A method for microbial fermentation production of raspberry ketone

Abstract

The present invention provides a method for producing raspberry ketone by microbial fermentation. Using agricultural production waste as the raw material for the fermentation medium, it adopts a one-step fermentation method with natural yeast from raspberry fruits to produce raspberry ketone, solving problems such as complex operation, long fermentation cycle, and high raw material prices. The operation steps include: S1: activation of the fermentation strain; S2: preparation of the fermentation seed liquid; S3: preparation of the sterilized raspberry leaf extract; S4: preparation of the sterilized bamboo shoot skin extract; S5: preparation of the fermentation medium; S6: production inoculation and fermentation of raspberry ketone; S7: obtaining the raspberry ketone product. The present invention effectively utilizes resources, reduces environmental pollution caused by discarded agricultural waste; the fermentation medium is green, environmentally friendly, and low-cost, and the produced raspberry ketone has high economic value, turning waste into treasure; the fermentation conditions are mild, with a short time, high efficiency, and high yield. The equipment required for industrial production is conventional fermentation equipment, with simple operation and strong feasibility.

Description

Technical Field

[0001] The present invention belongs to microbial fermentation synthesis in the field of biosynthesis, and particularly relates to a method for producing raspberry ketone by microbial fermentation. Background Art

[0002] Raspberry ketone, also known as Raspberry ketone (RK), with the chemical name of 4-(4-hydroxyphenyl)-2-butanone, is mainly present in raspberries and is widely used in perfumes, cosmetics, and can enhance the aroma of foods and beverages. Raspberry ketone has good bioactive functions, such as the regulatory effect on fatty liver, protecting and improving liver function, protecting the heart, regulating blood lipid and losing weight and reducing lipid, regulating blood sugar and anti-diabetic functions, antioxidant function, anti-inflammatory function, etc., and also has broad application prospects in the medical field.

[0003] The production of raspberry ketone mainly includes extraction method, chemical method, biological method, etc. The extraction method generally uses natural plant raw materials such as raspberry fruits and extracts them by solvent extraction method or with the assistance of other means. However, due to the low content of raspberry ketone in natural plant raw materials, the extraction yield is low, and the extraction process is complex, resulting in a high price of naturally extracted raspberry ketone, which is not suitable for large-scale industrial production. Chemical synthesis of raspberry ketone has the advantages of high yield and low price. Chemical synthesis can use natural raw materials, such as using natural anisaldehyde and natural acetone to catalyze aldol condensation to produce the precursor anisole acetone; subsequently, anisole acetone is converted to anisyl acetone by catalytic hydrogenation with nickel or palladium / carbon; finally, raspberry ketone is obtained through the demethylation reaction of anisyl acetone; however, this synthesis method is limited by factors such as raw material prices and the use of catalysts. Other chemical synthesis methods, such as using raw materials like phenol-butynol, phenol-methyl vinyl ketone, p-hydroxybenzaldehyde-acetone, methoxybenzyl chloride-ethyl acetoacetate, etc. for synthesis, these chemical synthesis methods will produce problems such as residues of toxic chemical reagents, residues of heavy metal catalysts, acid-base corrosion, by-products difficult to remove, and the product having an unpleasant odor, which affect the application of chemically synthesized raspberry ketone in fields such as medicine, food, and cosmetics; in addition, the environmental pollution problems caused by chemical synthesis cannot be ignored.

[0004] Compared with chemical synthesis, the biosynthesis method using enzymes and active substances produced by the life activities of microorganisms to produce target compounds shows special advantages due to the mild and safe conditions during the preparation process and the green and environmental protection production process, and has gradually attracted attention. The application of biosynthesis in the synthesis of raspberry ketone has also received attention.

[0005] Raspberry ketone is a derivative of phenylalanine produced through a complex and closely linked enzyme and regulatory system. Raspberry ketone is produced via the phenylpropanoid pathway, the precursors of which are aromatic amino acids. The biosynthesis of aromatic amino acids begins with the shikimate pathway. In the first step, dehydroquinate synthase condenses erythrose-4-phosphate derived from the pentose phosphate pathway and phosphoenolpyruvate derived from the glycolytic pathway, and further enzymatic steps generate the final product of this pathway, chorismic acid. Chorismate mutase 1 then converts chorismic acid to prephenic acid, and finally prephenic acid may biosynthesize phenylalanine in plants via the protocatechuic acid pathway. Phenylalanine may also be generated via the phenylpyruvic acid pathway in the cytoplasm under the action of chorismate mutase 2.

[0006] p-Coumaroyl-CoA is one of the substrates for the synthesis of raspberry ketone in plants. It is generated via the phenylpropanoid synthesis pathway. Phenylalanine ammonia-lyase catalyzes the deamination of phenylalanine to generate cinnamic acid, cinnamic acid hydroxylase catalyzes the conversion of cinnamic acid to p-coumaric acid, and p-coumaroyl-CoA ligase catalyzes the conversion of p-coumaric acid to p-coumaroyl-CoA. Since p-coumaric acid is inexpensive, it is usually added exogenously during the synthesis process to increase the yield of raspberry ketone.

[0007] Malonyl-CoA is another substrate for the synthesis of raspberry ketone. First, acetyl-CoA carboxylase catalyzes the conversion of acetyl-CoA to malonyl-CoA. One molecule of malonyl-CoA and one molecule of p-coumaroyl-CoA are decarboxylated and condensed under the catalysis of benzylideneacetone synthase to generate benzylideneacetone, and finally reduced under the catalysis of benzylideneacetone reductase to generate the target compound, raspberry ketone.

