CN111423999A - Antibacterial and deodorizing microbial preparation for pets and living environment thereof, and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of microbial preparations, in particular to a bacteriostatic and deodorizing microbial preparation for pets and a living environment of the pets. The microbial preparation comprises functional microbial agents of lactic acid bacteria, saccharomycetes and Neisseria; the lactic acid bacteria: yeast: the colony number ratio of the Netheriacico bacteria is (3-5): (8-11): (0.5 to 1.2). The invention also provides a preparation method and application of the bacteriostatic and odor-removing microbial preparation for the pets and the living environment thereof. The microbial agent has better effect of removing odor molecules, can keep low consumption of nutrient substances, can reduce harm caused by accumulation of metabolites, and can prolong the performance of the product for a long time by virtuous cycle.

Description

Antibacterial and deodorizing microbial preparation for pets and living environment thereof, and preparation method and application thereof

Technical Field

The invention relates to the technical field of microbial preparations, in particular to a bacteriostatic and odor-removing microbial preparation for pets and a living environment thereof, and a preparation method and application thereof.

Background

The increase of income increases the consumption of reducing pressure and dispatching solitary consumption, and the behavior of raising pets is more and more civilized and popular. According to statistics of Frost & Sullivan of the largest enterprise growth consulting company in the world, in 2018, the proportion of Chinese pet-raising users reaches 9978 thousands of users, and the proportion rises to 22%. In 2018, the number of pet cats is 6700 thousands, and the number of pet dogs is 7400 thousands. The proportion of pet-raising households per ten thousand in china in 2018 also increased from 16% to 22% in 2013. In addition to cats and dogs, chinese pet owners also breed 1.1 million other animals, including 8400 million aquatic animals, 900 million birds, 700 million reptiles, 700 million rodents, and the like.

While the living of the materials is increasingly satisfied, people pay more and more attention to the living quality, and a cleaner and healthier home environment is expected. When the pet and the person share one living environment, a certain difficulty is caused to maintain a clean and healthy home environment.

The main problems of peculiar smell and household sanitation are brought by pet raising. The pet odor sources mainly comprise oral cavity, skin, ears, anal gland, excrement and the like. For example, gastrointestinal problems, oral inflammation and the like can cause halitosis in pets; sebaceous gland secretions, skin infection and the like can cause various peculiar smells to be emitted from the body surface of the pet. At present, the effective ways to improve the home environment mainly include: keeping ventilation to naturally dissipate peculiar smell; cleaning the bottom plate, the wall surface, the pet tool and the like by adopting a chemical disinfectant/cleaning agent, such as drip, 84 disinfectant and the like; the air purification product is adopted to adsorb and cover the peculiar smell in the air.

Maintaining ventilation requires that all rooms be ventilated and that convection currents be established. The mode of keeping ventilation is greatly influenced by weather, for example, in winter, in order to keep indoor temperature, the door and the window are usually required to be closed; the south of China has humid air, doors and windows need to be closed to reduce the entrance of humid outdoor air into rooms in plum rain seasons every year, and the like. Chemical disinfectants and air purifiers generally have strong oxidation function or stimulation, and are not beneficial to the health of people after being continuously used.

In addition, pathogenic bacteria are a problem which is very troubling pet raising families, pet excrement and oral cavities contain a large amount of microorganisms, the microorganisms of the pet excrement and the oral cavities are spread in a cross mode when the pet licks the hair, then the microorganisms spread throughout the house environment along with the moving track or the hair falling off, and when the resistance level of the pet body is reduced to some extent, the pet body is very easy to be infected and attacked.

Disclosure of Invention

In view of the above, it is necessary to provide a bacteriostatic and odor-removing microbial preparation for pets and their living environments, and a preparation method and application thereof, so as to improve the quality of domestic living environments.

The invention is realized by the following technical scheme:

a bacteriostatic and odor-removing microbial preparation for pets and their living environment comprises functional bacteria lactobacillus, yeast and Nesterekoni (Nesterekonia); the lactic acid bacteria: yeast: the colony number ratio of the Netheriacico bacteria is (3-5): (8-11): (0.5 to 1.2).

