Method for preparing strepton F through fermentation of kitchen waste digestive juice and application of strepton F

Abstract

The invention discloses a method for preparing streptothricin F through fermentation of kitchen waste digestive juice and application of the streptothricin F. According to the method, the kitchen waste digestive juice and streptomyces lavendulae are subjected to aerobic co-fermentation and then subjected to liquid-phase purification, and a technical system for producing the agricultural antibiotic streptothricin F through fermentation of the kitchen waste digestive juice and separating and purifying the agricultural antibiotic streptothricin F is established. The agricultural antibiotic strepton F with high additional value and good antifungal effect is obtained, so that a new strategy is provided for realizing high-value utilization and harmless treatment of the kitchen waste digestive juice, the value of byproducts in the waste biomass utilization process is expected to be improved, the waste biomass utilization technical level is improved, and the method is worthy of popularization and application. The method has important significance on efficient and clean utilization and sustainable development of biomass.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural antibiotics, and in particular to a method for preparing streptothricin F by fermenting kitchen waste digestion liquid and an application thereof. Background Art

[0002] my country produces 50 million tons of kitchen waste every year, accounting for about 30% of domestic waste. If a large amount of kitchen waste is not properly disposed of, it will lead to a series of environmental problems such as waste accumulation, soil degradation, and water pollution. Therefore, achieving harmless and resource-based treatment of kitchen waste has become an urgent task for my country.

[0003] At present, common methods of kitchen waste disposal include landfill, incineration, composting and fermentation. However, landfill easily produces a large amount of leachate, which pollutes the land; the volatile organic compounds (VOCs) produced by incineration will cause serious secondary air pollution; the composting treatment cycle is long and the organic matter conversion rate is low. In contrast, anaerobic digestion to produce methane (i.e., biogas engineering) is considered to be the most effective way to utilize kitchen waste resources. Its byproduct digestate accounts for 80.1% of the total logistics and has extremely high agricultural value. It is known as "biofertilizer" and "biopesticide". However, the residual amounts of carbon, nitrogen and phosphorus in the digestate are high, and direct discharge will cause serious environmental burden and waste of resources.

[0004] Biological aerobic fermentation is a targeted production of high value-added products, which provides important support for the effective utilization of waste liquid. Studies have been conducted on the production of high value-added products such as agricultural antibiotics, polyhydroxyalkanoates, and lipid yeast proteins through aerobic biological fermentation of digestive fluid. However, studies have shown that the use of digestive fluid and Bacillus subtilis biofermentation requires the addition of exogenous carbon sources such as yeast extract and peptone, which has high production costs. In addition, in the production of agricultural antibiotics by co-fermentation of digestive fluid and actinomycetes, the antibacterial components have not been clearly identified and purified, resulting in low product purity and poor antibacterial effect, which urgently needs improvement.

[0005] Streptomycin F is an N-glycoside alkaline water-soluble agricultural antibiotic isolated from Streptomyces lilacinus in 1942. It mainly prevents and controls plant diseases by inhibiting RNA synthesis to kill plant pathogenic fungi, and has good broad-spectrum antifungal and antifungal effects. Compared with other agricultural antibiotics (such as Jinggangmycin, Kasugamycin, and Natamycin), Streptomycin F can effectively fight against highly resistant strains that interact with the 30S subunit of the 70S ribosome, and is the preferred choice among pesticide antibiotics. However, the technology of producing agricultural antibiotic Streptomycin F by directed biofermentation of kitchen waste digestion liquid has not been reported, and due to the complex components of kitchen waste digestion liquid, the separation and purification rules of agricultural antibiotic Streptomycin F in secondary fermentation liquid need to be further explored in the future to obtain high value-added Streptomycin F.

