Aspergillus fumigatus for degrading wax cane and application of aspergillus fumigatus

Abstract

The aspergillus fumigatus ZL2K2 strain is separated from soil of a sugarcane field of Guangxi guest, the preservation number of the aspergillus fumigatus ZL2K2 strain is GDMCC NO: 65138, the preservation date of the aspergillus fumigatus ZL2K2 strain is September 14, 2024, the preservation address is Guangzhou and Guangdong Microbial Culture Collection Center in China, Guangzhou and Guangdong Province, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation number of the aspergillus fumigatus ZL2K2 strain is CGMCC NO: 65138, and the preservation number is CGMCC NO: 65138. The strain is good in growth stability and high in wax cane degradation efficiency, and the wax cane degradation rate reaches 23.88%.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to a cane wax-degrading Aspergillus fumigatus and an application thereof. [Background technology]

[0002] Guangxi has abundant sugarcane resources, which are large in quantity and easy to obtain, and are therefore widely used. Sugarcane is an important raw material for sugar production and biomass energy, and is the most important raw material for sugar production in my country. It plays a pivotal role in my country's sugar production and plays a vital role in meeting the needs of people's daily life, food and beverage processing industry, and pharmaceutical manufacturing industry.

[0003] However, the byproducts of the sugarcane industry, bagasse and filter mud, decompose slowly and have low processing utilization rates, resulting in problems such as low product quality and difficulty in degradation when used in the papermaking industry; problems such as reduced quality and low safety when used in the production of fertilizers and animal feed; and problems such as poor clarification and filtration during production and processing in sugar factories, resulting in poor gloss and low yield of sugarcane products. The reason may be caused by the wax on the surface of sugarcane. Destroying the wax on the surface of sugarcane is the first step to more effectively utilize sugarcane and sugarcane byproducts.

[0004] The destruction of the wax on the surface of sugarcane is the first step to more effectively utilize sugarcane and degrade sugarcane byproducts. It is very necessary to take measures to reduce wax and clean wax. The main wax reduction methods are physical method, chemical method and microbial degradation method.

[0005] Physical method: mainly through hot water, hot oil and other heat treatment to remove wax. Although this method has high degradation efficiency, it will affect the loss of nutrients in the product during the sugar production process, seriously affect the processing of by-products, and reduce their utilization value. In addition, hot water and hot oil will also cause machine blockage. Chemical method: Chemical wax reducers are usually used to degrade wax, but chemical wax reducers are not only costly, but also affect the safety of the sugar production process, reduce sugar production, poor quality of by-product utilization, low reliability, poor safety, pollution of the environment and threats to workers' health. Microbial degradation method: Microbial degradation uses the metabolism of microorganisms in nature to remove organic matter, thereby achieving the purpose of degradation. Microbial degradation has the advantages of high efficiency, non-toxicity, no secondary pollution, high safety, easy operation, low cost, and environmental protection.

[0006] The invention uses the Aspergillus fumigatus screened by the inventors, which has the characteristics of good growth stability, high sugarcane wax degradation rate, etc. The invention lays a foundation for sugarcane wax degradation bacterial agents. [Summary of the invention]

[0007] In view of this, the object of the present invention is to provide a sugarcane wax-degrading Aspergillus fumigatus and application thereof.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] An Aspergillus fumigatus strain ZL2K2, whose preservation number is GDMCCNO: 65138, preservation date: September 14, 2024, preservation address: Guangdong Microbiological Culture Collection Center, Guangzhou, China, preservation unit: Guangdong Microbiological Culture Collection Center.

[0010] It is further explained that the Aspergillus fumigatus strain ZL2K2 is isolated from the soil of a sugarcane field in Laibin, Guangxi.

[0011] The present application also provides a microbial preparation comprising the Aspergillus fumigatus strain ZL2K2 as described above.

[0012] Further, the microbial preparation contains a viable count of not less than 1.0×10 6 fumigatus ZL2K2 cells per mL.

[0013] The present application also provides an application of the Aspergillus fumigatus ZL2K2 described above in degrading sugarcane wax.

[0014] The present application also provides a method for preparing the above-mentioned degraded cane wax, the method comprising the following steps:

[0015] The preserved Aspergillus fumigatus ZL2K2 was inoculated on the slant medium and cultured in a 37°C constant temperature incubator for 2 days to obtain activated strains. The strains were washed with sterile water to obtain a concentration of 1.0×10 6 The suspension of Aspergillus fumigatus ZL2K2 was prepared at a concentration of 100 μg / mL. The suspension was inoculated into the optimized fermentation medium in a shake flask. The culture was carried out at 37°C and a rotation speed of 180 r / min for 96 h. Samples were taken to calculate the degradation rate of sugarcane wax.

