CN114292791A - Composite microbial deodorant and application thereof - Google Patents
Composite microbial deodorant and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of environment-friendly deodorizers, in particular to a composite microbial deodorant and application thereof. The method comprises the following steps: 10-15 parts of bacillus subtilis, 10-15 parts of bacillus belgii, 3-5 parts of pseudomonas fluorescens, 3-5 parts of pseudomonas defensins, 1-2 parts of metarhizium anisopliae, 1-2 parts of hansenula polymorpha, 2-3 parts of lavender extract, 1-2 parts of complex enzyme, 0.1-0.2 part of xanthan gum and 50-70 parts of sucrose; adopting the bacillus subtilis with the preservation number of CGMCC No. 2108; adopting pseudomonas fluorescens with the preservation number of CGMCC No. 8820; adopts the preservation number of CGMCC No.18750 to defend pseudomonas. The composite microbial deodorant has high biological activity, rapid onset of action, stable quality and good deodorizing effect.
Description
Technical Field
The invention relates to the technical field of environment-friendly deodorizers, in particular to a composite microbial deodorant and application thereof.
Background
The livestock and poultry breeding industry in China is rapidly changed to ecological breeding, and along with the continuous improvement of the living quality and environmental awareness of people, how to efficiently and environmentally solve the problem of livestock and poultry excrement pollution becomes a current research hotspot. The livestock manure can generate a large amount of NH in the discharging and storing processes3、H2More than 200 malodorous gases such as S, osmyl, dimethyl sulfide and the like have strong pungent peculiar smell, not only have unpleasant feelings such as psychological aversion caused by influence on smell, but also have great harm to human bodies, and can enter human bodies through different ways such as respiratory tract, eyes, skin and the like, so that people are unconscious and uncomfortable. When the air purifier is placed in the air purifier for a long time, the air purifier greatly damages the nervous system of a human body and seriously affects the quality of the ambient air. How to effectively control and treat malodorous gases is increasingly gaining attention.
At present, the deodorant commonly used in livestock and poultry industry mainly comprises a physical deodorant, a chemical deodorant and a microbial deodorant. The physical deodorant absorbs the odor substances into micropores of the deodorant, but the deodorant is difficult to desorb after absorbing the odor substances, cannot be reused and is difficult to treat, so that secondary pollution to the environment is easily caused; the chemical deodorant has the characteristics of strong pertinence, quick response and the like, can cover odor, but cannot change the generation of the odor or prevent the odor from being emitted, and meanwhile, components such as used aromatic and the like sometimes bring unpleasant odor due to too strong shielding of spice and easily cause damage to objects and devices; the microbial deodorant converts substances with odor through the physiological metabolism of microorganisms, has the advantages of environmental protection and reutilization compared with a physical deodorant and a chemical deodorant, and becomes a main method for the research and application of odor prevention at home and abroad. However, the existing microbial deodorant still has the defects of single strain, low viable count, slow effect, non-lasting efficacy and the like, and is difficult to play a role in a short time and a long time.
The ecological breeding urgently needs a high-efficiency, safe and environment-friendly microorganism rapid deodorization technology.
Disclosure of Invention
The first purpose of the invention is to provide a compound microbial deodorant which can play a role in deodorization in a short time and reduce the stink generated in the stacking process of livestock and poultry manure. The invention also provides application of the composite microbial deodorant in deodorization of livestock and poultry manure or public places.
The invention provides a composite microbial deodorant which comprises the following raw materials in parts by weight: 10-15 parts of bacillus subtilis, 10-15 parts of bacillus belgii, 3-5 parts of pseudomonas fluorescens, 3-5 parts of pseudomonas defensins, 1-2 parts of metarhizium anisopliae, 1-2 parts of hansenula polymorpha, 2-3 parts of lavender extract, 1-2 parts of complex enzyme, 0.1-0.2 part of xanthan gum and 50-70 parts of sucrose;
wherein the bacillus subtilis adopts bacillus subtilis with the preservation number of CGMCC No. 2108; the pseudomonas fluorescens with the preservation number of CGMCC No.8820 is adopted as the pseudomonas fluorescens; the defensive pseudomonas adopts the defensive pseudomonas with the preservation number of CGMCC No. 18750.
Preferably, the Bacillus belgii is separated from a compost sample at 81-82 ℃ in a cow dung composting high-temperature stage; more preferably 81.5 deg.c.
Preferably, the viable count of the bacillus subtilis is 1 × 109～5.2×109CFU/g；
The viable count of the Bacillus belgii is 1 × 109～5×109CFU/g；
The viable count of the pseudomonas fluorescens is 1 multiplied by 108～5.5×108CFU/g；
The number of viable bacteria of the defense pseudomonas is 1 multiplied by 108～5.4×108CFU/g；
The viable count of the metarhizium anisopliae is 0.9 multiplied by 106～5×106CFU/g；
The number of viable bacteria of the Hansenula is 1 multiplied by 106～6.4×106CFU/g。
Preferably, the complex enzyme is a mixture of protease, cellulase and lipase; wherein the mass ratio of the protease to the cellulase to the lipase is 2:1:2-2.2:1.2: 2.2.
Preferably, the enzyme activities of the protease, the cellulase and the lipase in the compound enzyme are all 9.5-20.5 ten thousand U/g.
The composite microbial deodorant provided by the invention is applied to deodorization of livestock and poultry manure or public places.