[0008] The above is the pathway for the synthesis of raspberry ketone in plants. The method for biosynthesizing raspberry ketone using microorganisms is based on this pathway and the key enzymes in the pathway. Through genetic engineering means, the genes of microorganisms are edited, and the genes of related enzymes are transferred into microorganisms to obtain transgenic microorganisms capable of expressing the enzymes related to the raspberry ketone synthesis pathway, such as Escherichia coli, etc. Then, the transgenic microorganisms are used for fermentative production of raspberry ketone.

[0009] The currently reported biosynthesis methods mainly involve the fermentation synthesis using genetically engineered bacteria obtained from previous research. For example, in 2022, some researchers reported the synthesis of raspberry ketone in Escherichia coli by adding a simple carbon source. The reporters constructed a microbial platform for producing raspberry ketone from glucose; through genetic engineering and the optimization of phenylalanine ammonia-lyase, tyrosine and p-coumaric acid were produced at 19.3 g / L and 1.9 g / L respectively from glucose; the strain producing p-coumaric acid contained a plasmid to express p-coumaric acid ligase and benzylacetone synthase to produce raspberry ketone from glucose; by genetic and chemical operations to increase the content of malonyl-CoA in cells, the yield of raspberry ketone was improved; finally, fed-batch culture was carried out under optimal conditions to ferment and produce 62 mg / L of raspberry ketone using glucose as the carbon source. Another example is that Wang Chengcheng et al. reported the introduction of genes encoding p-coumaroyl-CoA ligase, benzylideneacetone synthase, and benzylideneacetone reductase from plants into Escherichia coli W3110, and successfully constructed the recombinant strain WCC-1; through the optimization of the initial induction time, the concentration of the inducer isopropyl β-D-thiogalactoside, and the induction temperature, the mass concentration of raspberry ketone produced by this strain in shake-flask fermentation can reach 125.86 mg / L; fed-batch fermentation was carried out in a 3 L fermenter, and the mass concentration of raspberry ketone increased to 178.13 mg / L. Another example is that in 2020, it was reported that a heterologous pathway for producing raspberry ketone from p-coumaric acid was successfully constructed in Corymebacterium glutamicum, and the resulting strain accumulated up to 99.80 mg / L of raspberry ketone.

[0010] Among the patent applications published by the Patent Office of the State Intellectual Property Office, the patent application with the application number 202011316680X (publication number: CN112391418A) discloses an industrial fermentation production method of raspberry ketone, which obtains raspberry ketone through fermentation in three stages using three microorganisms. In the first stage, Actinomycetes sp.OMK-74 is used to ferment p-coumaric acid as the main raw material to produce p-hydroxybenzaldehyde; in the second stage, Bacillus sp.OMK-75 is used as the fermentation strain, and p-hydroxybenzaldehyde synthesized in the first stage and then purified and acetone are used as the main raw materials to synthesize 4-hydroxybenzylideneacetone; in the third stage, the hydroxybenzylideneacetone synthesized in the second stage and then purified is used as the main raw material, and Saccharomyces OMK-75 is used as the fermentation strain for fermentation culture and then purified to obtain raspberry ketone. This method uses three different strains, undergoes fermentation culture in three stages, and undergoes three times of separation and purification to obtain the final product raspberry ketone; excluding the preparation and activation time of the seed liquid, the total culture time for fermenting raspberry ketone is about 300 hours, that is, 12.5 days (see its specific implementation method), and the intermediate product needs to be purified twice before the subsequent reaction can be carried out. Another patent application published by the Patent Office of the State Intellectual Property Office (patent application number: 2018106524422, publication number: CN108753852A) transfers 4-coumaroyl-CoA ligase, benzylacetone synthase, and benzylacetone reductase derived from plants parsley, Rheum palmatum, and raspberry into Escherichia coli for expression by genetic engineering means, and then obtains 70.62 mg / L of raspberry ketone under shake-flask fermentation conditions. The above methods have all achieved the microbial fermentation production of raspberry ketone, but currently, the microbial fermentation method still has disadvantages such as complex construction operations of genetically engineered bacteria in the early stage, high raw material prices, instability of genetically engineered bacteria, or the risk of long-term fermentation contamination, and is restricted in industrial applications. Summary of the Invention

[0011] The purpose of the present invention is to provide a microbial fermentation production method of raspberry ketone, which uses agricultural production waste as the raw material for the fermentation medium and adopts a one-step fermentation method with natural yeast isolated from raspberry fruits to produce raspberry ketone, so as to solve problems such as complex operation, long fermentation cycle, and high raw material prices.

[0012] The present invention is realized through the following technical solutions. A microbial fermentation production method of raspberry ketone, characterized in that the operation steps include S1: activation of the fermentation strain, S2: preparation of the fermentation seed liquid, S3: preparation of the sterilized raspberry leaf extract, S4: preparation of the sterilized bamboo shoot skin extract, S5: preparation of the fermentation medium, S6: production inoculation and fermentation of raspberry ketone, and S7: obtaining the raspberry ketone product.

[0013] The formula of the fermentation medium, by weight, consists of the following components: 250 - 450 parts of sterilized raspberry leaf extract, 200 - 450 parts of sterilized bamboo shoot skin extract, 0.2 - 2 parts of sterilized niacin mother liquor, 1 - 10 parts of sterilized methionine mother liquor, and the balance is made up to 1000 parts by weight of the total fermentation medium with sterilized water. After mixing evenly, adjust the pH to 4.5 - 6.5 under aseptic conditions;

[0014] The fermentation strain is Issatchenkia terricola WJL - G4, which was deposited on October 21, 2019 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, deposit number: CGMCC No. 18712.

[0015] The specific operation details are carried out according to the following plan.

[0016] S1: Activation of the fermentation strain.

