feeding the pigs with feed containing the above-mentioned probiotic composition and / or the above-mentioned herbal probiotic compound preparation at a final mass concentration of 0.5 to 2%.

[0042] Preferably, the feed is prepared using feed containing the above-mentioned probiotic composition and / or the above-mentioned southern Chinese medicinal herb probiotic compound preparation at a final mass concentration of 0.5%.

[0043] More preferably, the feed is prepared using feed containing the above-mentioned probiotic compound preparation of southern Chinese medicinal herbs at a final mass concentration of 0.5%.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention provides a probiotic composition, comprising, by weight, 1-2 parts of Lactobacillus rhamnosus, 1-2 parts of yeast, and 1-2 parts of Bacillus subtilis; wherein the concentration of Lactobacillus rhamnosus is 1*10. 10 ~5*1010 CFU / mL, yeast concentration 1*10 11 ~5*10 11 CFU / mL, the concentration of Bacillus subtilis is 5*10 8 ~1*10 9 CFU / mL. When the probiotic composition is mixed with feed and fed to pigs, it can effectively reduce the diarrhea index during feeding, improve the feed conversion ratio, and improve the growth performance of pigs, thus achieving effective prevention and treatment of swine diarrhea. Furthermore, this invention combines *Heliotropium indicum*, *Gnaphalium affine*, *Polygonum chinense*, *Impatiens balsamina*, *Gardenia jasminoides*, *Sophora flavescens*, *Citrus reticulata*, and *Poria cocos* with the probiotic composition for fermentation to obtain a compound preparation of southern Chinese medicinal herbs probiotics. When this compound preparation of southern Chinese medicinal herbs is mixed with feed and fed to pigs, it can more effectively reduce the diarrhea index during feeding, improve the feed conversion ratio, and improve the growth performance of pigs. Attached Figure Description

[0046] Figure 1 The graph shows the antibacterial test results of the fermentation broth of probiotic composition solutions with different ratios combined with traditional Chinese medicine liquid in Example 1.

[0047] Figure 2 The graph shows the antibacterial test results of fermentation broth containing different amounts of probiotic composition solutions combined with liquid from southern Chinese medicinal herbs in Example 1.

[0048] Figure 3 The graph shows the antibacterial test results of the fermentation broth at different fermentation times in Example 1.

[0049] Figure 4 The images shown are photographs of the jejunal villi of each group of nursery piglets in Example 3; a is a photograph of the jejunal villi of the control group nursery piglets, b is a photograph of the jejunal villi of the southern Chinese medicine group nursery piglets, c is a photograph of the jejunal villi of the probiotic group nursery piglets, and d is a photograph of the jejunal villi of the southern Chinese medicine-probiotic compound preparation group.

[0050] Figure 5 The images show the histopathological results of each group of weaned piglets in Example 3; a is the result of the jejunal villus length of each group of weaned piglets, b is the result of the jejunal crypt depth of each group of weaned piglets, and c is the ratio of the jejunal villus length to the jejunal crypt depth of each group of weaned piglets.

[0051] Figure 6 This is a graph showing the organ index results of each group of nursery piglets in Example 3;

[0052] Figure 7The following graph shows the results of immune level detection for each group of weaned piglets in Example 3; a represents the detection results of IgA expression level, b represents the detection results of IgG expression level, c represents the detection results of IgM expression level, d represents the detection results of IL-2 expression level, e represents the detection results of IL-6 expression level, f represents the detection results of IL-17 expression level, and g represents the detection results of TNF-α expression level.

[0053] Figure 8 The following figures show the results of detecting the expression of the JAK2 / STAT3 pathway in the jejunum of each group of weaned piglets: a) Western blot images of STAS3, p-STAT3, JAK2, p-JAK2, and IL-6 proteins; b) Relative expression levels of p-STAT3; c) Relative expression levels of p-JAK2; d) Relative expression levels of IL-6; e) Relative mRNA expression levels of STAT3; f) Relative mRNA expression levels of JAK2; and g) Relative mRNA expression levels of IL-6.