Further, the microbial preparation further comprises a substrate; the substrate mainly comprises nutrient substances (such as nutrient substances in a culture medium and the like) for maintaining survival of the functional bacteria agent, metabolites and derivatives of the functional bacteria during the culture process and the like.

Further, the lactic acid bacteria comprise cold-resistant lactic acid bacteria (L Lactobacillus harbinensis), Lactobacillus rhamnosus (L Lactobacillus rhamnous) and Lactobacillus chaff-like (L Lactobacillus parafarraginis), and the ratio of the number of the bacteria of the cold-resistant lactic acid bacteria, the Lactobacillus rhamnosus and the Lactobacillus chaff-like is (16-19): 5-7): 2-5;

the yeast includes Saccharomyces boulardii (Dekkera bruxellensis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Trichosporon flexneri (Cutaneotrichporon currus), and Candida ethanolica (Candida ethanolica); the Dekkera brussels: and (3) saccharomyces cerevisiae: trichosporon flexuosus: the colony number ratio of the Candida ethanolica is (79-83): (4-6.5): (3-6): (0.5 to 1.2).

Further, the total colony number of the microbial preparation is not less than 1 × 10⁸CFU/ml, preferably 1-1.8 × 10⁸CFU/ml。

The invention relates to a preparation method of a bacteriostatic and deodorant microbial preparation for pets and living environments thereof, which comprises the following steps:

1) respectively activating each functional bacterium;

2) respectively carrying out propagation culture to obtain first-grade seeds of each functional bacterium;

3) and mixing and culturing the functional bacteria obtained in the step 2).

Further, the functional microbial agents include lactic acid bacteria, yeast and Nesterekonia (Nesterenkonia).

Further, the culture method of each functional bacterium in the step 2) comprises the following steps: activating each functional bacterium according to conventional operation, culturing in a culture medium, adjusting the pH to 6.5, and culturing at 30-37 ℃ for 24-36 hours; the colonies are ready for use.

Further, in step 2), the yeast culture medium comprises the following components: 4-10% of molasses, 1-3% of corn steep liquor, 0.1-0.6% of protein, 1-2% of sodium chloride and the balance of water; the pH was 6.5;

the lactobacillus slant culture medium comprises the following components: 0.5-1.8% of peptone, 1-5% of beef extract, 2-10% of corn steep liquor, 2.8-4.5% of cane sugar, 0.3-1% of sodium acetate, 0.5-2% of magnesium sulfate, 1-8% of sodium chloride and the balance of water; the pH was 6.5;

the culture medium formula of the Netheria gasseri comprises the following components: 0.3% of sodium citrate, 0.2% of KCl and CaCl₂0.02% of eggWhite peptone 1%, MgSO4 & 7H₂O2%, agar 1.8% and NaCl 5%; pH 9.0.

Further, the step 3) comprises the steps of preparing the functional bacteria obtained by the culture in the step 2) into bacterial suspensions with equal concentration, mixing and culturing in a sterilized liquid culture medium, culturing at 30-37 ℃, and periodically checking until the number of bacterial colonies is not less than 1 × 10⁸CFU/ml.

Further, the functional bacterial suspension in the step 3) is prepared according to the ratio of lactic acid bacteria: yeast: the neisseria melitensis is (3-5): (8-11): (0.5 to 1.2) in a volume ratio. The strain mixed liquid is inoculated in a liquid culture medium according to the inoculation amount of 5 percent.

Further, the formula of the liquid culture medium for mixed culture in the step 3) is as follows: 10g of peptone, 5g of beef extract powder, 10g of sodium chloride, 3g of sodium citrate, 2g of KCl and CaCl₂0.2g、MgSO₄·7H₂Dissolving 20g of O in the sterilized water, continuously adding the sterilized water to 1000ml, adjusting the pH value to 4-6, and sterilizing.

The bacteriostatic and odor-removing microbial preparation for pets and living environments thereof can be directly used for household environments, particularly household environments of pet raising families. The product can also be applied to public places such as offices, markets, schools, hospitals and the like, and can also be directly applied to body surfaces, clothes and the like of pets and people.