Preferably, the public places are public toilets, garbage dumps, garbage transfer stations and hotels.
Preferably, the required composite microbial deodorant is calculated according to the volume ratio of the composite microbial deodorant to the target material to be deodorized being 1:10000-1:10100, then diluted by 10-10.2 times, uniformly sprayed on the target material, and uniformly stirred and mixed; more preferably, the required composite microbial deodorant is calculated according to the volume ratio of the composite microbial deodorant to the target material to be deodorized being 1:10000 (0.1 kg of deodorant is added per cubic meter), then diluted by 10 times, uniformly sprayed on the target material, and stirred and mixed uniformly.
The deodorization principle of the composite microbial deodorant provided by the invention is as follows:
firstly, the composite microbial deodorant provided by the invention comprises a composite enzyme and a plurality of microbial bacteria, and has the advantages of high biological activity, quick response and good deodorization effect. The livestock and poultry manure contains protein, carbohydrate and the like, the protein decomposes malodorous gases such as ammonia gas, hydrogen sulfide, methylamine and the like under the anaerobic condition, and the concentration of ammonia discharged into the air is too high to form acid precipitation, thereby causing water body and soil pollution. The decomposition of carbohydrates into greenhouse gases such as methane, carbon dioxide and the like causes global greenhouse effect. The invention adopts the compound enzyme as the scientifically compounded protease (the protease in the invention adopts neutral protease), cellulose and lipase, catalyzes and degrades macromolecular organic substances such as protein, cellulose, fat and other foul pollutants, catalyzes and degrades the macromolecular organic substances into micromolecular organic substances such as micromolecular monosaccharide, amino acid, fatty acid, acetic acid and the like which are easy to be decomposed by microorganisms, not only can reduce the generation of odor, but also can provide a nutrient substrate for flora. In addition, the substrate sucrose provided by the system provides starting energy for the strain before decomposition.
Secondly, various microbial bacteria in the composite microbial deodorant provided by the invention are synergistic, so that the composite microbial deodorant has strong deodorization effect. On the basis of the catalytic degradation of the composite enzyme, beneficial aerobic bacteria of bacillus subtilis CGMCC No.2108, bacillus belgii, pseudomonas fluorescens CGMCC No.8820 and defensive pseudomonas CGMCC No.18750 are used in a matching way, so that the malodorous pollutants are further degraded in a synergistic way, and the combined action of the four bacteria can degrade various malodorous pollutants which are difficult to degrade, and can prevent and control the reoccurrence of malodorous pollutants. Wherein the bacillus subtilis CGMCC No.2108 and the Bacillus beleisi mainly play a role in fixing and degrading H2S、NH3The two strains have wide growth temperature range, are resistant to high temperature, extreme pH and high salt, and can stably play a role in deodorization under the extreme conditions of multiple environments. Simultaneously withered grass budsActive substances such as subtilin, polymyxin, nystatin, brevibacillin and the like generated by the bacillus CGMCC No.2108 during the growth process have obvious inhibition effect on putrefying bacteria in garbage, and can reduce the generation of ammonia and hydrogen sulfide. In addition, pseudomonas fluorescens CGMCC No.8820 is a low-temperature bacterium, can normally grow at the low temperature of 5-10 ℃, can improve the bacterial count of bacillus subtilis and bacillus belgii and the effect of a deodorant at the low temperature, has an important auxiliary effect on degrading odor substances, is also an important disease biocontrol bacterium and rhizosphere growth-promoting bacterium, and can effectively colonize rhizosphere, generate secondary resistance metabolites and induce plant system resistance after excrement is decomposed and applied to the land. The composite material has an important auxiliary effect on degrading odor substances, and metabolites of the composite material contain various antibacterial substances, so that various harmful bacteria and plant pathogens can be inhibited. The defensive pseudomonas CGMCC No.18750 can improve the speed of decomposing odor by microorganisms and enzymes in the system and plays an important auxiliary role in the system. But also an important plant growth-promoting rhizobacteria, can secrete a plurality of active substances with broad-spectrum antibacterial capability, such as pyoluteorin, phenazine-1-carboxylic acid, nitropyrrolidin and the like, can also secrete some insecticidal toxins for defending pseudomonas besides antibacterial substances, and has more outstanding biological control effect compared with other pseudomonas. The Hansenula polymorpha can decompose and utilize saccharides, hydrogen sulfide, ammonia gas and the like in the environment, volatile fragrant substances such as alcohols, esters, phenols, ketones, aromatics and the like are generated in the metabolic process, odor substances are effectively covered, and the odor substances and the lavender extract are cooperated to effectively cover the odor and generate pleasant odor.
Thirdly, metarhizium anisopliae is a broad-spectrum pathogenic insect fungus, insects are killed by secreting toxin, the metarhizium anisopliae also has good infection and killing effects on fly eggs and larvae, and pests can be infected repeatedly in biological control, so that the method has incomparable advantages. Can effectively reduce the number of fly larvae in excrement and further improve the environment of a farm. The defense pseudomonads and the metarhizium anisopliae synergistically kill mosquito and fly larvae and ova, and the number of mosquitoes and flies is effectively reduced while deodorization is carried out.