[0017] Take the Issatchenkia terricola WJL - G4 strain preserved in the ampoule, inoculate it into the YPD liquid medium, and culture it in a 250 mL conical flask with a liquid loading of 30 mL at 25 - 28 °C, 120 - 250 rpm for 24 h. Then dilute it step - by - step to 10 -6 , suck 100 μL, spread it on the YPD solid medium and culture it at 25 - 28 °C for 24 h. Pick a single colony and inoculate it into the YPD slant medium, and culture it at 25 - 28 °C for 24 - 36 h to obtain the activated strain.

[0018] S2: Preparation of the fermentation seed liquid.

[0019] Pick a loop of the activated strain obtained in S1, inoculate it into a new YPD liquid medium, and culture it at 25 - 28 °C, 120 - 250 rpm under the condition that the liquid loading volume ratio is 10% - 25% for 24 h. Then add sterile glass beads and shake for 20 min to adjust the cell concentration to 1.0×10 7 - 5.0×10 7 CFU / mL to obtain the fermentation seed liquid.

[0020] S3: Preparation of the sterilized raspberry leaf extract.

[0021] The raspberry leaves are fresh leaves of Rubus idaeus L. in the genus Rubus of the Rosaceae family. After the fresh raspberry leaves are picked, they are washed with distilled water to remove surface impurities and drained to obtain washed raspberry leaves. The washed raspberry leaves are quickly frozen and stored at -15°C to -20°C to obtain frozen raspberry leaves. Take the washed raspberry leaves or the frozen raspberry leaves thawed at 25 - 45°C for 30 minutes, cut them into pieces and grind them to obtain a raspberry leaf grinding pulp. After adding distilled water according to the weight ratio of raspberry leaf grinding pulp to distilled water of 1:1 - 2, use a blender to beat the pulp for 1 - 3 minutes to obtain beaten raspberry leaves. Add cellulase to the beaten raspberry leaves, and the added weight of cellulase is 0.01 - 0.05% of the weight of the beaten raspberry leaves. After stirring evenly, cover a layer of plastic wrap on the container mouth, and enzymatically hydrolyze at a constant temperature of 35 - 55°C for 0.5 - 1.5 hours, then place it in an ultrasonic extractor, turn on the ultrasonic extractor to carry out simultaneous enzymatic hydrolysis and extraction for 0.2 - 0.5 hours. The set conditions of the ultrasonic extractor are an input power of 200 - 350 w and an ultrasonic frequency of 20 - 30 KHz. After the simultaneous enzymatic hydrolysis and extraction are completed, quickly raise the temperature to 90°C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, and the filtrate is sterilized by the pasteurization method, and then cooled to 22 - 28°C to obtain a sterilized raspberry leaf extract.

[0022] Preferably, the weight ratio of the raspberry leaf grinding pulp to distilled water is 1:1.4 - 1.6.

[0023] Preferably, the added weight of the cellulase is 0.02 - 0.03% of the weight of the beaten raspberry leaves, the enzymatic hydrolysis time is 0.8 - 1.2 hours, and the temperature is 42 - 46°C.

[0024] Preferably, the time for simultaneous enzymatic hydrolysis and extraction is 0.3 - 0.4 hours, and the set conditions of the ultrasonic extractor are an input power of 250 - 280 w and an ultrasonic frequency of 23 - 26 KHz.

[0025] S4: Preparation of sterilized bamboo shoot skin extract

[0026] The bamboo shoot skin is the inner fresh bamboo shoot skin obtained by removing the outermost part that has been fibrosed due to direct contact with air from the fresh tender shoots of perennial plants of the genus Phyllostachys in the Poaceae family. Take the inner fresh bamboo shoot skin, wash it with distilled water to remove impurities such as soil residues, drain the surface water, and obtain the washed bamboo shoot skin. Quick-freeze the washed bamboo shoot skin and store it at -15 to -20 °C to obtain the frozen bamboo shoot skin; take the washed bamboo shoot skin or the frozen bamboo shoot skin thawed at 25 - 45 °C for 30 minutes, cut it into pieces and grind it to obtain the ground bamboo shoot skin pulp. After adding distilled water according to the weight ratio of the ground bamboo shoot skin pulp to distilled water of 1:1 - 2, use a blender to beat the pulp for 1 - 3 minutes to obtain the bamboo shoot skin pulp. Add cellulase to the bamboo shoot skin pulp, and the added weight of cellulase is 0.01 - 0.05% of the weight of the bamboo shoot skin pulp. After mixing evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 35 - 52 °C, and enzymatically hydrolyze for 0.5 - 1.5 hours. Then place it in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time; the conditions for enzymatic hydrolysis and extraction at the same time are that the ultrasonic extractor is set at an input power of 200 - 400 w, an ultrasonic frequency of 15 - 20 KHz, and the temperature is kept constant at 35 - 52 °C. The time for enzymatic hydrolysis and extraction at the same time is 0.1 - 0.5 hours. Then, quickly raise the temperature to 90 °C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, sterilize the filtrate by pasteurization, and then cool it to 22 - 28 °C to obtain the sterilized bamboo shoot skin extract.

[0027] Preferably, the weight ratio of the ground bamboo shoot skin pulp to distilled water is 1:1.3 - 1.7.

[0028] Preferably, the added weight of the cellulase is 0.02 - 0.03% of the weight of the bamboo shoot skin pulp, the enzymatic hydrolysis time is 0.8 - 1.2 hours, and the temperature is 42 - 46 °C.

[0029] Preferably, the conditions for enzymatic hydrolysis and extraction at the same time are that the ultrasonic extractor is set at an input power of 280 - 300 w, an ultrasonic frequency of 16 - 17 KHz, the temperature is kept constant at 42 - 46 °C, and the time is 0.2 - 0.3 hours.