The invention has the beneficial effects that:

the microbial preparation is prepared by using a culture stock solution of microorganisms, functional live bacteria, nutrient substances of a culture medium, metabolites (such as various sugars, digestive enzymes, organic acids, antibacterial factors and the like) of the functional bacteria in the growth process and derivatives thereof are integrated, and the metabolic activities, the metabolites, the derivatives and the like of the functional bacteria are mutually promoted or restricted, so that all components are in a stable coexistence state, and the stability of the product performance is ensured.

The strain of the microbial preparation has wide energy source and tolerance to various environments, and can be mutually cooperated or restricted. Lactic acid bacteria, saccharomycetes and Neisseria ziliani strains with various advantages are compounded according to the proportion of the invention to form an advantageous group together, so that competitive inhibition among the bacteria with the advantages of the lactic acid bacteria, the saccharomycetes and the Neisseria ziliani strains can be avoided to a certain extent, and a sustainable and stable living environment is created.

The microbial preparation of the invention combines the functional bacteria, and can metabolize and degrade the peculiar smell components. In addition, metabolites of each functional bacteria (such as biological enzymes) also have the effect of degrading the odor components. As is well known, the viable bacteria quantity of the viable bacteria product is reduced along with the storage time until the colony number is reduced to a certain degree, and the bacterial colony quantity is reduced mainly because of the inhibition and toxic and side effects brought by the consumption of energy substances and the accumulation of metabolites.

Detailed Description

In order to better explain the problems to be solved, the technical solutions adopted and the beneficial effects achieved by the technical solutions of the present invention, further description will be given with reference to specific embodiments. It should be noted that the technical solutions of the present invention include, but are not limited to, the following embodiments.

The specific techniques or conditions not specified in the examples of the present invention are performed according to the techniques or conditions described in the literature in the art or according to the product specification. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained by commercial purchase and the like.

The bacteriostatic and odor-removing microbial preparation for pets and living environments thereof comprises functional bacteria lactic acid bacteria, saccharomycetes and Nesterekoni (Nesterekonia); the lactic acid bacteria: yeast: the colony number ratio of the Netheriacico bacteria is (3-5): (8-11): (0.5-1.2), preferably 3:10: 0.9.

In some embodiments, the microbial formulation further comprises a substrate; the substrate mainly comprises nutrient substances (such as nutrient substances in a culture medium and the like) for maintaining survival of the functional bacteria agent, metabolites and derivatives of the functional bacteria during the culture process, such as organic acid, enzymes, antibacterial peptide and the like.

In some embodiments, the lactic acid bacteria include Lactobacillus rhamnosus (L), Lactobacillus chaff (L), Lactobacillus rhamnosus (L), and Lactobacillus paracasei (16-19): 5-7): 2-5), preferably 17:6: 3.

The yeast includes Saccharomyces boulardii (Dekkera bruxellensis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Trichosporon flexneri (Cutaneotrichporon currus), and Candida ethanolica (Candida ethanolica); the Dekkera brussels: and (3) saccharomyces cerevisiae: trichosporon flexuosus: the colony number ratio of the Candida ethanolica is (79-83): (4-6.5): (3-6): (0.5 to 1.2); preferably 80:5: 4.2.

In some embodiments, the microbial preparation has a total colony count of 1-1.8 × 10⁸CFU/ml, preferably 1.3 × 10⁸CFU/ml。

The invention relates to a preparation method of a bacteriostatic and deodorant microbial preparation for pets and living environments thereof, which comprises the following steps:

1) respectively activating each functional bacterium;

2) respectively carrying out propagation culture to obtain first-grade seeds of each functional bacterium;

3) and mixing the functional bacteria obtained in the step 2) and culturing to obtain the functional bacteria.

In some embodiments, the culture method of each functional bacterium in step 2) is: activating each functional bacterium according to conventional operation, culturing in a culture medium, adjusting the pH to 6.5, and culturing at 30-37 ℃ for 24-36 hours; the colonies are ready for use.

