Fermentation method of traditional Chinese medicine residues

Abstract

The invention relates to the technical field of resource utilization of traditional Chinese medicine wastes, in particular to a fermentation method of traditional Chinese medicine residues. The method comprises the following steps: ultrasonically crushing the traditional Chinese medicine residue to obtain a crushed material, carrying out microwave vacuum drying on the crushed material to obtain a dried material, carrying out low-temperature plasma sterilization on the dried material to obtain a sterilized material, mixing the sterilized material with trichoderma reesei, carrying out aerobic fermentation to obtain an aerobic fermented material, mixing the aerobic fermented material with yarrowia lipolytica, and carrying out anaerobic fermentation. By adopting the method provided by the invention, the flavone retention rate of the traditional Chinese medicine residues is increased, and meanwhile, the grease yield is also increased.

Description

Technical Field

[0001] The present invention relates to the technical field of resource utilization of traditional Chinese medicine waste, and particularly to a fermentation method for traditional Chinese medicine residues. Background Art

[0002] At present, the pretreatment of traditional Chinese medicine residues mainly includes natural drying, direct composting, incineration, landfilling, and simple pulverization, and there are the following problems: 1. Low efficiency and restricted by the environment: Natural drying takes up to several days (the water content needs to be reduced from 60%-80% to below 10%), is easily affected by rainy weather and mildews, requires a large amount of space, and emits odors to pollute the environment. Manual operation: The degree of mechanization is low, the labor cost is high, and it is difficult to adapt to large-scale production. 2. Serious waste of resources: Traditional Chinese medicine residues contain effective components such as polysaccharides, flavonoids, alkaloids, and dietary fiber, but they are not extracted by traditional methods (such as composting and landfilling) and are directly discarded, resulting in waste of resources. Insufficient utilization of high added value: The potential in the fields of biofuels, pharmaceutical intermediates, feeds, etc. has not been developed. 3. Prominent environmental protection risks: The composting maturity period is long (2-3 months), the residual drugs inhibit the activity of microorganisms, and it is easy to produce malodorous gases (NH3, H2S) and leachate to pollute the soil and groundwater. Incineration: At low temperatures (<800°C), dioxins and nitrogen oxides are produced, and due to the high water content, additional fuel is required, and the energy consumption is high. Landfilling: Occupies land, anaerobic decomposition produces methane (the greenhouse effect is 25 times that of CO2), and the leachate contains heavy metals and organic substances to pollute groundwater. 4. Hygiene and safety hazards Traditional Chinese medicine residues may carry pathogens, eggs, and heavy metals, and traditional treatments cannot effectively remove them, posing safety risks when used as fertilizers / feeds. Dust is generated during the pulverization process, which is easy to cause respiratory diseases, and there are safety hazards in manual operation. 5. Lack of standardization: The process depends on experience, there are no unified standards for water content, treatment time, and environmental protection indicators, it is difficult to ensure stability, and it cannot meet the intensive needs of the modern traditional Chinese medicine industry. For example: The existing patent CN103583826A uses single hot air drying, and the energy consumption is as high as 2.8 kW·h / kg, and the problem of low flavonoid retention rate (≤60%) has not been solved. In the prior art, the strain selection of Trichoderma reesei and Yarrowia lipolytica has a significant impact on the fermentation efficiency. For example, Trichoderma reesei RUT-C30 is widely used in industrial biomass conversion due to its high cellulase activity (Reference 1), while Yarrowia lipolytica ATCC 20460 has become a preferred strain for microbial oil production due to its high oil production characteristics (Reference 2). The strain combination of the present invention solves the problems of low fermentation efficiency and single product of traditional single strains through synergistic effects.

