Oil pollution decomposing agent

Abstract

The invention discloses an oil decomposition agent. The oil decomposition agent is obtained by expanding and culturing a composite bacterium, wherein the expansion culture medium contains ginseng extract or modified ginseng extract, and ginseng contains abundant sugar substances, which provide nutrients for the growth of the composite bacterium and are easy to penetrate the microbial cell membrane after modification and be utilized by the microorganism, thereby improving the proliferation effect of the composite bacterium and further improving the oil decomposition ability. The bacterial agent disclosed by the invention has good oil decomposition ability.

Description

Technical Field

[0001] The invention relates to the technical field of grease decomposition, and in particular to an oil pollution decomposing agent. Background Art

[0002] The rapid development of the milk processing industry and the catering industry has caused a series of environmental problems, among which the environmental problems caused by oily wastewater are of vital importance. Oily wastewater contains high concentrations of oil and a large amount of suspended solids, and is one of the serious pollution sources in some places. In the process of catering wastewater treatment, microorganisms with high oil degradation ability are needed to solve difficult-to-degrade substances such as oil and protein. It is of great significance to separate and screen microorganisms with high oil degradation ability, develop efficient and low-cost microbial agents and use them in oil wastewater treatment.

[0003] Chinese patent 201810924259.3 discloses a method for preparing an environmentally friendly industrial grease decomposer. The method includes injecting polyoxyethylene alkyl ether, fatty acid alkanolamide, fatty alcohol polyoxypropylene polyoxyethylene ether, sodium citrate, sodium tartrate, sodium carboxymethyl propanol dioic acid, disodium ethylenediaminetetraacetic acid, glycerol and sodium tetradecyl sulfate into a mixing kettle, stirring the mixture with a stirrer, and keeping the obtained mixed solution for standby use; flushing the mixed solution into a reactor, adding 20-200 parts of deionized water, raising the temperature of the reactor to 44-55°C with a heating furnace, stirring the mixture with a stirrer during the heating process to fully mix the deionized water and the mixed solution, and keeping the mixed solution in the reactor for 1-3 hours to obtain the above-mentioned environmentally friendly industrial grease decomposer; the environmentally friendly industrial grease decomposer prepared by this method has a fast removal speed and good effect on long-chain grease, but the composition of the oily wastewater is complex and does not only contain long-chain grease, and the introduction of chemical substances causes secondary environmental pollution.

[0004] Therefore, the present invention provides an oil pollution decomposing agent which is easy to operate, low in cost and does not generate secondary pollution. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an oil pollution decomposing agent.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent through expansion culture.

[0008] The composite bacterial species is a mixture of one or more of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides. Preferably, the composite bacterial species is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of (1-10):(1-10):(1-10):(1-10):(1-10).

[0009] The seed culture medium comprises: 1-2 parts by weight of sodium chloride, 4-5 parts by weight of ammonium acetate, 0.1-0.4 parts by weight of magnesium nitrate, 0.1-0.5 parts by weight of calcium nitrate, 0.1-1.5 parts by weight of dipotassium hydrogen phosphate, 0.1-2 parts by weight of potassium dihydrogen phosphate, 8-12 parts by weight of yeast extract, 0.1-1.2 parts by weight of chitosan, and 900-1100 parts by weight of distilled water.

[0010] The culture conditions of the seed solution are: culture at 27-38°C, 60-180rpm, 800-1200lux light until the bacterial concentration reaches 10 8 -10 10 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 6.8-7.2.

[0011] The components of the expansion culture medium are: 8-12 parts by weight of beef extract, 7-15 parts by weight of peptone, 0.1-5.5 parts by weight of potassium dihydrogen phosphate, 0.1-3 parts by weight of dipotassium hydrogen phosphate, 900-1100 parts by weight of distilled water, and 550-650 parts by weight of hydrophilic porous polylactic acid particles.

[0012] The culture conditions of the expanded culture are: adding 8-20 mL of the seed solution to 1 L of the expanded culture medium, culturing at 27-38° C., 120-240 rpm, and 800-1200 lux; the pH of the expanded culture medium is 6.8-7.2; when the bacterial cell concentration reaches 10 14 -10 16 CFU / mL, centrifuge, and store the precipitate in a light-free environment with a relative humidity of (70-85)% and an oxygen content of (5-20)% to obtain the bacterial agent.