[0006] In addition, although studies have been conducted to isolate streptothricin components (including streptothricin F, E, D, C, B, A and X) from marine variants of Streptomyces lilacinus, existing separation and purification methods (such as ion exchange, activated carbon decolorization, etc.) can only increase the purity of streptothricin F to 60.8%-78.7%. Further improved methods, such as adding methanol desalting, desalting gel desalting, high-pressure liquid phase preparation and other steps after activated carbon decolorization, can increase the purity of streptothricin F to 98%, but the overall process is cumbersome, consumables are expensive, and it is easy to cause environmental pollution. Since there is no pure product of streptothricin F on the market, it is impossible to effectively qualitatively and quantitatively analyze streptothricin F. Therefore, it is urgent to explore a simple and easy-to-operate separation and purification method to extract streptothricin F from the secondary fermentation broth of kitchen waste. This will help to achieve the high-value utilization of kitchen waste biomass and promote the efficient and low-cost production and application of streptothricin F. Summary of the invention

[0007] The present invention provides a method for preparing streptothricin F by fermenting kitchen waste digestion liquid and its application, wherein the kitchen waste digestion liquid is treated and aerobic co-fermented with Streptomyces lilacinus to obtain streptothricin F, which helps to realize the high-value utilization of kitchen waste biomass and promote the efficient and low-cost production and application of streptothricin F. The technical scheme of the present invention is as follows:

[0008] A method for preparing streptothricin F by fermenting kitchen waste digestion liquid, comprising the following steps:

[0009] (1) mixing Gao's medium No. 1 or soluble starch with deammonified kitchen waste digestion liquid, sterilizing, and then adjusting the pH to 7.4-7.6 to obtain a sterile fermentation medium, wherein the deammonified kitchen waste digestion liquid accounts for 25-75% of the volume of the sterile fermentation medium, and the ammonia nitrogen content of the deammonified kitchen waste digestion liquid is 50-500 mg / L;

[0010] (2) inoculating Streptomyces lilacinus into a sterile fermentation medium, and performing aerobic fermentation at room temperature to obtain a fermentation liquid, wherein the aerobic fermentation time is 5 to 7 days, and the volume ratio of the inoculation amount of Streptomyces lilacinus is 5 to 15%;

[0011] (3) The fermentation broth is centrifuged and the supernatant is recovered, and then streptothricin F is separated.

[0012] In some possible implementations, the ammonia nitrogen content in the food waste digestate is 228 mg / L.

[0013] In some possible implementations, the method further includes the step (4) of purifying streptothricin F by preparative liquid chromatography, wherein the filler is a 5 μm-C18 chromatographic column filler, the mobile phase is a mixture of acetonitrile and trifluoroacetic acid in a volume ratio of 5:95, the injection volume is 0.5 to 1 mL, the flow rate is 12 to 15 mL / min, and the streptothricin F components are collected according to the peak time of streptothricin F, and concentrated and freeze-dried by a vacuum freeze dryer to obtain purified streptothricin F.

[0014] In some possible implementations, the operation of isolating streptothricin F in step (3) is to filter the supernatant with a sterile microporous filter membrane and retain the filtrate.

[0015] In some possible implementations, in step (2), aerobic fermentation is carried out under shaking culture at a rotation speed of 150 to 200 rpm.

[0016] In some possible implementations, the ammonia-free food waste digestate is obtained by removing ammonia nitrogen from the food waste digestate through membrane distillation.

[0017] A streptothricin F is prepared by the above method.

[0018] An antifungal composition, the active ingredient of which includes the streptothricin F mentioned above.

[0019] An antifungal composition, the active ingredient of which is the streptothricin F mentioned above.

[0020] The use of the streptothricin F in the preparation of an antifungal composition.

[0021] The present invention has at least the following beneficial effects:

[0022] (1) In the present invention, the kitchen waste digestion liquid is treated and aerobic co-fermented with Streptomyces lilacinus to obtain streptothricin F. The operation is simple and the obtained streptothricin F has good antibacterial properties.

[0023] (2) The present invention provides a new strategy for realizing high-value utilization and harmless treatment of kitchen waste digestate, which is expected to increase the value of by-products in the process of waste biomass utilization and improve the technical level of waste biomass utilization, which is of great significance to the efficient and clean utilization of biomass and sustainable development.

