Bacillus amyloliquefaciens E76 and application of composition thereof in pet food

Abstract

The invention relates to application of bacillus amyloliquefaciens E76 and a composition thereof in pet food, the bacillus amyloliquefaciens E76 is a microbial composition containing the bacillus amyloliquefaciens E76 and saccharomyces boulardii, and the bacillus amyloliquefaciens E76 comes from animal intestinal tracts and is combined with the saccharomyces boulardii according to a mass ratio of 2: 6 for use. By adding the composition to pet food, 1 * 10 < 7 > CFU of viable bacteria is contained in each gram of food. The preparation method of the pet food comprises the following steps: mixing the basic raw materials, extruding and puffing at 100-150 DEG C, cooling, adding the microbial composition, and finally drying until the moisture content is less than or equal to 10%. The composition can effectively improve intestinal health of pets, enhance immunity, promote digestive absorption and maintain intestinal microecological balance, has high stability and wide application prospects, and is suitable for daily feeding of dogs, cats and other pets.

Description

Technical Field

[0001] The present invention relates to the technical fields of pet food and microbial applications, and particularly to the application of a Bacillus amyloliquefaciens E76 and its composition in pet food. Background Art

[0002] With the improvement of people's living standards, pets have become important members of many families. The health status of pets directly affects their quality of life and the happiness of their owners. In recent years, pet intestinal health problems have become increasingly prominent, such as indigestion, diarrhea, constipation, etc., which seriously affect the growth and development and immune function of pets. Intestinal microecological balance is crucial for maintaining the health of pets. How to improve the pet intestinal microecology through nutritional intervention means has become a research hotspot.

[0003] Probiotics, as a safe and effective intestinal microecological regulator, have been widely used in the maintenance of intestinal health in humans and animals. Common probiotics include lactic acid bacteria, bifidobacteria, and bacillus, etc. Among them, bacillus is suitable for food processing and storage due to its heat resistance, acid and alkali resistance and other characteristics. However, at present, the types of probiotics for pet food on the market are limited, mainly concentrated in lactic acid bacteria and bifidobacteria, and there is less research on the application of bacillus in pet food.

[0004] Bacillus amyloliquefaciens is a probiotic with broad application prospects, with strong enzyme-producing activity, strong antibacterial ability, and immune promotion and other characteristics. In agriculture and animal husbandry, Bacillus amyloliquefaciens has been used in promoting plant growth, improving feed utilization rate and other aspects. However, the research on the application of Bacillus amyloliquefaciens in pet food to improve pet intestinal health is still in its infancy, and there is no in-depth report and mature product. Therefore, developing a method for applying Bacillus amyloliquefaciens to pet food is of great significance for improving pet intestinal health and enhancing its immunity. Summary of the Invention

[0005] The present invention provides a microbial composition that can be effectively applied in pet food, and its main components include Bacillus amyloliquefaciens E76 (deposit number: CGMCC NO. 32200) and Saccharomyces boulardii. The composition is made by mixing the strains in a mass ratio of 2:6, optimizing the efficacy ratio of each strain. Bacillus amyloliquefaciens E76 is derived from inside animals and the intestine, which helps to enhance its effect on pet intestinal health.

[0006] Technical Solution

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] Composition of the microbial composition:

[0009] Bacillus amyloliquefaciens E76 accounts for 25% of the composition by mass, and Saccharomyces boulardii accounts for 75%.

[0010] Application of the microbial composition in pet food:

[0011] The microbial composition is added to pet food so that each gram of pet food contains a total viable count of 1×10^7 CFU to ensure the activity and effect of probiotics.

[0012] Method for making pet food:

[0013] The present invention also relates to a method for making a pet food containing the microbial composition, and the steps include:

[0014] Mix chicken meal, sweet potato powder, potato powder, tapioca powder, fish meal, vitamin and mineral premix, prebiotic and other auxiliary materials to form a basic raw material mixture;

[0015] Extrusion-expand the basic raw material mixture at 100 - 150 °C to make granular pet food;

[0016] Cool the expanded pet food to room temperature;

[0017] Add the microbial composition to the cooled pet food at a mass ratio of 1:100 and mix evenly;

[0018] Dry the pet food added with the microbial composition at 40 °C until the moisture content ≤ 10%.

[0019] Application effect:

[0020] The microbial composition of the present invention can improve the intestinal microecological balance of pets, increase the number of beneficial bacteria in the intestine, reduce the number of harmful bacteria, and thus enhance the digestion and absorption ability, immunity and overall health status of pets.

[0021] Product stability:

[0022] The composition shows high stability during storage, is suitable for storage for 18 months under normal conditions, and maintains the effective number of viable bacteria.