[0016] In the present invention, it is further described that the slant culture medium is ZL culture medium; the formula of the ZL culture medium is: NH4NO3 3.00 g / L, KH2PO4 2.00 g / L, Na2HPO4 3.00 g / L, MgSO4 0.20 g / L, NaCl 1.00 g / L, cane wax 10 g / L, agar 20.00 g / L. Sterilize at 115°C under high pressure for 30 min.

[0017] In the present invention, it is further described that the fermentation medium formula after the shake flask optimization is: NH4NO33.00g / L, KH2PO42.00g / L, Na2HPO43.00g / L, MgSO40.20g / L, NaCl1.00g / L, cane wax 10g / L, lactose 25g / L, peptone 40g / L, sterilized at 121°C for 30min.

[0018] The present application also discloses the application of the above-mentioned Aspergillus fumigatus ZL2K2 in the fields of industry and agriculture.

[0019] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0020] The Aspergillus fumigatus strain ZL2K2 of the present invention is a strain isolated and screened from the soil of a sugarcane field in Laibin, Guangxi. The study found that the strain is an Aspergillus fumigatus strain ZL2K2 that degrades sugarcane wax. In order to study the ability of the strain to degrade sugarcane wax, a preliminary study of single factors was conducted. The applicant determined through the study that the optimal conditions for Aspergillus fumigatus strain ZL2K2 to degrade sugarcane wax are: NH4NO3 3.00 g / L, KH2PO4 2.00 g / L, Na2HPO4 3.00 g / L, MgSO4 0.20 g / L, NaCl 1.00 g / L, sugarcane wax 10 g / L, lactose 25 g / L, peptone 40 g / L; pH 6.0, inoculation amount 10%, culture temperature 37°C, culture time 96 hours. The sugarcane wax degradation rate reaches 23.88%

Brief Description of the Drawings

[0021] Figure 1 This is a morphological diagram of the Aspergillus fumigatus ZL2K2 strain on a plate according to an embodiment of the present application;

[0022] Figure 2 This is a graph showing the effect of different carbon sources on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0023] Figure 3 This is a graph showing the effect of different carbon source concentrations on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0024] Figure 4 This is a graph showing the effect of different nitrogen sources on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0025] Figure 5 This is a graph showing the effect of different nitrogen source concentrations on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0026] Figure 6 This is the effect of different pH on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0027] Figure 7 This is the effect of different inoculation amounts of the strain on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0028] Figure 8 This is a graph showing the effect of different fermentation temperatures on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax;

[0029] Fig. 9 This graph shows the effect of different fermentation times on the ability of Aspergillus fumigatus ZL2K2 to degrade sugarcane wax. [Specific implementation method]

[0030] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0031] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0032] 1. Screening of bacterial strains

[0033] Soil samples were taken from the sugarcane field in Laibin, Guangxi. ZL medium was used to form a screening medium to screen strains that degrade sugarcane wax. The process was as follows: Initial screening of strains: 1 g of soil was added to 9 mL of sterile saline to make 10 -1 The sample solution of concentration was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration, set aside for use. Take the last three dilutions, separate the strains on ZL medium plates by spreading, separate by streaking on the plates, purify repeatedly, and obtain pure cultures of the isolated strains. Store the plates in a 4°C refrigerator.

[0034] ZL culture medium is specifically ZL agar slant solid culture medium, and its formula is: NH4NO3 3.00 g / L, KH2PO4 2.00 g / L, Na2HPO4 3.00 g / L, MgSO4 0.20 g / L, NaCl 1.00 g / L, cane wax 10 g / L, and agar 20.00 g / L.

[0035] 2. Identification of bacterial strains and optimization of sugarcane wax degradation conditions

[0036] 1. Identification of bacterial species

[0037] (1) Observation of colony morphology: The slant bacteria were inoculated into a plate containing ZL medium and cultured at 37°C for 48 h. The colony morphology was observed.