[0024] (3) In some possible implementations, streptothricin F is purified by using an automated purification preparative liquid chromatograph, which is simple to operate and can obtain streptothricin F with a purity close to 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The concentration of streptothricin F after the food waste digestion liquid and Gao's medium No. 1 were mixed and fermented in different proportions for 5 days in Example 1;

[0026] Figure 2 The concentration of streptothricin F after the kitchen waste digestion liquid and soluble starch were mixed and fermented in different proportions for 5 days in Example 2;

[0027] Figure 3 The concentration of streptothricin F in Example 3 of the food waste digestion liquid and soluble starch fermented for different days;

[0028] Figure 4 The broad-spectrum antifungal activity of the fermentation liquid after 5 days of mixed fermentation of kitchen waste digestion liquid and soluble starch in Example 4;

[0029] Figure 5 The antifungal effect of the fermentation liquid after 5 days of fermentation of kitchen waste digestion liquid and soluble starch against Alternaria alternata in Example 4;

[0030] Figure 6 This is a preparative liquid chromatogram of streptothricin F after preparative liquid purification in Example 5;

[0031] Figure 7 The total ion current and mass spectrum of streptothricin F after liquid phase purification in Example 5;

[0032] Figure 8 The antifungal effect of streptothricin F against Alternaria alternata after liquid phase purification prepared in Example 5. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further illustrated and described below through specific implementation methods.

[0034] In the following examples, the water used can be one or more of distilled water, purified water, and drinking water; unless otherwise specified, the detection methods in the following examples are conventional detection methods; the reagents in the following examples are purchased from commercial channels unless otherwise specified.

[0035] Among them, the food waste digestate was provided by Ruikeji Renewable Energy Co., Ltd., Xiang'an District, Xiamen City, Fujian Province, and the parameters of the food waste digestate are shown in Table 1. Streptomyces lavendulae (BNCC153470), Alternaria (BNCC336535), Alternaria (BNCC123548), Fusarium graminearum (BNCC337560), and Thanatephorus cucumeris (BNCC356048) were provided by Beijing Beina Chuanglian Biotechnology Research Institute.

[0036] Gao's medium No. 1 includes 20 g / L soluble starch, 1.0 g / L KNO3, 0.5 g / L NaCl, 0.5 g / L K2HPO4, 0.5 g / L MgSO4·7H2O and 0.01 g / L FeSO4·7H2O.

[0037] Table 1 Key parameters of food waste digestate

[0038]

[0039] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0040] Example 1: Fermentation of food waste digestion liquid and Gao's medium No. 1 in different proportions to produce streptothricin F

[0041] (1) The ammonia nitrogen content of food waste digestate (FWD) was reduced from 2743 mg / L to 228 mg / L by membrane distillation (MD), and the deammonified food waste digestate (FWD-MD) was mixed with Gao's medium No. 1 at different volume ratios (100%:0%, 75%:25%, 50%:50%, 25%:75%, 0%:100%), and the pH was adjusted to 7.4; 100 mL of the mixed fermentation liquid was dispensed into each 500 mL conical flask and sterilized at 121°C for 20 min to obtain a sterile fermentation medium;

[0042] (2) inoculating 10% (V / V) Streptomyces lilacinus into a sterile fermentation medium, and aerobically fermenting in a shaking incubator at 30° C. and 200 rpm for 5 days to obtain a fermentation liquid;

[0043] (3) The fermentation broth was centrifuged at 8000 rpm for 10 min at 25°C, the supernatant was recovered and filtered through a 0.22 μm sterile microporous filter membrane, and the concentration of streptothricin F in the fermentation broth was detected by liquid phase.

[0044] The test results show (such as Figure 1 ), the yield of streptothricin F was the highest after aerobic fermentation of FWD-MD and Gao's medium No. 1 at a ratio of 50%:50%, and its concentration was 0.5727 g / L, which was 67.38 times higher than that of 100% untreated FWD fermentation, 7.92 times higher than that of 100% FWD-MD, and 1.40 times higher than that of 100% Gao's medium No. 1 fermentation broth.

