Probiotic and prebiotic composition for improving constipation of pets and application of probiotic and prebiotic composition

Abstract

The invention relates to a probiotic and prebiotic composition for improving constipation of pets and application of the probiotic and prebiotic composition. Active ingredients of the probiotic and prebiotic composition comprise probiotics and prebiotics, wherein the probiotics comprise bacillus pumilus, and the prebiotics comprise lactulose; the initial concentration of the probiotics is 0.5 * 10 < 10 >-1.5 * 10 < 10 > CFU / mL, the initial mass percent content of the prebiotics is 30%-50%, and the probiotic and prebiotics composition can remarkably promote small intestine peristalsis, increase the water content of excrement, improve the characteristics of the excrement, relieve constipation, improve the health condition of intestinal tracts and increase the defecation frequency, has good bowel relaxing effects, and can be used for preparing the probiotic and prebiotics composition. The invention has important application significance in preparation of drugs for improving animal constipation, drugs for improving animal intestinal peristalsis and drugs for promoting animal excretion.

Description

Technical Field

[0001] The present invention relates to the technical field of animal medicine, particularly to the technical field of drugs for improving constipation in small animals, and specifically to a probiotic prebiotic composition for improving constipation in pets and its application. Background Art

[0002] Constipation refers to the symptoms of reduced frequency of defecation, reduced fecal volume, hard feces or difficult defecation in animals. Constipation is a relatively common problem in pets, especially in cats and dogs. The causes of constipation are complex and diverse, and diet, stress, gastrointestinal diseases, drug use, etc. may all cause constipation. The longer the constipation lasts, the more difficult the treatment becomes. In severe cases, it can cause self-poisoning or secondary other diseases, worsening the condition.

[0003] Currently, the common treatment methods for animal constipation are drug treatment, and surgical treatment may be carried out in severe cases. Drug treatment, such as chemical irritant laxatives (such as bisacodyl), is likely to cause side effects such as intestinal dependence, dehydration, and electrolyte disorders, and the long-term use risk is significant; single probiotic preparations are affected by the digestive tract environment such as gastric acid and bile salts, with a low viable bacteria colonization rate and limited effects due to the lack of synergistic growth-promoting substrates; traditional prebiotics (such as oligosaccharides) have a single regulatory effect on the intestinal flora of some pets and are difficult to achieve the dual goals of rapid defecation and flora balance.

[0004] No effective solutions have been proposed for the problems in the current drug treatment for relieving animal constipation, such as easy induction of intestinal dependence, dehydration, electrolyte disorders, and low viable bacteria colonization rate and limited effects of single probiotic preparations. Summary of the Invention

[0005] The purpose of the present invention is to provide, in view of at least one deficiency in the prior art, a probiotic prebiotic composition for improving constipation in pets and its application, so as to solve the problems in the related art, such as easy induction of intestinal dependence, dehydration, electrolyte disorders, and low viable bacteria colonization rate and limited effects of single probiotic preparations in the drug treatment for relieving animal constipation.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention is to provide a probiotic prebiotic composition for improving constipation in pets. The active ingredients of the probiotic prebiotic composition include probiotics and prebiotics. Among them, the probiotics include Bacillus pumilus; the prebiotics include lactulose.

[0008] Further, the initial concentration of the probiotics is 0.5×10 10 ~1.5×10 10 CFU / mL, and the initial mass percentage content of the prebiotics is 30% - 50%.

[0009] Furthermore, the concentration of the probiotic is 1×10 10 CFU / mL, and the mass percentage content of the prebiotic is 50%.

[0010] Furthermore, the volume ratio of the prebiotic to the probiotic is 150 - 200:50 - 100.

[0011] Furthermore, the volume ratio of the prebiotic to the probiotic is 150:100.

[0012] Furthermore, the probiotic - prebiotic composition is obtained by mixing the components of the probiotic and the prebiotic according to the corresponding volume ratio. Among them, the initial concentration of the probiotic refers to the bacterial liquid concentration of the probiotic component before mixing; the initial mass percentage content of the prebiotic refers to the mass percentage content of the prebiotic component before mixing.

[0013] Furthermore, the Bacillus pumilus is named Bacillus pumilus NJAUYBN1 - 7, and its deposit number is CCTCC NO: M20242671, and the deposit date is November 29, 2024.

[0014] Furthermore, the RNA sequence of the Bacillus pumilus is as shown in SEQ ID NO: 1.

[0015] Furthermore, the survival rate of the Bacillus pumilus in the environment with pH 2 - pH 7 is greater than 50%.

[0016] Furthermore, the survival rate of the Bacillus pumilus in the pH 2 environment is greater than 50%.

[0017] Furthermore, when the bile salt concentration is 0.05% - 0.3%, the survival rate of the Bacillus pumilus is greater than 85%.

[0018] Furthermore, when the bile salt concentration is 0.3%, the survival rate of the Bacillus pumilus is greater than 85%.