[0054] Figure 9 The following figures show the results of detecting the expression of tight junction protein pathways in the jejunum of each group of weaned piglets: a) Western blot images of ZO-1, Occludin, and Claudin 1 proteins; b) Relative expression level of ZO-1 protein; c) Relative expression level of Occludin protein; d) Relative expression level of Claudin 1 protein; e) Relative expression level of ZO-1 mRNA; f) Relative expression level of Occludin mRNA; and g) Relative expression level of Claudin-1 mRNA. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0056] All animal experiments involved in the embodiments of this invention have been approved and confirmed by the ethics committee;

[0057] In this embodiment of the invention, the feed used to feed piglets is fresh piglet compound feed, which was purchased from Heilongjiang Sanhe Feed Co., Ltd., product number LM300;

[0058] The Lactobacillus rhamnosus used in this embodiment of the invention is Lactobacillus rhamnosus ATCC53103, the yeast is yeast ATCC204508, and the Bacillus subtilis is Bacillus subtilis ATCC23857.

[0059] Example 1: Preparation of a compound preparation of probiotics from southern Chinese medicinal herbs

[0060] I. Preparation of Liquid Herbs from Southern China

[0061] Mix 80g of monkey earring, 4.5g of twisted belly vine, 4.5g of fire charcoal mother, 4.5g of sap-repelling herb, 2.7g of gardenia, 1.4g of sophora flavescens, 1.4g of dried tangerine peel, and 1g of poria cocos evenly, soak in 800mL of water for 30 minutes, heat to a boil, then simmer for 1 hour. Filter to obtain dregs 1 and liquid 1. Add dregs 1 to 300mL of water and simmer for 0.5 hours. Filter to obtain dregs 2 and liquid 2. Add dregs 2 to 300mL of water, boil, and filter to obtain liquid 3.

[0062] Combine medicinal solutions 1, 2, and 3, centrifuge at 4000 r / min for 15 min, collect the supernatant, concentrate the supernatant to a crude drug concentration of 1 g / mL, sterilize at 121℃ for 20 min, and obtain 100 mL of southern medicinal liquid.

[0063] II. Activation of Probiotics

[0064] After thawing the frozen Lactobacillus rhamnosus, it was inoculated into MRS broth medium and cultured for 24 hours to obtain the cultured bacterial solution. The cultured bacterial solution was then inoculated into MRS broth medium at an inoculation rate of 2.5% (v / v) and cultured for 24 hours. The cultured bacterial solution after 24 hours was then inoculated into MRS broth medium at an inoculation rate of 2.5% (v / v) and cultured for 24 hours to obtain the activated Lactobacillus rhamnosus bacterial solution.

[0065] The activated Lactobacillus rhamnosus culture was inoculated into MRS broth at an inoculation rate of 2.5% (v / v). After thorough mixing, 200 μL of the broth containing the activated Lactobacillus rhamnosus was placed in a 96-well plate with three replicates. A blank broth was used as a control. The concentration of Lactobacillus rhamnosus was adjusted to OD using a UV spectrophotometer. 600 =0.5, yielding a Lactobacillus rhamnosus solution with a concentration of 1.76 × 10⁻⁵. 10 CFU / mL.

[0066] Lactobacillus rhamnosus was replaced with yeast, and MRS broth medium was replaced with YM broth medium. Following the above method, a yeast solution was obtained with a yeast concentration of 2.7 × 10⁻⁶. 11CFU / mL.

[0067] Lactobacillus rhamnosus was replaced with Bacillus subtilis, and MRS broth medium was replaced with LB broth medium. Following the above method, a Bacillus subtilis solution was obtained with a concentration of 9.5 × 10⁻⁶. 8 CFU / mL.

[0068] III. Determination of Probiotic Compound Preparations of Southern Chinese Herbal Medicines

[0069] 1. Experimental Methods

[0070] (1) Determination of probiotic ratio

[0071] The Lactobacillus rhamnosus solution, yeast solution, and Bacillus subtilis solution obtained in step two were compounded in mass ratios of 1:1:1, 1:2:2, 2:1:1, and 2:1:2, respectively, to obtain probiotic composition solutions with different ratios.

[0072] 3 mL of probiotic composition solutions with different ratios were mixed with 100 mL of the herbal liquid obtained in step one and then added to a sealed container. The mixture was fermented at 37°C for 24 h. The fermentation liquid was collected, and an in vitro Escherichia coli inhibition experiment was conducted using the paper disc diffusion method. The antibacterial results were recorded.