[0003] Therefore, there is a need to provide a fermentation method with high flavonoid retention rate and high oil yield for traditional Chinese medicine residues. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for fermenting traditional Chinese medicine residues. By using the method for fermenting traditional Chinese medicine residues provided by the present invention, the retention rate of flavonoids in traditional Chinese medicine residues is increased, and at the same time, the oil yield is also increased.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for fermenting traditional Chinese medicine residues, comprising the following steps:

[0007] 1) Ultrasonically crush the traditional Chinese medicine residues to obtain crushed materials;

[0008] 2) Microwave vacuum dry the crushed materials obtained in step 1) to obtain dried materials;

[0009] 3) Sterilize the dried materials obtained in step 2) by low-temperature plasma to obtain sterilized materials;

[0010] 4) Mix the sterilized materials obtained in step 3) with Trichoderma reesei and perform aerobic fermentation to obtain aerobic fermentation materials;

[0011] 5) Mix the aerobic fermentation materials obtained in step 4) with Yarrowia lipolytica and perform anaerobic fermentation.

[0012] Preferably, the frequency of ultrasonic crushing in step 1) is 20 kHz, and the particle size of the crushed materials is less than 1 mm.

[0013] Preferably, the traditional Chinese medicine residues in step 1) include Astragalus membranaceus traditional Chinese medicine residues and / or Ligustrum lucidum traditional Chinese medicine residues.

[0014] Preferably, the conditions for microwave vacuum drying in step 2) include: power of 800 W, pressure of -0.09 MPa, and time of 6 h.

[0015] Preferably, the conditions for low-temperature plasma sterilization in step 3) include: sterilizing for 10 min in an argon environment and at a voltage of 15 kV.

[0016] Preferably, the volume ratio of the sterilized materials to Trichoderma reesei in step 4) is 5:100;

[0017] The Trichoderma reesei is mixed with the sterilized materials in the form of a bacterial solution, and the number of spores of Trichoderma reesei in the bacterial solution is 5×10 9 CFU / L.

[0018] Preferably, the conditions for aerobic fermentation in step 4) include: supplying oxygen by introducing sterile air during the aerobic fermentation process, the introduction rate of the sterile air is 0.5 - 1.0 L / (L·min), the temperature is 35 °C, and the time is 48 h.

[0019] Preferably, the volume ratio of the aerobic fermentation material to Yarrowia lipolytica in step 5) is 100:3.

[0020] Yarrowia lipolytica (Yarrowia lipolytica ATCC 20460, deposit number ATCC 20460) is mixed with the aerobic fermentation material in the form of a bacterial solution, and the bacterial content of Yarrowia lipolytica in the bacterial solution is 1.5×10 9 CFU / L.

[0021] Preferably, the conditions for anaerobic fermentation in step 5) include: temperature is 28°C and time is 72 h.

[0022] Preferably, after anaerobic fermentation in step 5), an anaerobic fermentation material is obtained, and flavonoids in the anaerobic fermentation material are extracted by ethanol ultrasonic assistance;

[0023] The conditions for ethanol ultrasonic assistance extraction include: the power of ultrasonic crushing is 500 - 1000 W, the frequency is 20 kHz, the temperature is 50°C, the time is 30 min, and the ethanol is an ethanol solution with a volume percentage of 70% - 90%.

[0024] Advantages of the present invention:

[0025] 1. Upgrade of pretreatment technology

[0026] Microwave-vacuum co-drying: replacing traditional hot air drying, the drying time is shortened by 60% (from 15 hours → 6 hours), and the retention rate of active ingredients (flavonoids, polysaccharides) ≥ 90%.

[0027] Low-temperature plasma sterilization: no chemical residue, sterilization rate ≥ 99.9%, avoiding the destruction of active ingredients by high temperature.

[0028] 2. Screening of composite strains and two-stage fermentation

[0029] Strain combination: the combination of Trichoderma reesei + Yarrowia lipolytica realizes the efficient decomposition of cellulose and the synchronous production of microbial oil.

[0030] Fermentation process: the aerobic stage (48 hours, 35°C) rapidly decomposes cellulose; the anaerobic stage (72 hours, 28°C) accumulates oil, and the oil production rate is increased by 40%.

[0031] 3. Co-production technology of active ingredients

[0032] Ethanol ultrasonic assistance extraction: extracting flavonoids (extraction rate ≥ 85%) from the traditional Chinese medicine residue after anaerobic fermentation for use as feed additives.

[0033] Valorization of by-products: Biochar is prepared from the fermentation residue after ethanol ultrasonic-assisted extraction of anaerobic fermentation materials, and is safely disposed of after adsorbing heavy metals. Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments will be briefly introduced below.