[0019] Furthermore, taking 6 - week - old mice as the subjects, the administration method of the probiotic - prebiotic composition is oral administration at 0.2 - 0.3 mL / day.

[0020] Furthermore, taking 6 - week - old mice as the subjects, the administration method of the probiotic - prebiotic composition is oral administration at 0.25 mL / day.

[0021] In a specific pet experiment, the administration method of the probiotic - prebiotic composition is oral administration at 1 - 2 mL / kg, 1 - 2 times / day.

[0022] The second aspect of the present invention is to provide a therapeutic agent, which comprises the probiotic prebiotic composition as described in the first aspect.

[0023] The third aspect of the present invention is to provide an application of the probiotic prebiotic composition as described in the first aspect or the therapeutic agent as described in the second aspect, which comprises at least one of the following applications: the application in the preparation of a drug for improving animal constipation, the application in the preparation of a drug for improving animal intestinal peristalsis, and the application in the preparation of a drug for promoting animal defecation.

[0024] Adopting the above technical solutions, compared with the prior art, the present invention has the following technical effects:

[0025] The prebiotic (lactulose) and the probiotic (Bacillus) are compounded into a probiotic prebiotic composition of a synbiotic system. Lactulose, as a non-absorbable disaccharide, forms a hypertonic environment in the intestine, promotes the osmosis of water into the intestinal lumen, softens the feces, increases the fecal volume, and directly stimulates intestinal peristalsis; and accelerates the discharge of colonic contents by increasing the amount of intestinal lumen fluid. Bacillus pumilus can not only decompose undigested proteins, reduce fecal hardening, but also degrade plant fibers, reduce fecal viscosity, assist in lipid metabolism, improve fecal lubricity, occupy the intestinal mucosal attachment sites, consume oxygen (strictly aerobic characteristics), and inhibit the reproduction of pathogenic bacteria such as Clostridium.

[0026] Bacillus pumilus in the probiotic prebiotic composition of this application is isolated from feces after defecation by lactulose, compounded with lactulose in vitro and then administered by gavage. Lactulose selectively promotes the proliferation of Bacillus pumilus and inhibits pathogenic bacteria. The metabolites of Bacillus pumilus further decompose lactulose to generate short-chain fatty acids (SCFAs), bidirectionally activate intestinal peristalsis and mucosal repair, achieve the integrated effect of "short-term defecation + long-term conditioning", significantly shorten the defecation time of mice by maintaining fecal moisture and enhancing intestinal peristalsis, show a significant laxative effect, significantly improve the state of constipation, have a better defecation effect than adding only lactulose alone, can improve the functional constipation and intestinal microecological imbalance problems of small animals such as cats and dogs, and the pharmaceutical combination is safe and long-acting. It has important application value in the application in the preparation of a drug for improving animal constipation, the application in the preparation of a drug for improving animal intestinal peristalsis, and the application in the preparation of a drug for promoting animal defecation. The Bacillus pumilus involved in the present invention is named Bacillus pumilus NJAUYBN1-7, and its taxonomic name is Bacillus pumilus Bacillus pumilus and is hereinafter simply referred to as Bacillus pumilus. It is deposited in the China Center for Type Culture Collection, and its deposit number is CCTCC NO: M20242671. The deposit date is November 29, 2024, and the deposit address is Wuhan University, Wuhan, China. Description of the Drawings

[0027] The accompanying drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0028] Figure 1 It is a result graph of the acid resistance experiment of Bacillus pumilus in an embodiment of the present invention;

[0029] Figure 2 It is a result graph of the bile salt resistance experiment of Bacillus pumilus in an embodiment of the present invention;

[0030] Figure 3 It is a result graph of the influence of the probiotic prebiotic composition on the fecal morphology of normal mice in an embodiment of the present invention; wherein; A: normal group; B: lactulose group; C: TB1; D: TB2; E: TB3; F: TB4; G: TB5;

[0031] Figure 4 It is a result graph of the influence of the probiotic prebiotic composition on the fecal water content of normal mice in an embodiment of the present invention; wherein, different letters indicate significant differences (P<0.05);

[0032] Figure 5 It is a result graph of the influence of the probiotic prebiotic composition on the small intestine propulsion rate of normal mice in an embodiment of the present invention; wherein, different letters indicate significant differences (P<0.05);

[0033] Figure 6 It is a result graph of the influence of the probiotic prebiotic composition on the small intestine of the small intestine propulsion rate of normal mice in an embodiment of the present invention; wherein, A: NC; B: PC; C: TB1; D: TB2; E: TB3; F: TB4; G: TB5;

[0034] Figure 7 It is a result graph of the influence of the probiotic prebiotic composition on the fecal water content of constipated mice in an embodiment of the present invention; wherein, different letters indicate significant differences (P<0.05);