[0030] S5: Preparation of fermentation medium

[0031] First, prepare a sterilized nicotinic acid mother liquor and a sterilized methionine mother liquor. Weigh nicotinic acid and mix it according to the weight ratio of nicotinic acid:distilled water of 1:20 to obtain the nicotinic acid mother liquor; weigh methionine and mix it according to the weight ratio of methionine:distilled water of 1:20 to obtain the methionine mother liquor; sterilize both mother liquors at 121 °C for 15 minutes to obtain the sterilized nicotinic acid mother liquor and the sterilized methionine mother liquor respectively.

[0032] Measure and mix each component according to the formula weight parts and composition ratio of the fermentation medium:

[0033] 250 - 450 parts of sterilized raspberry leaf extract;

[0034] 200 - 450 parts of sterilized bamboo shoot skin extract;

[0035] 0.2 - 2 parts of sterilized niacin mother liquor;

[0036] 1 - 10 parts of sterilized methionine mother liquor;

[0037] The balance is made up to 1000 parts by weight of the total fermentation medium with sterilized water;

[0038] After mixing evenly, adjust the pH aseptically to 4.5 - 6.5 to obtain the fermentation medium.

[0039] Preferably, the weight parts of the sterilized raspberry leaf extract in the fermentation medium are 300 - 350 parts.

[0040] Preferably, the weight parts of the sterilized bamboo shoot skin extract in the fermentation medium are 250 - 350 parts.

[0041] Preferably, the weight parts of the sterilized niacin mother liquor are 1.0 - 1.2 parts.

[0042] Preferably, the weight parts of the sterilized methionine mother liquor are 5 - 6 parts.

[0043] Preferably, the pH of the fermentation medium is adjusted to 5.0 - 6.0.

[0044] S6: Production inoculation and fermentation of raspberry ketone.

[0045] Take the fermentation seed liquid obtained in step S2, and carry out production inoculation according to the weight ratio of the fermentation seed liquid to the fermentation medium of 1 - 10:100 to obtain a fermentation mixture, and then carry out fermentation culture of raspberry ketone. The conditions are: temperature 22 - 28 °C, rotation speed 120 - 250 rpm, liquid loading volume ratio 20% - 55%, fermentation time 20 - 48 h to obtain a raspberry ketone fermentation broth.

[0046] Preferably, in the production inoculation step, the weight ratio of the fermentation seed liquid to the fermentation medium is 6 - 7:10.

[0047] Preferably, in the fermentation step of raspberry ketone, the fermentation temperature is 24 - 26 °C, the rotation speed is 160 - 200 rpm, the liquid loading volume ratio is 35 - 40%, and the fermentation is carried out for 24 - 46 h.

[0048] Optionally, the fermentation of raspberry ketone adopts a fed-batch fermentation method. When the fermentation reaches 18 - 24 h after inoculation, the sterilized raspberry leaf extract and the sterilized bamboo shoot skin extract are respectively added to the fermenting fermentation mixture. The weight portion of the added sterilized raspberry leaf extract is 30 - 50 parts, and the weight portion of the added sterilized bamboo shoot skin extract is 20 - 50 parts.

[0049] Preferably, in the fed-batch fermentation, the weight portion of the added sterilized raspberry leaf extract is 42 - 45 parts, and the added weight portion of the sterilized bamboo shoot skin extract is 30 - 35 parts.

[0050] S7: Obtaining raspberry ketone product.

[0051] After the fermentation step of raspberry ketone is completed, the separation, extraction and purification of raspberry ketone are carried out. This step is carried out according to the known method, such as separating and purifying by preparative high performance liquid chromatography to obtain the raspberry ketone product.

[0052] Advantages of the present invention

[0053] The present invention makes full use of agricultural production wastes such as raspberry leaves and bamboo shoot skins, which not only effectively utilizes resources, reduces waste, but also reduces the environmental pollution caused by discarded agricultural wastes. At the same time, the cost of the fermentation medium is very low, and the produced raspberry ketone has high economic value, achieving the effect of turning waste into treasure.

[0054] The present invention only needs one-step fermentation to complete the fermentation production of raspberry ketone. The fermentation strain is a natural strain from raspberry fruits. The fermentation conditions are mild, the fermentation time is short, the efficiency is high, the yield is high, the fermentation medium is green and environment-friendly, and the equipment required for industrial production is conventional fermentation equipment, with simple operation and strong feasibility.

[0055] Biological preservation information

[0056] The terrestrial Issatchenkia terricola WJL-G4 was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 21, 2019. The preservation number: CGMCC No.18712. The proposed taxonomic name is: Issatchenkia terricola. The detection result is survival. The preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Specific embodiments

[0057] The following further describes the technical solutions of the present invention in conjunction with specific embodiments, but the present invention is not limited thereto.

[0058] Example 1

[0059] A method for microbial fermentation production of raspberry ketone, and the specific implementation plan is as follows:

[0060] S1: Activation of fermentation strains.

[0061] Take the Issatchenkia terricola WJL-G4 strain preserved in an ampoule, inoculate it into YPD liquid medium, and culture it in a 250 mL conical flask with a liquid loading of 30 mL at 28 °C and 160 rpm for 24 h. Then, dilute it stepwise to 10 -6 , pipette 100 μL, spread it on YPD solid medium and culture it at 28 °C for 24 h. Pick a single colony and inoculate it into YPD slant medium, and culture it at 28 °C for 24 h to obtain activated strains.

[0062] S2: Preparation of fermentation seed liquid.

[0063] Pick a loop of the activated strain obtained in S1, inoculate it into a new YPD liquid medium, and culture it in a 250 mL conical flask with a liquid loading of 30 mL at 28 °C and 160 rpm for 24 h. Then, add sterile glass beads and shake for 20 min to adjust the cell concentration to 1.0×10 7 CFU / mL to obtain fermentation seed liquid.