[0013] The preparation method of the hydrophilic porous polylactic acid particles is:

[0014] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 43-48°C and stir at 240-360 rpm for 20-40 min to obtain an enzyme treatment solution with a pH of 8.3-8.6 and a porcine pancreatic lipase concentration of 0.4-0.7 g / L;

[0015] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① are mixed in a mass ratio of (0.8-1.2): (2-4), stirred at 40-45°C and 240-360rpm for 10-16h, filtered and dried at 45-55°C for 4-6h to obtain the hydrophilic porous polylactic acid particles.

[0016] The preparation method of the porous polylactic acid particles is:

[0017] (1) At 90-96° C., polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of (1-1.5):(0.8-1.2):(6-9) and stirred at 540-660 rpm for 2-3.5 h to obtain a biphasic solution;

[0018] (2) At 28-35° C., n-pentanol and a precipitation aid were mixed in a mass ratio of (90-110):(0.25-0.3) and stirred at 540-660 rpm for 2-5 h to obtain a precipitant;

[0019] ⑶ Cool the two-phase solution obtained in step ⑴ to 45-55°C and stir at 300-420rpm, and add the precipitant obtained in step ⑵ and cooled to 1-5°C to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution and the precipitant is (0.8-1.5):(4-6).

[0020] The precipitation aid is one of xanthan gum and carbomer or a mixture of the two. Preferably, the precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of (0.1-5):(0.1-5).

[0021] The porous carrier can be used to store the bacteria in the porous structure of the carrier, thereby enhancing the stability of the obtained bacterial preparation and the ease of storage and transportation. When in use, the polylactic acid particles prepared by the present invention have a suitable surface tension range due to their unique surface structure, and therefore have good hydrophilicity and lipophilicity and spontaneously diffuse between the water and oil phases. At the same time, the bacteria are quickly sucked out of the porous carrier due to the polarity of water and the surface tension, and fully diffuse at the water-oil interface, and then carry out the physiological activity of decomposing oil.

[0022] Xanthan gum and carbomer have different molecular structures, and the ratio of polar groups to non-polar groups in their respective molecules is different, so that the two have different surface tensions in a two-phase solution obtained by stirring polylactic acid, cellulose acetate and n-butyl propionate. The difference in surface tension between the two makes the porous polylactic acid precipitated when n-pentanol is added have different surface morphologies; when xanthan gum and carbomer are compounded and used as precipitation aids, the average particle size distribution and wetting angle range of the porous polylactic acid obtained after proportion control are more conducive to the final bacterial agent being evenly distributed at the oil-water interface of the grease wastewater, which is convenient for the bacteria in the preparation to quickly diffuse out and reproduce and decompose grease, thereby improving the ability of the bacterial agent to decompose grease.

[0023] Polyfiber is an aliphatic polyester, with a large number of ester bonds in its molecular structure, poor hydrophilicity, and a long degradation cycle. Using porcine pancreatic lipase to modify polylactic acid can convert the ester bonds exposed in the outer layer of polylactic acid particles into polar hydroxyl and carboxyl groups, so that the obtained polylactic acid particles can be better distributed at the interface between the water phase and the oil phase, so that the bacteria in the particles can be better diffused at the oil-water interface, which is conducive to the rapid reproduction and increase of the bacteria. Moreover, when the decomposition activity occurs at the oil-water interface, the glycerol and low-level fatty acids in the decomposition products can quickly diffuse into the water to avoid the accumulation of decomposition products. The oil decomposition effect of the microbial agent obtained by hydrophilic modification of porous polylactic acid particles using porcine pancreatic lipase is better than that of using fluorescent Pseudomonas lipase, because porcine pancreatic lipase can achieve higher activity under milder and lower cost conditions due to its unique spatial molecular structure, and the ratio of polar groups to ester groups on the surface of the obtained polylactic acid particles is more moderate, which is more conducive to the uniform distribution of the microbial agent at the oil-water interface, and improves the ability of the microbial agent to decompose oil.

[0024] Carboxymethyl cellulose has more polar groups. The presence of these polar groups will result in particles with poor surface uniformity when porous polylactic acid particles are obtained by precipitation with n-pentanol. These particles with poor surface uniformity will rub and collide with each other and break and shatter during subsequent transportation and storage, that is, they will become ineffective before they are officially put into use. The proportion of polar groups in cellulose acetate is lower, and polylactic acid particles with better surface uniformity and easy transportation and storage can be precipitated, thereby improving the ability of the bacterial agent to decompose oil.

[0025] Furthermore, an oil decomposer is prepared by the following method: inoculating a composite bacterial strain into a seed culture medium to obtain a seed solution, and then inoculating the seed solution into an expansion culture medium to obtain the oil decomposer.