[0045] This is because the ammonia nitrogen concentration in the food waste digestion liquid is high and the bioavailable organic matter is small. If the food waste digestion liquid is directly used, it will inhibit the biological fermentation of Streptomyces lilacinus to produce streptothricin F. Therefore, in this embodiment, a part of the ammonia nitrogen in the food waste digestion liquid is removed by membrane distillation to reduce its ammonia nitrogen content to 228 mg / L, and after mixing with Gao's No. 1 to provide sufficient carbon source, biological fermentation is carried out to produce agricultural antibiotic streptothricin F, which significantly increases the concentration of streptothricin F in the fermentation liquid.

[0046] Example 2 Production of Streptothricin F by Mixing Kitchen Waste Digestion Liquid and Soluble Starch in Different Proportions

[0047] (1) The ammonia nitrogen content of food waste digestate (FWD) was reduced from 2743 mg / L to 228 mg / L by membrane distillation (MD), and the deammonified food waste digestate (FWD-MD) was mixed with 20 g / L soluble starch in different volume ratios (100%:0%, 75%:25%, 50%:50%, 25%:75%, 0%:100%), and the pH was adjusted to 7.4; 100 mL of the mixed fermentation liquid was dispensed into each 500 mL conical flask and sterilized at 121°C for 20 min to obtain a sterile fermentation medium;

[0048] (2) inoculating 10% Streptomyces lilacinus into a sterile fermentation medium, and aerobically fermenting on a shaking table at 30° C. and 200 rpm for 5 days to obtain a fermentation liquid;

[0049] (3) The fermentation broth was centrifuged at 8000 rpm for 10 min at 25° C., the supernatant was recovered and filtered through a 0.22 μm sterile microporous filter to obtain a secondary fermentation broth of kitchen waste, and the concentration of streptothricin F in the fermentation broth was detected by liquid chromatography.

[0050] The test results show (such as Figure 2 ), the concentration of streptothricin is the highest after aerobic fermentation of FWD-MD and soluble starch at a ratio of 50%:50%, and the concentration of streptothricin F is 0.5144 g / L after 5 days of fermentation of 50% FWD-MD + 50% soluble starch, which is slightly lower than the concentration of streptothricin F (0.5727 g / L) after 5 days of fermentation of 50% FWD-MD + 50% Gao's No. 1 medium. In Example 1, the carbon source of Gao's No. 1 medium is 20 g / L soluble starch. Therefore, in Design Case 2, soluble starch is directly used instead of Gao's No. 1 medium to mix with kitchen waste digestion liquid for aerobic fermentation for 5 days. As a result, the streptothricin yields of the two are almost equal. Considering the economic benefits, the design scheme of using 50% FWD-MD + 50% soluble starch to ferment and produce agricultural antibiotics is better.

[0051] Example 3 Changes in Streptothricin F Concentrations of Kitchen Waste Digestive Fluid and Soluble Starch Fermentation for Different Days

[0052] (1) The ammonia nitrogen in food waste digestate (FWD) was reduced from 2743 mg / L to 228 mg / L by membrane distillation (MD), and the deammonified food waste digestate (FWD-MD) was mixed with 20 g / L soluble starch at a ratio of 50%:50%, and the pH was adjusted to 7.4; 100 mL of the mixed fermentation liquid was dispensed into each 500 mL conical flask, and sterilized at 121°C for 20 min to obtain a sterile fermentation medium;

[0053] (2) inoculating 10% Streptomyces lilacinus into a sterile fermentation medium, and aerobically fermenting on a shaking table at 30° C. and 200 rpm / min for 0-8 days to obtain fermentation broths of different days;

[0054] (3) The fermentation broth was centrifuged at 8000 rpm / min for 10 min at 25° C., the supernatant was recovered and filtered through a 0.22 μm sterile microporous filter to obtain a secondary fermentation broth of kitchen waste, and the concentration of streptothricin F in the fermentation broth was detected by liquid phase chromatography.

