Spraying type microbial deodorant as well as preparation method and application thereof

Abstract

The invention provides a spraying type microbial deodorant and a preparation method and application thereof, and belongs to the technical field of microbial deodorization, the spraying type microbial deodorant is composed of a mixed microbial fermentation liquid and an acidifying agent; the mixed microbial fermentation liquid comprises bacillus subtilis, enterococcus faecium, lactobacillus casei and rhizopus oryzae; the acidifying agent is selected from one or more of anhydrous citric acid, malic acid, acetic acid, n-butyric acid, propionic acid, formic acid or lactic acid. After the microbial deodorant provided by the invention is sprayed to the livestock manure, the odor generated by the livestock manure can be quickly and effectively removed, the concentration of hydrogen sulfide and ammonia gas is reduced, and the contents of L-tryptophan, indole, indole-3-acetic acid and skatole in the manure are effectively reduced; the livestock manure and urine can be prevented from continuously generating odor in the subsequent placement process, and the deodorization effect is lasting.

Description

Technical Field

[0001] The invention relates to the technical field of microbial deodorization, and in particular to a spray-type microbial deodorant and a preparation method and application thereof. Background Art

[0002] In the process of livestock breeding, odor is easily generated due to the accumulation of feces and urine. The odor components mainly include ammonia and hydrogen sulfide, which are highly irritating and toxic, seriously affecting the normal life of breeding staff and surrounding residents, which is not conducive to health and easily pollutes the environment. At present, the deodorization methods are mainly combustion, oxidation, absorption, adsorption, neutralization and deodorization, etc., which have technical problems such as low efficiency, easy pollution, and incomplete deodorization. Although the use of microorganisms for deodorization is not easy to produce secondary pollution, the use of microorganisms for deodorization requires a period of reaction, and the processing speed is difficult to keep up with the large amount of livestock feces and urine output in the breeding farm every day. Therefore, it is hoped to provide a microbial deodorant with fast speed, high efficiency and thorough deodorization. Summary of the invention

[0003] The object of the present invention is to provide a spray-type microbial deodorant which can remove odor quickly, thoroughly and effectively for a long time.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a spray-type microbial deodorant, which is composed of mixed microbial fermentation liquid and acidulant;

[0006] The mixed microbial fermentation liquid includes Bacillus subtilis, Enterococcus faecium, Lactobacillus casei and Rhizopus oryzae;

[0007] The acidulant is selected from one or more of anhydrous citric acid, malic acid, acetic acid, n-butyric acid, propionic acid, formic acid or lactic acid.

[0008] Preferably, the final concentration of the Bacillus subtilis in the mixed microbial fermentation broth is: (1-2)×10 7 Pieces / mL;

[0009] The final concentration of Enterococcus faecium is: (1-3)×10 5 Pieces / mL;

[0010] The final concentration of Lactobacillus casei is: (3-5)×10 4 Pieces / mL;

[0011] The final concentration of Rhizopus oryzae is: (8-9)×10 4 Pieces / mL.

[0012] The present invention also provides a method for preparing the above-mentioned spray-type microbial deodorant: Bacillus subtilis, Enterococcus faecium, Lactobacillus casei and Rhizopus oryzae are cultured separately to obtain four single fermentation liquids, the four single fermentation liquids are mixed to obtain a mixed microbial fermentation liquid, and then an acidifier is added to adjust the pH of the microbial fermentation liquid to 5.2-5.4 to obtain the spray-type microbial deodorant.

[0013] Preferably, the culture medium formula used for culturing Bacillus subtilis is: 2-5g molasses, 8-12g soy protein, 2-3g beef extract, 3-5g sodium chloride and 0.8-1.2g potassium dihydrogen phosphate are added to every 1000mL distilled water, pH 6.8-7.2; the culture temperature is 30-35°C, and the culture is carried out for 20-48h.

[0014] Preferably, the culture medium formula used for culturing Enterococcus faecium is as follows: 30-35 g sucrose, 1-2 g yeast powder, 20-25 g soy protein, 10-15 g soybean meal, 0.1-0.2 g MgSO4, 0.01-0.03 g MnSO4 and 0.8-1.2 g potassium dihydrogen phosphate are added to every 1000 mL of distilled water, pH 5.8-6.2; the culture temperature is 35-38° C., and the culture is carried out for 20-24 hours.