[0038] (2) 16S rDNA Identification of the Strain: The purified strain was sent to Guangdong Provincial Microbiological Culture Collection Center for identification. Nucleotide BLAST analysis showed that the 16S rDNA sequence homology with the Aspergillus fumigatus strain was 99.7%, and it was preliminarily determined that the ZL2K2 strain was Aspergillus fumigatus.

[0039] The preservation information of the screened Aspergillus fumigatus ZL2K2 is as follows: the name is Aspergillus fumigatus ZL2K2, its classification name is Aspergillus fumigatus ZL2K2, the preservation number is GDMCC NO: 65138, the preservation date is September 14, 2024, the preservation address is: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the preservation unit is: Guangdong Provincial Microbial Culture Collection Center.

[0040] The morphological characteristics of Aspergillus fumigatus ZL2K2 on a plate according to the present invention are as follows: Figure 1 As shown, the surface of the colony formation on the culture medium is not smooth, rough, irregularly edged, slightly raised, opaque, with a small number of radial wrinkles and concentric rings; the texture is velvety to flocculent.

[0041] 2. Study on the optimization of sugarcane wax degradation conditions by Aspergillus fumigatus ZL2K2

[0042] The formula of the basic culture medium for cane wax degradation is: NH4NO3 3.00 g / L, KH2PO4 2.00 g / L, Na2HPO4 3.00 g / L, MgSO4 0.20 g / L, NaCl 1.00 g / L, cane wax 10 g / L, glucose 10 g / L, and peptone 10 g / L.

[0043] (1) Optimization of carbon source and its concentration

[0044] Aspergillus fumigatus ZL2K2 was inoculated with cane wax degradation basal medium, and the carbon source variables were sucrose, starch, lactose, glucose, and maltose. The liquid medium was prepared and cultured on a shaker at 35°C and 180 r / min for 72 hours; on this basis, the effect of lactose concentration (1, 1.5, 2, 2.5, 3%) on the degradation of cane wax by the strain was further explored, and the cane wax degradation rate was measured by sampling. The results are shown in Table 1 and Figure 2 , 3 .

[0045] Table 1 Effects of different carbon sources on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0046]

[0047] (2) Optimization of nitrogen source and its concentration

[0048] Aspergillus fumigatus ZL2K2 was inoculated with cane wax degradation basal medium, and beef extract, peptone, yeast powder, peanut cake powder, and soybean powder were selected as organic nitrogen sources in the medium. The liquid medium was prepared and cultured on a shaker at 35°C and a speed of 180r / min for 72h. On this basis, the effect of soybean powder concentration (1, 2, 3, 4, 5%) on the degradation of cane wax by the strain was further explored, and the cane wax degradation rate was measured by sampling. The results are shown in Table 2 and Figure 4 , Figure 5 .

[0049] Table 2 Effects of different nitrogen sources on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0050]

[0051] (3) Effect of different pH values ​​on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0052] Aspergillus fumigatus ZL2K2 was inoculated on the basis of the cane wax degradation basic medium, and the initial pH of the medium was adjusted to 4, 5, 6, 7, and 8, and the supernatant was taken to measure the enzyme activity. The liquid medium was prepared and incubated on a shaker at 35°C and a speed of 180r / min for 72h, and the cane wax degradation rate was measured. The results are shown in Table 3 and Figure 6 .

[0053] Table 3 Effect of different fermentation medium pH on the degradation ability of sugarcane wax by Aspergillus fumigatus ZL2K2

[0054] project pH(4) pH(5) pH(6) pH(7) pH(8) Degradation rate (%) 11.68 13.32 15.54 14.86 13.06

[0055] (4) Effects of different inoculation amounts on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0056] Aspergillus fumigatus ZL2K2 was inoculated on the basis of the cane wax degradation basic medium, and the inoculation amount was adjusted to 4, 6, 8, 10, and 12% respectively. The temperature was 35°C, the rotation speed was 180r / min, and the culture was carried out for 72h. The cane wax degradation rate was measured by sampling. The results are shown in Table 4 and Figure 7 .

[0057] Table 4 Effect of different inoculation amounts on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0058]

[0059] (5) Effects of different fermentation temperatures on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0060] Aspergillus fumigatus ZL2K2 was inoculated on the basis of the cane wax degradation basic medium, and the pH was 6.0, the inoculation amount was 10%, the rotation speed was 180r / min, and the temperatures were 31°C, 33°C, 35°C, 37°C, and 39°C for 72h, and the cane wax degradation rate was measured by sampling. The results are shown in Table 5 and Figure 8 .