[0026] Furthermore, the components of the expansion culture medium are: 8-12 parts by weight of beef extract, 7-15 parts by weight of peptone, 0.1-5.5 parts by weight of potassium dihydrogen phosphate, 0.1-3 parts by weight of dipotassium hydrogen phosphate, 900-1100 parts by weight of distilled water, 5-10 parts by weight of ginseng extract, and 550-650 parts by weight of hydrophilic porous polylactic acid particles.

[0027] The preparation method of ginseng extract comprises the following steps: drying ginseng and crushing it through 80-100 meshes to obtain powder; treating the powder at 0.1-0.3MPa and 100-130°C for 10-40min to obtain ginseng powder; drying the ginseng powder at 60-80°C for 1-3h to obtain dried ginseng powder; mixing the dried ginseng powder, enzyme and water in a mass ratio of 1:(0.1-0.5):(5-8), performing enzymolysis at 30-50°C for 6-12h, heating to 100°C to inactivate the enzyme for 1-2h after the enzymolysis is completed, filtering to obtain ginseng supernatant I and precipitate; concentrating the supernatant I under reduced pressure at a vacuum degree of 0.05-0.07MPa and a temperature of 60-80°C to a density of 1-1.15g / mL (25°C) to obtain a concentrated solution; and adding 9 5wt% ethanol, the volume ratio of ethanol to concentrated solution is (2-3):1, centrifugation to obtain precipitate, washing, freeze-drying to obtain a crude product; the crude product and water are mixed in a mass ratio of 1:(5-15) and ultrasonic extraction is performed for 30-50min, wherein the ultrasonic power is 800-1000W and the ultrasonic frequency is 20-30kHz, and then ethyl acetate is used for extraction, wherein the volume ratio of ethyl acetate to water is 1:1, and the aqueous phase is taken to obtain a crude product aqueous solution; the crude product aqueous solution and the mixed solution are mixed in a volume ratio of 3:(1-2), stirred at 500-700r / min for 30-50min, allowed to stand, centrifuged to obtain ginseng supernatant II, 95wt% ethanol is added to the ginseng supernatant II, the volume ratio of ethanol to ginseng supernatant II is (2-3):1, centrifugation to obtain precipitate, washing, freeze-drying to obtain a ginseng extract.

[0028] The mixed solution is a mixture of chloroform and acetone in a mass ratio of (1-3):(1-3).

[0029] The enzyme is one or a mixture of two or more of cellulase, alkaline protease and β-glucosidase. Preferably, the enzyme is a mixture of cellulase, alkaline protease and β-glucosidase in the mass ratio of (1-3):(1-3):(1-3).

[0030] Further preferably, the components of the expansion culture medium are: 8-12 parts by weight of beef extract, 7-15 parts by weight of peptone, 0.1-5.5 parts by weight of potassium dihydrogen phosphate, 0.1-3 parts by weight of dipotassium hydrogen phosphate, 900-1100 parts by weight of distilled water, 5-10 parts by weight of modified ginseng extract, and 550-650 parts by weight of hydrophilic porous polylactic acid particles.

[0031] The preparation method of the modified ginseng extract comprises the following steps:

[0032] S1 Preparation of ginseng extract: Dry ginseng and crush through 80-100 mesh to obtain powder; place the powder in 0.1-0.3MPa, 100-130℃ for 10-40min to obtain ginseng powder; dry the ginseng powder at 60-80℃ for 1-3h to obtain dry ginseng powder; mix the dry ginseng powder, enzyme and water in a mass ratio of 1:(0.1-0.5):(5-8), and perform enzymolysis at 30-50℃ for 6-12h. After the enzymolysis is completed, heat to 100℃ to inactivate the enzyme for 1-2h, filter and obtain supernatant I and precipitate; concentrate the supernatant I under reduced pressure at a vacuum degree of 0.05-0.07MPa and a temperature of 60-80℃ to a density of 1 -1.15g / mL (25°C) to obtain a concentrated solution; after cooling, 95wt% ethanol is added to the concentrated solution, the volume ratio of ethanol to the concentrated solution is (2-3):1, centrifugation is performed to obtain a precipitate, washing, and freeze-drying to obtain a crude product; the crude product and water are mixed in a mass ratio of 1:(5-15) and ultrasonic extraction is performed for 30-50min, wherein the ultrasonic power is 800-1000W and the ultrasonic frequency is 20-30kHz, and then ethyl acetate is used for extraction, wherein the volume ratio of ethyl acetate to water is 1:1, and the aqueous phase is obtained to obtain a crude product aqueous solution; the crude product aqueous solution is mixed with the mixed solution in a volume ratio of 3:(1-2), stirred at 500-700r / min for 30-50min, allowed to stand, centrifuged to obtain ginseng supernatant II, 95wt% ethanol is added to the ginseng supernatant II, the volume ratio of ethanol to ginseng supernatant II is (2-3):1, centrifugation is performed to obtain a precipitate, washing, and freeze-drying to obtain a ginseng extract.