[0055] The test results show (such as Figure 3 ), the synthesis of streptothricin F was effectively promoted in the early stage of fermentation, and the concentration of streptothricin F gradually increased with the fermentation time until it reached the maximum value on the 5th day of fermentation, with streptothricin being 0.5144 g / L. Subsequently, as time went on, the concentration of streptothricin F no longer increased and showed a slightly downward trend. The concentrations of streptothricin F on the 6th, 7th, and 8th days of fermentation were 0.5126 g / L, 0.4839 g / L, and 0.4255 g / L, respectively. This may be because streptothricin F is a secondary metabolite, and its degradation rate exceeds the synthesis rate.

[0056] Example 4 Antifungal effect of streptothricin F produced by fermentation of food waste digestion liquid and soluble starch

[0057] The volume ratio of FWD-MD to soluble starch in Example 2 was 50%:50%, and the four plant pathogenic fungi, Alternaria alternata, Alternaria alternata, Fusarium graminearum, and Rhizoctonia solani, were used to conduct qualitative antifungal experiments (such as Figure 4 ), then the 1000 μg / mL agricultural antibiotic fermentation broth was diluted 2-fold to prepare 1000, 500, 250, 125, 62.50, 31.25, 15.63, and 7.81 μg / mL concentrations, and the antifungal quantitative experiment on Alternaria alternata was carried out and the inhibition rate curve was drawn (such as Figure 5 ), calculate the half-maximum effect concentration EC of the drug safety index 50 .

[0058] The antifungal experimental method is the hyphae growth inhibition rate method. Different concentrations of antimicrobial agents were mixed with potato dextrose agar medium (1:9) and sterile plates were prepared in a clean bench. The blank control group was a mixture of sterile water and potato dextrose agar medium (1:9). Fungal blocks with a diameter of 6 mm were inoculated in the center of the sterile plate and cultured at 28°C for 10 days until the hyphae of the blank group covered the entire plate. The hyphae growth diameter was measured using a vernier caliper using the cross method. All experiments were repeated in 3 groups.

[0059] Calculation of mycelium growth inhibition rate:

[0060] Inhibition rate = [(mycelium growth diameter of control group - mycelium growth diameter of treatment group) / mycelium growth diameter of control group] * 100%.

[0061] The test results show (such as Figure 4 ), the fermentation broth had antibacterial effects on Alternaria, Alternaria, Fusarium graminearum, and Rhizoctonia solani, respectively. The mycelial growth diameters of Alternaria, Alternaria, Fusarium graminearum, and Rhizoctonia solani were 8.75mm, 42.20mm, 44.05mm, and 69.31mm, respectively, and the inhibition rates were as high as 99.05%, 57.97%, 55.63%, and 23.36%, respectively, with good broad-spectrum antifungal activity. Among them, the antibacterial effect on Alternaria was the best. In order to further determine the antibacterial effect of the fermentation broth on Alternaria, the 1000μg / mL fermentation broth was diluted 2 times 8 times to prepare 1000, 500, 250, 125, 62.50, 31.25, 15.63, and 7.81μg / mL concentrations, and antifungal quantitative experiments were carried out and inhibition rate curves were drawn (such as Figure 5 ). According to the inhibition rate curve, the EC of the fermentation broth against Alternaria alternata 50 =53.62μg / mL.

[0062] Example 5 Preparation of Liquid Phase Separation and Purification of Streptothricin F

[0063] The streptothricin F in Example 2 with a volume ratio of FWD-MD to soluble starch of 50%:50% was purified by preparative liquid chromatography-mass spectrometry and the purity of the purified streptothricin F was verified.