[0015] Preferably, the culture medium formula used when culturing Lactobacillus casei is: adding 2-3g fructose and 50-60mL skim milk to 1000mL LB liquid culture medium; pH 6.2-6.8; culture temperature is 35-37°C, and culture is performed for 48-72h.

[0016] Preferably, the culture medium formula used when culturing Rhizopus oryzae is: 0.8-1.2 g of dipotassium hydrogen phosphate, 2-3 g of sodium chloride, 0.2-0.5 g of magnesium sulfate, 0.01-0.03 g of ferrous chloride, 10-15 g of sucrose, 5-10 g of ammonium chloride are added to every 1000 mL of distilled water, and the pH is 7.2-7.5; the culture conditions are: 35-37° C., 48-96 h.

[0017] The present invention also provides application of the microbial deodorant in deodorizing livestock excrement and urine.

[0018] Preferably, the method for deodorizing livestock excrement and urine is: spraying the spray-type microbial deodorant on livestock excrement and urine, the spraying amount is 6-8% of the volume of livestock excrement and urine, and then supplementing the spraying once every 20-30 hours, spraying 4-6 times, and the supplementary spraying amount is 0.1-0.2% of the volume of livestock excrement and urine.

[0019] The microbial deodorant provided by the present invention can not only effectively remove the odor generated by feces, reduce the concentration of hydrogen sulfide and ammonia in fresh feces, but also prevent the continued generation of odor in poultry and livestock feces and urine during the subsequent placement process. The present invention can also effectively reduce the content of L-tryptophan, indole, indole-3-acetic acid and skatole in feces, and reduce the generation of odor. After the microbial deodorant provided by the present invention is sprayed on poultry and livestock feces and urine, the organic acid and enzyme in the deodorant will immediately react with the odor molecules to reduce the odor. At this time, the ammonia removal rate is 80-90%, and the hydrogen sulfide removal rate is about 60%. The present invention is designed according to the fermentation law of pig manure. About 5 days before fresh pig manure, the release amount of odor molecules increases significantly. In order to ensure that the deodorant reacts with the odor molecules, the present invention continuously replenishes the deodorant during this period to ensure the deodorization effect. Afterwards, as the organic acids and enzymes in the microbial deodorant are consumed, the pH rises, and livestock manure also contains rich organic matter, the microorganisms in the deodorant begin to grow rapidly, and the precursor substances for producing odor molecules are decomposed and utilized, reducing the generation of odor molecules and making the deodorization effect lasting. DETAILED DESCRIPTION

[0020] In the present invention, preferably, Bacillus subtilis is Bacillus subtilis B1, which is deposited in China Center for Type Culture Collection, with the deposit address at Wuhan University, the deposit number being CCTCC NO: M20241564, and the deposit date being July 15, 2024.

[0021] Lactobacillus casei is Lactobacillus casei Lvk1, which is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, the deposit number is CCTCCNO:M 20231752, and the deposit date is September 20, 2023.

[0022] Enterococcus faecium was purchased from Shandong Yihao Biotechnology Co., Ltd., and Rhizopus oryzae was purchased from Shandong Gaotai Chemical Technology Co., Ltd. The technical solution provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] (1) Cultivation of Bacillus subtilis B1:

[0025] Preparation of Bacillus subtilis B1 culture medium: Add 3 g molasses, 10 g soy protein, 3 g beef extract, 3 g sodium chloride and 1 g potassium dihydrogen phosphate to every 1000 mL of distilled water, pH 7.0;

[0026] Culture conditions: 35°C constant temperature, 150r / min shaking culture for 24h;

[0027] (2) Culture of Enterococcus faecium

[0028] Preparation of Enterococcus faecium culture medium: add 35g sucrose, 2g yeast powder, 20g soy protein, 15g soybean meal, 0.1g MgSO4, 0.01g MnSO4 and 1g potassium dihydrogen phosphate to every 1000mL distilled water, pH 6.0; culture conditions: constant temperature at 36℃, shaking at 150r / min for 48h;

[0029] (3) Cultivation of Lactobacillus casei Lvk1

[0030] Preparation of Lactobacillus casei Lvk1 culture medium: Add 3 g fructose and 60 mL skim milk to 1000 mL LB liquid medium; pH 6.5;

[0031] Culture conditions: 37℃ constant temperature static culture for 48h;

