A complex microbial agent of yarrowia lipolytica and application thereof in degrading oil

Abstract

The application discloses a kind of Yarrowia lipolytica complex microbial inoculant and its application in degrading oil and fat.The complex microbial inoculant comprises at least two of strain A, strain B and strain C; the strain A is Yarrowia lipolytica with the preservation number CICC@1444, the strain B is Yarrowia lipolytica with the preservation number CGMCC2.1300, and the strain C is Yarrowia lipolytica with the preservation number CGMCC2.1556.The complex microbial inoculant of the application has a high oil and fat degradation rate on oil and fat, especially for high-concentration oil and fat, and the oil and fat degradation rate of double-strain complex microbial inoculant can reach 29.5% at best, and the oil and fat degradation rate of three-strain complex microbial inoculant can reach 30% at best.

Description

Technical Field

[0001] This invention belongs to the field of microbial agents, specifically relating to a compound agent of Yersinia lipophila and its application in degrading oils. Background Technology

[0002] With the continuous improvement of people's living standards in my country, the amount of oily wastewater discharged from both household kitchens and catering kitchens is increasing day by day. Since grease is insoluble in water, direct discharge into sewers can cause blockages, and direct discharge can lead to environmental pollution. Therefore, the treatment of oily wastewater from catering has always been a widely concerned issue. Currently, the commonly used methods for treating oily wastewater are mainly physical and chemical methods. Physical methods generally require complex power structure design, complicated operating procedures, and large investments. Although chemical methods are low-cost, they are highly alkaline, not environmentally friendly, and may cause secondary pollution. In recent years, the newly developed biological method for degrading oily wastewater has the advantages of low cost, low investment, and no secondary pollution, and has attracted widespread attention from researchers at home and abroad in the field of oily wastewater degradation.

[0003] Yeast lipolyticum is found in bread and cheese and is a common yeast cell model organism. As a dimorphic yeast, its cell morphology can switch between yeast form, mycelial form, and pseudomycelial form. The genome of Yeast lipolyticum has been sequenced, containing 6 chromosomes, 20.5 MB, and a GC content of 49%. Due to its publicly available genome sequence, clear genetic background, genetic stability, large cell mass, high expression levels of heterologous proteins, and resistance to degradation, Yeast lipolyticum has been successfully developed into a novel yeast expression system and is considered GRAS (Generally Regulated as Safe), meaning it can be safely used in food and pharmaceutical production.

[0004] The main shortcomings of existing technologies are as follows:

[0005] 1. Current research is generally limited to the treatment of wastewater with low oil concentration (2g / mL), while the oil content of wastewater in actual kitchens is much higher than this. Therefore, it is necessary to explore the lipolysis effect of microorganisms in wastewater with high oil concentration.

[0006] 2. The efficiency of a single strain in degrading lipids is relatively low, so it is necessary to use multiple strains in combination to improve their ability to degrade lipids.

[0007] 3. Currently, the commonly used method for measuring the ability of microorganisms to degrade oils is extraction combined with ultraviolet spectrophotometry. This method determines the oil concentration through turbidity and calculates the oil degradation rate. However, the effect of microorganisms on oils in oily wastewater can be divided into degradation and emulsification. Experiments have shown that emulsified oils will revert to an insoluble state after centrifugation. Therefore, the above method cannot accurately reflect the actual amount of oil degraded by microorganisms, and the calculated oil degradation rate actually includes the effect of emulsification by biological emulsifiers.

[0008] 4. The extraction combined with ultraviolet spectrophotometry method is complex. Not only are the operational steps complicated, but it also requires multiple reagents, is environmentally unfriendly, time-consuming, and costly. Furthermore, this method is only suitable for testing low-concentration oils; the standard curve cannot show good linearity for oil concentrations above 2 g / L.

[0009] 5. Currently, most of the microorganisms reported in patents and literature that can be applied to the biological degradation of oils are Bacillus. There are relatively few articles and patents reporting on the degradation of oils by bacteria such as Yersinia lipophila, which can be safely used in food and pharmaceutical production.