The invention provides a compositeThe microbial deodorant has stable quality, long storage period and convenient use, firstly removes odor through the rapid catalytic degradation and odor conversion inhibition functions of the complex enzyme, and then deeply degrades odor pollutants through microbial flora. The bacillus subtilis and the Bacillus belgii play an important role in degradation, so the addition amount is the highest, and the number of living bacteria in the system is 1 multiplied by 109～5×109CFU/g. The pseudomonas fluorescens and the defending pseudomonas play important auxiliary roles, so the addition amount is the next time, the number of viable bacteria in the system is 1 multiplied by 108～5×108CFU/g; hansenula polymorpha and metarhizium anisopliae have important effects of producing fragrance and killing fly larvae, so that the number of viable bacteria in the system is 1 × 106～5×106CFU/g; the added xanthan gum is used as a dispersing agent, so that the prepared deodorant has good dispersibility, the suspension effect of the deodorant can be improved, and the deodorization effect is enhanced. The composite microbial deodorant provided by the invention is nontoxic, harmless, free of side effects, free of secondary pollution to the environment, wide in application range, capable of adapting to various temperature, salinity and pH ranges, suitable for livestock and poultry breeding and treatment plants, public toilets, garbage stations, garbage transfer stations, hotels and hotels, and capable of treating livestock and poultry excrement, human excrement and kitchen garbage.
The Bacillus subtilis (Bacillus subtilis) adopted by the invention is preserved in China general microbiological culture Collection center (CGMCC) in 13.7.2007, the preservation number is CGMCC No.2108, and the CGMCC is disclosed in Chinese patent document CN 101109010B; pseudomonas fluorescens (Pseudomonas florescens) is preserved in the China general microbiological culture Collection center of China general microbiological culture Collection center (CGMCC) at 2.17.2014, and the preservation number is CGMCC No.8820, which is disclosed in Chinese patent document CN 104152380B; defensive Pseudomonas (Pseudomonas proteins) is deposited in the common microorganism center of China general microbiological culture Collection center (CGMCC) at 2019, 10 and 28 months, with the preservation number of CGMCC No.18750, and is disclosed in Chinese patent document CN 112410247A. The growth temperature range of the bacillus subtilis CGMCC No.2108 is as follows: 20-55 ℃, pH range: 5-11, NaCl resistance range: 0 to 13 percent. The bacillus beleisi is separated from a heap sample at 81.5 ℃ in the high-temperature stage of cow dung compost, and the growth temperature range of the bacillus beleisi is as follows: 25-50 ℃, pH range 4-9, NaCl resistance range: 0-11% of acid-resistant, salt-resistant and high-temperature-resistant bacterial strain. Metarhizium anisopliae, Hansenula polymorpha, lavender extract, compound enzyme and xanthan gum are commercially available products. The lavender extract adopted by the invention can be replaced by essence.
In summary, the invention has the following advantages:
(1) the composite microbial deodorant provided by the invention contains the composite enzyme and a plurality of microbial bacteria, and has the advantages of high biological activity, quick response and good deodorization effect.
(2) The composite microbial deodorant provided by the invention is suitable for quickly deodorizing livestock and poultry manure, and can also be used for quickly deodorizing public toilets, garbage dumps, garbage transfer stations, hotels and restaurants.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A composite microbial deodorant is prepared from the following raw materials in parts by weight: 15 parts of bacillus subtilis, 15 parts of bacillus belgii, 5 parts of pseudomonas fluorescens, 5 parts of pseudomonas defensins, 2 parts of metarhizium anisopliae, 2 parts of hansenula polymorpha, 3 parts of lavender extract, 2 parts of complex enzyme, 0.2 part of xanthan gum and 51 parts of sucrose;
wherein the Bacillus subtilis adopts the Bacillus subtilis with the preservation number of CGMCC No. 2108; the pseudomonas fluorescens with the preservation number of CGMCC No.8820 is adopted as the pseudomonas fluorescens; the defense pseudomonads adopt the defense pseudomonads with the preservation number of CGMCC No. 18750;
the compound enzyme adopts a mixture of neutral protease, cellulase and lipase in a mass ratio of 2:1:2, wherein the enzyme activity of the neutral protease is 9.86 ten thousand U/g, the enzyme activity of the cellulase is 20.03 ten thousand U/g, and the enzyme activity of the lipase is 10.2 ten thousand U/g.
The bacillus belgii sample provided in this example was collected from a cow dung compost high temperature stage of 81.5 ℃ heap of qinhuang island lead kangdi agricultural technology limited company, and the sample was brought back to the laboratory and then strain isolation was completed within 24 hours: putting the sample into a sterile mortar, fully grinding the sample, taking 1g of the ground sample, putting the ground sample into sterile physiological saline, and performing activated shaking culture at 37 ℃ and 180r/min for 2 h; diluting the activated bacteria liquid to 10 of the total bacteria number-5、10-6After doubling, the strains are subjected to salt-resistant, acid-resistant and high-temperature-resistant strain separation in two modes of coating and streaking respectively, the strains are cultured in an LB (LB) culture medium (pH 5 and 10% NaCl) for 2 days at 45 ℃, and the strains are obtained after deodorization effect verification and are identified as Bacillus belgii by 16 s. The growth temperature range of the Bacillus belgii is as follows: 25-50 ℃, growth pH range of 4-9, NaCl resistance range: 0 to 11 percent.