[0073] (2) Determination of the amount of probiotics added

[0074] The Lactobacillus rhamnosus solution, yeast solution, and Bacillus subtilis solution obtained in step two are mixed in a mass ratio of 1:2:2 to obtain a probiotic composition solution.

[0075] The probiotic composition solution was mixed with 100 mL of the herbal liquid obtained in step one and then added to a sealed container. The mixture was fermented at 37°C for 24 hours. The fermentation liquid was collected for in vitro antibacterial experiments, and the antibacterial results were recorded.

[0076] The amounts of probiotic composition solution added were 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL, respectively.

[0077] (3) Determination of fermentation time

[0078] The Lactobacillus rhamnosus solution, yeast solution, and Bacillus subtilis solution obtained in step two are mixed in a mass ratio of 1:2:2 to obtain a probiotic composition solution.

[0079] Mix 3 mL of probiotic composition solution with 100 mL of the herbal liquid obtained in step one and add it to a sealed container. Ferment at 37°C for 48 h. Take the fermentation liquid at 12 h, 24 h, 36 h and 48 h respectively for in vitro antibacterial experiments and record the antibacterial results.

[0080] 2. Experimental Results

[0081] The antibacterial test results of probiotic composition solutions with different ratios combined with fermentation broth of traditional Chinese medicine are shown in the figure. Figure 1 As shown in the figure, the results indicate that when Lactobacillus rhamnosus solution, yeast solution, and Bacillus subtilis solution are mixed in a mass ratio of 1-2:1-2:1-2 and then mixed with the liquid of southern medicinal herbs for fermentation, the resulting fermentation broth all have excellent antibacterial effects. Moreover, the antibacterial effect is the best when Lactobacillus rhamnosus solution, yeast solution, and Bacillus subtilis solution are mixed in a mass ratio of 1:2:2.

[0082] The antibacterial test results of probiotic composition solutions with different addition amounts combined with fermentation broth of traditional Chinese medicine are shown in the figure below. Figure 2 As shown, the results indicate that when the amount of probiotic composition solution added is between 3 mL and 5 mL, the fermentation broth after mixing with the liquid of southern Chinese medicinal herbs exhibits a relatively excellent antibacterial effect; and when the amount of probiotic composition added is 3 mL, the antibacterial effect of the fermentation broth after mixing with the liquid of southern Chinese medicinal herbs is the best.

[0083] The antibacterial test results of fermentation broth at different fermentation times are shown in the figure below. Figure 3 As shown in the figure, the results indicate that the fermentation broth obtained after fermentation for 12–48 hours all have antibacterial effects; and the antibacterial effect is the best when the fermentation time is 24 hours.

[0084] IV. Preparation of Probiotic Compound Preparations from Southern Chinese Herbal Medicines

[0085] The Lactobacillus rhamnosus solution, yeast solution, and Bacillus subtilis solution obtained in step two are combined in a mass ratio of 1:2:2 to obtain a probiotic composition solution.

[0086] After mixing the probiotic composition solution with the liquid of southern Chinese medicinal herbs obtained in step one, the mixture is added to a sealed container and fermented at 37°C for 24 hours. The fermentation liquid is then collected to obtain the probiotic compound preparation of southern Chinese medicinal herbs.

[0087] Example 2: Effects of a compound preparation of probiotics from southern Chinese medicinal herbs on the growth performance of weaned piglets.

[0088] I. Experimental Methods

[0089] 1. Setup of experimental animals

[0090] Twenty-four healthy 7-day-old piglets of similar weight were selected. All piglets were vaccinated against foot-and-mouth disease, classical swine fever, and pseudorabies. They were raised separately in strictly disinfected pens, with free access to feed and antibiotic-free feed, as well as drinking water that had been sterilized at high temperatures.

[0091] 2. Freeze-drying preparation

[0092] The liquid of southern Chinese medicinal herbs obtained in step one of Example 1, the liquid probiotic composition obtained in step four, and the compound preparation of southern Chinese medicinal herbs and probiotics were respectively placed in an ultra-low temperature dryer for freeze drying to obtain freeze-dried southern Chinese medicinal herbs, freeze-dried probiotic composition, and freeze-dried compound preparation of southern Chinese medicinal herbs and probiotics.