[0033] S2 Preparation of modified ginseng extract: Concentrated sulfuric acid, n-butanol, ammonium sulfate, and ginseng extract are mixed in a mass ratio of (35-40):(25-35):(0.1-0.15):(0.4-0.8), cooled to 0°C, and stirred at 500-700r / min for 30-40min. Then, the pH value is adjusted to 7 with a 3-4mol / L sodium hydroxide aqueous solution, and then 95wt% ethanol is added for alcohol precipitation. The precipitate is collected and dissolved in water for dialyzation and freeze-dried to obtain a modified ginseng extract.

[0034] Ginseng contains abundant carbohydrates, which provide nutrients for the growth of composite bacteria and improve the utilization rate of materials. The inventors modified the ginseng extract with chemical groups. The ginseng extract modified with sulfuric acid may cause the hydrolysis of small molecules in the ginseng extract, further improve the solubility and redox properties of polysaccharides, so that they can easily penetrate the microbial cell membrane and be used by microorganisms, so that they can be better used by probiotics, promote the production of organic acids, inhibit the growth and reproduction of pathogens, help to ensure and improve the survival ability of probiotics, improve the proliferation effect of composite bacteria, and further improve the oil decomposition effect.

[0035] Beneficial effects of the invention: The invention provides an oil pollution decomposer, which contains Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides, wherein the composite bacteria have different degradation and decomposition capabilities for different substances, and Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides have synergistic effects and have good decomposition capabilities for oils with complex components. The oil pollution decomposer of the invention has good ability to decompose oil pollution. DETAILED DESCRIPTION

[0036] The above content of the present invention is further described in detail below in conjunction with specific implementation modes, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0037] Introduction of some raw materials in this application:

[0038] Bacillus subtilis, CGMCC: 1.9083, was purchased from China General Microbiological Culture Collection Center.

[0039] Bacillus licheniformis, CGMCC: 1.6510, was purchased from China General Microbiological Culture Collection Center.

[0040] Pseudomonas chengduensis, CGMCC: 1.15626, was purchased from China General Microbiological Culture Collection Center.

[0041] Pseudomonas xanthomarina: Pseudomonas xanthomarina, CGMCC: 1.10904, purchased from China General Microbiological Culture Collection Center.

[0042] Rhodobacter sphaeroides, CGMCC: 1.2569, was purchased from China General Microbiological Culture Collection Center.

[0043] Porcine pancreatic lipase, CAS: 9001-62-1, purchased from Shanghai Aladdin Co., Ltd., Enzyme commission (EC) Number: 3.1.1.3, purity: 15-35 units / mg, RTECS: TO9776500, item number: L111237-100g.

[0044] Polylactic acid, CAS: 26100-51-6, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: P169115-5g, molecular weight: 60000.

[0045] Cellulose acetate, CAS: 9012-09-3, was purchased from J&K Technology Co., Ltd., product number: C17782.

[0046] Carbomer, CAS: 54182-57-9, was purchased from Hubei Xinrunde Chemical Co., Ltd., product number: Carbomer 934.

[0047] Yeast extract, CAS: 8013-01-2, was purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A48126-500g, specification: BR.

[0048] Chitosan, CAS: 9012-76-4, was purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A14854-100g, specification: BR.

[0049] Beef extract, CAS: 68990-09-0, purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A21486-500g, specification: BR.

[0050] Peptone, CAS: 73049-73-7, purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A12846-500g, specification: BR.

[0051] Pseudomonas fluorescens lipase, CAS: 9001-62-1, purchased from Shanghai Aladdin Co., Ltd., Enzyme commission (EC) Number: 232-619-9, purity ≥ 20,000 U / g, RTECS: TO9776500, item number: L299014-50g.

[0052] Carboxymethyl cellulose, CAS: 9004-32-4, was purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A11895-250g, specification: BR.

[0053] Cellulase, enzyme activity: 10000U / g, CAS: 9012-54-8, product number: R014185, purchased from Shanghai Yien Chemical Technology Co., Ltd.

[0054] Alkaline protease, enzyme activity: 100,000 U / g, purchased from Shenzhen Jinfuyuan Biotechnology Co., Ltd.

