Bacillus subtilis ac-1 and application thereof

Abstract

The present application relates to the technical fields of microorganism and pet feed additive, and particularly relates to a bacillus subtilis AC-1 and application thereof. The bacillus subtilis AC-1 of the present application can reduce the odor of animal excrement, improve the appetite of animals, improve the health of animal fur color, improve the liver and kidney damage, and improve the immunity of animals.

Description

Technical Field

[0001] This invention relates to the field of microbial and pet food additive technology, and in particular to a Bacillus subtilis AC-1 and its applications. Background Technology

[0002] Probiotics play a vital role in animal gut health, promoting weight gain, improving gut microbiota, and reducing disease incidence. Therefore, many probiotics are used as feed additives in livestock and pet farming. However, there are many types of probiotics, such as Bacillus subtilis, Lactobacillus plantarum, and yeast. The mechanisms of action and effects of different probiotics vary greatly. Furthermore, even within the same type of probiotic, different strains exhibit significant functional differences, resulting in considerably different effects.

[0003] The pet industry is an emerging industry that has entered a period of stable and orderly growth. In 2020, the Chinese market size approached 300 billion yuan, and it is expected to continue to maintain steady growth in the future. However, many important issues in pet ownership still trouble pet owners and their pets.

[0004] One problem is the strong odor of cat feces, which seriously affects environmental hygiene and is a major concern for pet owners. To reduce the smell of cat feces, products have been developed, such as covering the feces with cat litter, but the effect is limited; the odor remains strong even after covering and shoveling. The future direction is to fundamentally reduce the odor of cat feces by improving pets' diets to promote healthier bowel movements. Currently, there are no reports of probiotic additives specifically designed to significantly reduce pet feces odor.

[0005] The second problem is that many pets are picky eaters, and many pet foods don't suit their tastes, leading to malnutrition in cats. To address this issue, various pet palatability enhancers have emerged, primarily composed of animal livers combined with various chemical flavorings, including aldehydes, alcohols, acids, ketones, and lipids. Animal livers are the detoxification site for animals, often accumulating toxins; long-term consumption is detrimental to pets' health, as is long-term use of chemical flavorings. Currently, there is a lack of healthy and effective probiotic palatability enhancers.

[0006] A shiny coat is an important indicator of a pet's health and a goal for pet owners. Many pets have dull, dry, frizzy coats (not caused by fright), lacking smoothness, and with hairs matted together. This is mainly caused by skin diseases, intestinal diseases, poor or unbalanced nutrient absorption, and low hair protein content. Currently, the main methods to improve a pet's coat's shine and smoothness are shampoos, conditioners, and nutritional supplements. However, while modern pet food contains a full range of nutrients, ensuring balanced nutrient absorption is crucial for animal health. Currently, there is a lack of probiotic additives that can significantly improve a pet's coat health.

[0007] Pets often forage and drink outdoors, consuming spoiled food such as decaying rat and bird carcasses, and rotten meat from garbage cans. This spoiled food can harm pets, one major culprit being kidney damage. Symptoms include lethargy, loss of appetite (or even complete anorexia), weight loss, vomiting, diarrhea, and frequent urination. Blood tests often show elevated creatinine levels. Sometimes, it can also damage liver cells, leading to elevated transaminase levels.

[0008] Many cat foods contain a lot of chicken liver. Animal livers are high in vitamin A, and long-term consumption can easily lead to vitamin A accumulation and cause vitamin A poisoning. The liver is the largest detoxification organ and is prone to accumulating toxic and harmful substances. Long-term consumption by pet cats may cause kidney damage.

[0009] Other factors that can cause liver and kidney damage in cats include moldy or spoiled starchy fillers such as grains and corn in some low-quality cat food, as well as excessive magnesium content, which can damage the kidneys. Cat food may also contain extremely high levels of protein or nutrients; long-term excessive protein intake can place a heavy burden on the liver and kidneys. The misuse of medications, such as antibiotics, can also easily lead to liver and kidney damage.

[0010] Therefore, adding probiotics that improve liver and kidney function in cats can help improve their overall health. Summary of the Invention

[0011] This invention provides Bacillus subtilis AC-1 and its applications to address the shortcomings of existing technologies in the lack of probiotics that can reduce animal fecal odor, improve animal appetite, improve animal coat health, improve liver and kidney damage, and enhance immunity.

