Deodorizing complex microbial inoculant special for pets as well as preparation method and application of deodorizing complex microbial inoculant

Abstract

The invention discloses a special deodorizing complex microbial inoculant for pets as well as a preparation method and application of the special deodorizing complex microbial inoculant, and relates to the technical field of biology. The active ingredients of the special deodorizing composite bacterial agent for the pets comprise a microorganism combination, and the microorganism combination comprises bifidobacterium animalis (Bifidobacterium animalis) F12 and Pichia burtonii (Hyphopichia burtonii) F24. The invention further discloses a preparation method of the special deodorizing composite bacterial agent for the pets. According to the invention, bifidobacterium animalis F12 and pichia pastoris F24 are separated and screened from fresh pet feces, and the two strains of bacteria are subjected to proportioning optimization to prepare the deodorizing composite bacterial agent. The deodorizing composite microbial agent provided by the invention is safe and pollution-free, and after the pet living area is treated by using the deodorizing composite microbial agent, the contents of NH3, H2S and odor molecules are obviously reduced, so that the living environment of pets can be effectively improved, and the space life quality of the living environment can be improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a deodorizing composite bacterial agent specifically for pets, and a preparation method and application thereof. Background Art

[0002] Pets are important partners in our lives. Whether they are dogs, cats or other small animals, they bring us a lot of joy and companionship. However, pets also bring some problems, one of which is the special smell of pets. It is undeniable that many pets will emit some odors. This is mainly due to factors such as pets' body hair, sweat glands and excrement. These odors will not only cause trouble to human living environment, but may also seriously affect neighborhood relations. Therefore, pet deodorants have become a magic weapon to solve the problem of pet odor. Pet deodorants, especially biological deodorants, are pure natural, quickly degraded, and very friendly to the health of small animals. They can not only effectively neutralize the odor of pets, but also make our living environment more fresh and comfortable.

[0003] Microbial deodorants have become a new type of pet deodorant product due to their pollution-free and long-lasting characteristics. Compared with biological enzyme deodorant products, they are more diverse, efficient and low-cost. Compared with chemical deodorants, microbial deodorants are rich in beneficial bacteria, which are more environmentally friendly and green, beneficial to the health of small animals, and extremely friendly to the human living environment.

[0004] The essence of microbial deodorization is to use microorganisms with specific functions to decompose some odorous substances into odorless substances or inhibit the production of odorous substances, so as to achieve the purpose of removing odors. In recent years, most domestic researchers have focused on the screening of single strains, while there are few studies on microbial agents composed of multiple microorganisms with different functions, and special deodorant products that are beneficial to pet health. Summary of the invention

[0005] The purpose of the present invention is to provide a pet-specific deodorizing composite bacterial agent and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. After the pet living area is treated with the deodorizing composite bacterial agent provided by the present invention, the content of NH3, H2S and odor molecules is significantly reduced, which can effectively improve the living environment of pets and improve the spatial living quality of human living environment.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a microbial combination with pet deodorizing effect, comprising Bifidobacterium animalis F12 and Hyphopichia burtonii F24;

[0008] The Bifidobacterium animalis F12 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 31, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 31414;

[0009] The Hyphopichia burtonii F24 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 31, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31415.

[0010] The present invention also provides the use of the above-mentioned microbial combination in preparing a deodorizing composite bacterial agent specifically for pets.

[0011] The present invention also provides a pet-specific deodorizing composite bacterial agent, wherein the active ingredients include the above-mentioned microbial combination.

[0012] The present invention also provides a method for preparing the above-mentioned pet-specific deodorizing composite bacterial agent, comprising the following steps:

[0013] The animal Bifidobacterium F12 and the Pichia bortonii F24 are inoculated into a fermentation medium for high-density fermentation to obtain a fermentation liquid A and a fermentation liquid B, respectively;

[0014] The fermentation liquid A and the fermentation liquid B are mixed evenly to obtain the pet-specific deodorizing composite bacterial agent.

[0015] Furthermore, the volume ratio of the fermentation liquid A to the fermentation liquid B is 1:1.

[0016] Furthermore, the fermentation medium consists of the following components: 2.5wt% molasses, 0.12wt% corn syrup powder, 0.33wt% ammonium sulfate, 0.07wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 0.05wt% calcium chloride and the balance water.