[0032] (4) Cultivation of Rhizopus oryzae

[0033] Preparation of Rhizopus oryzae culture solution: Add 1 g of dipotassium hydrogen phosphate, 2 g of sodium chloride, 0.3 g of magnesium sulfate, 0.02 g of ferrous chloride, 12 g of sucrose, and 5 g of ammonium chloride to every 1000 mL of distilled water, pH 7.3;

[0034] The culture conditions were: constant temperature culture at 36°C for 72 h;

[0035] A spray-type microbial deodorant:

[0036] The culture medium and culture conditions of the above four bacteria were used to culture Bacillus subtilis B1, Enterococcus faecium, Lactobacillus casei Lvk1 and Rhizopus oryzae respectively to obtain single fermentation broths of the four bacteria, which were mixed and then the final concentration of Bacillus subtilis B1 was adjusted to 2×10 7 The final concentration of Enterococcus faecium was 2×10 5 / mL, the final concentration of Lactobacillus casei Lvk1 was 4×10 4 / mL, and the final concentration of Rhizopus oryzae was 8×10 4 / mL to obtain a mixed microbial fermentation liquid, and then add an acidulant (composed of anhydrous citric acid and lactic acid in a mass volume ratio of 1:3) to adjust the pH of the mixed microbial fermentation liquid to 5.4 to obtain the spray-type microbial deodorant.

[0037] Example 2

[0038] Bacillus subtilis B1, Enterococcus faecium, Lactobacillus casei Lvk1 and Rhizopus oryzae were cultured using the culture broth and culture conditions of the four bacteria given in Example 1 to obtain single fermentation broths of the four bacteria, which were mixed and then the final concentration of Bacillus subtilis B1 was adjusted to 1×107 The final concentration of Enterococcus faecium was 1×10 5 / mL, the final concentration of Lactobacillus casei Lvk1 was 5×10 4 / mL, the final concentration of Rhizopus oryzae was 9×10 4 / mL to obtain a mixed microbial fermentation liquid, and then add (anhydrous citric acid and lactic acid in a mass volume ratio of 1:1) to adjust the pH of the mixed microbial fermentation liquid to 5.3 to obtain the spray-type microbial deodorant.

[0039] Example 3

[0040] Bacillus subtilis B1, Enterococcus faecium, Lactobacillus casei Lvk1 and Rhizopus oryzae were cultured using the culture broth and culture conditions of the four bacteria given in Example 1 to obtain single fermentation broths of the four bacteria, which were mixed and then adjusted to a final concentration of 1.5×10 7 The final concentration of Enterococcus faecium was 3×10 5 / mL, and the final concentration of Lactobacillus casei Lvk1 was 5×10 4 / mL, the final concentration of Rhizopus oryzae is: 8.5×10 4 / mL to obtain a mixed microbial fermentation liquid, and then add an acidulant (anhydrous citric acid and lactic acid in a mass volume ratio of 2:1) to adjust the pH of the mixed microbial fermentation liquid to 5.2 to obtain the spray-type microbial deodorant.

[0041] Example 4

[0042] The spray-type microbial deodorant described in Example 1 was sprayed onto fresh pig manure at a spraying amount of 6% of the volume of the pig manure, and then supplemented with spraying once every 24 hours at a supplementary spraying amount of 0.2% of the volume of livestock feces and urine, and sprayed 5 times.

[0043] Example 5

[0044] The spray-type microbial deodorant described in Example 1 was sprayed onto fresh pig manure at a spraying amount of 8% of the pig urine volume, and then supplemented with spraying once every 24 hours at a supplementary spraying amount of 0.1% of the urine volume, and sprayed 5 times.

[0045] Comparative Example 1

[0046] A microbial deodorant: Different from Example 1, the mixed microbial fermentation liquid in this comparative example is obtained by culturing Bacillus pumilus, Enterococcus faecium, Lactobacillus acidophilus and Trichoderma viride separately and then mixing, wherein Bacillus pumilus 1×10 8 The final concentration of Enterococcus faecium was 2×10 5 / mL, and the final concentration of Lactobacillus acidophilus was 4×10 4 / mL, and the final concentration of Trichoderma viride was 4×10 4 Pieces / mL.

[0047] Comparative Example 2

[0048] A microbial deodorant: Different from Example 1, the Bacillus subtilis and Lactobacillus casei in this comparative example were purchased from Shandong Yihao Biotechnology Co., Ltd. and used for feeding.