[0055] β-Glucosidase, enzyme activity: 30U / mg, source: almond, product number: G824159, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0056] Ginseng, Latin name: Panax ginseng CAMey, is a perennial herb of the genus Panax, family Araliaceae, Apiaceae, purchased from Bozhou Shouantang Pharmaceutical Co., Ltd.

[0057] Example 1

[0058] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0059] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0060] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0061] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0062] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0063] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0064] The preparation method of the hydrophilic porous polylactic acid particles is:

[0065] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0066] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0067] The preparation method of the porous polylactic acid particles is:

[0068] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0069] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0070] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0071] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0072] Example 2

[0073] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0074] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0075] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0076] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0077] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0078] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0079] The preparation method of the hydrophilic porous polylactic acid particles is:

[0080] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0081] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0082] The preparation method of the porous polylactic acid particles is:

[0083] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0084] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0085] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0086] The precipitation aid is xanthan gum.

[0087] Example 3

[0088] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0089] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0090] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0091] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0092] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0093] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0094] The preparation method of the hydrophilic porous polylactic acid particles is:

[0095] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0096] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0097] The preparation method of the porous polylactic acid particles is:

[0098] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0099] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0100] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0101] The precipitation aid is carbomer.

[0102] Comparative Example 1

[0103] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0104] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0105] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0106] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0107] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0108] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0109] The preparation method of the hydrophilic porous polylactic acid particles is:

[0110] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0111] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0112] The preparation method of the porous polylactic acid particles is:

[0113] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0114] (2) Stir n-pentanol at 600 rpm for 3 h at 30°C to obtain a precipitant;

[0115] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0116] Comparative Example 2

[0117] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0118] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0119] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0120] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0121] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of porous polylactic acid particles.

[0122] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0123] The preparation method of the porous polylactic acid particles is:

[0124] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0125] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0126] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0127] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0128] Comparative Example 3

[0129] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0130] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0131] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0132] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0133] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0134] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0135] The preparation method of the hydrophilic porous polylactic acid particles is:

[0136] ① Mix fluorescein Pseudomonas lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.0 and a fluorescein Pseudomonas lipase concentration of 0.5 g / L;

[0137] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0138] The preparation method of the porous polylactic acid particles is:

[0139] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0140] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0141] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0142] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0143] Comparative Example 4

[0144] A bacterial agent is prepared by the following method: a composite bacterial strain is inoculated into a seed culture medium for cultivation to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion cultivation to obtain the bacterial agent.

[0145] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0146] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0147] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0148] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0149] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0150] The preparation method of the hydrophilic porous polylactic acid particles is:

[0151] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0152] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0153] The preparation method of the porous polylactic acid particles is:

[0154] (1) At 95°C, polylactic acid, carboxymethyl cellulose and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0155] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0156] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0157] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0158] Example 4

[0159] An oil decomposer is prepared by the following method: inoculating a composite bacterial strain into a seed culture medium to obtain a seed liquid, and then inoculating the seed liquid into an expansion culture medium to obtain the oil decomposer.

[0160] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0161] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0162] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0163] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, 8 parts by weight of ginseng extract, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0164] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent.

[0165] The preparation method of the hydrophilic porous polylactic acid particles is:

[0166] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0167] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0168] The preparation method of the porous polylactic acid particles is:

[0169] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0170] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0171] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0172] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0173] The preparation method of the ginseng extract comprises the following steps: drying and crushing ginseng through 100 meshes to obtain powder; subjecting the powder to treatment at 0.3MPa and 130°C for 40min to obtain ginseng powder; drying the ginseng powder at 80°C for 3h to obtain dried ginseng powder; mixing the dried ginseng powder, enzyme and water in a mass ratio of 1:0.3:8, performing enzymolysis at 45°C for 12h, heating to 100°C for 2h to inactivate the enzyme after the enzymolysis is completed, filtering to obtain supernatant I and precipitate; performing reduced pressure concentration on the supernatant I at a vacuum degree of 0.07MPa and a temperature of 70°C to concentrate to a density of 1.10g / mL (25°C) to obtain a concentrated solution; and adding 95wt% of the concentrated solution after cooling. Ethanol, the volume ratio of ethanol to concentrated solution is 2:1, centrifuge to obtain precipitate, wash, freeze-dry to obtain a crude product; the crude product and water are mixed in a mass ratio of 1:10 and ultrasonically extracted for 50 minutes, wherein the ultrasonic power is 800W and the ultrasonic frequency is 25kHz, and then ethyl acetate is used for extraction, wherein the volume ratio of ethyl acetate to water is 1:1, and the aqueous phase is taken to obtain a crude product aqueous solution; the crude product aqueous solution is mixed with the mixed solution in a volume ratio of 3:1, stirred at 700r / min for 50 minutes, allowed to stand, centrifuged to obtain supernatant II, 95wt% ethanol is added to supernatant II, the volume ratio of ethanol to supernatant II is 3:1, centrifuge to obtain precipitate, wash, freeze-dry to obtain ginseng extract. The mixed solution is a mixture of chloroform and acetone in a mass ratio of 3:1. The enzyme is a mixture of cellulase, alkaline protease, and β-glucosidase in a mass ratio of 1:1:1.