[0017] Furthermore, the rotation speed of the high-density fermentation culture is 120 rpm, and the ventilation volume is 0.8-1.0 vvm.

[0018] Furthermore, the high-density fermentation culture is carried out at a temperature of 30° C. for 5-7 days.

[0019] The present invention also provides application of the above-mentioned microbial combination or pet-specific deodorizing composite bacterial agent in pet deodorizing.

[0020] The present invention also provides a pet deodorizing method, comprising the steps of diluting the above-mentioned pet-specific deodorizing composite bacterial agent and spraying the pet's living environment.

[0021] The present invention discloses the following technical effects:

[0022] The present invention separates and screens animal Bifidobacterium F12 and Pichia bortonii F24 from fresh pet feces, and prepares a deodorizing composite bacterial agent from the two strains after optimizing the ratio. The deodorizing composite bacterial agent provided by the present invention is safe and pollution-free. After the deodorizing composite bacterial agent is used to treat the pet living area, the content of NH3, H2S and odor molecules is significantly reduced, which can effectively improve the living environment of pets and improve the spatial living quality of human living environment. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Isolation, purification and screening of ammonia removal bacteria

[0030] 1.1. Enrichment, separation and purification of ammonia-removing bacteria

[0031] Take 10 mL of fresh pet feces and add it to 90 mL of liquid selective medium for ammonia removal bacteria ((NH4)2SO4 0.8 g·L -1 ) and cultured at 30°C and 120 rpm for 1 day. Then, 10 mL was transferred to a new 90 mL liquid selective medium for ammonia removal bacteria ((NH4)2SO41 g·L -1 ) After 1 day of culture, transfer 10 mL to a new 90 mL liquid selective medium for ammonia removal bacteria ((NH4)2SO42 g·L -1 ). Take 1 mL of the enriched bacterial solution for gradient dilution. -4 , 10 -5 , 10 -6 0.1 mL of bacterial solution was inoculated onto a solid selective medium plate for ammonia removal bacteria, and each gradient was repeated three times, and inverted and cultured at 30°C for 2 days. A single colony was selected for repeated streaking purification until pure strains were obtained without foreign bacteria observed under a microscope. Finally, 121 strains were isolated and purified, and then the ammonia removal rate was determined.

[0032] Formula of liquid selective culture medium for ammonia-removing bacteria: glucose 5 g, K2HPO4 0.5 g, NaCl 0.5 g, Na2CO3 1 g, MgSO4·7H2O 0.5 g, Na2CO3 1 g, (NH4)2SO4 0.68 g, FeSO4·7H2O 0.01 g, distilled water 1000 mL, pH natural.

[0033] 1.2. Screening of ammonia-removing bacteria

[0034] The isolated and purified single strains were activated respectively, inoculated into a 250mL Erlenmeyer flask containing 100mL of deammonia bacteria screening medium at a 5% inoculum, shaken thoroughly, and cultured continuously at 30°C and 180rpm for 7 days, supplemented with 0.08g (NH4)2SO4 every other day to eliminate the bacteria that cannot utilize NH4. + -N microorganisms, and finally screened out the two strains with the highest ammonia removal efficiency, F12 and F24, with ammonia removal rates of 92.1% and 70.3%, respectively.

[0035] The formula of the ammonia-removing bacteria screening culture medium is the same as the above-mentioned ammonia-removing bacteria liquid selection culture medium formula.

[0036] 2. Screening of hydrogen sulfide-degrading bacteria

[0037] Preliminary screening test: After activating the strains isolated and purified in part 1.1, inoculate them into 500mL wide-mouth bottles containing 200mL desulfurization bacteria screening medium at a 5% inoculum size, place a 20mL sterile beaker containing 6mL of 25% (v / v) H2SO4 in the wide-mouth bottle, then add 2g of FeS to the beaker, cover the bottle with a stopper, and seal it with a sealing film. After culturing at 30°C and 180r / min for 5 days, observe whether the bacterial solution is turbid. If it is turbid, it means that the strain inoculated in this bottle has the ability to degrade H2S.