[0010] Patent CN201610878211.4 - A compound microbial agent and its application using ultraviolet spectrophotometry to test the lipolysis rate. In a culture medium with a soybean oil content of 4 g / L, the lipolysis rates of *Candida albicans* WSH-Z06, *Pseudomonas aeruginosa* BBE, and *Streptococcus* WSH-24 before domestication were 42.2%, 33.7%, and 28%, respectively; CN201710027747.X - A microbial agent for lipolysis and its preparation method using *Rhizopus sinensis* and *Trichoderma tumefaciens* at a ratio of 1:4 (… A V / V (volume / volume) compound was used, and the oil degradation rate was measured to be 77.5% in a culture medium with an olive oil content of 2 g / L at 30℃ and 150 rpm for 60 h using ultraviolet spectrophotometry. CN201810792925.2 – A highly efficient microbial compound agent and method for degrading oils and its application – uses four strains of Bacillus licheniformis, Bacillus subtilis, Candida utilis, and Candida albicans in a compound at an oil content of 1% (V / V), with oil degradation rates ranging from 35% to 60% for each formulation. Similar patents, while showing higher oil degradation rates from microorganisms, all use ultraviolet spectrophotometry for testing, with extremely low oil content in the samples, and the strains used are not certified as safe for consumption. Furthermore, their application is limited to industrial wastewater treatment. Summary of the Invention

[0011] To address the shortcomings of existing technologies, such as low oil degradation efficiency, complex operation processes, environmental unfriendliness, time-consuming nature, and high cost associated with single-strain oil degradation, this invention provides a compound microbial agent of *Yarrowia lipolytica* and its application in oil degradation. The compound microbial agent of this invention exhibits a high oil degradation rate, particularly for high-concentration oils. The optimal oil degradation rate for a two-strain compound agent reaches 29.5%, while the optimal oil degradation rate for a three-strain compound agent reaches 30%. This compound microbial agent is suitable for testing the ability of microorganisms to degrade oils at high oil concentrations and for application research of microorganisms in near-real-world kitchen environments.

[0012] The first aspect of the present invention provides a microbial composition comprising at least two of strains A, B, and C, wherein strain A is Yarrowia lipolytica with accession number CICC@1444, strain B is Yarrowia lipolytica with accession number CGMCC2.1300, and strain C is Yarrowia lipolytica with accession number CGMCC2.1556.

[0013] In one embodiment, the microbial composition comprises any one of the following:

[0014] (1) Strains A and B;

[0015] (2) Strains A and C;

[0016] (3) Strains B and C;

[0017] (4) Strain A, strain B and strain C.

[0018] Preferably, the volume ratio of strain A to strain B is 1:(0.5-2), for example 1:1, and the volume ratio of strain A to strain C is 1:(0.5-2), for example 1:1.

[0019] In a preferred embodiment, the strain exists in the form of a bacterial solution.

[0020] A second aspect of the present invention provides a compound microbial agent comprising the microbial composition described in the first aspect of the present invention.

[0021] The third aspect of the present invention provides a method for preparing the compound microbial agent described in the second aspect of the present invention, wherein the compound microbial agent is obtained by inoculating the microbial composition into a culture medium suitable for its growth and shaking it.

[0022] Preferably, the microbial composition is inoculated at an inoculation rate of 2% to 6%, where % is a volume percentage.

[0023] Preferably, the culture medium contains 20 g / L peptone and 10 g / L yeast extract.

[0024] Preferably, the shaking culture is carried out at 28°C and 200 r / min for 60 h.

[0025] The fourth aspect of the present invention provides a method for degrading oils, wherein the degradation is carried out using strain A, the microbial composition described in the first aspect of the present invention, or the compound microbial agent described in the second aspect of the present invention, wherein strain A is Yarrowia lipolytica with accession number CICC@1444.

[0026] Preferably, the concentration of the oil is 1-20%, for example 5%, 10% or 15%, where % is a volume percentage.

[0027] Preferably, the oil is corn oil, rapeseed oil, olive oil, soybean oil, or peanut oil.

[0028] Preferably, the compound microbial agent is obtained by the preparation method described in the fourth aspect of the present invention.

[0029] The fifth aspect of the present invention provides the application of strain A, the microbial composition described in the first aspect of the present invention, or the compound microbial agent described in the second aspect of the present invention in the degradation of oils or the preparation of oil-degrading preparations, wherein strain A is Yarrowia lipolytica with the preservation number CICC@1444.