[0035] Figure 1 It is a flowchart of the present invention. Detailed Embodiments

[0036] The present invention provides a fermentation method for traditional Chinese medicine residues, comprising the following steps:

[0037] 1) Ultrasonically crush the traditional Chinese medicine residues to obtain crushed materials;

[0038] 2) Microwave vacuum dry the crushed materials obtained in step 1) to obtain dried materials;

[0039] 3) Sterilize the dried materials obtained in step 2) by low-temperature plasma to obtain sterilized materials;

[0040] 4) Mix the sterilized materials obtained in step 3) with Trichoderma reesei and perform aerobic fermentation to obtain aerobic fermentation materials;

[0041] 5) Mix the aerobic fermentation materials obtained in step 4) with Yarrowia lipolytica and perform anaerobic fermentation.

[0042] The present invention ultrasonically crushes the traditional Chinese medicine residues to obtain crushed materials. In the present invention, the traditional Chinese medicine residues preferably include Astragalus membranaceus traditional Chinese medicine residues and / or Ligustrum lucidum traditional Chinese medicine residues. The present invention has no special limitation on the source of the traditional Chinese medicine residues, and the traditional Chinese medicine residues obtained after conventional extraction of traditional Chinese medicine can be used. In the present invention, the frequency of the ultrasonic crushing is preferably 20 kHz, and the particle size of the crushed materials is preferably less than 1 mm.

[0043] The present invention microwave vacuum dries the obtained crushed materials to obtain dried materials. In the present invention, the conditions for the microwave vacuum drying preferably include: power of 800 W, pressure of -0.09 MPa, and time of 6 h. The present invention has no special limitation on other conditions for the microwave vacuum drying, and conventional conditions can be used.

[0044] The obtained dried material is subjected to low-temperature plasma sterilization to obtain a sterilized material. In the present invention, the conditions for the low-temperature plasma sterilization preferably include: sterilization for 10 min in an argon environment at a voltage of 15 kV. The present invention has no special limitation on other conditions for the low-temperature plasma sterilization, and those skilled in the art can adopt the conventional ones.

[0045] The obtained sterilized material in the present invention is mixed with Trichoderma reesei and then subjected to aerobic fermentation to obtain an aerobic fermentation material. In the present invention, the volume ratio of the sterilized material to Trichoderma reesei is preferably 5:100, and after adjusting the moisture content to 60%-70%, aerobic fermentation is carried out. In the present invention, Trichoderma reesei is preferably mixed with the sterilized material in the form of a bacterial solution, and the spore count of Trichoderma reesei in the bacterial solution is preferably 5×10 9 CFU / L. In the present invention, Trichoderma reesei is preferably Trichoderma reesei RUT-C30 strain (deposit number: ATCC 56765), which is a highly efficient cellulose-decomposing strain modified by multiple mutageneses and has the following advantages: outstanding enzyme-producing ability: it can secrete highly active cellulases (such as exoglucanase, endoglucanase and β-glucosidase), and the cellulose degradation efficiency is 2-3 times higher than that of the original strain QM6a (literature support: Appl Microbiol Biotechnol, 2010, 87: 1439-1451); wide industrial application: it has been used in fields such as biomass energy and feed additives, and the technology maturity is high. The present invention has no special limitation on the preparation method of the Trichoderma reesei bacterial solution, and those skilled in the art can prepare it by using the conventional method for preparing the Trichoderma reesei bacterial solution. In the present invention, the conditions for the aerobic fermentation preferably include: during the aerobic fermentation process, oxygen is supplied by introducing sterile air, the introduction rate of the sterile air is 0.5-1.0 L / (L·min), the temperature is 35 °C, and the time is 48 h.