[0064] S3: Preparation of sterilized raspberry leaf extract.

[0065] The raspberry leaves are fresh leaves of Rubus idaeus L. of the genus Rubus in the Rosaceae family. After picking the fresh raspberry leaves, wash them with distilled water to remove surface impurities and drain the water to obtain washed raspberry leaves. Quick-freeze the washed raspberry leaves and store them at -20 °C to obtain frozen raspberry leaves. Take the frozen raspberry leaves thawed at 25 °C for 30 min, cut them into pieces and grind them to obtain raspberry leaf pulp. Add distilled water according to the weight ratio of raspberry leaf pulp to distilled water of 1:2, and then use a blender to beat the pulp for 2 min to obtain beaten raspberry leaves. Add cellulase to the beaten raspberry leaves, and the added weight is 0.02% of the weight of the beaten raspberry leaves. Stir evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 40 °C and enzymatically hydrolyze for 1.5 h. Then, place it in an ultrasonic extractor and set the input power to 300 w and the ultrasonic frequency to 30 KHz. Enzymatically hydrolyze and extract simultaneously for 0.5 h, then quickly raise the temperature to 90 °C to inactivate the enzyme for 5 minutes. Then, filter it with 8 layers of gauze, and sterilize the filtrate by pasteurization under the conditions of 80 °C and 15 minutes. After that, cool it to 28 °C to obtain sterilized raspberry leaf extract.

[0066] S4: Preparation of sterilized bamboo shoot skin extract

[0067] The bamboo shoot skin is the fresh bamboo shoot skin obtained from the new tender shoots of perennial plants of the genus Phyllostachys in the Poaceae family. The outermost part that has been fibrosed by direct contact with air is removed to obtain the inner fresh bamboo shoot skin. The inner fresh bamboo shoot skin is washed with distilled water to remove impurities such as soil residues, drained of surface moisture, quick-frozen, and stored at -20°C to obtain frozen bamboo shoot skin. Take the frozen bamboo shoot skin thawed at 25°C for 30 minutes, cut it into pieces and grind it to obtain bamboo shoot skin grinding pulp. After adding distilled water according to the weight ratio of bamboo shoot skin grinding pulp to distilled water of 1:2, use a blender to beat the pulp for 3 minutes to obtain bamboo shoot skin pulp. Add cellulase to the bamboo shoot skin pulp, and the added weight is 0.02% of the weight of the bamboo shoot skin pulp. After mixing evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 50°C and enzymatically hydrolyze for 1.5 hours, and then place it in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set with an input power of 200w, an ultrasonic frequency of 20KHz, and the temperature is kept constant at 50°C. After enzymatic hydrolysis and extraction for 0.5 hours, quickly raise the temperature to 90°C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, and the filtrate is sterilized by pasteurization under the conditions of 75°C and 20 minutes, and then cooled to 28°C to obtain the sterilized bamboo shoot skin extract.

[0068] S5: Preparation of fermentation medium

[0069] Weigh nicotinic acid and dissolve and mix it evenly according to the weight ratio of nicotinic acid: distilled water of 1:20 to obtain a nicotinic acid mother liquor; weigh methionine and dissolve and mix it evenly according to the weight ratio of methionine: distilled water of 1:20 to obtain a methionine mother liquor; sterilize both mother liquors at 121°C for 15 minutes to obtain the sterilized nicotinic acid mother liquor and the sterilized methionine mother liquor respectively.

[0070] Measure and mix each component according to the following weight parts and ratios: 450 parts of sterilized raspberry leaf extract; 450 parts of sterilized bamboo shoot skin extract; 2 parts of sterilized nicotinic acid mother liquor; 10 parts of sterilized methionine mother liquor; make up to a total weight of 1000 parts with sterilized water, mix evenly, and adjust the pH to 6.5 under sterile conditions to obtain the fermentation medium.

[0071] S6: Production inoculation and fermentation of raspberry ketone.

[0072] Take the fermentation seed liquid obtained in step S2 and carry out production inoculation according to the weight ratio of the fermentation seed liquid to the fermentation medium of 10:100 to obtain a fermentation mixture, and then carry out fermentation culture of raspberry ketone. The conditions are a temperature of 28°C, a rotation speed of 250 rpm, a loading volume ratio of 50%, and fermentation for 48 hours to obtain a raspberry ketone fermentation broth. After detection, the concentration of raspberry ketone in the fermentation broth is 1054 mg / L.

[0073] S7: Obtaining of raspberry ketone product.

[0074] After the fermentation step of raspberry ketone is completed, the separation, extraction and purification of raspberry ketone are carried out, which are carried out according to the known methods to obtain the raspberry ketone product.

[0075] Example 2

[0076] The method for microbial fermentation production of raspberry ketone is as follows in specific operation steps.

[0077] S1: Activation of the fermentation strain.

[0078] Take the Issatchenkia terricola WJL-G4 strain preserved in the ampoule bottle, inoculate it into the YPD liquid medium, culture it in a 250 mL conical flask with a liquid loading of 30 mL at 25 °C and 160 rpm for 24 h, then dilute it step by step to 10 -6 , suck 100 μL, spread it on the YPD solid medium and culture it at 25 °C for 24 h, pick a single colony and inoculate it into the YPD slant medium, culture it at 25 °C for 36 h to obtain the activated strain.

[0079] S2: Preparation of the fermentation seed liquid.

[0080] Pick a loop of the activated strain obtained in S1, inoculate it into a new YPD liquid medium, culture it in a 250 mL conical flask with a liquid loading of 35 mL at 26 °C and 160 rpm for 24 h, then add sterile glass beads, shake for 20 min, and adjust the cell concentration to 5.0×10 7 CFU / mL to obtain the fermentation seed liquid.