[0035] Figure 8 It is a result graph of the influence of the probiotic prebiotic composition on the fecal morphology of constipated mice in an embodiment of the present invention; wherein, A: NC; B: MC; C: PC; D: TB-L; E: TB-M; F: TB-H;

[0036] Figure 9 It is a result graph of the influence of the probiotic prebiotic composition on the first black feces time of constipated mice in an embodiment of the present invention; wherein, different letters indicate significant differences (P<0.05);

[0037] Figure 10It is a result graph of the effect of the probiotic prebiotic composition on the intestinal propulsion rate of constipated mice in an embodiment of the present invention; wherein, different letters indicate significant differences (P<0.05);

[0038] Figure 11 It is an intestinal result graph of the effect of the probiotic prebiotic composition on the intestinal propulsion rate of constipated mice in an embodiment of the present invention; wherein, A: NC; B: MC; C: PC; D: TB-L; E: TB-M; F: TB-H;

[0039] Figure 12 It is a picture of beagle dogs administered with drugs and their defecation conditions after intervention in an embodiment of the present invention;

[0040] Figure 13 It is the defecation condition of constipated cats with megacolon after constipation intervention in an embodiment of the present invention;

[0041] Figure 14 It is the defecation condition of constipated dogs after intervention in an embodiment of the present invention after surgery. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually measured according to national standards. The experimental materials not indicated the sources in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is all conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standard, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise stated, all parts are by weight and all percentages are by mass percentage. Unless otherwise defined or explained, all the professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention.

[0043] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not a limitation of the present invention.

[0044] For those not specially marked with the manufacturer in the embodiments, they can be obtained conventionally through commercial purchase.

[0045] The present invention is exemplarily described below.

[0046] Example 1 - Isolation and Identification of Bacillus pumilus

[0047] This example relates to a preferred isolation and identification of Bacillus pumilus, and its isolation and screening include the following steps:

[0048] The experimental dogs were fasted for 24 h, and lactulose was used to intubate the experimental dogs. Fresh dog feces were aseptically collected, 1 g of which was suspended in physiological saline, heated at 80 °C for 10 minutes (to kill non-spore bacteria), allowed to stand and filtered to obtain the supernatant, which was gradient-diluted and then spread on LB solid medium, and cultured at 37 °C for 24 hours. The bacterial colonies were examined under a microscope and photographed for recording.

[0049] The purified colonies were picked with a sterile toothpick and inoculated into a liquid test tube, and cultured at 37 °C for 48 h. 1.6 mL of the liquid after 48 h of culture was aliquoted into glycerol tubes, labeled, mixed evenly, and stored at -80 °C.

[0050] A bacterial genomic DNA extraction kit (purchased from Beyotime Biotechnology Co., Ltd., Nanjing) was used, and genomic DNA was extracted from the strains after rescreening according to the instructions. Using the extracted DNA as a template, the 16S rRNA gene was amplified by PCR with universal primers, and the amplified 16S rRNA gene fragment was sequenced. The measured sequence was BLAST-aligned in the GenBank database of NCBI to determine that it was Bacillus.

[0051] The Basic Local Alignment Search Tool (BLAST) was used for sequence alignment. The complete RNA sequence information of Bacillus pumilus is shown in SEQ ID NO: 1, as follows:

[0052]

[0053] The above-mentioned Bacillus pumilus NJAUYBN1-7 has been deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: M20242671 and the deposit date of November 29, 2024.

[0054] Example 2 - Acid and Bile Salt Tolerance Experiments of Bacillus pumilus

[0055] Based on the Bacillus pumilus CCTCC M 20242672 isolated in Example 1, acid and bile salt tolerance experiments were conducted in this example.

[0056] 1. Acid Tolerance

[0057] OD600 is the absorbance of the bacterial culture solution at a wavelength of 600 nm and can be used to measure the growth density of bacteria.

[0058] In this example, 4 mL of LB liquid medium with different pH values (2, 3, 4, 5, 6, 7) was prepared using concentrated hydrochloric acid. 200 μL of the Bacillus pumilus bacterial solution was added to the media with different pH values and cultured in an incubator at 37 °C for 2 h to simulate the gastric acid environment. Then, the OD values of the bacterial solutions with different pH values were measured using a spectrophotometer at a wavelength of 600 nm to analyze their growth conditions. pH = 7 is the optimal pH value for Bacillus pumilus. The results are as Figure 1 shown.

[0059] As Figure 1 can be seen, when pH < 3, the growth density of Bacillus pumilus begins to decrease significantly, but when pH = 2, the survival rate of Bacillus pumilus is still about 50%, indicating that the Bacillus pumilus obtained in this application has a certain ability to break through the gastric acid barrier.