The number of viable bacteria of each kind of bacteria in the composite microbial deodorant provided in this embodiment is as follows:
the viable count of Bacillus subtilis is 5.2 × 109CFU/g，
The viable count of Bacillus belgii is 4.8 × 109CFU/g，
The viable count of the pseudomonas fluorescens is 5.5 multiplied by 108CFU/g，
The number of viable bacteria for protecting against Pseudomonas is 5.4 × 108CFU/g，
The viable count of Metarhizium anisopliae is 4.6 × 106CFU/g，
The number of viable bacteria of Hansenula polymorpha is 6.4 × 106CFU/g。
The preparation method of the composite microbial deodorant of the embodiment is as follows:
(1) respectively fermenting the bacillus subtilis and the bacillus belgii by LB culture medium liquid to a stable stage, respectively adding dextrin accounting for 4% of the mass of the bacteria liquid and anhydrous sodium sulphate accounting for 6% of the mass of the bacteria liquid, and then spraying powder at 150 ℃ to prepare bacillus subtilis powder and bacillus belgii powder; fermenting the liquid of LB culture medium for pseudomonas fluorescens and the liquid of YPD culture medium for hansenula polymorpha to a stationary phase, centrifuging the liquid of LB culture medium and YPD culture medium for 10-20min at 8000rpm of 5000-; culturing the defensive pseudomonas by using an LB culture medium to a stationary phase, inoculating the defensive pseudomonas into a defensive pseudomonas solid fermentation culture medium for culturing to obtain a defensive pseudomonas solid fermentation product, and drying to obtain defensive pseudomonas bacteria powder; culturing Metarhizium anisopliae with PDA culture medium to stationary phase, inoculating into Metarhizium anisopliae solid fermentation culture medium, culturing to obtain Metarhizium anisopliae solid fermentation product, and drying to obtain Metarhizium anisopliae powder; the culture temperatures of the bacillus subtilis and the bacillus belgii are both 37 ℃, the culture temperature of the metarhizium anisopliae is 25 ℃, and the culture temperatures of the pseudomonas fluorescens, the pseudomonas defensins and the hansenula are all 30 ℃;
the LB culture medium comprises the following formula: 5.0g/L yeast extract, 10.0g/L, NaCI 5.0.0 g/L tryptone and 1.0g/L glucose, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 30 min;
the formula of the PDA culture medium is as follows: 20.0g/L of sucrose and 200g/L of potatoes, adjusting the pH value to 7.5-8.0, and sterilizing at 121 ℃ for 30 min;
the YPD medium formula is as follows: yeast extract 10.0g/L, peptone 20.0g/L, glucose 20.0g/L, adjusting pH value to 7.2-7.4, sterilizing at 115 deg.C for 30 min;
the formula of the culture medium for defending the pseudomonas solid fermentation is as follows: 10g/L of glucose, 6g/L of maltose, 6g/L of peptone, 5g/L of ammonium sulfate, 0.3g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 0.3g/L of dipotassium phosphate, 70g/L of rice hulls, 70g/L of corn flour, 12g/L of soybean meal, 300g/L of bran and 30g/L of light calcium carbonate, adjusting the pH value to 7.2-7.4, and sterilizing at 115 ℃ for 30 min; the water content of the solid fermentation medium is 50% of the weight of the solid fermentation medium;
the formula of the metarhizium anisopliae solid fermentation culture medium is as follows: 300g/L of wheat bran, 150g/L of corn flour, 3g/L of monopotassium phosphate, 3g/L of magnesium sulfate and 0.1g/L of copper sulfate, adjusting the pH value to 5.5-6.0, and sterilizing at 121 ℃ for 30 min; the water content of the solid fermentation medium is 50% of the weight of the solid fermentation medium; (2) uniformly mixing the prepared bacillus subtilis powder, pseudomonas fluorescens powder, pseudomonas defense powder, bacillus belgii powder, metarhizium anisopliae powder and hansenula polymorpha powder, and then adding lavender extract, compound enzyme and xanthan gum and uniformly mixing to obtain the compound microbial deodorant.
The application of the composite microbial deodorant provided by the embodiment is deodorization in livestock and poultry manure or public places, and the public places are public toilets, garbage dumps, garbage transfer stations and hotels. It is used in deodorization according to the following method: calculating the required dosage of the composite microbial deodorant according to the volume ratio of the composite microbial deodorant to the target material to be deodorized being 1:10000, then diluting the dosage by 10 times with water, uniformly spraying the dosage on the target material, and uniformly stirring and mixing the dosage.
The complex enzyme and the prepared complex microbial deodorant in the embodiment 1 of the invention are tested as follows:
test I, compound enzyme auxiliary deodorization effect test
The method comprises the steps of mixing bacterial liquids obtained by culturing bacillus subtilis and bacillus belief for two days according to the mass ratio of 15%, inoculating pig manure, adding cellulase (purchased from Shandong and Zhongkang-sourced bioscience Co., Ltd.), lipase (purchased from Shandong and Zhongkang-sourced bioscience Co., Ltd.), neutral protease (purchased from Shandong and Zhongkang-sourced bioscience Co., Ltd.) and complex enzyme containing the three enzymes into the pig manure according to the final concentration of 50U/g, culturing at 35 ℃, measuring experimental data every 24h by using a SMART SOR AR8500 ammonia detector and a BH-90 hydrogen sulfide detector, and obtaining the measurement results shown in tables 1-2.