[0093] 3. Grouping and processing of experimental animals

[0094] Twenty-four 7-day-old weaned piglets were randomly divided into four groups of six piglets each: a control group, a traditional Chinese medicine group, a probiotic group, and a traditional Chinese medicine-probiotic compound preparation group. The specific treatments for each group are as follows:

[0095] Control group: fed with feed twice daily on an ad libitum basis.

[0096] The only difference between the southern medicinal herb group and the control group was that the feed was replaced with feed containing 0.5% (w / w) freeze-dried southern medicinal herbs; all other treatments were exactly the same.

[0097] The only difference between the probiotic group and the control group was that the feed was replaced with feed mixed with 0.5% (w / w) freeze-dried probiotic composition; all other treatments were exactly the same.

[0098] The only difference between the Southern Chinese Medicine-Probiotic Compound Preparation Group and the control group was that the feed was replaced with feed mixed with 0.5% (w / w) freeze-dried Southern Chinese Medicine Probiotic Compound Preparation; all other treatments were exactly the same.

[0099] 4. Growth performance index testing

[0100] Each group of 7-day-old weaned piglets was fed continuously for 21 days according to the corresponding treatment method, and the following indicators were calculated:

[0101] (1) Average daily weight gain: Before and after feeding, the piglets in each group were weighed and the total weight gain and average daily weight gain of each group of piglets during the feeding process were calculated.

[0102] (2) Diarrhea index: During the feeding process, the feces of each group of weaned piglets were scored daily according to the feces scoring table shown in Table 1, and the diarrhea index of each group of weaned piglets was calculated according to Formula I.

[0103] Table 1. Stool Scoring Table

[0104] degree Appearance Initial water content in feces score normal Strips or granules ＜70 0 Mild Soft stool, formed 70～75 1 moderate The stool is thick and unformed, with no separation of fecal matter and water. 75～80 2 serious The liquid is not formed, and there is separation of fecal matter and water. ＞80 3

[0105] Formula I: Diarrhea Index = (Sum of diarrhea scores of all weaned piglets in the group during the feeding period) / (Number of feeding days * Number of weaned piglets in the group);

[0106] (3) Feed intake and feed conversion ratio: During the feeding process, the feed intake of each group of weaned piglets was recorded daily, the total feed intake during the feeding process and the average daily feed intake of each group of weaned piglets were calculated, and the feed conversion ratio of each group of weaned piglets was calculated according to Formula II.

[0107] Formula II: Feed conversion ratio = (total feed intake of each group of weaned piglets during the feeding process) / (total weight gain of each group of weaned piglets during the feeding process).

[0108] II. Experimental Results

[0109] The growth performance results of each group of weaned piglets are shown in Table 2, where growth performance includes average daily weight gain, average daily feed intake, feed conversion ratio and diarrhea index.

[0110] Table 2. Growth performance results of each group of weaned piglets

[0111] Average daily weight gain / g Average daily feed intake / g Meat-to-fat ratio Diarrhea Index control group 196.67 192.76 0.98 0.022 Southern Chinese Medicine Group <![CDATA[237.94 a ]]> 194.42 <![CDATA[0.82 a ]]> <![CDATA[0.003 a ]]> Probiotic group <![CDATA[217.06 b ]]> 194.61 0.9 <![CDATA[0.008 a ]]> Southern Chinese medicinal herbs-probiotic compound preparation group <![CDATA[247.78 a ]]> 194.73 <![CDATA[0.8 a ]]> <![CDATA[0.002 a ]]>

[0112] Note: In the table, "a" indicates a significant difference from the control group, and "b" indicates a significant difference from the Southern Chinese Medicine-Probiotic Compound Preparation group.

[0113] The results showed that, compared with the control group, adding freeze-dried traditional Chinese medicine, freeze-dried probiotic composition, or freeze-dried traditional Chinese medicine-probiotic compound preparation to the feed could effectively improve the average daily weight gain of weaned piglets and reduce the feed conversion ratio and diarrhea index during the feeding process. This indicates that the traditional Chinese medicine liquid, probiotic composition, and traditional Chinese medicine-probiotic compound preparation in Example 1 can all improve the growth performance of weaned piglets and prevent diarrhea in weaned piglets. Furthermore, the weaned piglets in the traditional Chinese medicine-probiotic compound preparation group not only had a significantly higher average daily weight gain than other groups, but also had a significantly lower feed conversion ratio and diarrhea index, indicating that the traditional Chinese medicine-probiotic compound preparation has a more superior effect on improving the growth performance of weaned piglets.