[0060] 2. Bile Salt Tolerance

[0061] In this example, 4 mL of bile salt-containing LB medium with different concentrations (0%, 0.05%, 0.1%, 0.2%, 0.3%) was prepared using porcine bile salt. 200 μL of the Bacillus pumilus bacterial solution was added to the media with different bile salt concentrations and cultured in an incubator at 37 °C for 2 h to simulate the intestinal environment. Then, the OD values of the bacterial solutions with different bile salt concentrations were measured using a spectrophotometer at a wavelength of 600 nm to analyze their growth conditions. The results are as Figure 2 shown.

[0062] As Figure 2 can be seen, compared with the control group (0% bile salt), the growth density of Bacillus pumilus decreased with the increase of bile salt concentration, but when the bile salt concentration was 0.3%, the survival rate could still reach 85%, indicating that the Bacillus pumilus obtained in this application has a certain bile salt tolerance.

[0063] Example 3 - Preparation of Probiotic - Prebiotic Composition

[0064] This example relates to the preparation of a preferred probiotic - prebiotic composition.

[0065] The active ingredients of the probiotic - prebiotic composition include probiotics and prebiotics. Among them, the probiotics include Bacillus pumilus, and the prebiotics include lactulose; the initial concentration of the probiotics is 0.5×10 10 ~1.5×10 10 CFU / mL, and the initial mass percentage content of the prebiotics is 30% - 50%.

[0066] Further, the probiotics are the Bacillus pumilus CCTCC M 20242672 isolated in Example 1.

[0067] Further, the volume ratio of lactulose to the Bacillus pumilus bacterial liquid in the probiotic - prebiotic composition is 150 - 200:50 - 100.

[0068] Bacterial liquid preparation: The Bacillus pumilus NJAUYBN1 - 7 (preservation number: CCTCC M20242672) in Example 1 was placed in an LB liquid medium and cultured at 37°C. By the plate counting method, the bacterial liquid was serially diluted, spread on plates, and after cultivation, plates with 30 - 300 colonies were selected for plate counting to obtain a bacterial liquid with a concentration of 1×10 10 CFU / mL.

[0069] Preparation of probiotic - prebiotic composition: The probiotic - prebiotic composition is obtained by mixing the probiotic component and the prebiotic component in a corresponding volume ratio. The corresponding probiotic - prebiotic compositions are prepared according to the following component ratios. Specific examples are as follows:

[0070] Probiotic - prebiotic composition 1 (200 μL of 30% lactulose + 50 μL of bacterial liquid): Mix 200 μL of a lactulose solution with a mass percentage content of 30% and 50 μL of bacterial liquid to obtain the probiotic - prebiotic composition.

[0071] Probiotic - prebiotic composition 2 (150 μL of 30% lactulose + 100 μL of bacterial liquid): Mix 150 μL of a lactulose solution with a mass percentage content of 30% and 100 μL of bacterial liquid to obtain the probiotic - prebiotic composition.

[0072] Probiotic - prebiotic composition 3 (200 μL of 50% lactulose + 50 μL of bacterial liquid): Mix 200 μL of a lactulose solution with a mass percentage content of 50% and 50 μL of bacterial liquid to obtain the probiotic - prebiotic composition.

[0073] Probiotic-prebiotic composition 4 (150 μL 50% lactulose+100 μL bacterial solution): 150 μL of 50% lactulose solution by mass percentage was mixed with 100 μL bacterial solution to obtain a probiotic-prebiotic composition.

[0074] Example 4 - Experiment on improving constipation in mice

[0075] This example uses the probiotic-prebiotic composition prepared in Example 3 to verify the efficacy of the probiotic-prebiotic composition in improving constipation in mice through a laxative test, a fecal water content test, and a small intestinal motility test.

[0076] 1. Experimental Animals and Materials

[0077] 54 6-week-old SPF-grade healthy male Kunming mice were purchased from Qinglongshan Experimental Animal Center. During the feeding period, mice in each group were allowed to drink water freely. The feeding environment was: half-cycle lighting during the day and night, constant humidity, and temperature controlled at 22-25°C. Commercial lactulose (Shaanxi Hanshi Liangfang Biotechnology Co., Ltd.); loperamide hydrochloride capsules (Xi'an Janssen Pharmaceutical Co., Ltd.); gum arabic (Zhejiang Yinuo Biotechnology Co., Ltd.); activated carbon powder (Tianjin Dingshengxin Chemical Co., Ltd.); lactulose (Anhui Yixinyuan Biotechnology Co., Ltd.).

[0078] 2. Drug Preparation

[0079] Preparation of loperamide hydrochloride suspension (1 mg / mL): loperamide hydrochloride (2 mg / capsule), take 50 capsules, take out the drug powder in the capsules and mix well, add sterile water to 100 mL to obtain loperamide hydrochloride suspension, and prepare it before use.

[0080] Activated carbon suspension: Weigh 50g of gum arabic, add 400mL of water, and boil until the solution is transparent. Weigh 25g of activated carbon, add it to the above solution and boil it three times. After the solution cools, add water to make up to 500mL to obtain activated carbon suspension (50g / L), and store it in a refrigerator at 4℃.