[0064] Among them, the automatic purification preparative liquid chromatograph used in this embodiment is Waters 2545, the chromatographic preparative column is filled with 5μmC18 filler, 19mm×150mm, the mobile phase is (5%) acetonitrile: (95%) trifluoroacetic acid, the flow rate is 12mL / min, 10min, the detection wavelength is 200nm, the sample volume is 1mL, and the streptothricin F component is collected according to the streptothricin F peak time setting.

[0065] The test results show (such as Figure 6-7 ), there were no other impurity peaks on the preparative liquid chromatogram and total ion flow diagram of streptothricin F collected by the automatic purification preparative liquid phase, indicating that high-purity streptothricin F with a purity close to 100% can be separated by the automatic purification preparative liquid phase. The high-purity streptothricin was concentrated and lyophilized through a vacuum freeze dryer to obtain purified streptothricin with a purity close to 100% in the form of a white solid powder. In order to further determine the antibacterial effect of pure streptothricin F against Alternaria alternata, 10 μg / mL high-purity streptothricin was diluted 2 times 8 times to prepare concentrations of 10, 5, 2.5, 1.25, 0.62, 0.31, 0.16, and 0.08 μg / mL, and an antifungal quantitative experiment was carried out and an inhibition rate curve was drawn (such as Figure 8 ). According to the inhibition rate curve, the EC of pure streptothricin F against Alternaria alternata 50 =0.66μg / mL.

[0066] In this specification, the schematic representation of the above terms should not be understood to necessarily be directed to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments described in this specification. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Those of ordinary skill in the art can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing streptothricin F by fermenting kitchen waste digestion liquid, characterized in that: The following steps are involved: (1) mixing Gao's medium No. 1 or soluble starch with deammonified kitchen waste digestion liquid, sterilizing, and then adjusting the pH to 7.4-7.6 to obtain a sterile fermentation medium, wherein the deammonified kitchen waste digestion liquid accounts for 25-75% of the volume of the sterile fermentation medium, and the ammonia nitrogen content of the deammonified kitchen waste digestion liquid is 50-500 mg / L; (2) inoculating Streptomyces lilacinus into a sterile fermentation medium, and performing aerobic fermentation at room temperature to obtain a fermentation liquid, wherein the aerobic fermentation time is 5 to 7 days, and the volume ratio of the inoculation amount of Streptomyces lilacinus is 5 to 15%; (3) The fermentation broth is centrifuged and the supernatant is recovered, and then streptothricin F is separated.

2. The method according to claim 1, characterized in that The ammonia nitrogen content in the kitchen waste digestion liquid is 228 mg / L.

3. The method according to claim 1, characterized in that The method further comprises the step (4) of purifying streptothricin F by preparative liquid chromatography, wherein the filler is a 5 μm-C18 chromatographic column filler, the mobile phase is a mixture of acetonitrile and trifluoroacetic acid in a volume ratio of 5:95, the injection volume is 0.5 to 1 mL, the flow rate is 12 to 15 mL / min, and the streptothricin F components are collected according to the peak time of streptothricin F, and concentrated and freeze-dried by a vacuum freeze dryer to obtain purified streptothricin F.

4. The method according to any one of claims 1 to 3, characterized in that: The operation of isolating streptothricin F in step (3) is to filter the supernatant with a sterile microporous filter membrane and retain the filtrate.

5. The method according to any one of claims 1 to 3, characterized in that: In step (2), the aerobic fermentation is carried out under shaking culture at a rotation speed of 150 to 200 rpm.

6. The method according to any one of claims 1 to 3, characterized in that: The ammonia-nitrogen-free kitchen waste digestion liquid is obtained by removing ammonia nitrogen from the kitchen waste digestion liquid through a membrane distillation method.

7. A streptothricin F, characterized in that Prepared by the method described in any one of claims 1 to 6.

8. An antifungal composition, characterized in that The effective ingredient thereof includes the streptothricin F as claimed in claim 7.

9. An antifungal composition, characterized in that The active ingredient is the streptothricin F described in claim 7.

10. Use of the streptothricin F according to claim 7 in the preparation of an antifungal composition.

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