[0114] Example 3: Effects of a compound preparation of probiotics from southern Chinese medicinal herbs on the immune function of weaned piglets.

[0115] I. Experimental Methods

[0116] As shown in Example 2, control group, traditional Chinese medicine group, probiotic group and traditional Chinese medicine-probiotic compound preparation group were set up respectively. After feeding the 7-day-old nursery piglets of each group for 21 consecutive days according to the corresponding treatment method, the nursery piglets of each group were euthanized and their intestines were examined by histopathology. One nursery piglet from each group was randomly selected to photograph and observe its jejunal villi, and the length of the jejunal villi and the crypt depth of each nursery piglet were measured.

[0117] Simultaneously, the weight of the heart, liver, kidneys, and spleen of each group of piglets was measured, and the ratio of each organ weight to the total weight of the weaned piglets was calculated to obtain organ indices, including the heart index, liver index, kidney index, and spleen index.

[0118] Next, the levels of IgA, IgM, IgG, IL-6, IL-2, IL-17 and TNF-α in each group of weaned piglets were detected using ELISA kits from Shanghai Enzyme-Linked Biotechnology (models: MM-090501, MM-040301, MM-038301, MM-042101, MM-041801, MM-040201, and MM-7784001) according to the kit instructions.

[0119] Next, jejunal proteins were extracted from each group of weaned piglets. Proteins were separated by SDS-PAGE and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane. The membrane was blocked at 25°C with Tris-buffered saline (TBST) containing 5% (w / w) skim milk powder for 1 hour. After incubation with primary antibodies (as shown in Table 3) at 4°C for 16 hours, the membrane was thoroughly washed with TBST and then shaken at 25°C for 1 hour with HRP-conjugated secondary antibodies (1:5000, v / v) (Kangwei Century, CW0103S / CW0102S). Protein bands were then detected using an enhanced chemiluminescence (ECL) assay kit and analyzed using ImageJ (version 1.51j8) software to obtain the expression of tight junction proteins and the JAK2 / STAT3 pathway in the jejunum of each group of weaned piglets.

[0120] Table 3 Primary Antibody Information

[0121] Antibody brand Item number JAK2 Cell signaling 3230T P-STAT3 Cell signaling 9145T P-JAK2 Cell signaling 4406T Claudin 1 PROTEINTECH 13050-1-AP Occludin PROTEINTECH 13409-1-AP ZO-1 PROTEINTECH 21773-1-AP STAT3 ABClonal A1192 GAPDH bioworld MB001

[0122] mRNA was extracted from each group of weaned piglets. Using the extracted mRNA, the mRNA was reverse transcribed into cDNA using the HiScript III RTSuperMix for quantitative PCR kit (Vazyme, China) according to its instructions. qPCR amplification primers for Occludin, ZO-1, Claudin 1, β-actin, IL-6, STAT3, and JAK2 were designed based on NCBI Gene Bank, as shown in Table 4.

[0123] Table 4. Upstream and downstream primers for qPCR amplification

[0124]

[0125] 1 μL of cDNA was amplified using ChamQ Universal SYBR Quantitative PCR Master Mix (Vazyme, China) with the upstream and downstream primers shown in Table 4. Real-time fluorescence PCR was performed on a real-time fluorescence quantitative PCR instrument (LightCycler480, Roche, USA) to obtain the relative expression levels of mRNA for STAT3, JAK2, ZO-1, Occludin, Claudin 1, and IL-6 proteins.

[0126] II. Experimental Results

[0127] Photographs of the jejunal villi of each group of weaned piglets are shown below. Figure 4 As shown, a is a photograph of the jejunal villi of the control group piglets, b is a photograph of the jejunal villi of the traditional Chinese medicine group piglets, c is a photograph of the jejunal villi of the probiotic group piglets, and d is a photograph of the jejunal villi of the traditional Chinese medicine-probiotic compound preparation group.