[0081] 3. Experimental Grouping and Treatment

[0082] Eighteen ICR mice (purchased from Qinglongshan Experimental Animal Center) were raised in an environment with a temperature of (22±2)°C and a humidity of 60%±10%. The animal laboratory had 12 hours of light and dark alternation every day. Before the experiment, the mice were adaptively raised for 7 days to make them adapt to the surrounding growth environment, and they were allowed to eat and drink freely during this period. After 7 days, the mice were divided into a blank group (NC), a positive control group (commercial lactulose, PC), a TB1 group, a TB2 group, a TB3 group, a TB4 group, and a TB5 group, a total of 6 groups, with 3 mice in each group. To screen for appropriate concentrations of the preparation for subsequent experiments, the mice in the blank group were gavaged with 0.25 mL of normal saline, the mice in the positive control group were gavaged with 0.25 mL of lactulose, and the TB1, TB2, TB3, TB4, and TB5 groups were gavaged with different concentrations of the probiotic prebiotic composition according to Table 1, and the concentration of Bacillus pumilus bacterial solution was 1.0×10 10 CFU / mL.

[0083] Table 1 Gavage doses for mice in each group

[0084]

[0085] 4. Laxative test

[0086] 4.1 Sample collection

[0087] After gavage using the above treatment method was completed, the time for the first soft stool to be excreted, the number of fecal pellets excreted within 4 hours, the duration of soft stools in each group of mice were recorded, and the fecal characteristics were observed and scored according to the Bristol Stool Form Scale (Table 2).

[0088] Table 2 Bristol Stool Form Scale

[0089]

[0090] 4.2. Result analysis

[0091] The fecal diagrams of each group of mice are shown in Figure 3 , where A: normal group; B: lactulose group; C: TB1; D: TB2; E: TB3; F: TB4; G: TB5. The scores are shown in Table 3. It can be seen that compared with the blank group, the feces of the mice in the dose groups were significantly more moist, and as the concentration of the probiotic prebiotic composition increased, the fecal moisture also increased, and the number of fecal pellets increased significantly. It was proved that the lactulose - Bacillus composition has the effect of maintaining fecal moisture and improving constipation.

[0092] Table 3 Effects of lactulose - Bacillus compound laxative solution on the number of feces excreted by mice and scores

[0093]

[0094] As can be seen from Table 4, compared with the positive control group, with the change of the concentration of the preparation group, the onset time of lactulose-bacillus composition for defecation in mice was significantly shortened, while the duration was significantly increased.

[0095] Table 4 Effects of lactulose-bacillus compound laxative solution on the fecal excretion time of normal mice

[0096]

[0097] 5. Detection of fecal water content

[0098] Fecal moisture content is an important indicator to judge the effect of moistening the intestine and relieving constipation. When feces stay in the intestine for too long, the water in the feces is continuously absorbed by the intestine. When the moisture content is too low, constipation is very likely to occur. When feces can pass through the intestine quickly, the water content retained in the feces will increase. Therefore, fecal moisture content also reflects the speed of intestinal peristalsis to a certain extent.

[0099] 5.1 Sample collection

[0100] After gavage of each group of mice using the above treatment method, collect the feces within 4 h after the mice defecate softly, weigh and record the wet weight W1 of the feces, and then put the feces of each group of mice into a blower for high-temperature drying (100 °C) and dehydration for 6 h. Weigh again and record the dry weight W2; calculate the fecal moisture content R of the mice = (W1 - W2) / W1 × 100%.

[0101] 5.2 Result analysis

[0102] The fecal moisture content of each group of mice is shown in Figure 4 . From Figure 4 it can be seen that different letters indicate significant differences (P < 0.05). The fecal moisture content of the mice in the dose group was significantly higher than that of the normal group and the positive control group, proving that the lactulose-bacillus composition has the effect of maintaining fecal moisture and improving constipation.

[0103] 6. Small intestine motility test

[0104] After gavage of ink to mice, the propulsion speed of the ink in the intestine can also reflect the effect of intestinal peristalsis in mice. The more the ink is propelled, that is, the higher the propulsion rate, the stronger the intestinal peristalsis ability of the mice and the weaker the degree of constipation.

[0105] 6.1 Sample collection

[0106] After the laxative test was completed in each group of mice using the above treatment method, the mice were fasted but allowed to drink water for 16 h. Then, each mouse was orally administered 0.25 mL of activated carbon suspension. After 20 min, the mice were sacrificed by cervical dislocation. The abdominal cavity of the mice was quickly opened, and the entire intestine from the pylorus to the anus was taken out. After being straightened without tension, the length of the intestine from the pylorus to the junction of the ileum and cecum was measured as L1; then, the length of the intestine from the pylorus to the advancing length of the activated carbon in the intestine was measured as L2, and the intestinal propulsion rate D of the mice was calculated as D = L2 / L1 × 100%.