TABLE 1 different enzymes vs NH3Influence of production amount
TABLE 2 different enzyme pairs H2Influence of S production
As is clear from tables 1 and 2, neutral protease, lipase and cellulase were treated with NH3And H2The production of S has obvious auxiliary reduction effect, the effect of the compound enzyme containing the three enzymes is optimal, and the deodorization effect of the strain can be effectively enhanced.
Test two, suspension ratio in different dispersants
And (4) performing performance tests on different dispersants on the basis of the optimal addition amount. Adding four dispersants of 1 thousandth xanthan gum, 3 thousandth sodium carboxymethylcellulose, 3 thousandth sodium tripolyphosphate and 3 thousandth PEG6000 into the composite microbial deodorant, preparing related reagents according to the national standard GBT 14825-.
TABLE 3 different dispersant suspension ratios
| Dispersing agent | Blank space | Xanthan gum | Sodium carboxymethylcellulose | Sodium tripolyphosphate | PEG6000 |
| Adding amount of | 0 | 1‰ | 3‰ | 3‰ | 3‰ |
| Suspension rate | 57.63％ | 94.83％ | 73.30％ | 59.56％ | 57.78％ |
As can be seen from table 3, compared with the blank and the other three dispersants, the suspension rate of xanthan gum is the highest, reaching 94.83%, which shows that the addition of a proper amount of xanthan gum can effectively improve the suspension ability and the dispersion degree of the microbial deodorant in water.
Third, testing the effect on the toxicity of housefly larvae
Diluting the composite microbial deodorant by 300 times with water, adding 1mL of the diluted composite microbial deodorant into a 9mm culture dish, adding about 20mL of PDA semisolid culture medium, and uniformly mixing to obtain the mixed culture medium. 20 housefly larvae are picked, washed by sterile water, inoculated into the cooled mixed culture medium and cultured at 25 ℃. Mortality of housefly larvae was observed every 24h and the test results are shown in table 4.
TABLE 4 killing effect on housefly larvae
As can be seen from Table 4, the mortality of the housefly larvae reaches 48% after the composite microbial deodorant is used for 48 hours, which indicates that the composite microbial deodorant has a good inhibition effect on the housefly reproduction.
Test of the promoting effects of Pseudomonas fluorescens on Bacillus subtilis and Bacillus belgii
Preparing a liquid LB culture medium, inoculating strains with the same quantity according to different treatments in the table 5, culturing for 2 days at the constant temperature of 30 +/-0.5 ℃, and counting the bacillus subtilis and the bacillus belgii respectively.
TABLE 5 number of Bacillus subtilis and Bacillus belgii inoculated alone and co-cultured
As is clear from Table 5, the number of viable bacteria of Pseudomonas fluorescens was effectively increased in the co-cultured state with Bacillus subtilis and Bacillus belgii.
Example 2
A composite microbial deodorant is prepared from the following raw materials in parts by weight: 13 parts of bacillus subtilis, 13 parts of bacillus belgii, 4 parts of pseudomonas fluorescens, 4 parts of pseudomonas defensins, 1.5 parts of metarhizium anisopliae, 1.5 parts of hansenula polymorpha, 2.5 parts of lavender extract, 1.5 parts of complex enzyme, 0.15 part of xanthan gum and 59 parts of sucrose;
wherein the Bacillus subtilis adopts the Bacillus subtilis with the preservation number of CGMCC No. 2108; the pseudomonas fluorescens with the preservation number of CGMCC No.8820 is adopted as the pseudomonas fluorescens; the defense pseudomonads adopt the defense pseudomonads with the preservation number of CGMCC No. 18750;
the compound enzyme adopts a mixture of neutral protease, cellulase and lipase in a mass ratio of 2:1:2, wherein the enzyme activity of the neutral protease is 9.9 ten thousand U/g, the enzyme activity of the cellulase is 19.3 ten thousand U/g, and the enzyme activity of the lipase is 9.8 ten thousand U/g.
The bacillus belgii sample provided in this example was collected from a cow dung compost high temperature stage of 81.5 ℃ heap of qinhuang island lead kangdi agricultural technology limited company, and the sample was brought back to the laboratory and then strain isolation was completed within 24 hours: putting the sample into a sterile mortar, fully grinding the sample, taking 1g of the ground sample, putting the ground sample into sterile physiological saline, and performing activated shaking culture at 37 ℃ and 180r/min for 2 h; diluting the activated bacteria liquid to 10 of the total bacteria number-5、10-6After doubling, the strains are subjected to salt-resistant, acid-resistant and high-temperature-resistant strain separation in two modes of coating and streaking respectively, the strains are cultured in an LB (LB) culture medium (pH 5 and 10% NaCl) for 2 days at 45 ℃, and the strains are obtained after deodorization effect verification and are identified as Bacillus belgii by 16 s. The growth temperature range of the Bacillus belgii is as follows: 25-50 ℃, growth pH range of 4-9, NaCl resistance range: 0 to 11 percent.