[0012] This invention screened and identified a strain of Bacillus subtilis from soil, named Bacillus subtilis AC-1, and submitted it for deposit. The specific deposit information is as follows:

[0013] Accession number: CGMCC NO.26448, classification name: Bacillus subtilis, depository: China General Microbiological Culture Collection Center (CGMCC) of Institute of Microbiology, Chinese Academy of Sciences, depository address: No.3, No.1 Beichen West Road, Chaoyang District, Beijing, 100101, deposit date: January 12, 2023.

[0014] The present invention further provides a microbial agent comprising Bacillus subtilis AC-1, a fermentation broth containing AC-1, and / or its metabolites.

[0015] The microbial agent can be in various forms, such as Bacillus subtilis AC-1 fermentation broth or Bacillus subtilis AC-1 fermentation broth prepared into microbial powder, etc.

[0016] The present invention also provides a composition comprising the Bacillus subtilis AC-1 or the bacterial agent.

[0017] According to the composition, the composition is a feed additive or a pet palatability enhancer.

[0018] Preferably, Bacillus subtilis AC-1 fermentation broth is added to the feed.

[0019] The present invention also provides a method for preparing a composition, which involves mixing AC-1 fermentation culture broth with pet food, drying, and pulverizing.

[0020] Preferably, the preparation method of the composition includes: culturing AC-1 cells in LB medium at 35°C and 200 rpm for 48 hours to achieve a bacterial count of 3 × 10⁻⁶. 9 Take 50ml of the product and mix it with 100g of cat food (Yidi Cat Food, complete all-stage food, Hebei Chongjia Feed Co., Ltd.). Dry at 55℃, grind into powder, and store at room temperature away from light.

[0021] The present invention also provides the application of the Bacillus subtilis AC-1, the bacterial agent, or the composition in animal husbandry.

[0022] According to the application, the animal is a pet.

[0023] Preferably, the animal is a feline or canine.

[0024] More preferably, the animal is a cat.

[0025] The present invention also provides the use of the Bacillus subtilis AC-1, the bacterial agent, or the composition in improving animal appetite.

[0026] Preferably, its use in the preparation of animal feed, feed additives or palatability enhancers.

[0027] Preferably, the palatability enhancer is a cat food palatability enhancer.

[0028] The present invention also provides the use of the Bacillus subtilis AC-1, the inoculum, or the composition in any of the following:

[0029] 1) Increase the total protein level in animal hair;

[0030] 2) Improves the shine of animal fur;

[0031] 3) Improves the smoothness of animal fur.

[0032] The present invention also provides the use of the Bacillus subtilis AC-1, the inoculum, or the composition in any of the following:

[0033] 1) Improves diarrhea in animals;

[0034] 2) Reduce the odor of animal feces;

[0035] Preferably, it reduces the odor of feces from animals with diarrhea;

[0036] 3) Reduce the water content of animal feces;

[0037] 4) Reduce the content of H2S, NH3, or VOCs in animal feces;

[0038] 5) Improves the shape of animal feces.

[0039] The present invention also provides the use of the Bacillus subtilis AC-1, the inoculum, or the composition in any of the following:

[0040] 1) Improves liver and kidney damage in animals;

[0041] 2) Improve animal immunity;

[0042] 3) Reduce the levels of creatinine or alanine aminotransferase in animal serum;

[0043] 4) Increase serum albumin levels.

[0044] The beneficial effects of this invention are:

[0045] On the one hand, this invention provides the application of Bacillus subtilis AC-1 in pet breeding. Bacillus subtilis AC-1 can effectively reduce the content of VOC (volatile organic compounds), NH3, and H2S in pet cat feces, reduce fecal moisture, improve fecal shape, and further reduce the odor of pet feces, which is beneficial to the health of the breeding environment.

[0046] On the other hand, the present invention provides an application of Bacillus subtilis AC-1 in pet breeding. Bacillus subtilis AC-1 can effectively promote pets' appetite and encourage them to eat cat food. AC-1 can be used as a pet appetite stimulant.

[0047] Thirdly, this invention provides an application of Bacillus subtilis AC-1 in pet breeding. Bacillus subtilis AC-1 can effectively improve the health of pet hair, promote the accumulation of pet hair protein, and make the coat shiny and the hair soft.