In some embodiments, the medium composition for culturing the yeast in step 2) is: 4-10% of molasses, 1-3% of corn steep liquor, 0.1-0.6% of protein, 1-2% of sodium chloride and the balance of water; the pH was 6.5;

in some embodiments, the medium for culturing the lactic acid bacteria in step 2) comprises: 0.5-1.8% of peptone, 1-5% of beef extract, 2-10% of corn steep liquor, 2.8-4.5% of cane sugar, 0.3-1% of sodium acetate, 0.5-2% of magnesium sulfate, 1-8% of sodium chloride and the balance of water; the pH was 6.5;

in some embodiments, the medium formulation for culturing said neisseria meningitidis in step 2) is: 0.3% of sodium citrate, 0.2% of KCl and CaCl₂0.02%, peptone 1%, MgSO4 & 7H₂O2% and agar 1.8%; pH 9.0.

In some embodiments, the specific operation of step 3) includes preparing bacterial suspensions with equal concentration from each functional bacterium obtained by culturing in step 2), mixing and culturing in sterilized liquid culture medium, culturing at 30-37 ℃, and periodically checking until the colony number is not less than 1 × 10⁸CFU/ml.

In some embodiments, the functional bacterial suspension in step 3) is prepared according to the ratio of lactic acid bacteria: yeast: the neisseria melitensis is (3-5): (8-11): (0.5 to 1.2) in a volume ratio. The strain mixed liquid is inoculated in a liquid culture medium according to the inoculation amount of 5 percent.

In some embodiments, the liquid medium formulation for the mixed culture in step 3) is: 10g of peptone, 5g of beef extract powder, 10g of sodium chloride, 3g of sodium citrate, 2g of KCl and CaCl₂0.2g、MgSO₄·7H₂Dissolving 20g of O in the sterilized water, continuously adding the sterilized water to 1000ml, adjusting the pH value to 4-6, and sterilizing.

The bacteriostatic and odor-removing microbial preparation for pets and living environments thereof can be directly used in home environments, can also be applied to public places such as offices, markets, schools, hospitals and the like, and can also be directly used on the body surfaces, clothes and the like of pets and people.

Verification test

Test sample (microbial preparation of the invention) preparation:

1) and activating each functional bacterium (: the functional bacteria include lactobacillus, yeast, and Neisseria; wherein the lactobacillus comprises cold-resistant lactobacillus, lactobacillus rhamnosus and lactobacillus chaff-like bacteria according to the colony count of 17:6: 3; the yeast comprises Dekkera bruxellensis, Saccharomyces cerevisiae, neomyces flexuosus and Candida ethanolica with the colony number of 80:5: 4.2.

2) Respectively carrying out propagation culture to obtain first-grade seeds of each functional bacterium: the components of the culture medium of the lactobacillus are as follows: 1% of peptone, 5% of beef extract, 8% of corn steep liquor, 3.5% of sucrose, 0.3% of sodium acetate, 1% of magnesium sulfate, 1% of sodium chloride and the balance of water; the pH was 6.5; culturing at 37 deg.C for 24 hr; the culture medium of the yeast comprises the following components: 6% of molasses, 1% of corn steep liquor, 0.6% of protein, 1% of sodium chloride and the balance of water; the pH was 6.5; culturing at 37 deg.C for 24 hr; the culture medium formula of the Neisseria ziliani is as follows: 0.3% of sodium citrate, 0.2% of KCl and CaCl₂0.02%, peptone 1%, MgSO4 & 7H₂O2% and agar 1.8%; incubated at 37 ℃ for 24 hours at pH 9.0.

3) Mixing and culturing the functional bacteria obtained in the step 2), namely preparing the functional bacteria obtained in the step 2) into bacterial suspensions respectively, mixing the three bacterial suspensions according to the ratio of the number of the bacterial colonies of lactobacillus, saccharomycetes and Nicejun junceae bacteria of 3:10:0.9, inoculating the bacterial mixture into a liquid culture medium according to the inoculation amount of 5 percent, culturing at 37 ℃, and periodically checking until the number of the bacterial colonies is not less than 1 × 10⁸CFU/ml.

The formula of the liquid culture medium is as follows: 10g of peptone, 5g of beef extract powder, 10g of sodium chloride, 3g of sodium citrate, 2g of KCl and CaCl₂0.2g、MgSO₄·7H₂Dissolving O20 g in sterilized water, adding sterilized water to 1000ml, adjusting pH to 5, and sterilizing.