The number of viable bacteria of each kind of bacteria in the composite microbial deodorant provided in this embodiment is as follows:
the viable count of Bacillus subtilis is 3.2 × 109CFU/g，
The viable count of Bacillus belgii is 3.1 × 109CFU/g，
The viable count of the pseudomonas fluorescens is 2.8 multiplied by 108CFU/g，
The number of viable bacteria for protecting against Pseudomonas is 2.9 × 108CFU/g，
The viable count of Metarhizium anisopliae is 3.0 × 106CFU/g，
The number of viable bacteria of Hansenula polymorpha is 3.5 × 106CFU/g。
The preparation method of the composite microbial deodorant of the embodiment is as follows:
(1) respectively fermenting the bacillus subtilis and the bacillus belgii by LB culture medium liquid to a stable stage, respectively adding dextrin accounting for 4% of the mass of the bacteria liquid and anhydrous sodium sulphate accounting for 6% of the mass of the bacteria liquid, and then spraying powder at 150 ℃ to prepare bacillus subtilis powder and bacillus belgii powder; fermenting the liquid of LB culture medium for pseudomonas fluorescens and the liquid of YPD culture medium for hansenula polymorpha to a stationary phase, centrifuging the liquid of LB culture medium and YPD culture medium for 10-20min at 8000rpm of 5000-; culturing the defensive pseudomonas by using an LB culture medium to a stationary phase, inoculating the defensive pseudomonas into a defensive pseudomonas solid fermentation culture medium for culturing to obtain a defensive pseudomonas solid fermentation product, and drying to obtain defensive pseudomonas bacteria powder; culturing Metarhizium anisopliae with PDA culture medium to stationary phase, inoculating into Metarhizium anisopliae solid fermentation culture medium, culturing to obtain Metarhizium anisopliae solid fermentation product, and drying to obtain Metarhizium anisopliae powder; the culture temperatures of the bacillus subtilis and the bacillus belgii are both 37 ℃, the culture temperature of the metarhizium anisopliae is 25 ℃, and the culture temperatures of the pseudomonas fluorescens, the pseudomonas defensins and the hansenula are all 30 ℃;
the LB culture medium comprises the following formula: 5.0g/L yeast extract, 10.0g/L, NaCI 5.0.0 g/L tryptone and 1.0g/L glucose, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 30 min;
the formula of the PDA culture medium is as follows: 20.0g/L of sucrose and 200g/L of potatoes, adjusting the pH value to 7.5-8.0, and sterilizing at 121 ℃ for 30 min;
the YPD medium formula is as follows: yeast extract 10.0g/L, peptone 20.0g/L, glucose 20.0g/L, adjusting pH value to 7.2-7.4, sterilizing at 115 deg.C for 30 min;
the formula of the culture medium for defending the pseudomonas solid fermentation is as follows: 10g/L of glucose, 6g/L of maltose, 6g/L of peptone, 5g/L of ammonium sulfate, 0.3g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 0.3g/L of dipotassium phosphate, 70g/L of rice hulls, 70g/L of corn flour, 12g/L of soybean meal, 300g/L of bran and 30g/L of light calcium carbonate, adjusting the pH value to 7.2-7.4, and sterilizing at 115 ℃ for 30 min; the water content of the solid fermentation medium is 50% of the weight of the solid fermentation medium;
the formula of the metarhizium anisopliae solid fermentation culture medium is as follows: 300g/L of wheat bran, 150g/L of corn flour, 3g/L of monopotassium phosphate, 3g/L of magnesium sulfate and 0.1g/L of copper sulfate, adjusting the pH value to 5.5-6.0, and sterilizing at 121 ℃ for 30 min; the water content of the solid fermentation medium is 50% of the weight of the solid fermentation medium; (2) uniformly mixing the prepared bacillus subtilis powder, pseudomonas fluorescens powder, pseudomonas defense powder, bacillus belgii powder, metarhizium anisopliae powder and hansenula polymorpha powder, and then adding lavender extract, compound enzyme and xanthan gum and uniformly mixing to obtain the compound microbial deodorant.
The application of the composite microbial deodorant provided by the embodiment is deodorization in livestock and poultry manure or public places, and the public places are public toilets, garbage dumps, garbage transfer stations and hotels. It is used in deodorization according to the following method: calculating the required dosage of the composite microbial deodorant according to the volume ratio of the composite microbial deodorant to the target material to be deodorized being 1:10000, then diluting the dosage by 10 times with water, uniformly spraying the dosage on the target material, and uniformly stirring and mixing the dosage.
Example 3
A composite microbial deodorant is prepared from the following raw materials in parts by weight: 10 parts of bacillus subtilis, 10 parts of bacillus belgii, 3 parts of pseudomonas fluorescens, 3 parts of pseudomonas defensins, 1 part of metarhizium anisopliae, 1 part of hansenula polymorpha, 2 parts of lavender extract, 1 part of complex enzyme, 0.1 part of xanthan gum and 69 parts of sucrose;
wherein the Bacillus subtilis adopts the Bacillus subtilis with the preservation number of CGMCC No. 2108; the pseudomonas fluorescens with the preservation number of CGMCC No.8820 is adopted as the pseudomonas fluorescens; the defense pseudomonads adopt the defense pseudomonads with the preservation number of CGMCC No. 18750;
the compound enzyme adopts a mixture of neutral protease, cellulase and lipase in a mass ratio of 2:1:2, wherein the enzyme activity of the neutral protease is 10.3 ten thousand U/g, the enzyme activity of the cellulase is 18.8 ten thousand U/g, and the enzyme activity of the lipase is 9.5 ten thousand U/g.
