Probiotics for improving immunity of pets and application thereof

Abstract

The invention discloses a bacterial strain for improving immunity of pets, the bacterial strain is Lactobacillus acidophilus M 20250331-LCX, the bacterial strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.34179, the preservation date is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the telephone number is 010-64807355. When the strain is used for preparing pet food to feed pets, the immunity of the pets can be remarkably improved, for example, the morbidity of intestinal diseases can be reduced, and the excrement odor of the pets can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to probiotics capable of improving pet immunity and applications thereof. Background Art

[0002] With the rapid development of the pet health industry, the scientific management of pet immunity has become a research hotspot. The immune system of pets (dogs, cats, etc.) is susceptible to environmental stress, nutritional imbalance, aging and pathogenic infection, which can lead to a decline in immunity and induce digestive, skin or chronic diseases. Traditional solutions mostly rely on drugs or nutritional supplements, but long-term use may be accompanied by side effects or dependence. Therefore, probiotics, as a safe and natural immunomodulator, have received widespread attention in recent years. Probiotics are defined as "live microorganisms that can bring health benefits to the host when consumed in adequate amounts." In the pet field, common probiotic strains include: Lactobacillus (such as Lactobacillus acidophilus, Lactobacillus rhamnosus), Bifidobacterium (such as Bifidobacterium animalis), Enterococcus (such as Enterococcus faecalis), Bacillus (such as Bacillus subtilis), and yeast (such as Saccharomyces boulardii).

[0003] Probiotics enhance pet immunity through various pathways. For example, they strengthen the intestinal barrier by colonizing the intestinal mucosa, competitively inhibiting pathogens, promoting mucin secretion, and strengthening the physical barrier. Immune cell activation: Metabolites (such as short-chain fatty acids) stimulate the intestinal lymphoid tissue (GALT), increasing macrophage and natural killer cell (NK) cell activity and immunoglobulin (such as IgA) secretion. Anti-inflammatory signaling regulation: downregulating pro-inflammatory factors (such as TNF-α and IL-6) and upregulating anti-inflammatory factors (such as IL-10) to alleviate chronic inflammatory responses. Maintaining microbial balance: inhibiting the excessive proliferation of harmful bacteria, repairing intestinal microecological imbalances, and reducing the immune burden caused by endotoxins entering the bloodstream.

[0004] CN117844713A discloses a composite probiotic for improving immunity, comprising Bifidobacterium animalis subsp. lactis BLa80, Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA05. CN111494480A discloses a fermented Chinese medicine for pigs that improves immunity, comprising multiple probiotics.

[0005] The inventors of the present application screened and obtained a strain of Lactobacillus acidophilus with strong acid resistance. Through further mutagenesis and domestication, its extracellular polysaccharide secretion ability was improved, and its use in pet food can significantly enhance the immunity of pets. Summary of the Invention

[0006] On the one hand, the present invention provides a strain for improving the immunity of pets, the strain being Lactobacillus acidophilus M 20250331-LCX, which is deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration, with a deposit number of CGMCC No. 34179 and a deposit date of April 14, 2025. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

[0007] On the other hand, the present invention also provides a method for culturing the above-mentioned strain, which comprises the step of culturing the strain using a culture medium.

[0008] In one embodiment, the culture medium is MRS medium.

[0009] In one embodiment, the MRS medium formula (per liter) is: peptone 10.0g, beef extract powder 5.0g, yeast extract powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, ammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, agar 15.0g, Tween 80 1.0g, L-cysteine ​​0.5g, pH 6.4.

[0010] In one embodiment, the culture temperature is 30°C-40°C, preferably, 37°C.

[0011] In one embodiment, the culture time is 24h-72h, for example, 36h, 48h.

[0012] In one embodiment, the pH of the culture is 2.0-8.0, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0.

[0013] On the other hand, the present invention also provides a pet food, which contains the above-mentioned strain.

[0014] In one embodiment, the content of the above strain in the pet feed is 1×10 9 -1×10 10 CFU / kg.

[0015] In one embodiment, the pet feed further comprises other basic feeds for feeding pets.

[0016] On the other hand, the present invention also provides the use of the above strain in preparing pet food or pet feed.

[0017] On the other hand, the present invention also provides the use of the above strain in preparing food or feed for improving the immunity of pets.

[0018] On the other hand, the present invention also provides a method for improving the immunity of a pet, comprising the step of feeding the pet with a feed containing the above-mentioned strain.

[0019] In one embodiment, the dosage of the strain is 1×10 9 CFU-1×10 10 CFU; for example, 1 × 10 9 CFU-2×10 9 CFU.

[0020] In the present invention, improving immunity includes reducing the incidence of gastrointestinal diseases in pets and reducing pet fecal odor.

[0021] In one embodiment, the pet is selected from cats and dogs, preferably, cats.