[0023] Beneficial effects

[0024] The microbial composition of the present invention has the following beneficial effects:

[0025] Intestinal health: The strains in the composition have a synergistic regulatory effect on the intestinal microecology and can significantly improve the intestinal health level of pets.

[0026] High stability: The optimized strain ratio and process parameters significantly improve the strain survival rate during pet food processing and storage, ensuring the effectiveness of probiotics.

[0027] Good safety: The strains used are all safe food-grade microorganisms. It has been proven by experiments that they have no toxic or side effects on pets and are suitable for long-term feeding.

[0028] Wide applicability: It is applicable to the daily feeding of various pets such as dogs and cats, meeting the health needs of different pets. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 Schematic diagram of the pet food production process flow for Embodiment 3 of the present invention;

[0031] Figure 2 Schematic diagram of the preparation process of the microbial composition for Embodiment 2 of the present invention. Detailed Embodiments

[0032] The following will describe the present invention in detail in combination with the drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; moreover, the drawing part is only for more specifically describing the embodiments and is not intended to specifically limit the present invention.

[0033] It should be pointed out that in the specification, when referring to "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc., it indicates that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes such specific features, structures or characteristics. In addition, when combining an embodiment to describe a specific feature, structure or characteristic, implementing such a feature, structure or characteristic in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0034] Generally, terms can be understood at least in part from their use in context. For example, depending at least in part on the context, the term "one or more" as used herein can be used to describe any feature, structure, or property in a singular sense, or can be used to describe a combination of features, structures, or properties in a plural sense. Additionally, the term "based on" can be understood to not necessarily be intended to convey a set of exclusive factors, but rather can alternatively, depending at least in part on the context, allow for the existence of other factors that are not necessarily explicitly described.

[0035] The present invention relates to a microbial composition containing Bacillus amyloliquefaciens E76 (deposit number: CGMCC NO. 32200) and Saccharomyces boulardii, and its application in pet food. Bacillus amyloliquefaciens E76 was isolated from the animal body and intestine, and the effect of enhancing pet intestinal health is achieved through an optimized combination.

[0036] The microbial composition is formulated in a mass ratio of 2:6 to ensure the synergistic effect of each strain. The composition is added to pet food so that each gram of the food contains 1×10^7 CFU of viable bacteria to meet the health needs of the pet's gastrointestinal tract.

[0037] In the present invention, the method for preparing pet food includes the following steps:

[0038] Mixing of basic raw materials: Mix chicken powder, sweet potato powder, potato powder, tapioca powder, fish meal, vitamin and mineral premix, prebiotic, and other auxiliary materials to form a basic raw material mixture.

[0039] Extrusion and puffing: Extrude and puff the basic raw material mixture at 100 - 150 °C to make granular pet food.

[0040] Cooling: Cool the puffed pet food to room temperature.

[0041] Addition of probiotic composition: Add the microbial composition to the cooled pet food at a ratio of 1:100 and mix evenly.

[0042] Drying: Dry at 40 °C until the moisture content ≤ 10%.

[0043] The final pet food can effectively improve the intestinal microecological balance of pets, enhance the digestion and absorption ability and immune function.

[0044] Example 1: Selection and culture of strains

[0045] 1.1 Selection of strains

[0046] Bacillus amyloliquefaciens E76

[0047] Depository Institution: General Microbiology Center of China Microbial Culture Collection Management Committee; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit Number: CGMCC NO. 32200; Deposit Date: October 14, 2024

[0048] Source: Isolated from the body and intestine of live animals and has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC).

[0049] Saccharomyces boulardii

[0050] 1.2 Cultivation of Strains

[0051] Medium Preparation:

[0052] Bacillus amyloliquefaciens E76: Use LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0).

[0053] Saccharomyces boulardii: Use YPD medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L).

[0054] Cultivation Conditions:

[0055] Bacillus amyloliquefaciens E76: 37°C, 180 rpm, cultivate for 12 hours.

[0056] Saccharomyces boulardii: 30°C, 180 rpm, cultivate for 24 hours.

[0057] Bacterial Liquid Preparation:

[0058] Centrifuge the cultured bacterial liquid of each strain (8000 rpm, 10 minutes), discard the supernatant, resuspend with sterile normal saline, and adjust the bacterial concentration to 1×10^10 CFU / mL.

[0059] Example 2: Preparation of Microbial Composition

[0060] 2.1 Composition Ratio

[0061] Ratio Principle: Determine the optimal ratio according to the functional characteristics of each strain.

[0062] Specific Ratio:

[0063] Bacillus amyloliquefaciens E76: 25%

[0064] Saccharomyces boulardii: 75%

[0065] Mixing Method:

[0066] Mix the two bacterial liquids according to the above ratio, stir well, and prepare a mixed bacterial liquid.