[0128] The histopathological test results of each group of weaned piglets are shown in the figure below. Figure 5 As shown, a is the result of jejunal villus length in each group of weaned piglets, b is the result of jejunal crypt depth in each group of weaned piglets, and c is the ratio of jejunal villus length to jejunal crypt depth in each group of weaned piglets.

[0129] The results showed that, compared with the control group, the length of jejunal villi, the depth of jejunal crypts, and the ratio of jejunal villi length to jejunal crypt depth were significantly increased in piglets fed with the corresponding feeds in the traditional Chinese medicine group, the probiotic group, and the traditional Chinese medicine-probiotic compound preparation group. Furthermore, there were no significant differences in the length of jejunal villi, the depth of jejunal crypts, and the ratio of jejunal villi length to jejunal crypt depth in piglets in the traditional Chinese medicine group and the probiotic group. However, the length of jejunal villi, the depth of jejunal crypts, and the ratio of jejunal villi length to jejunal crypt depth in piglets in the traditional Chinese medicine-probiotic compound preparation group were significantly higher than those in the other three groups.

[0130] This indicates that feeding nursery piglets with feed containing the probiotic composition of Example 1 or the probiotic compound preparation of southern Chinese medicinal herbs of Example 1 can increase the length of the jejunal villi, improve the absorption efficiency of nutrients, and promote intestinal health.

[0131] The organ index results for each group of weaned piglets are shown in the following figure. Figure 6 As shown, the results indicated that there were no significant differences in the heart, kidney, and spleen indices of weaned piglets among the control group, the traditional Chinese medicine group, the probiotic group, and the traditional Chinese medicine-probiotic compound preparation group. Although the liver index of weaned piglets in the traditional Chinese medicine-probiotic compound preparation group was slightly higher, this was due to the greater weight gain of the weaned piglets in this group.

[0132] The results of the immune level test for each group of weaned piglets are shown in the figure below. Figure 7 As shown, a represents the detection results of IgA expression level, b represents the detection results of IgG expression level, c represents the detection results of IgM expression level, d represents the detection results of IL-2 expression level, e represents the detection results of IL-6 expression level, f represents the detection results of IL-17 expression level, and g represents the detection results of TNF-α expression level.

[0133] The results showed that, compared with the control group, the expression level of IgM in the probiotic group was significantly increased, and the expression levels of IL-2, IL-17 and TNF-α were significantly decreased; the expression levels of IgA, IgG and IgM in the traditional Chinese medicine-probiotic compound preparation group were significantly increased, and the expression levels of IL-2, IL-6, IL-17 and TNF-α were significantly decreased.

[0134] The results show that feeding nursery piglets with feed containing the probiotic composition of Example 1 or the probiotic compound preparation of southern Chinese medicinal herbs of Example 1 can improve the immunity of nursery piglets and enhance their resistance to external factors.

[0135] The results of detecting the expression of the JAK2 / STAT3 pathway in the jejunum of each group of weaned piglets are shown in the figure below. Figure 8As shown, a) is the Western blot map of STAS3, p-STAT3, JAK2, p-JAK2 and IL-6 proteins; b) is the relative expression level of p-STAT3; c) is the relative expression level of p-JAK2; d) is the relative expression level of IL-6; e) is the relative mRNA expression level of STAT3; f) is the relative mRNA expression level of JAK2; and g) is the relative mRNA expression level of IL-6.

[0136] The results showed that, compared with the control group, the protein levels of P-STAT3 / STAT3, P-JAK2 / JAK2, and IL-6 in weaned piglets were significantly downregulated in the traditional Chinese medicine group, the probiotic group, and the traditional Chinese medicine-probiotic compound preparation group. Moreover, the protein levels of the above proteins were significantly upregulated in the traditional Chinese medicine-probiotic compound preparation group compared with the traditional Chinese medicine and probiotic group.

[0137] The expression results of tight junction protein pathways in the jejunum of each group of weaned piglets are shown in the figure below. Figure 9 As shown, a) is the Western blot map of ZO-1 protein, Occludin protein, and Claudin 1 protein; b) is the relative expression level of ZO-1 protein; c) is the relative expression level of Occludin protein; d) is the relative expression level of Claudin 1 protein; e) is the relative expression level of ZO-1 mRNA; f) is the relative expression level of Occludin mRNA; and g) is the relative expression level of Claudin 1 mRNA.