[0038] After activating a single strain that can make the bacterial solution turbid in the initial screening test, it was inoculated into a conical flask containing 100 mL of desulfurization bacteria screening culture medium at an inoculation rate of 5%, 10 mL of DMSO was added to the culture medium, and then the conical flask was sealed with a sterilized rubber stopper. After culturing at 30°C and 180 r / min for 2 days, a filter paper strip soaked in lead acetate solution was inserted into the gas in the upper layer of the conical flask to observe whether the filter paper turned black. If it turned black, it meant that DMSO had been decomposed into H2S. The strain F12 with desulfurization effect was finally screened out by the above method.

[0039] Desulfurization bacteria screening culture medium formula: glucose 5g, K2HPO40.5 g, KNO31 g, MgCl20.5 g, NaCl0.5g, NH4Cl 0.5g, Na2CO31 g, FeCl20.01 g, distilled water 1000mL, pH natural.

[0040] The strain F12 was identified as belonging to Bifidobacterium animalis and the strain F24 was identified as belonging to Hyphopichia burtonii.

[0041] 3. Strain preservation

[0042] Bifidobacterium animalis F12 was deposited at the General Microbiology Center of China Microorganism Culture Collection Administration on July 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31414;

[0043] Hyphopichia burtonii F24 was deposited at the General Microbiology Center of China National Culture Collection Administration on July 31, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31415.

[0044] Example 2

[0045] MRS liquid culture medium formula: peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween 801.0mL, dipotassium hydrogen phosphate 2g, sodium acetate 5g, diammonium citrate 2g, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.25g, distilled water 1000mL, pH 6.2-6.6 (solid culture medium plus agar 20g).

[0046] Potato liquid culture medium formula: 200g potato, 20g glucose, 1000mL distilled water, pH 7.0-7.2 (solid culture medium plus 20g agar).

[0047] The fermentation medium formula: 2.5wt% molasses, 0.12wt% corn syrup powder, 0.33wt% ammonium sulfate, 0.07wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, 0.05wt% calcium chloride and the balance water.

[0048] The preparation method of the deodorizing composite bacterial agent comprises the following steps:

[0049] 1) Solid slant culture: F12 was inoculated in MRS solid medium, and F24 was inoculated in potato solid medium, and cultured at 30°C for 2 days to activate the strains.

[0050] 2) Primary seed culture: The activated strains in step 1) were inoculated into liquid culture medium (F12 was inoculated into MRS liquid culture medium, and F24 was inoculated into potato liquid culture medium), and cultured at 30°C and 180 rpm for 2 days. 600 When the value reaches 0.6-0.8, stop culturing and obtain the first-level seeds.

[0051] 3) Secondary seed culture: The primary seeds obtained in step 2) were inoculated in 10 L Erlenmeyer flasks at a 10% inoculation rate, and the culture medium was the same as that in step 2), and cultured at 30° C. for 2 days to obtain secondary seed solution.

[0052] 4) The F12 secondary seed liquid obtained in step 3) was inoculated into a 100 L fermentation tank containing 30 L fermentation medium at an inoculum size of 10%, and high-density fermentation culture was carried out at 30° C., 120 rpm, and aeration volume of 0.8 vvm (0.8-1.0 vvm can achieve the same effect) for 5 days (up to 7 days) to obtain fermentation liquid A.

[0053] 5) The F24 secondary seed liquid obtained in step 3) was inoculated into a 100 L fermentation tank containing 30 L fermentation medium at an inoculum size of 10%, and high-density fermentation culture was carried out at 30° C., 120 rpm, and aeration volume of 0.8 vvm (0.8-1.0 vvm can achieve the same effect) for 5 days (up to 7 days) to obtain fermentation liquid B.

[0054] 6) The fermentation liquid A and the fermentation liquid B are mixed evenly at a volume ratio of 1:1 to obtain a deodorizing composite bacterial agent.

[0055] Experimental Example 1

[0056] The deodorizing effect of the deodorizing composite bacterial agent obtained in Example 2 on pet cats, Maine Coon, was measured.

[0057] 50g of Maine feces and sawdust mixed with cat litter were placed in a 1000mL beaker, the water content was adjusted to 50%, and sterilized at 121°C for 30min, then the deodorizing composite bacterial agent prepared in Example 2 was inoculated at 10% inoculum, and fully mixed, and a 50mL small beaker containing 20mL of dilute sulfuric acid solution and a 50mL small beaker containing 20mL of zinc acetate-sodium acetate solution were placed in the above-mentioned 1000mL beaker, and the 1000mL beaker was sealed with a double-layer plastic film and placed in a 30°C constant temperature room for fermentation and culture. Each treatment was repeated 3 times, and the release of ammonia and hydrogen sulfide was detected every 3d during the culture process by taking out the small beaker.