[0067] Example 3: Preparation of Pet Food

[0068] 3.1 Formulation Design

[0069] Basic formulation (per 1000 g):

[0070] Chicken meal: 645 g

[0071] Fish: 55 g

[0072] Sweet potato powder: 34 g

[0073] Cassava powder: 32 g

[0074] Potato powder: 20 g

[0075] Other excipients: 214 g

[0076] Addition of microbial composition:

[0077] Dosage: Make each gram of pet food contain 1×10^7 CFU of viable bacteria.

[0078] Addition method: After the food is cooled.

[0079] 3.2 Processing Technology

[0080] Mixing: Mix all the raw materials in the basic formulation evenly.

[0081] Extrusion and puffing: Process through a twin-screw extruder at 100 - 150 °C, and set the screw speed at 50 Hz to prepare granular pet food.

[0082] Cooling: Cool the puffed food to room temperature.

[0083] Addition of probiotic composition: Add the microbial composition to the cooled food at a ratio of 1:100 and mix evenly.

[0084] Drying and packaging: Dry at 40 °C until the moisture content ≤ 10%.

[0085] Example 4: Product Detection and Analysis

[0086] 4.1 Detection of Viable Bacteria Count

[0087] Detection standard: Detect and analyze the product according to the "General Rules for Counting Viable Probiotics" (Standard No.: T / CHC1009 - 2023) issued by the China Health Care Association.

[0088] Detection method:

[0089] Sample treatment: Take 10 g of the sample, add 90 mL of sterile normal saline, shake well, and make a dilution of 10 -1 times dilution.

[0090] Gradient dilution: Serial 10-fold dilutions were performed up to 10 -7 .

[0091] Culture medium selection:

[0092] Bacillus amyloliquefaciens E76: Nutrient agar medium.

[0093] Saccharomyces boulardii: Yeast extract - glucose - chloramphenicol medium.

[0094] Coating and culturing: Samples of appropriate dilution were spread on the corresponding culture medium plates.

[0095] Culturing conditions:

[0096] Bacteria: 37°C, 24 - 48 hours.

[0097] Yeast: 30°C, 48 hours.

[0098] Counting: Plates with colony counts between 30 - 300 were selected to calculate the viable cell count.

[0099] Test results:

[0100] Table 1 Test results of viable cell counts of each strain

[0101] Strain Viable count (CFU / g) Bacillus amyloliquefaciens E76 0.25×10^7 Saccharomyces boulardii 0.75×10^7 Total 1.0×10^7

[0102] Result analysis: The viable cell counts of each strain met the design requirements, and the total viable cell count reached 1.0×10^7 CFU / g.

[0103] 4.2 Nutrient component detection

[0104] Detection standards:

[0105] Moisture: GB / T6435 - 2014

[0106] Crude protein: GB / T6432 - 2018

[0107] Crude fat: GB / T6433 - 2006

[0108] Crude fiber: GB / T6434 - 2022

[0109] Ash: GB / T6438 - 2007

[0110] Test results:

[0111] Table 2 Analysis results of pet food nutrient components (dry matter basis, %)

[0112] Ingredient Content (%) Moisture 6.1 Crude protein 45.6 Crude fat 22.0 Crude fiber 1.6 Ash 8.0

[0113] Result analysis: The indicators of various nutritional components meet the national standards for pet food and meet the nutritional needs of pets.

[0114] Example 5: Pet live feeding experiment

[0115] 5.1 Experimental design

[0116] Experimental animals: 32 healthy adult cats were selected and randomly divided into a control group and an experimental group. The experimental group was further divided into two single-strain groups and a microbial composition group, and each group was fed the corresponding pet food.

[0117] 5.2 Observation indicators

[0118] Food intake, weight change, fecal characteristics, health status.

[0119] Intestinal flora analysis: 16S rRNA gene high-throughput sequencing technology was used.

[0120] For pet food and feed research, fecal trait scores often include the following key indicators:

[0121] Shape: Evaluate whether the feces are formed. For example, strip-shaped feces receive a higher score, while scattered or watery feces receive a lower score.

[0122] Color: Usually, healthy colors (such as brown) receive a higher score, while abnormal colors (such as black, green) receive a lower score.

[0123] Hardness / consistency: Moderate hardness receives a higher score, while too hard or too soft receives a lower score.

[0124] Odor: In a healthy state, the fecal odor should be moderate, and a foul odor receives a lower score.

[0125] Common scoring criteria (full score 10 points)

[0126] A commonly used scoring reference standard is:

[0127] 8 - 10 points: The shape, color, and hardness are normal, the odor is moderate, and the fecal state meets the health standards.

[0128] 5 - 7 points: The feces are softer or have a slightly abnormal odor, but the formation is normal.