[0030] Preferably, the concentration of the oil is 1-20%, for example 5%, 10% or 15%, where % is a volume percentage.

[0031] Preferably, the oil is corn oil, rapeseed oil, olive oil, soybean oil, or peanut oil.

[0032] Preferably, the compound microbial agent is obtained by the preparation method described in the fourth aspect of the present invention.

[0033] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0034] The reagents and raw materials used in this invention are all commercially available.

[0035] The positive and progressive effects of this invention are as follows:

[0036] 1. Compound bacterial agents have a high oil degradation rate, especially for high concentrations of oil. Compared with single strains, they have a statistically significant effect. Among them, the oil degradation rate of two-strain compound bacterial agents can reach 29.5%, and the oil degradation rate of three-strain compound bacterial agents can reach 30%, indicating that compounding can effectively improve the oil degradation rate of bacterial strains.

[0037] 2. This method is suitable for testing the ability of microorganisms to degrade oils at high oil concentrations and is applicable to research on the application of microorganisms in a near-real kitchen setting. Attached Figure Description

[0038] Figure 1 The study investigated the lipid degradation rate and lipase activity of yeast experimental strains in oil-containing culture media.

[0039] Figure 2 This is a schematic diagram for measuring the remaining oil level in a centrifuge tube.

[0040] Figure 3 This is a schematic diagram showing the lipid solubility of strain CICC@1444 in different types of household vegetable oils.

[0041] Figure 4 This is a schematic diagram showing the lipolysis rate of strain CICC@1444 under different inoculum amounts. Detailed Implementation

[0042] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0043] Specific technical solution of the present invention:

[0044] Experimental Objective

[0045] A method for evaluating the ability of bacterial strains to degrade lipids under ultra-high oil content conditions is provided. The strain with the highest lipid degradation rate under ultra-high oil content conditions was screened from six species of Yersinia lipophila, and the consistency of the results with the results of lipase activity assay was verified.

[0046] This study provides six methods for preparing compound bacterial agents by combining two strains of Yersinia lipophila to degrade lipids, and screens out the two strain combinations with the highest lipid degradation rate under ultra-high oil content conditions; or screens out the three strain combinations with the highest lipid degradation rate under ultra-high oil content conditions.

[0047] Experimental reagents

[0048] 1. YPD culture medium

[0049] Add 20 g / L glucose, 10 g / L yeast extract, and 20 g / L tryptone. If necessary, add 20 g / L agar powder. Pour the culture medium into a shake flask according to the above formula, place it in an autoclave, and sterilize at 121°C for 20 minutes.

[0050] 2. Sugar-free YPD medium

[0051] Yeast extract 10g / L, peptone 20g / L, according to the above culture medium formula, are placed into Erlenmeyer flasks and placed in an autoclave for sterilization at 121℃ for 20min.

[0052] 3. Experimental oil

[0053] The vegetable oils used in the experiment were mainly purchased from the market, including peanut oil (Jinlongyu), rapeseed oil (Jinlongyu), corn oil (Jinlongyu), olive oil (Jinlongyu), and soybean oil (Jinlongyu).

[0054] Experimental protocol

[0055] 1. Six methods for preparing lipophilic yeast agents:

[0056] Six strains of *Yarrowia lipolyticis* (strain sources are shown in Table 1) were streaked onto solid culture dishes and incubated at 37°C for 24–48 h. Single colonies were picked and transferred to centrifuge tubes containing YPD liquid medium. These centrifuge tubes were then placed in an air-bath constant-temperature shaker at 28°C and 200 rpm for 24–48 h until the culture OD reached its maximum. 600 =1, remove the bacterial agent from the shaker for later use.

[0057] Table 1 Information on the strains used in the experiment

[0058]

[0059] 2. Six methods for degrading lipids using single strains of Yersinia lipophila:

[0060] Six strains of Yersinia lipophila were selected as experimental strains, and OD was... 600 Six types of lipophilic Yersinia (blank control plus sugar-free YPD) were added at an inoculum of 2% (v / v) to 98 mL of sugar-free YPD medium (peptone 20 g / L, yeast extract 10 g / L) with an oil content of 10% (v / v). The medium was sealed with sterile sealing film and placed in an air bath constant temperature shaker at 28℃ and 200 rpm for 60 h. The lipid degradation rate was then tested (each experimental group was set up in parallel).