[0081] S3: Preparation of the sterilized raspberry leaf extract.

[0082] The raspberry leaves are the fresh leaves of Rubus idaeus L. of the Rosaceae family. After the fresh raspberry leaves are picked, they are washed with distilled water to remove the surface impurities and drained, and the washed raspberry leaves are obtained. Take the washed raspberry leaves, cut them into pieces and grind them to obtain the raspberry leaf grinding pulp. After adding distilled water according to the weight ratio of raspberry leaf grinding pulp to distilled water of 1:1, use a blender to beat the pulp for 2 min to obtain the beaten raspberry leaves. Add cellulase to the beaten raspberry leaves, and the added weight is 0.02% of the weight of the beaten raspberry leaves. After stirring evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 45 °C, enzymatically hydrolyze for 1.0 h, then place it in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set at an input power of 300 w, an ultrasonic frequency of 25 KHz, keep the temperature constant at 45 °C, enzymatically hydrolyze and extract for 0.2 h at the same time, then quickly raise the temperature to 90 °C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, and the filtrate is pasteurized under the conditions of 80 °C and 15 minutes, and then cooled to 26 °C to obtain the sterilized raspberry leaf extract.

[0083] S4: Preparation of sterilized bamboo shoot skin extract

[0084] The bamboo shoot skin is the fresh outer skin of the tender shoots of perennial bamboos of the genus Phyllostachys in the family Poaceae, and the inner fresh bamboo shoot skin obtained by removing the outermost part that has been fibrosed due to direct contact with air. Take the inner fresh bamboo shoot skin, wash it with distilled water to remove impurities such as soil residues, drain the surface water, and obtain the washed bamboo shoot skin; take the washed bamboo shoot skin, cut it into pieces and grind it to obtain a bamboo shoot skin grinding pulp. After adding distilled water according to the weight ratio of bamboo shoot skin grinding pulp to distilled water of 1:1, use a blender to beat the pulp for 2 minutes to obtain a bamboo shoot skin pulp. Add cellulase to the bamboo shoot skin pulp, and the added weight is 0.02% of the weight of the bamboo shoot skin pulp. After mixing evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 45 °C, enzymatically hydrolyze for 1.5 h, and then place it in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set at an input power of 250 w and an ultrasonic frequency of 20 KHz, keep the temperature constant at 45 °C, enzymatically hydrolyze and extract for 0.5 h at the same time, then quickly raise the temperature to 90 °C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, and the filtrate is pasteurized under the conditions of 80 °C and 15 minutes, and then cooled to 26 °C to obtain the sterilized bamboo shoot skin extract.

[0085] S5: Preparation of fermentation medium

[0086] Weigh nicotinic acid and mix it according to the weight ratio of nicotinic acid:distilled water of 1:20 to obtain a nicotinic acid mother liquor; weigh methionine and mix it according to the weight ratio of methionine:distilled water of 1:20 to obtain a methionine mother liquor; sterilize both mother liquors at 121 °C for 15 minutes to obtain the sterilized nicotinic acid mother liquor and the sterilized methionine mother liquor respectively.

[0087] Measure and mix each component according to the following weight parts and ratios: 450 parts of sterilized raspberry leaf extract; 400 parts of sterilized bamboo shoot skin extract; 2 parts of sterilized nicotinic acid mother liquor; 9 parts of sterilized methionine mother liquor; make up to a total weight of 1000 parts with sterilized water, mix evenly, and adjust the pH to 5.5 aseptically to obtain the fermentation medium.

[0088] S6: Production inoculation and fermentation of raspberry ketone

[0089] Take the fermentation seed liquid obtained in step S2, and carry out production inoculation according to the weight ratio of the fermentation seed liquid to the fermentation medium of 5:100 to obtain a fermentation mixture, and then carry out fermentation culture of raspberry ketone. The conditions are: temperature 26 °C, rotation speed 180 rpm, liquid loading volume ratio 35%, after fermentation for 24 h, carry out fed-batch fermentation, add 45 parts by weight of the sterilized raspberry leaf extract and 35 parts by weight of the sterilized bamboo shoot skin extract to the fermentation mixture, and then continue to ferment for 24 h to obtain a raspberry ketone fermentation broth. After detection, the concentration of raspberry ketone in the fermentation broth is 1695 mg / L.

[0090] S7: Raspberry ketone product obtained.

[0091] After the raspberry ketone fermentation step is completed, extraction, separation and purification of raspberry ketone are carried out to obtain a raspberry ketone product.

[0092] Example 3

[0093] A method for microbial fermentation production of raspberry ketone, and the specific implementation steps are as follows.

[0094] S1: Activation of fermentation strains.

[0095] Take the Issatchenkia terricola WJL-G4 strain preserved in an ampoule, inoculate it into YPD liquid medium, culture it in a 250 mL conical flask with a liquid loading volume of 30 mL at 28 °C and 180 rpm for 24 h, then dilute it step by step to 10 -6 , suck 100 μL, coat it on YPD solid medium and culture it at 28 °C for 24 h, pick a single colony and inoculate it into YPD slant medium, culture it at 28 °C for 24 h to obtain an activated strain.

[0096] S2: Preparation of fermentation seed liquid.

[0097] Pick a loop of the activated strain obtained in S1, inoculate it into a new YPD liquid medium, culture it in a 250 mL conical flask with a liquid loading volume of 40 mL at 28 °C and 180 rpm for 24 h, then add sterile glass beads, shake for 20 min, and adjust the cell concentration to 4×10 7 CFU / mL to obtain a fermentation seed liquid.