[0129] 1 - 4 points: The feces are too soft, diluted, or have an obvious abnormal odor, which may indicate digestive problems or health abnormalities.

[0130] 5.3 Experimental results

[0131] The data after 8 weeks of feeding are as follows:

[0132]

[0133] The microbial composition group has better effects than the control group and the single strain group in terms of body weight gain, improvement of fecal traits, and abundance of intestinal beneficial bacteria.

[0134] Example 6: Safety evaluation

[0135] 6.1 Acute toxicity test

[0136] By the method of single - dose gavage with a high - dose microbial composition, no toxic reaction was observed. The experimental standards and results are as follows:

[0137]

[0138] 6.2 28 - day oral toxicity test

[0139] Conducted according to GB15193.22 - 2014 "National Food Safety Standard 28 - day Oral Toxicity Test", the results showed that there were no adverse reactions in the experimental group, the liver and kidney functions were normal, and no abnormalities were found in the main organ tissues.

[0140] Example 7: Product stability

[0141] 7.1 Storage stability

[0142] Stored at 25°C and 4°C for 18 months, and the viable bacteria count was detected monthly.

[0143] The product was detected and analyzed according to the "General Rules for Probiotic Viable Bacteria Count" (Standard No.: T / CHC1009 - 2023) issued by the China Health Care Association.

[0144] The results are as follows:

[0145] Storage time (months) Viable count at 25°C storage (CFU / g) Viable count at 4°C storage (CFU / g) 0 1.0×10^7 1.0×10^7 3 8.5×10^6 9.0×10^6 6 7.0×10^6 8.5×10^6 12 5.5×10^6 8.0×10^6 18 5.0×10^6 8.0×10^6

[0146] After 18 months, the viable bacteria quantity remained above 50% of the initial value under the storage condition of 25°C and above 80% of the initial value at 4°C, showing good stability.

[0147] Example 8: Evaluation of application effect

[0148] Consumer trial survey

[0149] Subjects: 50 pet owners, and the pet species include dogs and cats.

[0150] Trial time: 4 weeks.

[0151] Survey method: After the trial period, fill out a questionnaire.

[0152] Feedback results:

[0153] Table 8 Statistical results of consumer feedback

[0154]

[0155]

[0156] Result analysis: Most pet owners reported an improvement in their pets' health conditions and a high level of satisfaction with the product.

[0157] The present invention significantly improves the intestinal health and immune function of pets by applying a microbial composition consisting of Bacillus amyloliquefaciens E76 (CGMCC NO. 32200) and Saccharomyces boulardii to pet food. The results of various tests and experiments show that the microbial composition has good stability and safety and broad application prospects.

[0158] The present invention covers any alternatives, modifications, equivalent methods, and schemes made within the spirit and scope of the present invention. To enable the public to have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention. However, those skilled in the art can fully understand the present invention without these detailed descriptions. In addition, well-known methods, processes, procedures, components, and circuits are not described in detail to avoid unnecessary confusion to the essence of the present invention.

[0159] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens E76, with the preservation number of CGMCC NO. 32200, is isolated from the animal body and intestine, and has been preserved in the China General Microbiological Culture Collection Center (CGMCC). Its application in pet food is used to improve the intestinal health and immune function of pets.

2. The application according to claim 1, wherein The Bacillus amyloliquefaciens E76 (preservation number: CGMCC NO. 32200) and Saccharomyces boulardii are combined to form a microbial composition.

3. The microbial composition according to claim 2, wherein The mass ratio of the Bacillus amyloliquefaciens E76 to Saccharomyces boulardii is 2:

6.

4. The microbial composition according to claim 2 or 3, characterized in that, The composition is added to pet food so that each gram of the food contains a total viable count of 1×10^7 CFU.

5. According to the application described in claim 4, the manufacturing method of the pet food includes the following steps: (1) Mix chicken meal, sweet potato powder, potato powder, tapioca powder, fish meal, vitamin and mineral premix, prebiotics and other auxiliary materials to form a basic raw material mixture; (2) Extrude and expand the basic raw material mixture at 100-150 °C to make granular pet food; (3) Cool the extruded pet food to room temperature; (4) Add the microbial composition to the cooled pet food at a mass ratio of 1:100 and mix evenly; (5) Dry the pet food added with the microbial composition at 40 °C until the moisture content ≤ 10%.

6. According to the application described in any one of claims 1-5, the microbial composition can improve the intestinal microecological balance of pets, increase the number of beneficial bacteria, reduce the number of harmful bacteria, and enhance the immunity and overall health of pets.

7. A microbial composition containing Bacillus amyloliquefaciens E76 and Saccharomyces boulardii, characterized in that, Mix in a mass ratio of 2:6, suitable for adding to pet food.

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