[0061] 3. Six combinations of two-to-one combinations of lipolytic Yeast and their methods for degrading lipids:

[0062] First, six types of lipophilic Yeast were paired up for experiments. In each pair, OD was measured separately. 600Two types of lipophilic Yersinia (blank control plus sugar-free YPD) were mixed at a 1:1 (V / V) ratio and inoculated at 1% (V / V) in 98 mL of sugar-free YPD medium (20 g / L peptone and 10 g / L yeast extract) with an oil content of 10% (V / V). The medium was sealed with sterile sealing film and incubated in an air bath at 28°C and 200 rpm for 60 h. The lipid degradation rate was then tested (parallel experiments were set up for each experimental group).

[0063] 4. Evaluation method for the ability of bacterial strains to degrade lipids under ultra-high oil content conditions:

[0064] After the degradation reaction is complete, transfer the liquid in the conical flask to a 50 mL centrifuge tube and centrifuge at a low speed of 4000 r / min for 10 min. Transfer the supernatant oil layer in the centrifuge tube to a 15 mL measuring centrifuge tube. Transfer the intermediate layer to a 2 mL centrifuge tube and centrifuge at a high speed of 12000 r / min for 10 min. Transfer the supernatant oil layer obtained from this centrifugation to the corresponding 15 mL measuring centrifuge tube. Measure and record the volume of the remaining oil (accurate to 0.1 mL).

[0065] To further qualitatively verify the lipid degradation ability of the strain and the comparability after quantification, the lipid degradation rate is defined as κ, which is calculated according to formula (1.1).

[0066]

[0067] In the formula: V0 is the initial refueling volume, mL;

[0068] V1 represents the remaining oil volume in the control group, in mL;

[0069] V2 represents the remaining oil volume in the experimental group, in mL.

[0070] 5. Lipase Activity Test Method

[0071] Using polyvinyl alcohol-ol olive oil emulsion as the substrate, 4 mL of the substrate was mixed with 5 mL of phosphate buffer solution and preheated in a 40°C water bath for 5 min. Then, 1 mL of fermentation supernatant was added to form a 10 mL reaction system, which was heated in a 40°C water bath. The reaction was carried out in a shaker at 200 rpm for 15 min (timed precisely from the start of enzyme addition), and then 10 mL of 95% ethanol was added to terminate the reaction. The amount of fatty acids hydrolyzed by the enzyme was determined by titration with a 0.1 mol / L sodium hydroxide standard solution. Three drops of 1% phenolphthalein were added as an indicator, and the titration endpoint was reached when the liquid turned a light pink color and maintained this color for 3 seconds. The amount of sodium hydroxide consumed was recorded, and the enzyme activity was calculated by comparing it with the control sample. The control sample did not contain fermentation broth, but all other parameters were the same, and the determination was repeated three times. The lipase activity value was calculated according to formula (1.2).

[0072] X = (BA) × 5 × n (1.2)

[0073] In the formula: X represents the lipase activity of the sample, U / mL;

[0074] B represents the volume of NaOH standard solution required for the titration of the sample group, in mL;

[0075] A represents the volume of NaOH standard solution required for the blank group to complete the titration, in mL;

[0076] n is the dilution factor of the enzyme solution to be tested in the experiment.

[0077] Example 1: Six methods for testing the lipid degradation ability of Yersinia lipophila

[0078] The experimental strains were inoculated at a rate of 2% (v / v) into 98 mL of sugar-free YPD medium (20 g / L peptone, 10 g / L yeast extract) with an oil content of 10% (v / v). The medium was sealed with sterile sealing film and incubated in a shaker at 28°C and 200 rpm for 60 hours. After 60 hours, the conical flasks were removed from the shaker. At this point, the lower layer of the flask was a milky white homogeneous bacterial solution, and the upper layer was a yellow emulsion of oil. The solution in the conical flasks was shaken well and transferred to two 50 mL centrifuge tubes. The tubes were centrifuged at 4000 rpm for 10 minutes. At this point, the centrifuge tubes contained four layers: the top layer was a clear oil layer; the second layer was a layer of lipid degradation products, which was an emulsion; the third layer was a light brown liquid bacterial layer; and the bottom layer was a flocculent bacterial layer. Transfer the topmost oil layer from the centrifuge tube to a 15mL measuring centrifuge tube, and the second layer to multiple 2mL centrifuge tubes. Centrifuge all 2mL tubes again at 12000rpm for 5 minutes. At this point, the centrifuge tubes will have three layers: the top layer is a clear oil layer, the second layer is a dense solid layer of lipid degradation products, and the third layer is a light brown liquid layer of bacterial culture. Then, transfer the supernatant oil layer from the 2mL centrifuge tubes to the corresponding 15mL measuring centrifuge tubes. Figure 2 The volume of remaining oil is measured and recorded (accurate to 0.1 mL).