[0049] Comparative Example 3

[0050] A microbial deodorant: Different from Example 1, the acidulant in this comparative example is composed of formic acid and malic acid in a volume ratio of 1:1.

[0051] Comparative Example 4

[0052] A microbial deodorant: Different from Example 1, the acidulant in this comparative example is lactic acid.

[0053] Test Example 1

[0054] 1. Experimental groups:

[0055] (1) Feces group:

[0056] Example 4 group: 1 kg of fresh pig manure, deodorized according to the method given in Example 4;

[0057] Comparative Example 1: 1 kg of fresh pig manure was sprayed with the microbial deodorant described in Comparative Example 1 and deodorized according to the method of Example 4;

[0058] Comparative Example 2: 1 kg of fresh pig manure was sprayed with the microbial deodorant described in Comparative Example 1, and deodorized according to the method of Example 4;

[0059] Comparative Example 3: 1 kg of fresh pig manure was sprayed with the microbial deodorant described in Comparative Example 3, and deodorized according to the method of Example 4;

[0060] Positive control group: 1 kg of fresh pig manure, sprayed with microbial deodorant (purchased from Dongguan Wante Chemical Co., Ltd.), and deodorized using the method described in Example 4;

[0061] Blank control group: 1 kg of fresh pig manure without any treatment. Each time other groups were sprayed with microbial deodorant, this control group was sprayed with the same volume of water.

[0062] (2) Urine group:

[0063] Example 4 group: 1 L of fresh urine, deodorized according to the method given in Example 4;

[0064] Comparative Example 1: 1 L of fresh urine was sprayed with the microbial deodorant described in Comparative Example 1, and deodorization was performed according to the method of Example 4;

[0065] Comparative Example 2 group: 1 L of fresh urine was sprayed with the microbial deodorant described in Comparative Example 2, and deodorized according to the method of Example 4;

[0066] Comparative Example 3 group: 1L of fresh urine was sprayed with the microbial deodorant described in Comparative Example 3, and deodorized according to the method of Example 4;

[0067] Positive control group: 1L of fresh urine, sprayed with microbial deodorant (purchased from Dongguan Wante Chemical Co., Ltd.), and deodorized using the method described in Example 4;

[0068] Blank control group: 1L of fresh urine without any treatment. Each time other groups were sprayed with microbial deodorant, this control group was sprayed with the same volume of water.

[0069] 2. Test methods:

[0070] Three parallel tests were conducted for each treatment group. The NH3 release was measured by Nessler's reagent colorimetric method and the H2S release was measured by methylene blue method. The concentrations of hydrogen sulfide and ammonia in each treatment group were measured on the 1st, 5th, 10th and 20th day of treatment. The results are shown in Tables 1 and 2:

[0071] Table 1 Hydrogen sulfide and ammonia release in feces group (mg / m 3 )

[0072]

[0073]

[0074] Table 2 Hydrogen sulfide and ammonia release in urine group (mg / m 3 )

[0075]

[0076] As can be seen from Table 1 and Table 2, the microbial deodorant provided by the present invention can effectively remove the odor produced by feces, reduce the concentration of hydrogen sulfide and ammonia produced by fresh feces, and has better technical effects than Comparative Examples 1 to 4 and commercially available microbial deodorants. The present invention takes effect quickly. After spraying on livestock feces and urine, the organic acids and enzymes in the deodorant will immediately react with the odor molecules to reduce the odor. At this time, the ammonia removal rate is 80-90%, and the hydrogen sulfide removal rate is about 60%. At the same time, because the organic acids and enzymes in the microbial deodorant are consumed, the pH rises, and the livestock feces also contain rich organic matter. The microorganisms in the deodorant begin to grow rapidly, and the precursor substances for the production of odor molecules are decomposed and utilized, which reduces the generation of odor molecules and has a lasting deodorizing effect.

[0077] Test Example 2

[0078] The experimental groups were as shown in Experimental Example 1. After each experimental group was sprayed 5 times, 100 g of feces was taken and 9 times the volume of methanol was added. After stirring evenly, the contents of L-tryptophan, indole, indole-3-acetic acid and skatole in the feces (fresh weight) were detected by high performance liquid chromatography. The results are shown in Table 3:

[0079] Table 3 Contents of L-tryptophan, indole, indole-3-acetic acid and skatole in feces (μg / g)

[0080]

[0081]

[0082] As can be seen from Table 2, the microbial deodorant provided by the present invention can effectively reduce the content of L-tryptophan, indole, indole-3-acetic acid and skatole in feces, and reduce the odorous substances in feces, which is significantly improved compared with the existing product technology.