First, safety evaluation

1.1 acute oral toxicity test

And (4) the prepared sample is sent to a microbiological analysis and detection center of Guangdong province to complete the test.

1.1.1 sample treatment

The test sample (5.0 g) was weighed and diluted to 20m L by adding sterile water.

1.1.2 animal sources

SPF-grade mice of KM variety 20, male and female halves, offered by southern medical university. The weight range is 18-22g, the production license number of the experimental animal is as follows: SCXK (Yue) 2016-; quality certification numbering: 44002100021034.

1.1.3 detection conditions

The animal breeding environment is room temperature (20-26) deg.C, relative humidity is (40-70)%, group cage breeding is carried out, 10 animals are cultured in each cage, and the license number of experimental animals is SYXK (Yue) 2016-.

1.1.4 feed sources

Provided by Beijing Huafukang Biotechnology GmbH. The feed production license number is SCXK (Jing) 2014-; the quality certification number is 1103221900010119.

1.1.5 Experimental methods

The detection basis is as follows: the disinfection specifications (2002 edition) 2.3.1 acute oral toxicity test selects the dose: the dose was designed to be 5000mg/kg BW.

The method briefly comprises the steps of setting 1 dose group according to test requirements, wherein each group of animals comprises 20 animals, each half of the animals comprises a male animal and a female animal, fasting the animals for 16h before the test, freely drinking water, performing oral gavage for 1 time, wherein the average gavage amount is 0.4m L/20 g & BW each time, continuously fasting the animals for 2h after the gavage, then feeding normal diet, observing for 14 days, observing for 1 time every day, and recording the death condition of the mice.

Table 1 acute oral toxicity test table

1.1.6 results of the experiment

During the observation period of 14d, no obvious signs of poisoning were seen in the experimental animals, and no animal death occurred.

TABLE 2 acute oral toxicity test results

According to the results, the microbial preparation is judged to be of actual non-toxic grade according to the classification of acute oral toxicity in the disinfection technical specification.

1.2 acute skin irritation/Corrosion test

The prepared sample is sent to Guangzhou customs technical center to complete the test.

1.2.1 test samples

The invention provides an odor-removing microbial preparation (product name: pet freshener, batch number: 2021.08.12).

1.2.2 animals and feeding conditions

Animal sources: 4 ordinary-grade New Zealand white rabbits are provided by Dongxin Hua laboratory animal farms in Guangzhou city Huadu district, the weight range is 2.21-2.48kg, and the production license number of the laboratory animal is as follows: SCXK (Yue) 2019-: 44007600006702.

feeding conditions are as follows: the feed is provided by the medical experimental animal center of Guangdong province. After the animals are introduced, the animals are raised in a single cage in an animal room of the center, and are used after the quarantine is qualified, and the experimental animals use license numbers: SYXK (yue) 2018-: 18 ℃ -26 ℃, relative humidity: 40 to 70 percent.

1.2.3 test methods

The two sides of the spine of the back of the rabbit are cut off 24 hours before the test, the skin surface is not damaged in the cutting process, and the hair removal range is respectively 3cm × 3 cm.

The test method comprises the steps of taking 0.5ml of a test object, smearing the test object on the skin of one side, smearing the test object with the area of 2.5cm × 2.5.5 cm, covering the test object with two layers of gauze (2.5cm × 2.5.5 cm) and a layer of cellophane, and fixing the test object by using a non-irritant adhesive tape and a bandage, wherein the skin of the other side is not treated as a control, a closed test is adopted, the application time is 4h, the test object is removed by using pure water after the test is finished, the result is observed, the rabbit is graded according to a skin irritation/corrosion standard in a skin irritation/corrosion test table 1 of technical Specification for cosmetic safety (2015 edition), and the control area and the test area are treated in the same.

1.2.4 evaluation of results

Skin reactions at the applied parts were observed at 1, 24, 48, and 72 hours after removal of the test substances, skin reaction scores were made according to the skin irritation/corrosion test table 1 of the technical standards for cosmetic safety (2015 th edition), comprehensive evaluations were made on the average values of the test animal scores, skin irritation intensity was determined according to the highest average value of the scores at each observation time point of 24, 48, and 72 hours according to the skin irritation/corrosion test table 2 of the technical standards for cosmetic safety (2015 th edition), and symptoms other than irritation were observed.