[0107] 6.2 Result Analysis

[0108] The results of the small intestine propulsion rate of each group of mice are shown in Figure 5 and Figure 6 . Among them, Figure 5 is the result graph of the effect of the lactulose-bacillus composition on the small intestine propulsion rate of normal mice; Figure 6 is the intestinal graph of the effect of the lactulose-bacillus composition on the small intestine propulsion rate of normal mice, where A: NC; B: PC; C: TB1; D: TB2; E: TB3; F: TB4; G: TB5. From Figure 5 and Figure 6 , it can be seen that compared with the normal group and the positive control group, the intestinal peristalsis of the mice in the group intragastrically administered with the probiotic prebiotic composition was significantly enhanced, indicating that the lactulose-bacillus composition has the effect of moistening the intestine and relieving constipation. Among them, different letters indicate significant differences (P < 0.05).

[0109] Example 5 - Construction of a mouse functional constipation model and optimization of the concentration of the probiotic prebiotic composition

[0110] This example relates to a specific example of constructing a mouse functional constipation model.

[0111] Animal models play an important role in the study of the mechanism of functional constipation. Different research purposes have different choices of modeling animals and modeling methods. Drug modeling is the main method for functional constipation modeling, and loperamide hydrochloride is one of the commonly used drugs. Loperamide hydrochloride is a peripheral opioid receptor agonist, and its mechanism is to inhibit intestinal water secretion and colonic peristalsis, delay fecal evacuation time and intestinal lumen transport, and present constipation symptoms by reducing the number, weight and water content of animal fecal particles.

[0112] In this experiment, a mouse functional constipation model was established using loperamide hydrochloride. After modeling with loperamide hydrochloride, the number of fecal pellets excreted by the mice within 6 h and the water content of the feces were significantly reduced compared with the blank control group, indicating that the modeling was successful.

[0113] 1. Construction of a mouse functional constipation model

[0114] After one week of adaptive cultivation, 36 mice were randomly divided into 6 groups of 6 mice each, namely the normal group (NC), the model group (MC), the low-dose TB group (TB-L), the medium-dose TB group (TB-M), the high-dose TB group (TB-H), and the positive control group (commercial lactulose treatment group, PC). During the experiment, the NC group and the MC group were intragastrically administered 0.25 mL of normal saline daily; from day 1 to day 4, in addition to the NC mice, all the other mice were intragastrically administered 0.25 mL of loperamide hydrochloride suspension (1 mg / mL) twice a day (at 9:00 am and 4:00 pm) to induce a functional constipation model in mice. After the intragastric administration on day 4, all the mice in each group were fasted but allowed to drink water for 16 h. From day 5 to day 11, the TB-L, TB-M, and TB-H groups were intragastrically administered 0.25 mL of the lactulose-bacillus composition daily according to different concentrations (Table 5), and the lactulose group was intragastrically administered 0.25 mL of lactulose daily. After the intragastric administration on day 11, all the mice in each group were fasted but allowed to drink water for 16 h, and then each mouse was intragastrically administered 0.25 mL of activated carbon suspension.

[0115] Table 5 Intragastric administration doses for mice in each group

[0116]

[0117] 2. Laxative test

[0118] Sample collection

[0119] After the intragastric administration of ink, record the time when the first black stool was excreted in each group of mice, the number of feces excreted within 6 h, and observe and score the fecal characteristics according to the Bristol Stool Form Scale (Table 2).

[0120] 3. Detection of fecal water content

[0121] After the intragastric administration of ink, collect the feces within 6 h after the first black stool of the mice, weigh and record the wet weight W1 of the feces, and then put the feces of each group of mice into a blower for high-temperature drying (100 °C) and dehydration for 6 h. Weigh again and record the dry weight W2; calculate the fecal moisture content R of the mice = (W1 - W2) / W1 × 100%.

[0122] 4. Small intestine motility experiment

[0123] After the modeling and treatment experiment, the mice were fasted but allowed to drink water for 16 h. Then each mouse was intragastrically administered 0.25 mL of activated carbon suspension, and sacrificed by cervical dislocation 30 min later. Quickly open the abdominal cavity of the mice, take out the entire intestine from the pylorus to the anus, straighten it without tension, and measure the length of the intestine from the pylorus to the junction of the ileum and cecum as L1; then measure the length of the intestine from the pylorus to the advancing length of the activated carbon in the intestine as L2, and calculate the intestinal propulsion rate D of the mice = L2 / L1 × 100%.

[0124] 5. Results and analysis

[0125] The moisture content of feces is an important indicator for evaluating the success of constipation model establishment. The weakened intestinal peristalsis function of constipated animals and the dryness of feces lead to a decrease in the moisture content of feces.