[0138] The results showed that, compared with the control group, the protein levels of ZO-1, Occludin, and Claudin 1 in the southern Chinese medicine group, the probiotic group, and the southern Chinese medicine-probiotic compound preparation group were significantly increased. Moreover, the protein levels of these proteins were significantly upregulated in the southern Chinese medicine-probiotic compound preparation group compared with the southern Chinese medicine and probiotic group.

[0139] The results show that traditional Chinese medicine, probiotics, and the combination of traditional Chinese medicine and probiotics can all improve the intestinal immune function of weaned piglets through JAK2 / STAT3 signaling and tight junction proteins. Compared with the other two groups, the combination of traditional Chinese medicine and probiotics has a more significant improvement effect.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A probiotic composition, characterized in that, It contains 1 to 2 parts by weight of Lactobacillus rhamnosus, 1 to 2 parts of yeast and 1 to 2 parts of Bacillus subtilis; The concentration of *Lactobacillus rhamnosus* was 1*10. 10 ~5*10 10 CFU / mL, yeast concentration 1*10 11 ~5*10 11 CFU / mL, the concentration of Bacillus subtilis is 5*10 8 ~1*10 9 CFU / mL.

2. The probiotic composition according to claim 1, characterized in that, It contains 1 part Lactobacillus rhamnosus, 2 parts yeast and 2 parts Bacillus subtilis by weight.

3. The use of the probiotic composition according to any one of claims 1 to 2 in the preparation of a medicament for the prevention and treatment of swine diarrhea.

4. The application according to claim 3, characterized in that, The pigs in question are weaned piglets.

5. The use of the probiotic composition according to any one of claims 1 to 2 in the preparation of a medicament for improving the immunity of pigs.

6. A compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea, characterized in that, The composition contains a southern medicinal herb composition and the probiotic composition according to any one of claims 1 to 2; The mass-volume ratio of the herbal composition and the probiotic composition according to any one of claims 1 to 2 is 100:3 to 5; the herbal composition contains, by mass parts, 60 to 80 parts of *Eriocaulon buergerianum*, 4 to 11 parts of *Tinospora sinensis*, 4 to 11 parts of *Polygonum chinense*, 4 to 11 parts of *Impatiens balsamina*, 2 to 8 parts of *Gardenia jasminoides*, 1 to 6 parts of *Sophora flavescens*, 1 to 6 parts of *Citrus reticulata*, and 1 to 3 parts of *Poria cocos*.

7. The probiotic compound preparation of southern Chinese medicinal herbs according to claim 6, characterized in that, The mass-to-volume ratio of the herbal composition and the probiotic composition according to any one of claims 1 to 2 is 100g:3mL.

8. The use of the probiotic composition according to any one of claims 1 to 2 and / or the compound preparation of southern Chinese medicinal herbs according to any one of claims 6 to 7 in reducing the feed conversion ratio of pigs.

9. A method for preparing a compound preparation of probiotics from southern Chinese medicinal herbs for the prevention and treatment of swine diarrhea, characterized in that, Based on parts by weight, the following steps are included: S1. Mix the southern medicinal materials with water and boil thoroughly. Separate the solid and liquid, collect the liquid and concentrate it to a concentration of 0.5 g / mL to 2 g / mL to obtain the medicinal liquid. The composition of the southern medicinal herbs, by mass parts, contains 60-80 parts of monkey ear ring, 4-11 parts of twisted belly vine, 4-11 parts of fire charcoal mother, 4-11 parts of savior, 2-8 parts of gardenia, 1-6 parts of sophora flavescens, 1-6 parts of tangerine peel and 1-3 parts of poria cocos. S2. Add the probiotic composition according to any one of claims 1 to 2 to the medicinal liquid obtained in step S1, and after full fermentation, a southern Chinese medicinal herb probiotic compound preparation is obtained; the mass-volume ratio of the probiotic composition according to any one of claims 1 to 2 to the southern Chinese medicinal herb composition in step S1 is 3 to 5 mL: 100 g.

10. The preparation method according to claim 9, characterized in that, The full fermentation mentioned in step S2 specifically refers to fermentation at 25–37°C for 12–48 hours.

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