[0022] The present invention screens out a strain of Lactobacillus acidophilus with strong acid resistance, and improves the production capacity of extracellular polysaccharides through further optimal mutagenesis; using it to prepare pet food can significantly improve the immunity of pets, for example, it can reduce the incidence of intestinal diseases and reduce pet fecal odor. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 .The results of the incidence of gastrointestinal diseases in adult cats fed different feeds.

[0024] Figure 2 .The results of ammonia content in feces of adult cats fed different feeds. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0026] In order to make the experimental content and technical solutions of the present invention more clearly explained, the present invention is described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0027] Example 1. Screening of acid-resistant Lactobacillus acidophilus

[0028] Lactobacillus acidophilus is a commonly used probiotic strain. Improving its acid resistance can significantly enhance its survival rate and colonization efficiency in the pet's gastrointestinal tract, thereby amplifying its immunomodulatory effect. In this example, the acid resistance of nine Lactobacillus acidophilus strains collected in the laboratory was evaluated using the following method.

[0029] In this embodiment, the MRS medium formula (per liter) is: peptone 10.0 g, beef extract powder 5.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, agar 15.0 g, Tween 80 1.0 g, L-cysteine ​​0.5 g, pH 6.4.

[0030] Lactobacillus acidophilus was cultured in MRS medium (pH 6.4) at 37°C for 20 hours to the logarithmic phase. Then, the culture was inoculated into MRS medium with a pH of 3.5 at a 2% inoculum size. The OD600 was measured after 18 hours of culture. The results are shown in the following table:

[0031] Test strain (Lactobacillus acidophilus) <![CDATA[OD after 18 hours of cultivation 600 > LSD34 1.357 LCX12 0.914 ZXY09 0.578 LSD02 1.645 LCX14 2.131 ZND01 1.112 ZXY13 0.541 ZXY15 0.687 LCX11 1.024

[0032] As shown in the table above, most of the tested Lactobacillus acidophilus strains had poor adaptability to a pH 3.5 environment. For example, the OD values ​​of ZXY09, ZXY13, and ZXY15 were less than 1.0. Among these strains, LCX14 showed the strongest growth ability, indicating strong acid tolerance and the ability to penetrate the gastric acid barrier.

[0033] The exopolysaccharide (EPS) produced by Lactobacillus acidophilus is one of its key functional metabolites, playing multiple roles in pet immune regulation, intestinal health, and pathogen defense. The production of exopolysaccharides by strains is also an important indicator for evaluating the effectiveness of Lactobacillus acidophilus in improving immunity. In this embodiment, the exopolysaccharide content of the above-mentioned acid-tolerant Lactobacillus acidophilus strains (LCX14, LSD34, and LSD02) was measured.

[0034] The exopolysaccharide content of the strain was determined using the following method:

[0035] Lactobacillus acidophilus (LCX14, LSD34, LSD02) was inoculated into MRS liquid medium and fermented at 37°C for 24 hours. 50 mL of culture was taken and placed in a boiling water bath for 15 minutes. The culture was cooled to room temperature and 80% trichloroacetic acid was added to a final concentration of 4%. The culture was allowed to stand overnight at 4°C and centrifuged at 12,000 g for 20 minutes. The supernatant was placed in a dialysis bag and dialyzed for 24 hours and then for 36 hours. The water was changed four times and the volume was fixed to obtain a sample containing extracellular polysaccharides (EPS) for later use.

[0036] The sulfuric acid-phenol method (polysaccharide is dehydrated under the action of concentrated sulfuric acid to generate furfural, which can be condensed with phenol to develop color) is used to determine the content of extracellular polysaccharide (EPS) of Lactobacillus acidophilus; the extracellular polysaccharide sample obtained by the above dialysis is determined by the sulfuric acid-phenol method to obtain the content of EPS. A blank culture medium is used as a control to deduct background interference. When measuring specifically: 1 mL sample + 5 mL 6% phenol + 25 mL concentrated sulfuric acid is used, and the mixture is allowed to stand at room temperature for 30 minutes, and the OD value is measured. 490 The polysaccharide concentration was calculated based on the standard curve.

[0037] The exopolysaccharide concentrations of Lactobacillus acidophilus LCX14, LSD34, and LSD02 were measured to be 255 mg / L, 270 mg / L, and 108 mg / L, respectively. As can be seen, the exopolysaccharide concentrations of different Lactobacillus acidophilus strains vary. LCX14, which has the strongest acid tolerance, had an intermediate exopolysaccharide yield, while LSD34, which has the weakest acid tolerance, had the highest exopolysaccharide yield.

[0038] Example 2. Acclimation of Lactobacillus acidophilus LCX14

[0039] To enhance the exopolysaccharide production capacity of Lactobacillus acidophilus LCX14, this method employed a combined UV-induced and chemical mutagenesis approach. Specifically, the mutagenesis was performed using UV irradiation (30 seconds, 15W) followed by treatment with 0.5% diethyl sulfate (DES). Initial strain screening was performed using the colony stringing method: strains were cultured on solid MRS medium at 37°C for 48 hours. An inoculating needle was then applied to a single colony and slowly pulled outward. The colonies of different strains were compared to see if they exhibited distinct stringing patterns. Lactic acid bacteria exhibiting continuous stringing were screened.