[0174] Example 5

[0175] The method is basically the same as Example 4, except that the modified ginseng extract is used to replace the ginseng extract.

[0176] The preparation method of the modified ginseng extract comprises the following steps: S1 preparation of ginseng extract: drying ginseng and crushing it through 100 meshes to obtain powder; placing the powder under 0.3MPa and 130°C for 40min to obtain ginseng powder; drying the ginseng powder at 80°C for 3h to obtain dried ginseng powder; mixing the dried ginseng powder, enzyme and water in a mass ratio of 1:0.3:8, performing enzymolysis at 45°C for 12h, heating to 100°C for 2h to inactivate the enzyme after the enzymolysis is completed, filtering to obtain supernatant I and precipitate; performing reduced pressure concentration on the supernatant I at a vacuum degree of 0.07MPa and a temperature of 70°C to concentrate it to a density of 1.10g / mL (25°C) to obtain a concentrated solution; after cooling, adding the concentrated solution to the Add 95wt% ethanol, the volume ratio of ethanol to concentrated solution is 2:1, centrifuge to obtain precipitate, wash, freeze-dry to obtain crude product; mix crude product and water in a mass ratio of 1:10 and perform ultrasonic extraction for 50 minutes, wherein the ultrasonic power is 800W and the ultrasonic frequency is 25kHz, then extract with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1:1, and take the aqueous phase to obtain crude product aqueous solution; mix the crude product aqueous solution with the mixed solution in a volume ratio of 3:1, stir at 700r / min for 50 minutes, let stand, centrifuge to obtain supernatant II, add 95wt% ethanol to supernatant II, the volume ratio of ethanol to supernatant II is 3:1, centrifuge to obtain precipitate, wash, freeze-dry to obtain ginseng extract. The mixed solution is a mixture of chloroform and acetone in a mass ratio of 3:1. The enzyme is a mixture of cellulase, alkaline protease, and β-glucosidase in a mass ratio of 1:1:1.

[0177] Preparation of S2 modified ginseng extract: concentrated sulfuric acid, n-butanol, ammonium sulfate, and ginseng extract were mixed in a mass ratio of 38:29:0.13:0.6, cooled to 0°C, and stirred at 600 r / min for 40 min. The pH was then adjusted to 7 with a 4 mol / L sodium hydroxide aqueous solution. 95 wt% ethanol was then added for alcohol precipitation. The precipitate was collected and dissolved in water for dialyzation and freeze-dried to obtain a modified ginseng extract.

[0178] Test Example 1

[0179] Test of the ability to decompose grease: According to GB / T 22500-2008 "Standard for the determination of ultraviolet absorbance of animal and vegetable oils", the ultraviolet spectrophotometry of animal and vegetable oils in the grease wastewater before and after treatment with the bacterial agents obtained in the above embodiments and comparative examples was measured, and the grease concentration before and after treatment was calculated according to the absorbance standard curve. Among them, the initial grease concentration was 30 mg / L. The grease wastewater was treated with the bacterial agents obtained in the above embodiments and comparative examples at 30°C, 1000 lux, and 300 rpm. The treatment time was 48 hours, and the mass ratio of the bacterial agent to the grease wastewater was 1:50. Grease decomposition rate = (initial grease concentration - grease concentration after treatment) / initial grease concentration × 100%. The test results are shown in Table 1.