[0048] Fourthly, this invention provides an application of Bacillus subtilis AC-1 in pet breeding. Bacillus subtilis AC-1 can effectively improve liver and kidney function in cats and enhance immunity. It also effectively reduces serum creatinine and alanine aminotransferase levels and increases serum albumin levels. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0050] Example 1: Preparation method of Bacillus subtilis AC-1 fermentation broth

[0051] The Bacillus subtilis AC-1 used in this embodiment was derived from soil, and its specific preservation information is as follows:

[0052] Accession number: CGMCC NO.26448, classification name: Bacillus subtilis, depository: China General Microbiological Culture Collection Center (CGMCC) of Institute of Microbiology, Chinese Academy of Sciences, depository address: No.3, No.1 Beichen West Road, Chaoyang District, Beijing, 100101, deposit date: January 12, 2023.

[0053] This embodiment provides a method for preparing Bacillus subtilis AC-1 fermentation broth, which specifically includes the following steps:

[0054] (1) After activating Bacillus subtilis AC-1 strain on LB plate, single clones were picked and inoculated into 100 mL of LB medium and cultured overnight on a shaker at 35℃ and 200 rpm.

[0055] (2) The above-mentioned Bacillus subtilis AC-1 was then inoculated into 2L of LB medium and cultured on a shaker at 35℃ and 150rpm for 48 hours. After that, the fermentation broth was collected and stored at room temperature for later use. The bacterial culture was diluted with LB medium to 3×10⁻⁶. 9 / ml.

[0056] Example 2: Bacillus subtilis AC-1 as a feed additive

[0057] This embodiment provides a method for preparing feed using Bacillus subtilis AC-1 as a feed additive, as detailed below:

[0058] The Bacillus subtilis AC-1 fermentation broth obtained in Example 1 (concentration 3×10⁻⁶) was used to ferment the Bacillus subtilis AC-1 broth. 9 Mix 50ml of ( / ml) cat food with 100g of cat food (Yidi cat food, complete all-stage cat food, Hebei Chongjia Feed Co., Ltd.), dry at 55℃ until the moisture content is less than 12%, then grind into powder (60-100 mesh) to obtain powdered feed. Package and store at room temperature in a dry place. The powdered feed contains approximately 1×10⁻⁶ Bacillus subtilis. 9 / g.

[0059] Example 3: Bacillus subtilis AC-1 as a cat attractant

[0060] Twenty British Shorthair kittens (1 year old, male, Beijing Xinuoyin Biotechnology Co., Ltd.) were purchased from the market and fed Yidi cat food (complete all-stage food, Hebei Chongjia Feed Co., Ltd.) for one week as an adaptation diet, 80 grams of cat food per day, twice a day, 40 grams each time. After one week, the formal experiment began. On the first feeding of cat food each day, 10 cats were fed only cat food, and the feeding time was recorded for 3 consecutive days. The remaining 10 cats were also fed cat food plus 1 gram of Bacillus subtilis AC-1 powder (sprinkled on top of the first feeding), and the feeding time was recorded for 3 consecutive days.

[0061] Table 1. Effects of AC-1 additives on cat food.

[0062]

[0063] As can be seen from Table 1, AC-1 bacterial powder has a significant appetite-stimulating effect on cats.

[0064] Example 4: Effects of AC-1 bacterial powder on cat fur

[0065] Twenty 1-year-old male British Shorthair cats were purchased from the market (Beijing Xinuoyin Biotechnology Co., Ltd.). They were fed Yidi cat food (complete, all-stage general-purpose food, Hebei Chongjia Feed Co., Ltd.) for one week as an acclimatization diet, 80 grams of cat food per day, twice a day, 40 grams each time. After one week, the formal experiment began. The experimental group consisted of 10 cats. Each day, 1 gram of Bacillus subtilis AC-1 powder was sprinkled on the cat food during the first feeding, and this was continued for more than 70 days. Simultaneously, 10 cats served as a control group, feeding only cat food. At the end of the feeding period, the brightness and smoothness of the cats' fur (back) were visually observed and recorded. Approximately 500 mg of hair from the left hindquarters of each cat was collected for total protein content determination (Kjeldahl method).

[0066] Record metrics:

[0067] Cat fur shine: Dry and dull, grade I; with some shine, grade II; shiny fur, grade III;

[0068] Fur smoothness: Fur that is not frizzy due to fright or stress is marked as I; normal smoothness (fur is matted together) is marked as II; fluffy and smooth fur is marked as III.

[0069] The results are shown in Table 2.

[0070] Table 2 shows the effect of AC-1 additives on cat coat.

[0071]

[0072] The results in the table above show that AC-1 bacterial powder can increase the total protein level in cat fur, effectively improving the shine and smoothness of cat fur.