[0040] In this example, five mutant strains with more obvious stringing phenomenon than LCX14 were screened, and the acid resistance and exopolysaccharide production performance of the five mutant strains were tested using the method of Example 1. The results are shown in the following table:

[0041]

[0042] As can be seen from the above table, the extracellular polysaccharide production of the strains initially screened after mutagenesis was increased to varying degrees compared with LCX14, among which the extracellular polysaccharide production of mutant strain-2 and mutant strain-4 was increased more significantly; however, it can also be seen from the above table that the acid resistance of the strains changed to varying degrees after mutagenesis, among which the acid resistance of mutant strain-1 and mutant strain-4 was significantly reduced.

[0043] Overall, the mutagenic strain-2 had the best effect and was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) with the deposit number CGMCC No. 34179 and the deposit date of April 14, 2025. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Tel: 010-64807355, and the strain name is Lactobacillus acidophilus M 20250331-LCX.

[0044] As can be seen from the above, whether it is acid resistance or the ability to produce extracellular polysaccharides, the mutant strain-2 showed better results. The applicant further verified through mouse experiments whether the mutant strain-2 can increase the level of immunoglobulins compared with the LCX14 wild-type strain.

[0045] Twenty-one male mice (6 weeks old) of comparable health were selected. They were housed in a PF environment with a standard light cycle (12 hours light / 12 hours dark), constant temperature and humidity, free access to water, and fed a diet without probiotics. After one week of adaptive feeding, they were randomly divided into three groups (control group, LCX14 group, and mutagenized strain-2 group), with seven mice in each group.

[0046] LCX14 group and mutant strain-2 group were gavaged with 1×10 9 CFU / mouse (once daily). A control group received an equal volume of purified water daily for 4 weeks. After the feeding trial, the mice were fasted for 4-6 hours, and blood was collected from the posterior ocular venous plexus. The blood was allowed to coagulate at room temperature for 30-60 minutes. The supernatant serum was centrifuged and collected. Serum IgG levels were measured using an ELISA kit, and the results are as follows:

[0047] Group IgG content (mg / ml) control group 2.09±0.18 LCX14 group 2.34±0.10 Mutagenesis strain-2 group 2.99±0.14

[0048] As shown in the table above, the LCX14 group showed a slight increase in IgG levels compared to the control group, while the mutant-2 group showed a significant increase in IgG levels compared to both the control and LCX14 groups. This indicates that the mutant-2 group significantly improves immunity compared to the wild-type LCX14.

[0049] Example 3. Application of Lactobacillus acidophilus in improving immunity of pets

[0050] In this embodiment, Lactobacillus acidophilus mutant strain-2 (Lactobacillus acidophilus M 20250331-LCX in Example 2) was added to the daily cat food to ensure that the cat's consumption was 1×10 9 -2×10 9CFU / day to observe its effect on the intestinal health of pets; the control group chose daily cat food without Lactobacillus acidophilus.

[0051] The above cat food groups (cat food with added Lactobacillus acidophilus) and control groups (cat food without added Lactobacillus acidophilus) were used for cat feeding trials. Ten adult cats were fed each type of cat food. The incidence of gastrointestinal diseases in the adult cats and the average ammonia content in their feces were recorded. The data were obtained from the test results of 30 adult cats followed up for 480 hours. Figure 1 and Figure 2 shown.

[0052] like Figure 1 As shown in Figure 2, the incidence of gastrointestinal diseases in adult cats was significantly reduced when the experimental group of cats was fed with cat food compared with the control group. Figure 2 As shown, when the experimental group of cats was fed with cat food, the average ammonia content in the feces of the adult cats was significantly lower than that in the control group.

[0053] The above experiments show that adding Lactobacillus acidophilus M 20250331-LCX to feed is beneficial to reducing the incidence of pet diseases and enhancing the immunity of pets.

[0054] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A strain for improving the immunity of pets, the strain being Lactobacillus acidophilus M20250331-LCX, which is deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration, with a deposit number of CGMCC No. 34179 and a deposit date of April 14, 2025. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

2. A method for culturing the strain according to claim 1, comprising the step of culturing the strain using a culture medium.

3. The method according to claim 2, characterized in that The culture medium is MRS culture medium.

4. The method according to claim 2, characterized in that The culture temperature is 30°C-40°C, preferably 37°C.

5. The method according to claim 2, characterized in that The culture time is 24h-72h.

6. The method according to claim 2, characterized in that The pH of the culture is 2.0-8.

0.

7. A pet food comprising the strain according to claim 1.

8. Use of the strain according to claim 1 in preparing pet food or pet feed.

9. Use of the strain according to claim 1 in preparing food or feed for improving immunity of pets.

10. A method for improving the immunity of a pet, comprising the step of feeding the pet with a feed containing the strain according to claim 1.

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