[0046] The obtained aerobic fermentation material in the present invention is mixed with Yarrowia lipolytica and then subjected to anaerobic fermentation. In the present invention, the volume ratio of the aerobic fermentation material to Yarrowia lipolytica is preferably 100:3. In the present invention, Yarrowia lipolytica is preferably mixed with the aerobic fermentation material in the form of a bacterial solution, and the bacterial content of Yarrowia lipolytica in the bacterial solution is preferably 1.5×10 9CFU / L. The yeast strain Yarrowia lipolytica ATCC 20460 (deposit number: ATCC 20460) used in the present invention has the following characteristics: strong oil accumulation ability: under nitrogen-limited conditions, the intracellular oil content can reach 40% - 50% of the dry weight (literature support: Biotechnol Adv, 2015, 33: 1177 - 1193); wide substrate adaptability: it can utilize a variety of carbon sources (such as glucose, glycerol and short-chain fatty acids), and has strong synergism with the cellulose degradation products of Trichoderma reesei. The present invention has no special limitation on the preparation method of the Yarrowia lipolytica bacterial liquid, and those skilled in the art can prepare it by using the conventional method for preparing the Yarrowia lipolytica bacterial liquid. In the present invention, the conditions of the anaerobic fermentation preferably include: the temperature is 28 °C and the time is 72 h.

[0047] In the present invention, after the anaerobic fermentation, an anaerobic fermentation material is obtained, and flavonoids in the anaerobic fermentation material are extracted by ethanol ultrasonic assistance; the conditions of the ethanol ultrasonic assistance extraction include: the power of ultrasonic crushing is 500 - 1000 W, the frequency is 20 kHz, the temperature is 50 °C, and the time is 30 min. The ethanol is an ethanol solution with a volume percentage content of 70% - 90%. In the present invention, biochar is prepared from the fermentation residue obtained after the ethanol ultrasonic assistance extraction of the anaerobic fermentation material. The preparation method of the biochar preferably includes the following steps: carbonizing the fermentation residue at a temperature of 500 °C for 2 - 4 h to obtain biochar. In the present invention, the specific surface area of the biochar is preferably 300 - 500 m 2 / g, with a high density of surface oxygen-containing functional groups (-COOH, -OH), strong ion exchange ability, and preferably adsorbing Pb 2+ and Cr 6+ , and still having a high adsorption rate after multiple cycles.

[0048] To further illustrate the present invention, the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] A fermentation method for Astragalus residue, the steps are as follows:

[0051] 1) Ultrasonic crushing 100 kg of Astragalus residue at a frequency of 20 kHz to obtain a crushed material with a particle size less than 1 mm;

[0052] Among them, the moisture content of the Astragalus residue is 75%, and the mass percentage content of flavonoids is 1.5%;

[0053] 2) Microwave vacuum drying the crushed material obtained in step 1) to obtain a dried material;

[0054] Among them, the conditions for microwave vacuum drying are as follows: power is 800 W, pressure is -0.09 MPa, and time is 6 h; the moisture content of the dried material is 4.8%; the flavonoid content in the dried material is 1.38% (retention rate 92%); the flavonoid content after drying: 1.38% (retention rate 92% after drying). Calculation method: Flavonoid retention rate during drying = (flavonoid content after drying / original flavonoid content) × 100% = (1.38% / 1.5%) × 100% = 92%; The determination of flavonoid content uses spectrophotometry (UV-Vis), referring to the flavonoid component detection standard in the 2020 edition of the Chinese Pharmacopoeia. Using rutin as the reference substance, the detection wavelength is 510 nm.

[0055] 3) Sterilize the dried material obtained in step 2) by low-temperature plasma to obtain a sterilized material;

[0056] Among them, the conditions for low-temperature plasma sterilization are as follows: sterilize for 10 min in an argon environment and at a voltage of 15 kV;

[0057] 4) Mix the sterilized material obtained in step 3) with Trichoderma reesei liquid (Trichoderma reesei RUT-C30 strain (deposit number: ATCC 56765)), and then perform aerobic fermentation at 35 °C for 48 h to obtain an aerobic fermentation material;

[0058] Among them, the volume ratio of the sterilized material to the Trichoderma reesei liquid is 5:100, and the number of Trichoderma reesei spores in the liquid is 5×10 9 CFU / L.