[0061] Table 5 Effect of different fermentation temperatures on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0062] project 31℃ 33℃ 35℃ 37℃ 39℃ Degradation rate (%) 15.55 16.84 17.92 19.72 14.74

[0063] (6) Effects of different fermentation times on the degradation of sugarcane wax by Aspergillus fumigatus ZL2K2

[0064] Aspergillus fumigatus ZL2K2 was inoculated on the basis of the cane wax degradation basic medium, and cultured for 72, 84, 96, 108, and 120 hours at a temperature of 37°C, pH 6.0, and a rotation speed of 180 r / min, and samples were taken to measure the cane wax degradation rate. The results are shown in Table 6 and Fig. 9 .

[0065] Table 6 Effect of different fermentation times on the degradation capacity of sugarcane wax by Aspergillus fumigatus ZL2K2

[0066]

[0067]

[0068] (7) Verification test

[0069] Based on the optimal result of the single factor experiment, a verification test was conducted and samples were taken to measure the degradation rate of sugarcane wax. The results are shown in Table 5.

[0070] Table 7 Test verification results

[0071] project Test 1 Test 2 Test 3 average value Degradation rate (%) 23.83 24.06 23.75 23.88

[0072] Through Tables 1 to 7 and Figures 2 to 9The single factor optimization and validation test data of sugarcane wax degradation rate can determine that the optimal conditions for Aspergillus fumigatus ZL2K2 to degrade sugarcane wax are: NH4NO3 3.00 g / L, KH2PO4 2.00 g / L, Na2HPO4 3.00 g / L, MgSO4 0.20 g / L, NaCl 1.00 g / L, sugarcane wax 10 g / L, lactose 25 g / L, peptone 40 g / L; pH 6.0, inoculation 10%, culture temperature 37 ° C, culture time 96 h. The degradation rate of sugarcane wax reached 23.88%

[0073] The degradation rate of sugarcane wax was determined by weighing method.

[0074] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An Aspergillus fumigatus strain ZL2K2, whose deposit number is GDMCC NO: 65138, deposit date: September 14, 2024, deposit address: Guangdong Microbiological Culture Collection Center, Guangzhou, China, deposit unit: Guangdong Microbiological Culture Collection Center.

2. The Aspergillus fumigatus according to claim 1 ( Aspergillus fumigatus ) strain ZL2K2, characterized in that The strain was isolated from the soil of a sugarcane field in Laibin, Guangxi.

3. A method comprising the Aspergillus fumigatus ( Aspergillus fumigatus )Microbial preparation of strain ZL2K2.

4. The microbial preparation according to claim 3, characterized in that The number of viable bacteria in the microbial preparation is not less than 1.0×10 6 fumigatus ZL2K2 cells per mL.

5. Use of Aspergillus fumigatus ZL2K2 as claimed in claim 1 in degrading sugarcane wax.

6. A method for preparing the degraded sugarcane wax as claimed in claim 5, characterized in that: The method comprises the following steps: The preserved Aspergillus fumigatus ZL2K2 was inoculated on the slant medium and cultured in a 37°C constant temperature incubator for 2 days to obtain activated strains. The strains were washed with sterile water to obtain a concentration of 1.0×10 6 The suspension of Aspergillus fumigatus ZL2K2 was prepared at a concentration of 100 μg / mL. The suspension was inoculated into the optimized fermentation medium in a shake flask. The culture was carried out at 37°C and a rotation speed of 180 r / min for 96 h. Samples were taken to calculate the degradation rate of sugarcane wax.

7. The method according to claim 6, characterized in that The slant culture medium is ZL culture medium; the formula of ZL culture medium is: NH4NO3 3.00 g / L, KH2PO4 2.00 g / L, Na2HPO4 3.00 g / L, MgSO4 0.20 g / L, NaCl 1.00 g / L, cane wax 10 g / L, agar 20.00 g / L. Sterilize at 115°C under high pressure for 30 minutes.

8. The method according to claim 6, characterized in that The fermentation medium formula after shake flask optimization is: NH4NO3 3.00g / L, KH2PO4 2.00g / L, Na2HPO4 3.00g / L, MgSO4 0.20g / L, NaCl 1.00g / L, cane wax 10g / L, lactose 25g / L, peptone 40g / L, sterilized at 121°C for 30min.

9. Use of the Aspergillus fumigatus ZL2K2 according to claim 1 in industrial and agricultural fields.

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