1.2.5 test results

TABLE 3 record of the results of the acute skin irritation test of the test substances on rabbits

Whether there are other symptoms than skin irritation: none.

According to the results, the microbial preparation of the invention is judged to be in a nonirritant grade according to skin irritation strength grading in disinfection technical Specification.

1.3 acute eye irritation test

The prepared sample is sent to Guangzhou customs technical center to complete the test.

1.3.1 materials and methods

And (3) testing a sample: the invention provides an odor-removing microbial preparation (product name: pet freshener, batch number: 2021.08.12).

1.3.2 animal and feeding environments

3 common-grade New Zealand white rabbits with the weight of 2.24 kg-2.36 kg are provided by a Guangzhou Huadu Huazhou Dongxin laboratory animal farm, and the production license number of the laboratory animal is as follows: SCXK (yue) 2019-: no.44007600006702, the feed was provided by the medical laboratory animal center in Guangdong province. The animal is purchased and then raised in a single cage in an animal room, and is used after the quarantine is qualified, and the license number of the experimental animal is as follows: SYXK (yue) 2018-: 18 ℃ to 26 ℃, relative humidity: 40 to 70 percent.

1.3.3 test methods

(1) Both eyes of the test rabbits were examined 24h before the start of the test (including the use of sodium fluorescein), and the eligible rabbits were examined for the test.

(2) Slightly pulling the lower eyelid of one eye of the rabbit, dripping the test object with the thickness of 0.1m L into the conjunctival sac to allow the upper and lower eyelids to be closed passively for 1s to prevent the test object from being lost, leaving the other eye as a control without treatment, and not rinsing the eye within 24h after dripping the test object.

(3) The eyes of the animals were examined at 1h, 24h, 48h and 72h of instillation of the test substance. After 24h observation and recording, all animals were further examined for their eyes with sodium fluorescein. Eye irritation response scores were recorded in each examination according to the evaluation criteria for eye damage in acute eye irritation/corrosivity test table 1 in technical standards for cosmetic safety (2015), and the eye irritation intensity of the test subject was graded and determined according to the highest scores and recovery times at each observation time of 24h, 48h and 72h and according to the acute eye irritation/corrosivity test table 3 in technical standards for cosmetic safety (2015).

1.3.4 test results

TABLE 4 acute eye irritation test results for rabbits

Attached: whether there are other effects than ocular: none.

From the above results, it was found that the microbial preparation for removing an offensive odor of the present invention is non-irritating.

1.4 acute inhalation toxicity test

The prepared sample is sent to Guangzhou customs technical center to complete the test.

Detection standard: reference GB/T21605-

The experimental environment is as follows: SPF-grade laboratory animal house, license number for use of laboratory animals: SYXK (yue) 2018-: the room temperature is 22 +/-1 ℃, and the humidity is 60 +/-5%.

Experimental animals and feed: 20 SPF-level Kunming mice, each half of which is male and female, and the weight of which is 18.0-20.0 g. The experimental animals and the feed are purchased from the medical experimental animal center of Guangdong province, and the production license numbers of the experimental animals are as follows: SCXK (yue) 2018-; animal certification number: no. 4400720006608.

Sample treatment: the microbial preparation stock solution prepared as described above was used as a test substance.

The contamination concentration of the sample: the formal test adopts a one-time limit method, and the contamination concentration is set to be 5000mg/m³20 experimental animals (male and female halves) were inhaled once for 4 h.

The test steps are as follows:

1) the infection mode is as follows: adopting a dynamic contamination method, and inhaling contamination for 4h by using an HOPE-MED 8050 contamination device at a time;

2) contamination conditions: the inlet flow rate is about 3.6m³Per h, the volume of the contamination cabinet is 0.3m³Cumulative intake of 14.7m³The temperature in the cabinet is 22 +/-1 ℃, the humidity in the cabinet is 55-85%, the oxygen concentration is 20 +/-0.5%, the relative density of the liquid phase for the test is 1004mg/m L, and the food is fasted and forbidden during the contamination period and is normally taken after the completion.