[0126] As Figure 7 shown, the fecal moisture content of mice in the model group was significantly lower than that of mice in the normal group (different letters indicate significant differences (P<0.05)), indicating successful modeling. After 7 days of treatment, the fecal moisture content of mice intragastrically administered with the lactulose-bacillus composition increased compared with that of MC mice, with a significant difference, and the fecal moisture content of TB-H mice was close to that of NC mice, indicating that the lactulose-bacillus composition can relieve constipation symptoms by increasing the fecal moisture content of mice.

[0127] During the experiment, the body weight, diet, activity, and defecation of mice in each group were normal. Among them, the surface moisture of feces in MC group mice decreased, and the fecal particles were fine. After treatment with lactulose and the lactulose-bacillus composition, the constipation of mice was improved, the fecal moisture of mice increased, the fecal particles gradually increased from fine, and the fecal characteristics of the TB-M group were close to those of the normal group. The results are as Figure 8 shown in and Table 6; among them, A: NC; B: MC; C: PC; D: TB-L; E: TB-M; F: TB-H; different letters indicate significant differences (P<0.05).

[0128] As can be seen from the above, the lactulose-bacillus composition can improve the fecal characteristics of mice.

[0129] Table 6 Effects of lactulose-bacillus composition on the number of feces excreted by constipated mice and scoring

[0130]

[0131] As Figure 9 can be seen, compared with the NC group, the time for the first black feces to be excreted by mice in the NC group was significantly shorter than that in the MC group, and the number of fecal grains within 6 h in the MC group decreased significantly (P<0.05), indicating that this modeling method is reasonable and the mouse constipation model was successfully established.

[0132] In addition, the time for the first black feces to be excreted by mice in the PC group, TB-L group, and TB-M group was also significantly higher than that in the NC group (P<0.05), but the difference in the time for the first black feces to be excreted by mice in the TB-H group compared with the NC group was not significant (P>0.05). Compared with the MC group, the time for the first black feces to be excreted by mice in the TB-H group was significantly shortened (P<0.05), and the number of fecal grains at 6 h increased significantly (P<0.05), and the difference compared with the NC group was not significant (P>0.05).

[0133] The results of the intestinal ink propulsion rate of each group of mice are as follows Figures 10 - 11 shown, among which, Figure 10 Figure 10 is the result graph of the effect of the lactulose - Bacillus composition on the intestinal propulsion rate of constipated mice. Different letters indicate significant differences (P<0.05); Figure 11 Figure 11 is the intestinal graph of the effect of the lactulose - Bacillus composition on the intestinal propulsion rate of constipated mice; among which, A: NC; B: MC; C: PC; D: TB - L; E: TB - M; F: TB - H. It can be seen from the figure that the ink propulsion rate of the MC group is significantly lower than that of the NC group, so it shows that this modeling method is feasible and the modeling is successful.

[0134] In addition, compared with the MC group, the intestinal ink propulsion rates of the TB - M and TB - H groups of mice were significantly increased (P<0.05); there was no significant difference in the intestinal ink propulsion rates of the TB - M and TB - H groups of mice compared with the NC group (P>0.05). Therefore, the high - dose lactulose - Bacillus composition has a beneficial effect on the small intestine motility of mice and can effectively promote intestinal motility in mice.

[0135] After gavage of constipated mice with lactulose - Bacillus compositions at different doses, the constipation function of the mice was significantly improved. Among them, when the probiotic prebiotic composition contained 50% lactulose and the volume ratio of the Bacillus pumilus bacterial liquid with a concentration of 1×10 10 CFU / mL was 150:100, the effect was the most significant, which could significantly promote the small intestine propulsion of mice, increase the number and water content of black feces excreted in 6 hours, and show a good effect of moistening the intestine and relieving constipation.

[0136] Example 6 - Pet verification experiment

[0137] This example is based on the probiotic prebiotic composition prepared in Example 3, and involves a specific intervention of administering the probiotic prebiotic composition to pets with different constipation backgrounds to verify the efficacy of the probiotic prebiotic composition in improving constipation.

[0138] The probiotic prebiotic composition uses 50% lactulose and the Bacillus pumilus bacterial liquid with a concentration of 1×10 10 CFU / mL, and the volume ratio of lactulose to the Bacillus pumilus bacterial liquid is 150:100.

[0139] 1. Intervention on constipated beagle dogs

[0140] Case background: A 16 - kg beagle dog suffered from constipation due to improper diet, manifested as no defecation for 2 days, abdominal distension and pain (the abdomen was tense on palpation), restlessness, and decreased appetite.

[0141] Intervention plan: Orally administer the above - mentioned probiotic prebiotic composition twice (dose 2 mL / kg), with an interval of 3 hours.

[0142] Intervention effect: Six hours after the last administration, the dog was observed to have active soft stools (see Figure 12 ). Later monitoring showed that the defecation frequency recovered from once every three days to once a day, and the abdominal palpation tension decreased significantly; the water content of the feces returned to normal on the third day.