[0079] Calculation of oil degradation rate: The oil degradation rate is defined as κ, and its calculation method is shown in equation (1.1):

[0080]

[0081] Where V0 is the initial refueling volume (mL); V1 is the remaining oil volume of the control group (mL); and V2 is the remaining oil volume of the experimental group (mL).

[0082] The results of the lipolysis rate and lipase activity of six lipolytic yeasts tested using this method at a 10% (v / v) oil concentration are as follows: Figure 1As shown, the strain with the best lipid degradation rate was CICC@1444, with a lipid degradation rate of 22.5%. CICC@1444 also exhibited the highest lipase activity, reaching 8.25 U / mL. A positive correlation was found between the lipid degradation rate and enzyme activity of the six *Yarrowia lipolytica* strains, demonstrating that this method can effectively test the lipid degradation ability of *Yarrowia lipolytica*.

[0083] Example 2: Test of the lipid degradation ability of CICC@1444 strain in different household vegetable oils

[0084] Since different types of oils have different unsaturated fatty acid contents and chemical compositions, and the sensitivity of oils to the growth and metabolism of strains also varies, the oils contained in oily wastewater in real-world scenarios are also diverse. Therefore, the strain CICC@1444, which has the highest oil degradation rate in Example 1, was selected to test the oil degradation rate of different types of household vegetable oils.

[0085] CICC@1444 was inoculated at a rate of 2% (v / v) into 98 mL of sugar-free YPD medium (20 g / L peptone, 10 g / L yeast extract) containing corn oil, rapeseed oil, olive oil, soybean oil, and peanut oil, respectively, with an oil content of 10% (v / v). The medium was sealed with sterile sealing film and incubated in a shaker at 28°C and 200 rpm for 60 h. After 60 h, the conical flasks were removed from the shaker, and the oil degradation rate was tested. The specific testing method is described in Example 1. The oil degradation rate results were obtained from... Figure 3 As shown, CICC@1444 exhibits the highest oil degradation rate of 37.5% for peanut oil.

[0086] Example 3: Effect of inoculum size on the lipid degradation ability of CICC@1444 strain

[0087] The inoculum size affects the growth and reproduction rate of the strain in the environment. Therefore, the strain CICC@1444, which had the highest oil degradation rate in Example 1, was selected to test its ability to degrade oil under different inoculum sizes. CICC@1444 strain was added to 98 mL of sugar-free YPD medium (20 g / L peptone, 10 g / L yeast extract) with an oil content of 10% (V / V) at inoculum sizes of 1%, 2%, 4%, 6%, and 8%. The flasks were sealed with sterile sealing film and incubated in a constant temperature shaker at 28℃ and 200 rpm for 60 h. After 60 h, the conical flasks were removed from the shaker for oil degradation rate testing. The specific testing method is described in Example 1.

[0088] Oil degradation rate results are as follows Figure 4As shown, the degradation rate of lipids by the strain varied under different inoculum amounts. When the inoculum amount was less than 1%, the lipid degradation rate was 15%; when the inoculum amount was between 2% and 6%, the lipid degradation rate exceeded 20%; and when the inoculum amount was greater than 6%, the lipid degradation rate began to decrease. When the inoculum amount exceeded a certain range, the bacterial cells rapidly multiplied and grew, and the mycelia or mycelial masses formed by the cells aggregated, reducing the dissolved oxygen level in the environment and further affecting the growth of the strain and the activity of its metabolites. Therefore, this explains why the relationship between inoculum amount and lipid degradation rate is not linear, and why there exists an optimal range for inoculum amount. In this experiment, under the condition that other factors remained constant, the optimal inoculum amount was 2%–6%.