3) Observing period and index, recording poisoning symptom and death condition after 14 days of experimental observation, weighing live animals every week during observation period, weighing live animals after observation period, performing autopsy after death, calculating L C₅₀。

Results of the experiment

No obvious toxic symptom and death of the experimental animal can be observed within 14 days of observation, no abnormality of the general anatomy of the experimental animal can be observed, so that the female and male mice can inhale L C infected with toxin once for 4h₅₀>5000mg/m³. The results of this acute inhalation toxicity test are shown in Table 5.

TABLE 5 test results of acute inhalation toxicity of test substances on mice

L C of the microbial preparation for removing peculiar smell of the invention₅₀4h is more than 5000mg/m³。

1.5 detection of harmful bacteria

According to the technical specification for safety of cosmetics (2015 edition), the microbial preparation prepared by the method is subjected to harmful bacteria detection, and the target harmful bacteria are as follows: heat-resistant coliform bacteria, staphylococcus aureus and pseudomonas aeruginosa. None of the results were detected.

According to the regulations of GB4789.4-2016 and GB4789.6-2016, the microbial preparation of the invention is subjected to harmful bacteria detection, and target harmful bacteria are as follows: salmonella, Escherichia coli. None of the results were detected.

Second, functional verification

1. Removal rate of hydrogen sulfide

With reference to the regulations of GB/T11742-89 and QB/T2761-2006, two sets of tests are respectively set up: the first group is a blank test chamber 1 (blank chamber 1) and a sample test chamber 1 (sample chamber 1); the second group is a blank test chamber 2 (blank chamber 2) and a sample test chamber 2 (sample chamber 2). All test chambers have a volume of 1.5m³。

The test method comprises the following steps: the blank test chamber and the sample test chamber of each group are filled with hydrogen sulfide gas with the same initial concentration, and then the prepared microbial preparation sample is added into each sample test chamber according to the standard specification, and no sample is added into the blank test chamber. After standing for 24 hours, the hydrogen sulfide gas concentrations in the respective chambers were measured as shown in Table 6.

TABLE 6 record of experimental data on hydrogen sulfide removal

The test is finished by the microbiological detection center of Guangdong province.

2. Removal rate of ammonia

With reference to the regulations of GB/T11742-89 and QB/T2761-2006, two sets of tests are respectively set up: the first group is a blank test chamber 1 (blank chamber 1) and a sample test chamber 1 (sample chamber 1); the second group is a blank test chamber 2 (blank chamber 2) and a sample test chamber 2 (sample chamber 2). All test chambers have a volume of 1.5m³。

The test method comprises the following steps: and introducing ammonia gas with the same initial concentration into the blank test chamber and the sample test chamber of each group, adding the prepared microbial preparation sample into each sample test chamber according to standard regulation, wherein no sample is added into the blank test chamber. After standing for 24 hours, the ammonia gas concentrations in the chambers were measured as shown in Table 7.

TABLE 7 record of experimental data on ammonia removal rate

The test is finished by the microbiological detection center of Guangdong province.

3. Escherichia coli bacteriostasis experiment

The results of the bacteriostasis experiments on the Escherichia coli are carried out according to the specification of QB/T2738-2012 (7.3) and are shown in Table 8.

TABLE 8 antibacterial protocol for Escherichia coli

As can be seen from the above experiments, the microbial preparation of the present invention has an inhibitory effect of more than 99% on Escherichia coli.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A bacteriostatic odor-removing microbial preparation for pets and their living environments is characterized by comprising lactic acid bacteria, yeast and Neisseria; the lactic acid bacteria: yeast: the colony number ratio of the Netheriacico bacteria is (3-5): (8-11): (0.5 to 1.2).

2. The bacteriostatic odor-reducing microbial preparation for pets and living environments thereof according to claim 1, wherein said lactic acid bacteria comprise cold-resistant lactic acid bacteria, lactobacillus rhamnosus, lactobacillus chaff-like bacteria; the cold-resistant lactic acid bacteria: lactobacillus rhamnosus: the ratio of the number of colonies of the Lactobacillus chaff-like bacteria is (16-19): (5-7): 2-5).