[0083] From the comprehensive test examples 1 and 2, it can be seen from comparative example 1 that the deodorizing effects of different strains are not the same, and the microbial combination provided by the present invention has a better technical effect than other combinations; it can be seen from comparative example 2 that the use of Bacillus subtilis B1 and Lactobacillus casei Lvk1 can further enhance the technical effect of the microbial deodorant; it can be seen from comparative examples 3 and 4 that the technical effects of different acidulants are also different, among which the combination of anhydrous citric acid and lactic acid has a better technical effect than other combinations.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A spray-type microbial deodorant, characterized in that: It is composed of mixed microbial fermentation liquid and acidulant; The mixed microbial fermentation liquid includes Bacillus subtilis, Enterococcus faecium, Lactobacillus casei and Rhizopus oryzae; The acidulant is selected from one or more of anhydrous citric acid, malic acid, acetic acid, n-butyric acid, propionic acid, formic acid or lactic acid.

2. The microbial deodorant according to claim 1, characterized in that The final concentration of the Bacillus subtilis in the mixed microbial fermentation broth is: (1-2)×10 7 Pieces / mL; The final concentration of Enterococcus faecium is: (1-3)×10 5 Pieces / mL; The final concentration of Lactobacillus casei is: (3-5)×10 4 Pieces / mL; The final concentration of Rhizopus oryzae is: (8-9)×10 4 Pieces / mL.

3. A method for preparing the spray-type microbial deodorant according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: culturing Bacillus subtilis, Enterococcus faecium, Lactobacillus casei and Rhizopus oryzae respectively to obtain four single fermentation liquids, mixing the four single fermentation liquids to obtain the mixed microbial fermentation liquid, and then adding an acidulant to adjust the pH of the mixed microbial fermentation liquid to 5.2-5.4 to obtain the spray-type microbial deodorant.

4. The preparation method according to claim 3, characterized in that: The culture medium formula used for culturing Bacillus subtilis is as follows: 2-5 g molasses, 8-12 g soy protein, 2-3 g beef extract, 3-5 g sodium chloride and 0.8-1.2 g potassium dihydrogen phosphate are added to every 1000 mL of distilled water, pH 6.8-7.2; the culture temperature is 30-35° C. and the culture is carried out for 20-48 hours.

5. The preparation method according to claim 3, characterized in that: The culture medium formula used for culturing Enterococcus faecium is as follows: add 30-35g sucrose, 1-2g yeast powder, 20-25g soybean protein, 10-15g soybean meal, 0.1-0.2g MgSO4, 0.01-0.03g MnSO4 and 0.8-1.2g dipotassium hydrogen phosphate to every 1000mL distilled water, pH 5.8-6.2; the culture temperature is 35-38°C and the culture is carried out for 48-72h.

6. The preparation method according to claim 3, characterized in that: The culture medium formula used when culturing Lactobacillus casei is as follows: adding 2-3 g of fructose and 50-60 mL of skim milk to 1000 mL of LB liquid culture medium; pH 6.2-6.8; culture temperature 35-37° C., and culture for 48-72 hours.

7. The preparation method according to claim 3, characterized in that: The culture medium formula used when culturing Rhizopus oryzae is as follows: 0.8-1.2 g of dipotassium hydrogen phosphate, 2-3 g of sodium chloride, 0.2-0.5 g of magnesium sulfate, 0.01-0.03 g of ferrous chloride, 10-15 g of sucrose, 5-10 g of ammonium chloride are added to every 1000 mL of distilled water, and the pH value is 7.2-7.5; the culture conditions are: 35-37° C., 48-96 h.

8. Use of the microbial deodorant according to claim 1 or 2 or the microbial deodorant obtained by the preparation method according to any one of claims 3 to 7 in deodorizing livestock manure and urine.

9. The use according to claim 8, characterized in that The method for deodorizing livestock excrement is as follows: spraying the spray-type microbial deodorant on livestock excrement, with a spraying amount of 6-8% of the mass or volume of the livestock excrement, and then supplementing the spraying once every 20-30 hours, and spraying 4-6 times.

10. The use according to claim 10, characterized in that The supplementary spraying amount is 0.1-0.2% of the volume of poultry and livestock manure and urine.

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