[0143] 2. Intervention on constipation in cats with megacolon

[0144] Case background: A 5-kg adult cat with idiopathic megacolon, who had long relied on manual defecation and had not defecated for nearly three days.

[0145] Intervention plan: Single-dose oral administration of 10 mL of the above probiotic prebiotic composition (dose 2 mL / kg), combined with abdominal massage.

[0146] Intervention effect: Three hours after administration, the bowel sounds increased; feces were discharged 8 hours later ( Figure 13 ); There were 2 spontaneous bowel movements within 48 hours, and the fecal hardness approached normal.

[0147] 3. Intervention on postoperative constipation in dogs

[0148] Case background: An 8-kg Teddy dog had poor defecation due to the side effects of surgical anesthesia and had not defecated 48 hours after surgery, accompanied by mild vomiting.

[0149] Intervention plan: Oral administration of 10 mL of the above probiotic prebiotic composition (dose 1.25 mL / kg), used continuously for 2 days, once a day.

[0150] Intervention effect: Six hours after the first administration, yellowish-brown soft stools were discharged ( Figure 14 ), and no undigested food residues were found in the feces; Twenty-four hours later, the dog resumed spontaneous eating, and the frequency of bowel sounds recovered from once per minute to 5 times.

[0151] As can be seen from the above, the probiotic prebiotic composition involved in this example can effectively improve pet constipation.

[0152] In summary, the present invention relates to a probiotic prebiotic composition for improving pet constipation and its application. The laxative test, fecal water content detection, and small intestine motility test were respectively carried out on healthy mice and constipation model mice using lactulose combined with Bacillus pumilus CCTCC M 20242672, which verified that it can significantly promote small intestine peristalsis, increase fecal water content rate, improve fecal properties, relieve constipation, improve the health status of the intestine, increase the frequency of defecation, has a good effect of moistening the intestine and relieving constipation, and can significantly improve the constipation state of mice. Traditional laxatives use 50% lactulose, while for the probiotic prebiotic composition prepared by this application, the concentration of lactulose only needs to be 30% to achieve and even exceed the effect of traditional 50% lactulose. It utilizes that lactulose can be preferentially metabolized by Bacillus pumilus to produce short-chain fatty acids (SCFAs, such as acetic acid and butyric acid); and SCFAs can reduce the intestinal pH (to 5.5 - 6.0), thereby inhibiting the growth of pathogenic bacteria (such as Escherichia coli and Salmonella), and then optimizing the colonization environment of probiotics, and having a better intervention effect on constipated pets. Therefore, the probiotic prebiotic composition involved in this application has important application significance and research value in the applications of preparing drugs for improving animal constipation, preparing drugs for improving animal intestinal peristalsis, and preparing drugs for promoting animal defecation.

[0153] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all the equivalent replacements and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A probiotic prebiotic composition for improving pet constipation, characterized in that, The active ingredients of the probiotic prebiotic composition include probiotics and prebiotics; wherein, the probiotics include Bacillus pumilus, and the prebiotics include lactulose.

2. The probiotic prebiotic composition according to claim 1, wherein The initial concentration of the probiotic is 0.5×10 10 ~1.5×10 10 CFU / mL, and the initial mass percentage content of the prebiotic is 30% - 50%.

3. The probiotic prebiotic composition according to claim 1, characterized in that, The volume ratio of the prebiotics to the probiotics is 150-200:50-100.

4. The probiotic prebiotic composition according to claim 1, wherein The Bacillus pumilus is named Bacillus pumilus NJAUYBN1-7, and its preservation number is CCTCC NO: M20242671, and the preservation date is November 29, 2024.

5. The probiotic prebiotic composition according to claim 4, wherein The RNA sequence of the Bacillus pumilus is shown as SEQ ID NO:

1.

6. The probiotic prebiotic composition according to claim 4, wherein The survival rate of the Bacillus pumilus in the environment of pH 2-pH 7 is greater than 50%.

7. The probiotic prebiotic composition according to claim 4, characterized in that, When the bile salt concentration of the Bacillus pumilus is 0.05%-0.3%, the survival rate is greater than 85%.

8. The probiotic prebiotic composition according to claim 1, wherein Taking 6-week-old mice as the object, the administration method of the probiotic prebiotic composition is oral administration at 0.2-0.3 mL / day.

9. A therapeutic agent, characterized in that, The therapeutic agent comprises the probiotic prebiotic composition according to any one of claims 1-8.

10. Use of a probiotic prebiotic composition according to any one of claims 1-8 or a therapeutic agent according to claim 9, the use comprising at least one of the following uses: use in the preparation of a drug for improving animal constipation, use in the preparation of a drug for improving animal intestinal peristalsis, and use in the preparation of a drug for promoting animal defecation.

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