[0059] 5) Mix the aerobic fermentation material obtained in step 4) with Yarrowia lipolytica liquid, and then perform anaerobic fermentation at 28 °C for 72 h to obtain an anaerobic fermentation material, and the oil yield is 23%; The oil is synthesized and accumulated intracellularly by Yarrowia lipolytica (Yarrowia lipolytica ATCC 20460) during the anaerobic fermentation stage by metabolizing soluble carbon sources (such as glucose, short-chain fatty acids) in the Chinese medicine residue. Calculation method: Determination of oil production: Use the Soxhlet extraction method (refer to national standard GB5009.6-2016), use n-hexane as the solvent, extract the total lipids in the anaerobic fermentation material, and weigh after drying to calculate the oil yield. Calculation formula:

[0060] Oil yield (%) = mass of extracted oil (g) / dry weight of anaerobic fermentation material (g) × 100%

[0061] Among them, the volume ratio of the aerobic fermentation material to the Yarrowia lipolytica liquid is 100:3, and the bacterial content of Yarrowia lipolytica in the liquid is 1.5×10 9 CFU / L.

[0062] 6) The anaerobic fermentation material obtained in step 5) is subjected to ethanol ultrasonic-assisted extraction to obtain flavonoids, and fermentation residues are obtained. The total retention rate of flavonoids: refers to the total retention rate of flavonoids from the original medicinal residues to after ethanol ultrasonic extraction (i.e., the final extraction rate), including cumulative losses in the whole process such as drying, sterilization, and fermentation. The final flavonoid extraction amount: 1.28% (total retention rate 85.3%) = (1.28% / 1.5%) × 100% = 85.3%;

[0063] Among them, the conditions for ethanol ultrasonic-assisted extraction are: the temperature is 50 °C, the time is 30 min, the power is 800 W, and 80% ethanol solution (v / v);

[0064] 7) The fermentation residues obtained in step 6) are carbonized at 500 °C for 3 hours to obtain biochar, and the adsorption rate of Pb 2+ is 87% (first adsorption). After adsorption, the biochar is desorbed with 0.1 M HNO3, and the adsorption-desorption is repeated 5 times. After 5 cycles, the adsorption rate still remains 82%. The adsorption rate is measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900), referring to the standard HJ 700-2014.

[0065] 8) The above data are the average value ± standard deviation (SD) of three independent experiments, and the experimental repeatability error ≤ 5%.

[0066] Example 2

[0067] A fermentation method for Ligustrum lucidum medicinal residues, the steps are as follows:

[0068] 1) 100 kg of Ligustrum lucidum medicinal residues are ultrasonically crushed at a frequency of 20 kHz to obtain crushed materials with a particle size less than 1 mm;

[0069] Among them, the moisture content of the Ligustrum lucidum medicinal residues is 64%, and the mass percentage content of flavonoids is 3.2%;

[0070] 2) The crushed materials obtained in step 1) are subjected to microwave vacuum drying to obtain dried materials;

[0071] Among them, the conditions for microwave vacuum drying are: the power is 800 W, the pressure is -0.09 MPa, and the time is 6 h; the moisture content of the dried materials is 5.2%; the content of flavonoids in the dried materials is 2.94% (retention rate 91.9%). The calculation method for the flavonoid content after drying: 2.94% (retention rate 91.9% after drying) = (2.94% / 3.2%) × 100% = 91.9%;

[0072] 3) The dried materials obtained in step 2) are subjected to low-temperature plasma sterilization to obtain sterilized materials;

[0073] Among them, the conditions for low-temperature plasma sterilization are: sterilization for 10 min in an argon environment with a voltage of 15 kV;

[0074] 4) After mixing the sterilized material obtained in step 3) with Trichoderma reesei liquid, aerobic fermentation is carried out at a temperature of 35 °C for 48 h to obtain an aerobic fermentation material;

[0075] Among them, the volume ratio of the sterilized material to the Trichoderma reesei liquid is 5:100, and the number of spores of Trichoderma reesei in the liquid is Yarrowia lipolytica Yarrowia lipolytica is 5×10 9 CFU / L.

[0076] 5) After mixing the aerobic fermentation material obtained in step 4) with Yarrowia lipolytica liquid, anaerobic fermentation is carried out at a temperature of 28 °C for 72 h to obtain an anaerobic fermentation material, and the oil yield is 22%;

[0077] Among them, the volume ratio of the aerobic fermentation material to the Yarrowia lipolytica liquid is 100:3, and the bacterial content of Yarrowia lipolytica in the liquid is 1.5×10 9 CFU / L.