3. The bacteriostatic odor-reducing microbial preparation for pets and living environments thereof according to claim 1, wherein said yeasts comprise Dekkera bruxellensis, Saccharomyces cerevisiae, Neomyceliophthora curvata, Candida ethanolica; the Dekkera brussels: and (3) saccharomyces cerevisiae: trichosporon flexuosus: the colony number ratio of the Candida ethanolica is (79-83): (4-6.5): (3-6): (0.5 to 1.2).

4. The bacteriostatic odor-reducing microbial preparation for pets and living environments thereof according to claim 1, wherein the total colony count of the microbial preparation is not less than 1 × 10⁸CFU/ml。

5. A preparation method of a bacteriostatic and odor-removing microbial preparation for pets and living environments thereof is characterized by comprising the following steps:

1) respectively activating each functional bacterium; the functional microbial inoculum comprises lactic acid bacteria, saccharomycetes and Neisseria;

2) respectively carrying out amplification culture on the functional bacteria in the step 1) to obtain first-grade seeds of the functional bacteria;

3) and (3) performing mixed culture on the primary seeds of the functional bacteria obtained in the step 2).

6. The method for preparing bacteriostatic and odor-removing microbial preparation for pets and living environments thereof according to claim 5, wherein said lactic acid bacteria comprise cold-resistant lactic acid bacteria, Lactobacillus rhamnosus, Lactobacillus chaff-like bacteria; the cold-resistant lactic acid bacteria: lactobacillus rhamnosus: the ratio of the number of colonies of the Lactobacillus chaff-like bacteria is (16-19): (5-7): 2-5); the yeast comprises Dekkera bruxellensis, Saccharomyces cerevisiae, neomyces flexuosus and Candida ethanolica; the Dekkera brussels: and (3) saccharomyces cerevisiae: trichosporon flexuosus: the colony number ratio of the Candida ethanolica is (79-83): (4-6.5): (3-6): (0.5 to 1.2).

7. The method for preparing bacteriostatic and deodorant microbial preparations for pets and living environments thereof according to claim 5, wherein in the step 2), the yeast culture medium comprises the following components: 4-10% of molasses, 1-3% of corn steep liquor, 0.1-0.6% of protein, 1-2% of sodium chloride and the balance of water; the pH was 6.5;

the lactic acid bacteria culture medium comprises the following components: 0.5-1.8% of peptone, 1-5% of beef extract, 2-10% of corn steep liquor, 2.8-4.5% of cane sugar, 0.3-1% of sodium acetate, 0.5-2% of magnesium sulfate, 1-8% of sodium chloride and the balance of water; the pH was 6.5;

the culture medium formula of the Netheria gasseri comprises: 0.3 percent of sodium citrate, 0.2 percent of KCl and CaCl₂0.02%, peptone 1%, MgSO4 & 7H₂O2%, agar 1.8% and sodium chloride 5%; pH 9.0.

8. The method for preparing the bacteriostatic and odor-removing microbial preparation for the pets and the living environments thereof as claimed in claim 5, wherein the step 3) comprises respectively preparing the functional bacteria cultured in the step 2) into bacterial suspensions, mixing and inoculating the bacterial suspensions into a liquid culture medium, and culturing at 30-37 ℃ until the colony number is not less than 1 × 10⁸CFU/ml.

9. The method for preparing bacteriostatic and odor-removing microbial preparation for pets and living environments thereof according to claim 8, wherein the bacterial suspension of the functional bacteria in the step 3) is prepared according to the following steps of lactic acid bacteria: yeast: the neisseria melitensis is (3-5): (8-11): (0.5-1.2) mixing in a volume ratio; then inoculating the strain into a liquid culture medium for culture according to the inoculation amount of 5%.

10. The method for preparing bacteriostatic and deodorant microbial preparations for pets and living environments thereof according to claim 8, wherein the liquid medium is prepared by the following steps: 10g of peptone, 5g of beef extract powder, 10g of sodium chloride, 3g of sodium citrate, 2g of KCl and CaCl₂0.2g、MgSO₄·7H₂Dissolving 20g of O in the sterilized water, continuously adding the sterilized water to 1000ml, adjusting the pH value to 4-6, and sterilizing.