[0180] Table 1 Oil decomposition rate of the bacterial agent of the present invention

[0181] Oil decomposition rate (%) Example 1 94.2 Example 2 92.8 Example 3 91.5 Comparative Example 1 84.1 Comparative Example 2 74.0 Comparative Example 3 87.6 Comparative Example 4 87.4 Example 4 95.1 Example 5 96.3

[0182] Test Example 2

[0183] Test of the distribution of bacterial agent carrier particles in water-oil mixture: animal and plant mixed oil and distilled water were mixed at a mass ratio of 1:1 at 65°C and stirred at 600rpm for 1h to obtain a water-oil mixture; the animal and plant mixed oil was composed of lard and peanut oil at a mass ratio of 1:1. When the water-oil mixture was naturally cooled to 25°C, the bacterial agents obtained in the above-mentioned embodiments and comparative examples were added respectively and stirred at 600rpm for 30min; the mass ratio of the bacterial agent to the water-oil mixture was 1:10. Then, after standing at 25°C for 2h, the carrier particles distributed in the oil phase, water phase, and water-oil interface were counted respectively, and the proportion of carrier particles in the three states was calculated, and the proportion = (the number of carrier particles in the oil phase or water phase or water-oil interface) / the total number of carrier particles × 100%.

[0184] Table 2 Proportion of the bacterial agent of the present invention distributed in the oil phase, water phase, and carrier particles at the interface between water and oil phases

[0185]

[0186]

[0187] The oil degradation rate of Example 1 (using a mixture of xanthan gum and carbomer in a mass ratio of 1:1 as the precipitation aid) = 94.2%, and the water-oil two-phase ratio = 88.4% are significantly better than those of Example 2-3 (using one of xanthan gum and carbomer as the precipitation aid) = (91.5-92.8)%, and the water-oil two-phase ratio = (80.5-83.3)%. This is because: the molecular structures of xanthan gum and carbomer are different, and the ratios of polar groups and non-polar groups contained in their respective molecules are different, so that the two are in the presence of polylactide. The two-phase solution obtained by stirring acid, cellulose acetate and n-butyl propionate has different surface tensions. The difference in surface tension between the two makes the porous polylactic acid precipitated when n-pentanol is added have different surface morphologies; when xanthan gum and carbomer are compounded as precipitation aids, the average particle size distribution and wetting angle range of the porous polylactic acid obtained after proportional regulation are more conducive to the final bacterial agent being evenly distributed at the oil-water interface of the grease wastewater, which is convenient for the bacteria in the preparation to quickly diffuse out and reproduce and decompose grease, thereby improving the ability of the bacterial agent to decompose grease.

[0188] The oil degradation rate of Example 1 (hydrophilic porous polylactic acid particles prepared by a specific method) = 94.2%, the water-oil two-phase ratio = 88.4% is significantly better than that of Comparative Example 2 (polylactic acid particles are not hydrophilically modified) and Comparative Example 3 (hydrophilic porous polylactic acid particles are prepared using Pseudomonas fluorescens lipase) = (74.0-87.6)%, the water-oil two-phase ratio = (25.4-74.9)%, this is because: polylactic acid fibers belong to aliphatic polyesters, there are a large number of ester bonds in the molecular structure, and the hydrophilicity is poor. , and will lead to a longer degradation cycle; the use of porcine pancreatic lipase to modify polylactic acid can convert the ester bonds exposed in the outer layer of polylactic acid particles into polar hydroxyl and carboxyl groups, so that the obtained polylactic acid particles can be better distributed at the interface between the water phase and the oil phase, so that the bacteria in the particles can be better diffused at the oil-water interface, which is conducive to the rapid reproduction and increase of the bacteria; and when the decomposition activity occurs at the oil-water interface, the glycerol and low-level fatty acids in the decomposition products can quickly diffuse into the water to avoid the accumulation of decomposition products. The oil decomposition effect of the microbial agent obtained by hydrophilic modification of porous polylactic acid particles using porcine pancreatic lipase is better than that of using fluorescent Pseudomonas lipase, because porcine pancreatic lipase can achieve higher activity under milder and lower cost conditions due to its unique spatial molecular structure, and the ratio of polar groups to ester groups on the surface of the obtained polylactic acid particles is more moderate, which is more conducive to the uniform distribution of the microbial agent at the oil-water interface, and improves the ability of the microbial agent to decompose oil.

[0189] The oil degradation rate of Example 1 (preparing porous polylactic acid particles using cellulose acetate) = 94.2%, the water-oil two-phase ratio = 88.4% is significantly better than the oil degradation rate of Comparative Example 1 (not using the precipitation aid) and Comparative Example 4 (using carboxymethyl cellulose) = (84.1-87.4)%, the water-oil two-phase ratio = (72.5-73.3)%, this is because: carboxymethyl cellulose has more polar groups, the presence of these polar groups will result in the use of n-pentanol precipitation to obtain porous polylactic acid particles. The particles with poor surface uniformity will rub and collide with each other during subsequent transportation and storage, and break and crush, that is, they have already failed before they are officially put into use; while the proportion of polar groups in cellulose acetate is low, and polylactic acid particles with better surface uniformity and convenient transportation and storage can be precipitated, thereby improving the ability of the bacterial agent to decompose oil.