[0078] 6) The anaerobic fermentation material obtained in step 5) is subjected to ethanol ultrasonic-assisted extraction of flavonoids therein, and the final flavonoid extraction amount of the fermentation residue is: 2.75% (total retention rate 85.9%) = (2.75% / 3.2%) × 100% = 85.9%;

[0079] Among them, the conditions for ethanol ultrasonic-assisted extraction are: temperature is 50 °C, time is 30 min, power is 800 W, 80% ethanol solution (v / v);;

[0080] 7) The fermentation residue obtained in step 6) is carbonized at 500 °C for 3 hours to obtain biochar, and the adsorption rate of Pb 2+ is 85% (first adsorption). After adsorption, the biochar is desorbed with 0.1 M HNO3, and the adsorption-desorption is repeated 5 times. After 5 cycles, the adsorption rate still remains 80%. The adsorption rate is measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900), referring to the standard HJ 700-2014.

[0081] 8) The above data are the average value ± standard deviation (SD) of three independent experiments, and the experimental repeatability error ≤ 5%

[0082] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation method for Chinese medicine residues, characterized in that, It includes the following steps: 1) Ultrasonically crush the Chinese medicine residues to obtain crushed materials; 2) Microwave vacuum dry the crushed materials obtained in step 1) to obtain dried materials; 3) Sterilize the dried materials obtained in step 2) by low-temperature plasma to obtain sterilized materials; 4) Mix the sterilized materials obtained in step 3) with Trichoderma reesei and perform aerobic fermentation to obtain aerobically fermented materials; 5) Mix the aerobically fermented materials obtained in step 4) with Yarrowia lipolytica and perform anaerobic fermentation.

2. The fermentation method according to claim 1, characterized in that The frequency of ultrasonic crushing in step 1) is 20 kHz, and the particle size of the crushed materials is less than 1 mm.

3. The fermentation method according to claim 1, wherein The Chinese medicine residues in step 1) include Astragalus membranaceus Chinese medicine residues and / or Ligustrum lucidum Chinese medicine residues.

4. The fermentation method according to claim 1, characterized in that, The conditions for microwave vacuum drying in step 2) include: power of 800 W, pressure of -0.09 MPa, and time of 6 h.

5. The method according to claim 1, wherein The conditions for low-temperature plasma sterilization in step 3) include: sterilization for 10 min in an argon environment and at a voltage of 15 kV.

6. The method according to claim 1, wherein The volume ratio of the sterilized materials to Trichoderma reesei in step 4) is 5:100; The Trichoderma reesei is mixed with the sterilized material in the form of a bacterial solution, and the spore count of Trichoderma reesei in the bacterial solution is 5×10 9 CFU / L.

7. The method according to claim 1, wherein The conditions for aerobic fermentation in step 4) include: oxygen supply is carried out by introducing sterile air during the aerobic fermentation process, the introduction rate of the sterile air is 0.5 - 1.0 L / (L·min), the temperature is 35°C, and the time is 48 h.

8. The method according to claim 1, characterized in that, The volume ratio of the aerobically fermented materials to Yarrowia lipolytica in step 5) is 100:3; The Yarrowia lipolytica is mixed with the aerobic fermentation material in the form of a bacterial liquid, and the bacterial content of Yarrowia lipolytica in the bacterial liquid is 1.5×10 9 CFU / L.

9. The method according to claim 1, wherein The conditions for anaerobic fermentation in step 5) include: temperature of 28°C and time of 72 h.

10. The method according to claim 1, wherein After anaerobic fermentation in step 5), anaerobically fermented materials are obtained, and flavonoids in the anaerobically fermented materials are extracted by ethanol ultrasonic assistance; The conditions for ethanol ultrasonic assistance extraction include: the power of ultrasonic crushing is 500 - 1000 W, the frequency is 20 kHz, the temperature is 50°C, the time is 30 min, and the ethanol is an ethanol solution with a volume percentage content of 70% - 90%.

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