The bacillus belgii sample provided in this example was collected from a cow dung compost high temperature stage of 81.5 ℃ heap of qinhuang island lead kangdi agricultural technology limited company, and the sample was brought back to the laboratory and then strain isolation was completed within 24 hours: placing the sample in a sterile mortar, grinding the sample thoroughly, placing 1g into sterile physiological saline, and adding into 37 deg.CActivated and shake cultured for 2h at the temperature of 180 r/min; diluting the activated bacteria liquid to 10 of the total bacteria number-5、10-6After doubling, the strains are subjected to salt-resistant, acid-resistant and high-temperature-resistant strain separation in two modes of coating and streaking respectively, the strains are cultured in an LB (LB) culture medium (pH 5 and 10% NaCl) for 2 days at 45 ℃, and the strains are obtained after deodorization effect verification and are identified as Bacillus belgii by 16 s. The growth temperature range of the Bacillus belgii is as follows: 25-50 ℃, growth pH range of 4-9, NaCl resistance range: 0 to 11 percent.
The number of viable bacteria of each kind of bacteria in the composite microbial deodorant provided in this embodiment is as follows:
the viable count of Bacillus subtilis is 1.2 × 109CFU/g，
The viable count of Bacillus belgii is 1.4 × 109CFU/g，
The viable count of the pseudomonas fluorescens is 1.1 multiplied by 108CFU/g，
The number of viable bacteria for protecting against Pseudomonas is 1.2 × 108CFU/g，
The viable count of Metarhizium anisopliae is 0.9 × 106CFU/g，
The number of viable bacteria of Hansenula polymorpha is 1.0 × 106CFU/g。
The preparation method of the composite microbial deodorant of the embodiment is as follows:
(1) respectively fermenting the bacillus subtilis and the bacillus belgii by LB culture medium liquid to a stable stage, respectively adding dextrin accounting for 4% of the mass of the bacteria liquid and anhydrous sodium sulphate accounting for 6% of the mass of the bacteria liquid, and then spraying powder at 150 ℃ to prepare bacillus subtilis powder and bacillus belgii powder; fermenting the liquid of LB culture medium for pseudomonas fluorescens and the liquid of YPD culture medium for hansenula polymorpha to a stationary phase, centrifuging the liquid of bacterium 5000-8000rpm for 10-20min to obtain precipitates, and freeze-drying the precipitates by cold air to obtain pseudomonas fluorescens powder and hansenula polymorpha powder; culturing the defensive pseudomonas by using an LB culture medium to a stationary phase, inoculating the defensive pseudomonas into a defensive pseudomonas solid fermentation culture medium for culturing to obtain a defensive pseudomonas solid fermentation product, and drying to obtain defensive pseudomonas bacteria powder; culturing Metarhizium anisopliae with PDA culture medium to stationary phase, inoculating into Metarhizium anisopliae solid fermentation culture medium, culturing to obtain Metarhizium anisopliae solid fermentation product, and drying to obtain Metarhizium anisopliae powder; the culture temperatures of the bacillus subtilis and the bacillus belgii are both 37 ℃, the culture temperature of the metarhizium anisopliae is 25 ℃, and the culture temperatures of the pseudomonas fluorescens, the pseudomonas defensins and the hansenula are all 30 ℃;
the LB culture medium comprises the following formula: 5.0g/L yeast extract, 10.0g/L, NaCI 5.0.0 g/L tryptone and 1.0g/L glucose, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 30 min;
the formula of the PDA culture medium is as follows: 20.0g/L of sucrose and 200g/L of potatoes, adjusting the pH value to 7.5-8.0, and sterilizing at 121 ℃ for 30 min;
the YPD medium formula is as follows: yeast extract 10.0g/L, peptone 20.0g/L, glucose 20.0g/L, adjusting pH value to 7.2-7.4, sterilizing at 115 deg.C for 30 min;
the formula of the culture medium for defending the pseudomonas solid fermentation is as follows: 10g/L of glucose, 6g/L of maltose, 6g/L of peptone, 5g/L of ammonium sulfate, 0.3g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 0.3g/L of dipotassium phosphate, 70g/L of rice hulls, 70g/L of corn flour, 12g/L of soybean meal, 300g/L of bran and 30g/L of light calcium carbonate, adjusting the pH value to 7.2-7.4, and sterilizing at 115 ℃ for 30 min; the water content of the solid fermentation medium is 50% of the weight of the solid fermentation medium;
the formula of the metarhizium anisopliae solid fermentation culture medium is as follows: 300g/L of wheat bran, 150g/L of corn flour, 3g/L of monopotassium phosphate, 3g/L of magnesium sulfate and 0.1g/L of copper sulfate, adjusting the pH value to 5.5-6.0, and sterilizing at 121 ℃ for 30 min; the water content of the solid fermentation medium is 50% of the weight of the solid fermentation medium;
(2) uniformly mixing the prepared bacillus subtilis powder, pseudomonas fluorescens powder, pseudomonas defense powder, bacillus belgii powder, metarhizium anisopliae powder and hansenula polymorpha powder, and then adding lavender extract, compound enzyme and xanthan gum and uniformly mixing to obtain the compound microbial deodorant.
The application of the composite microbial deodorant provided by the embodiment is deodorization in livestock and poultry manure or public places, and the public places are public toilets, garbage dumps, garbage transfer stations and hotels. It is used in deodorization according to the following method: calculating the required dosage of the composite microbial deodorant according to the volume ratio of the composite microbial deodorant to the target material to be deodorized being 1:10000, then diluting the dosage by 10 times with water, uniformly spraying the dosage on the target material, and uniformly stirring and mixing the dosage.