[0073] Example 5: Effects of AC-1 bacterial powder on pet cat feces

[0074] Thirty British Shorthair cats (1 year old, male) were purchased from the market and acclimatized for one week with Yidi cat food (complete all-stage food, Hebei Chongjia Feed Co., Ltd.), 80 grams of cat food per day, twice a day, 40 grams each time. After one week, the formal experiment began. Each day, on the first feeding of cat food, the cats were fed only raw ribbonfish (40 grams) instead of cat food for 1-3 days, until at least 20 cats had loose stools, exhibiting varying degrees of diarrhea, foul odor, and mucus and foam on the stool. Then, 10 cats with loose stools were fed only cat food (control group), while the other 10 cats were fed cat food plus 1 gram / day of AC-1 probiotic powder (experimental group) for 5 consecutive days. The dryness and wetness of the cat feces and the odor were recorded daily. On the 5th day, cat feces were collected to observe and test the fecal shape, moisture, odor, white blood cells, red blood cells, epithelial cells, and changes in NH3, H2S, and VOCs in the feces. The results are shown in Table 5.

[0075] Table 3. Effects of AC-1 bacterial powder on cat feces.

[0076]

[0077]

[0078] Note: **, P < 0.01, the difference is extremely significant.

[0079] As can be seen from the table above, AC-1 probiotic powder can significantly reduce the odor (H2S, NH3 and VOC content) of feces in cats with diarrhea, improve the appearance of feces, and change them from unformed to normal or more normal. It has a significant effect on improving diarrhea caused by indigestion.

[0080] Example 6: Improvement of feline renal function by AC-1 bacterial powder

[0081] This experiment was conducted with the assistance of a pet hospital in Beijing. The hospital provided 40 cats: 20 male Ragdoll cats, aged 2 years; and 20 male British Shorthair cats, also aged 2 years. All 20 Ragdoll cats had low serum albumin and high alanine aminotransferase levels, with other biochemical indicators normal. All 20 British Shorthair cats had high serum creatinine levels, with other biochemical indicators normal. They were fed Yidi cat food (complete, all-stage general-purpose food, Hebei Chongjia Feed Co., Ltd.) for one week as an adaptation diet, 100 grams of cat food per day, twice a day, 50 grams each time. One week later, the formal experiment began. Ten Ragdoll cats were fed normal cat food with 1 gram of AC-1 probiotic powder added daily, while ten Ragdoll cats served as a control group, eating only normal cat food. Ten British Shorthair cats were fed normal cat food with 1 gram of AC-1 probiotic powder added daily, while ten British Shorthair cats served as a control group, eating only normal cat food. After 60 days, serum albumin and alanine aminotransferase levels were measured in 20 Ragdoll cats, and serum creatinine levels were measured in 20 British Shorthair cats. The results are shown in Table 4.

[0082] Table 4 Effects of AC-1 bacterial powder on biochemical indicators of cat blood

[0083]

[0084] Note: **, P < 0.01, the difference is extremely significant.

[0085] This table only lists the indicators that showed significant changes in the pet hospital's tests. Other indicators were normal and showed no significant changes before and after the experiment, so they are not listed.

[0086] As can be seen from the table above, AC-1 probiotic powder can improve the liver and kidney function of cats with high creatinine or transaminase levels, significantly reducing these two indicators. It can also improve low albumin levels, which is beneficial for enhancing the pet's immunity.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis AC-1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 26448.

2. A microbial agent, characterized in that, Including the Bacillus subtilis of claim 1 ( Bacillus subtilis AC-1 and / or fermentation broth containing Bacillus subtilis AC-1.

3. A composition, characterized in that, Including the Bacillus subtilis of claim 1 ( Bacillus subtilis AC-1 or the bacterial agent as described in claim 2.

4. The composition according to claim 3, characterized in that, The composition is a feed additive or a pet palatability enhancer.

5. The Bacillus subtilis strain described in claim 1 ( Bacillus subtilis The use of the microbial agent of claim 2 or the composition of any one of claims 3-4 in the preparation of cat food, feed additives or palatability enhancers.

6. The Bacillus subtilis of claim 1 ( Bacillus subtilis The use of the microbial agent of claim 2 or the composition of any one of claims 3-4 in any of the following: 1) Increase the total protein level in cat fur; 2) Improves the shine of the cat's fur; 3) Improves the smoothness of the cat's fur; The application does not involve the treatment of diseases.

7. The Bacillus subtilis strain according to claim 1 ( Bacillus subtilis The use of AC-1, the bacterial agent of claim 2, or the composition of any one of claims 3-4 in the preparation of a medicine to improve feline diarrhea caused by indigestion.

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