[0089] Example 4: Method for degrading oils with a two-strain compound bacterial agent and test of oil degradation rate.

[0090] First, six types of lipophilic Yeast were paired up for experiments. In each pair, OD was measured separately. 600 Two types of lipophilic Yeasts (=1) were mixed at a 1:1 (V / V) ratio and inoculated at 1% (V / V) each (total inoculation amount of 2%) into 98 mL of sugar-free YPD medium (20 g / L peptone, 10 g / L yeast extract) with an oil content of 10% (V / V). The medium was sealed with sterile sealing film and placed in an air bath constant temperature shaker at 28℃ and 200 rpm for 60 h. After that, the conical flask was removed from the shaker and the oil degradation rate was tested. The specific test method is described in Example 1. Table 2 shows the lipid degradation rates of the paired strains. The results indicate that pairwise pairing of strains can improve their lipid degradation ability to a certain extent. Table 2 shows that the optimal combination for lipid degradation was CICC@1444 and CGMCC2.1300, with a lipid degradation rate of 29.5%. The lipid degradation rate of the combination of CGMCC2.1300 and CGMCC2.1556 reached 1.75 times the average lipid degradation rate of the two single strains. To demonstrate the significant effect of paired strain pairing on improving lipid degradation rate, a statistical significance analysis was performed on the lipid degradation rates of single strains and paired strain pairings. From a statistical perspective, a P-value less than 0.05 indicates a significant difference, and a P-value less than 0.01 indicates an extremely significant difference.

[0091] Table 2. Lipid degradation rates of six different Yersinia lipophila compound inoculants.

[0092] 90811 15.5％ 56209 14％ 12％ 1444 18.5％ 23.5％ 22.5％ 53987 18.5％ 22.5％ 19.5％ 10％ 2.1300 15％ 14.5％ 29.5％ 16％ 15％ 2.1556 24.5％ 21％ 27.5％ 15％ 28.5％ 17.5％

[0093] Table 3. One-way ANOVA of single strain CGMCC2.1300 and the two-strain compound inoculum CGMCC2.1300 & CGMCC2.1556

[0094] OK 273.375 1 273.375 50.86047 0.0191 18.51282 error 10.75 2 5.375 total 299.875 5

[0095] Table 4. One-way ANOVA of single strain CGMCC2.1300 and the two-strain compound inoculum CICC@1444 & CGMCC2.1300

[0096] OK 301.0417 1 301.0417 118.4426 0.008337 18.51282 error 5.083333 2 2.541667 total 314.2083 5

[0097] Table 5. One-way ANOVA of single strain CGMCC2.1556 and the two-strain compound inoculum CICC@1444&CGMCC2.1556

[0098] OK 287.0417 1 287.0417 22.00958 0.042556 18.51282 error 26.08333 2 13.04167 total 353.2083 5

[0099] As shown in Table 3-5, the P-values ​​for both single and double strains are less than 0.05, which proves that the combination of double strains can effectively improve the lipid degradation rate of the strains.

[0100] Example 5: Preferred method for degrading oils using a three-strain compound bacterial agent and oil degradation rate test.

[0101] As shown in Examples 1 and 4, strain 1444 exhibited the best single-strain lipolysis ability, while the two-strain combinations showed relatively good results: 90811 & 2.1556, 1444 & 2.1300, 1444 & 2.1556, and 2.1300 & 2.1556. Therefore, this invention integrates the above strains into three three-strain combinations and verifies their lipolysis rates. These combinations include 90811 & 1444 & 2.1556, 90811 & 2.1300 & 2.1556, and 1444 & 2.1300 & 2.1556. In each combination experiment, OD... 600 Three types of Yersinia lipophila were mixed in a 1:1:1 (V / V) ratio and inoculated at 0.67% (V / V) each (total inoculation amount of 2%) into 98 mL of sugar-free YPD medium (20 g / L peptone, 10 g / L yeast extract) with an oil content of 10% (V / V). The medium was sealed with sterile sealing film and placed in an air bath constant temperature shaker at 28℃ and 200 rpm for 60 h. After that, the conical flask was removed from the shaker and the oil degradation rate was tested. The specific test method is described in Example 1. The results are shown in Table 6. The lipid degradation rate of the 90811&1444&2.1556 combination was 20%, the 90811&2.1300&2.1556 combination was 25%, and the 1444&2.1300&2.1556 combination was 30%. These results indicate that the preferred three-strain combination 1444&2.1300&2.1556 can further improve the lipid degradation ability of *Yarrowia lipolytica*. The lipid degradation rates of the other two three-strain combinations decreased instead of increasing. A significant difference analysis was performed on the lipid degradation rates of the 1444&2.1300&2.1556 combination and the single strain CGMCC2.1300. The results are shown in Table 7.