Comparative example 1
A deodorant is substantially the same as the deodorant in example 1, except that: the Bacillus subtilis was replaced with a commonly commercially available Bacillus subtilis.
Comparative example 2
A deodorant is substantially the same as the deodorant in example 1, except that: 0 part of pseudomonas fluorescens.
Comparative example 3
A deodorant is substantially the same as the deodorant in example 1, except that: 0 part of defensive pseudomonas.
Comparative example 4
A deodorant is substantially the same as the deodorant in example 1, except that: bacillus belgii was replaced with the common commercial Bacillus belgii.
The deodorizing effect test was performed on the composite microbial deodorant prepared in examples 1 to 3 of the present invention and the deodorant prepared in comparative examples 1 to 4, and the deodorizing effect test was performed after the microbial deodorant was dissolved in an appropriate amount of water (0.1 kg of microbial deodorant was dissolved in 1L of water) and uniformly sprayed in one ten-thousandth ratio (0.1 kg of microbial deodorant was added per cubic meter) according to the volume of the excrements of livestock and poultry, and the deodorizing effect test was performed after the uniform mixing and stirring, and the test results are shown in tables 6 and 7.
TABLE 6 deodorizing Effect of microbial deodorant of different composition
TABLE 7 deodorization Effect detection Table after spraying for 3-7 days
The composite microbial deodorant provided in examples 1 to 3 of the present invention has a better deodorizing effect than the deodorant provided in comparative examples 1 to 4, and the ammonia gas removal rate, the hydrogen sulfide removal rate, and the odor concentration reduction rate of the composite microbial deodorant provided in examples 1 to 3 are higher than those of comparative examples 1 to 4.
As can be seen from table 6, the composite microbial deodorant provided in the embodiment of the present invention has an excellent effect of removing ammonia and hydrogen sulfide, wherein the composite microbial deodorant provided in example 1 has the best effect, the odor concentration is reduced by 63% after being sprayed for 2 hours, the removal rate of ammonia reaches 94% after being sprayed for 24 hours, and the removal rate of hydrogen sulfide reaches 95%. The composite microbial deodorant provided in examples 1-3 was detected to have an odor concentration reduced by more than 80% after spraying for 24 hours, which is much greater than the deodorant provided in comparative examples 1-4.
It can be seen from table 7 that the composite microbial deodorant provided in examples 1 to 3 of the present invention still has excellent deodorizing effects after being sprayed for 3 days, 5 days, and 7 days, and the odor reduction rate after 7 days is still more than 76%, which is much higher than that of comparative examples 1 to 4.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. The composite microbial deodorant is characterized by comprising the following raw materials in parts by weight: 10-15 parts of bacillus subtilis, 10-15 parts of bacillus belgii, 3-5 parts of pseudomonas fluorescens, 3-5 parts of pseudomonas defensins, 1-2 parts of metarhizium anisopliae, 1-2 parts of hansenula polymorpha, 2-3 parts of lavender extract, 1-2 parts of complex enzyme, 0.1-0.2 part of xanthan gum and 50-70 parts of sucrose;
wherein the bacillus subtilis adopts bacillus subtilis with the preservation number of CGMCC No. 2108; the pseudomonas fluorescens with the preservation number of CGMCC No.8820 is adopted as the pseudomonas fluorescens; the defensive pseudomonas adopts the defensive pseudomonas with the preservation number of CGMCC No. 18750.
2. The composite microbial deodorant according to claim 1, wherein the Bacillus belgii is Bacillus belgii separated from a heap sample of cow dung compost at a high temperature of 81 ℃ to 82 ℃.
3. The composite microbial deodorant according to claim 2,
the viable count of the bacillus subtilis is 1 multiplied by 109～5.2×109CFU/g；
The viable count of the Bacillus belgii is 1 × 109～5×109CFU/g；
The viable count of the pseudomonas fluorescens is 1 multiplied by 108～5.5×108CFU/g；
The number of viable bacteria of the defense pseudomonas is 1 multiplied by 108～5.4×108CFU/g；
The viable count of the metarhizium anisopliae is 0.9 multiplied by 106～5×106CFU/g；
The number of viable bacteria of the Hansenula is 1 multiplied by 106～6.4×106CFU/g。
4. The compound microbial deodorant according to claim 3, wherein the compound enzyme is a mixture of protease, cellulase and lipase; wherein the mass ratio of the protease to the cellulase to the lipase is 2:1:2-2.2:1.2: 2.2.
5. The compound microbial deodorant according to claim 4, wherein the enzyme activities of the protease, the cellulase and the lipase in the compound enzyme are all 9.5-20.5 ten thousand U/g.
6. Use of the composite microbial deodorant according to any one of claims 1 to 5 for deodorizing animal excrements or public places.
7. The use according to claim 6, wherein the public places are public toilets, refuse dumps, refuse transfer stations, hotels and restaurants.
8. The use of claim 6, wherein the required composite microbial deodorant is calculated according to the volume ratio of the composite microbial deodorant to the target material to be deodorized being 1:10000-1:10100, then diluted by 10-10.2 times, uniformly sprayed on the target material, stirred and mixed uniformly.
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