[0190] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, a person skilled in the art should understand that the technical solutions of the invention may be modified or replaced by equivalents without departing from the scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. An oil pollution decomposing agent, characterized in that: The oil decomposition agent is prepared by the following method: the composite bacteria are inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain an oil decomposition agent; The components of the expansion culture medium are: beef extract, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, distilled water, modified ginseng extract, and hydrophilic porous polylactic acid particles; The preparation method of the modified ginseng extract comprises the following steps: S1 Preparation of ginseng extract: Dry ginseng and crush it through 80-100 mesh to obtain powder; place the powder in 0.1-0.3MPa, 100-130℃ for 10-40min to obtain ginseng powder; dry the ginseng powder at 60-80℃ for 1-3h to obtain dry ginseng powder; mix the dry ginseng powder, enzyme and water in a mass ratio of 1:(0.1-0.5):(5-8), and hydrolyze at 30-50℃ for 6-12h. After the hydrolysis is completed, heat to 100℃ to inactivate the enzyme for 1-2h, filter and obtain ginseng supernatant I and precipitate; concentrate the supernatant I under reduced pressure at a vacuum degree of 0.05-0.07MPa and a temperature of 60-80℃ to a density of 1-1.15g / mL (25℃) to obtain a concentrated solution; add 95wt% ethanol to the concentrated solution after cooling, and the volume ratio of ethanol to the concentrated solution is ( 2-3):1, centrifuge to obtain a precipitate, wash, and freeze-dry to obtain a crude product; mix the crude product and water in a mass ratio of 1:(5-15) and perform ultrasonic extraction for 30-50 minutes, wherein the ultrasonic power is 800-1000W and the ultrasonic frequency is 20-30kHz, and then extract with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1:1, and take the aqueous phase to obtain a crude product aqueous solution; mix the crude product aqueous solution with the mixed solution in a volume ratio of 3:(1-2), stir at 500-700r / min for 30-50min, let stand, centrifuge to obtain ginseng supernatant II, add 95wt% ethanol to the ginseng supernatant II, the volume ratio of ethanol to ginseng supernatant II is (2-3):1, centrifuge to obtain a precipitate, wash, and freeze-dry to obtain a ginseng extract; the mixed solution is a mixture of chloroform and acetone in a mass ratio of (1-3):(1-3); S2 Preparation of modified ginseng extract: Concentrated sulfuric acid, n-butanol, ammonium sulfate, and ginseng extract are mixed in a mass ratio of (35-40):(25-35):(0.1-0.15):(0.4-0.8), cooled to 0°C, stirred at 500-700r / min for 30-40min, and then adjusted to pH=7 with 3-4mol / L sodium hydroxide aqueous solution, and then added with 95wt% ethanol for alcohol precipitation, the precipitate is collected and dissolved in water for dialysis and freeze-drying to obtain the modified ginseng extract; the preparation method of the hydrophilic porous polylactic acid particles is: ① Mix and stir porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 43-48°C to obtain an enzyme treatment solution; ② Mixing the porous polylactic acid particles and the enzyme treatment solution obtained in step ①, stirring, filtering and drying the precipitate to obtain the hydrophilic porous polylactic acid particles; The preparation method of the porous polylactic acid particles is: (1) Mixing polylactic acid, cellulose acetate and n-butyl propionate and stirring to obtain a two-phase solution; (2) mixing n-pentanol and a precipitation aid and stirring to obtain a precipitating agent; ⑶ Cooling and stirring the two-phase solution obtained in step ⑴, and adding the precipitant obtained in step ⑵ to obtain the porous polylactic acid particles.

2. The oil pollution decomposing agent according to claim 1, characterized in that: The composite bacterial species is a mixture of multiple species selected from Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides.

3. The oil pollution decomposing agent according to claim 1, characterized in that: The components of the seed culture medium are: sodium chloride, ammonium acetate, magnesium nitrate, calcium nitrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, yeast extract, chitosan and distilled water.

4. The oil pollution decomposing agent according to claim 1, characterized in that: The culture conditions of the expansion culture are: adding the seed solution to the expansion culture medium and culturing under light; Centrifuge and save the precipitate to obtain the bacterial agent.

5. The oil pollution decomposing agent according to claim 1, characterized in that: The enzyme is one or a mixture of two or more of cellulase, alkaline protease and beta-glucosidase.

6. The oil pollution decomposing agent according to claim 1, characterized in that: The precipitation aid is one of xanthan gum and carbomer or a mixture of the two.

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