[0102] Table 6. Significance Analysis of the Effects of the Three-Plant Compound Treatment

[0103]

[0104] Table 7. One-way ANOVA of single strain CGMCC2.1300 and three-strain compound bacterial agents CICC@1444, CGMCC2.1556, and CGMCC2.1300

[0105] OK 360.375 1 360.375 961 0.001039 18.51282 error 0.75 2 0.375 total 361.875 5

[0106] As shown in Table 7, the P-value of the single three strains is less than 0.01, which proves that the combination of two strains can effectively improve the lipid degradation rate of the strains.

Claims

1. A microbial composition, characterized in that, The microbial composition is selected from any one of the following groups: (1) Strains A and B; (2) Strain B and strain C; (3) Strains A, B and C; The strain A is *Yamaceae lipolytica* with accession number CICC 1444. Yarrowia lipolytica The strain B is *Saccharomyces lipolyticus* with accession number CGMCC2.1300. Yarrowia lipolytica The strain C is *Saccharomyces lipolyticus* with accession number CGMCC2.1556. Yarrowia lipolytica ).

2. The microbial composition according to claim 1, characterized in that, When the microbial composition is group (1), the volume ratio of strain A to strain B is 1:(0.5-2). And / or, the strain exists in the form of a bacterial suspension.

3. The microbial composition according to claim 2, characterized in that, When the microbial composition is group (1), the volume ratio of strain A to strain B is 1:

1.

4. A compound microbial agent comprising the microbial composition as described in any one of claims 1-3.

5. The method for preparing the compound microbial agent as described in claim 4, characterized in that, The compound bacterial agent is obtained by inoculating the microbial composition into a culture medium suitable for its growth and shaking it.

6. The preparation method according to claim 5, characterized in that, The microbial composition is inoculated at an inoculum of 2% to 6%, where % is a volume percentage; and / or, the culture medium contains 20 g / L peptone and 10 g / L yeast extract; and / or, the shaking culture is carried out at 28°C and 200 r / min for 60 h.

7. A method for degrading oils, characterized in that, Degradation is carried out using the microbial composition as described in any one of claims 1-3 or the compound microbial agent as described in claim 4.

8. The method as described in claim 7, characterized in that, The concentration of the oil is 1-20%, where % is a volume percentage; And / or, the oil is corn oil, rapeseed oil, olive oil, soybean oil or peanut oil; And / or, the compound microbial agent is obtained by the preparation method as described in claim 5 or 6.

9. The method as described in claim 8, characterized in that, The concentration of the oil is 5%, where % is a volume percentage.

10. The method as described in claim 8, characterized in that, The concentration of the oil is 10%, where % is a volume percentage.

11. The method as described in claim 8, characterized in that, The concentration of the oil is 15%, where % is a volume percentage.

12. The use of the microbial composition according to any one of claims 1-3 or the compound microbial agent according to claim 4 in the degradation of oils or the preparation of oil-degrading preparations.

13. The application as described in claim 12, characterized in that, The concentration of the oil is 1-20%, where % is a volume percentage; And / or, the oil is corn oil, rapeseed oil, olive oil, soybean oil or peanut oil; And / or, the compound microbial agent is obtained by the preparation method as described in claim 5 or 6.

14. The application as described in claim 13, characterized in that, The concentration of the oil is 5%, where % is a volume percentage.

15. The application as described in claim 13, characterized in that, The concentration of the oil is 10%, where % is a volume percentage.

16. The application as described in claim 13, characterized in that, The concentration of the oil is 15%, where % is a volume percentage.

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