[0098] S3: Preparation of sterilized raspberry leaf extract.

[0099] The raspberry leaves are fresh leaves of Rubus idaeus L. of the genus Rubus in the Rosaceae family. After the fresh raspberry leaves are picked, they are washed with distilled water to remove surface impurities and drained to obtain washed raspberry leaves. The washed raspberry leaves are taken, cut into pieces and ground to obtain a raspberry leaf grinding pulp. After adding distilled water according to the weight ratio of raspberry leaf grinding pulp to distilled water of 1:1.5, it is beaten into pulp with a blender for 1.5 min to obtain a beaten raspberry leaf. Cellulase is added to the beaten raspberry leaf, and the added weight is 0.04% of the weight of the beaten raspberry leaf. After stirring evenly, a layer of plastic wrap is covered on the container mouth, and the temperature is kept constant at 40 °C. After enzymatic hydrolysis for 1 h, it is placed in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set at an input power of 350 w and an ultrasonic frequency of 25 kHz, and the temperature is kept constant at 40 °C. After enzymatic hydrolysis and extraction for 0.2 h at the same time, it is quickly heated to 90 °C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is pasteurized under the conditions of 75 °C and 25 minutes, and then cooled to 26 °C to obtain a sterilized raspberry leaf extract.

[0100] S4: Preparation of sterilized bamboo shoot skin extract

[0101] The bamboo shoot skin is the fresh outer skin of the new tender shoots of the perennial plant bamboo of the genus Bambusa in the Poaceae family, and the inner fresh bamboo shoot skin obtained after removing the outermost part that has been fibrosed and in direct contact with the air. The inner fresh bamboo shoot skin is taken and washed with distilled water to remove impurities such as soil residues, and the surface water is drained and frozen at -18 °C to obtain frozen bamboo shoot skin; the frozen bamboo shoot skin thawed at 35 °C for 30 min is taken, cut into pieces and ground to obtain a bamboo shoot skin grinding pulp. After adding distilled water according to the weight ratio of bamboo shoot skin grinding pulp to distilled water of 1:1.5, it is beaten into pulp with a blender for 1.5 min to obtain a bamboo shoot skin pulp. Cellulase is added to the bamboo shoot skin pulp, and the added weight is 0.03% of the weight of the bamboo shoot skin pulp. After mixing evenly, a layer of plastic wrap is covered on the container mouth, and the temperature is kept constant at 45 °C. After enzymatic hydrolysis and extraction for 1.5 h at the same time, it is placed in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set at an input power of 250 w and an ultrasonic frequency of 20 kHz, and the temperature is kept constant at 45 °C. After enzymatic hydrolysis and extraction for 0.2 h at the same time, it is quickly heated to 90 °C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is pasteurized under the conditions of 80 °C and 15 minutes, and then cooled to 26 °C to obtain a sterilized bamboo shoot skin extract.

[0102] S5: Preparation of fermentation medium

[0103] The preparation methods of the sterilized niacin stock solution and the sterilized methionine stock solution are the same as those in Example 1.

[0104] Weigh and mix each component according to the following parts by weight and ratios: 250 parts of sterilized raspberry leaf extract; 200 parts of sterilized bamboo shoot skin extract; 0.2 part of sterilized niacin mother liquor; 2 parts of sterilized methionine mother liquor; make up to a total of 1000 parts by weight with sterilized water, mix evenly, and adjust the pH aseptically to 4.5 to obtain the fermentation medium.

[0105] S6: Production inoculation and fermentation of raspberry ketone.

[0106] Take the fermentation seed liquid obtained in step S2, carry out production inoculation according to the ratio of the fermentation seed liquid to the fermentation medium of 2:100 by weight to obtain a fermentation mixture, and then carry out fermentation culture of raspberry ketone. The conditions are: temperature 26°C, rotation speed 150 rpm, liquid filling volume ratio 45%, after fermentation for 24 h, obtain raspberry ketone fermentation broth; after detection, the concentration of raspberry ketone in the fermentation broth is 916 mg / L.

[0107] S7: Obtaining of raspberry ketone product.

[0108] After the raspberry ketone fermentation step is completed, carry out extraction, separation and purification of raspberry ketone to obtain a raspberry ketone product.

[0109] Example 4

[0110] A method for microbial fermentation production of raspberry ketone, the specific implementation plan is as follows.

[0111] S1: Activation of fermentation strains.

[0112] Take the Issatchenkia terricola WJL-G4 strain preserved in an ampoule, inoculate it into YPD liquid medium, culture it in a 250 mL conical flask with a liquid filling volume of 30 mL at 28°C, 160 rpm for 24 h, then dilute it stepwise to 10 -6 , pipette 100 μL, spread it on YPD solid medium and culture it at 28°C for 24 h, pick a single colony and inoculate it into YPD slant medium, culture it at 28°C for 24 h to obtain an activated strain.

[0113] S2: Preparation of fermentation seed liquid.

[0114] Pick a loop of the activated strain obtained in S1, inoculate it into a new YPD liquid medium, culture it in a 250 mL conical flask with a liquid filling volume of 45 mL at 28°C, 160 rpm for 24 h, add sterile glass beads, shake for 20 min, and adjust the cell concentration to 3.5×10 7 CFU / mL to obtain the fermentation seed liquid.

[0115] S3: Preparation of sterilized raspberry leaf extract.