[0058] The results showed that the deodorizing bacteria agent could remove 99.24% of NH3 and 98.95% of H2S from Maine Coon feces.

[0059] Experimental Example 2

[0060] The deodorizing effect of the deodorizing composite bacterial agent obtained in Example 2 on Maine coon sheds was measured.

[0061] The deodorizing composite bacterial agent prepared in Example 2 was diluted 10 times with water and heated at 0.02 kg / m 2 The cat house was sprayed with a dosage of 100 mg / kg, and the spraying frequency was 1 time / day at 14:00 in the afternoon. The test data was recorded with a handheld ammonia meter. At 2 pm, especially after going to the toilet, the cat house had a strong smell, and the ammonia data was measured at 9.6 ppm. After spraying the deodorizing bacteria agent, the ammonia measurement data immediately dropped to 1.1 ppm. It can be seen that the deodorizing composite bacteria agent of the present invention has a significant effect on removing ammonia. After spraying the deodorizing composite bacteria agent, Maine Coon had no abnormal reaction and began to forage and take a nap.

[0062] Experimental Example 3

[0063] The deodorizing effect of the deodorizing composite bacterial agent obtained in Example 2 on golden retriever was measured.

[0064] Golden retrievers are large dogs with heavy body odor. Golden retrievers returning home from outside often have a special smell, especially after raining. The deodorizing composite bacterial agent prepared in Example 2 was diluted 10 times with water and sprayed on the body of the golden retriever returning from outside. The spraying frequency was 1 time / day. The smell was graded into 5 levels, the smell of the golden retriever just arriving home was graded as 5, and the smell after spraying the deodorizing bacteria was graded as 1-2. It can be seen that the deodorizing composite bacterial agent of the present invention has a significant effect on deodorizing the golden retriever, and after spraying the deodorizing composite bacterial agent, the golden retriever had no abnormal reaction and was as active as usual.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A microbial combination with pet deodorizing effect, It is characterized in that Includes Bifidobacterium animalis F12 and Hyphopichia burtonii F24; The animal Bifidobacterium F12 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on July 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31414; The Pichia pastoris F24 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 31, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31415.

2. Use of the microbial combination as claimed in claim 1 in the preparation of a deodorizing compound bacterial agent for pets.

3. A pet-specific deodorizing compound bacterial agent, It is characterized in that The active ingredient comprises the microbial combination according to claim 1.

4. A method for preparing the pet-specific deodorizing composite bacterial agent as claimed in claim 3, It is characterized in that The following steps are involved: The animal Bifidobacterium F12 and the Pichia bortonii F24 are inoculated into a fermentation medium for high-density fermentation to obtain a fermentation liquid A and a fermentation liquid B, respectively; The fermentation liquid A and the fermentation liquid B are mixed evenly to obtain the pet-specific deodorizing composite bacterial agent.

5. The preparation method according to claim 4, It is characterized in that The volume ratio of the fermentation liquid A to the fermentation liquid B is 1:

1.

6. The preparation method according to claim 4, It is characterized in that The fermentation medium consists of the following components: 2.5 wt% molasses, 0.12 wt% corn syrup powder, 0.33 wt% ammonium sulfate, 0.07 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.05 wt% calcium chloride and the balance water.

7. The preparation method according to claim 4, It is characterized in that The rotation speed of the high-density fermentation culture is 120 rpm, and the ventilation volume is 0.8-1.0 vvm.

8. The preparation method according to claim 4, It is characterized in that The high-density fermentation culture is carried out at a temperature of 30° C. for 5 to 7 days.

9. Use of the microbial combination as claimed in claim 1 or the pet-specific deodorizing composite bacterial agent as claimed in claim 3 in pet deodorization.

10. A method for deodorizing pets. It is characterized in that The method comprises the steps of diluting the pet-specific deodorizing composite bacterial agent as claimed in claim 3 and spraying the diluting agent on the living environment of the pet.