[0116] The raspberry leaves are fresh leaves of Rubus idaeus L. of the genus Rubus in the Rosaceae family. After the fresh raspberry leaves are picked, they are washed with distilled water to remove surface impurities and drained to obtain washed raspberry leaves. The washed raspberry leaves are quick-frozen and stored at -20°C to obtain frozen raspberry leaves. Take the frozen raspberry leaves thawed at 25°C for 30 minutes, cut them into pieces and grind them to obtain raspberry leaf grinding pulp. After adding distilled water according to the weight ratio of raspberry leaf grinding pulp to distilled water of 1:2, use a blender to beat the pulp for 1.8 minutes to obtain beaten raspberry leaves. Add cellulase to the beaten raspberry leaves, and the added weight is 0.03% of the weight of the beaten raspberry leaves. After stirring evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 50°C, enzymatically hydrolyze for 1.4 hours, and then place it in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set at an input power of 350 w and an ultrasonic frequency of 20 KHz, keep the temperature constant at 50°C, enzymatically hydrolyze and extract for 0.2 hours at the same time, quickly raise the temperature to 90°C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, and the filtrate is pasteurized under the conditions of 80°C and 15 minutes, and then cooled to 26°C to obtain the sterilized raspberry leaf extract.

[0117] S4: Preparation of sterilized bamboo shoot skin extract

[0118] The bamboo shoot skin is the fresh outer skin of the new shoots of the perennial plant bamboo of the genus Phyllostachys in the Poaceae family. The inner fresh bamboo shoot skin obtained after peeling the bamboo shoot skin from the fresh young shoots and removing the outermost part that has been fibrosed by direct contact with the air. Take the inner fresh bamboo shoot skin and wash it with distilled water to remove impurities such as soil residues, and drain the surface water to obtain washed bamboo shoot skin. Take the washed bamboo shoot skin, cut it into pieces and grind it to obtain bamboo shoot skin grinding pulp. After adding distilled water according to the weight ratio of bamboo shoot skin grinding pulp to distilled water of 1:2, use a blender to beat the pulp for 1.5 minutes to obtain bamboo shoot skin pulp. Add cellulase to the bamboo shoot skin pulp, and the added weight is 0.05% of the weight of the bamboo shoot skin pulp. After mixing evenly, cover a layer of plastic wrap on the container mouth, keep the temperature constant at 45°C, enzymatically hydrolyze and extract for 1.5 hours at the same time, and then place it in an ultrasonic extractor for enzymatic hydrolysis and extraction at the same time. The conditions are that the ultrasonic extractor is set at an input power of 280 w and an ultrasonic frequency of 19 KHz, keep the temperature constant at 45°C, enzymatically hydrolyze and extract for 0.3 hours at the same time, quickly raise the temperature to 90°C to inactivate the enzyme for 5 minutes, then filter with 8 layers of gauze, and the filtrate is pasteurized under the conditions of 80°C and 15 minutes, and then cooled to 26°C to obtain the sterilized bamboo shoot skin extract.

[0119] S5: Preparation of fermentation medium

[0120] The preparation methods of the sterilized nicotinic acid mother liquor and the sterilized methionine mother liquor are the same as those in Example 1.

[0121] Measure and mix each component according to the following parts by weight and ratios: 250 parts of sterilized raspberry leaf extract; 200 parts of sterilized bamboo shoot skin extract; 0.2 part of sterilized niacin mother liquor; 1 part of sterilized methionine mother liquor; make up to a total of 1000 parts by weight with sterilized water. After mixing evenly, adjust the pH aseptically to 6.5 to obtain the fermentation medium described above.

[0122] S6: Production inoculation and fermentation of raspberry ketone.

[0123] Take the fermentation seed liquid obtained in step S2, and carry out production inoculation according to the ratio of the weight of the fermentation seed liquid to the fermentation medium of 1:100 to obtain a fermentation mixture, and then carry out the fermentation culture of raspberry ketone. The conditions are: temperature 26°C, rotation speed 220 rpm, liquid loading volume ratio 50%, ferment for 48 h to obtain a raspberry ketone fermentation broth. After testing, the concentration of raspberry ketone in the fermentation broth is 874 mg / L.

[0124] S7: Obtaining of raspberry ketone product.

[0125] After the raspberry ketone fermentation step is completed, carry out separation, extraction and purification to obtain a raspberry ketone product.

[0126] Example 5

[0127] A method for microbial fermentation production of raspberry ketone, and the specific implementation plan is as follows.

[0128] The operations of steps S1 - S5 are the same as those in Example 3.

[0129] S6: Production inoculation and fermentation of raspberry ketone.

[0130] Take the fermentation seed liquid obtained in step S2, and carry out production inoculation according to the ratio of the weight of the fermentation seed liquid to the fermentation medium of 2:100 to obtain a fermentation mixture, and then carry out the fermentation culture of raspberry ketone. The conditions are: temperature 26°C, rotation speed 150 rpm, liquid loading volume ratio 45%, after fermenting for 24 h, carry out fed-batch fermentation, add 30 parts by weight of the sterilized raspberry leaf extract and 20 parts by weight of the sterilized bamboo shoot skin extract to the fermentation mixture, and then continue to ferment for 24 h to obtain a raspberry ketone fermentation broth. After testing, the concentration of raspberry ketone in the fermentation broth is 1429 mg / L.

[0131] S7: Obtaining of raspberry ketone product.

[0132] After the raspberry ketone fermentation step is completed, carry out extraction, separation and purification of raspberry ketone to obtain a raspberry ketone product.

[0133] Example 6

[0134] A method for microbial fermentation production of raspberry ketone, and the specific implementation scheme is as follows.

[0135] The operations of steps S1 - S5 are the same as those in Example 4.

[0136] S6: Production inoculation and fermentation of raspberry ketone.

[0137] Take the fermentation seed liquid obtained in step S2, and carry out production inoculation according to the ratio of the weight of the fermentation seed liquid to the fermentation medium being 1:100, to obtain a fermentation mixture, and then carry out fermentation culture of raspberry ketone. The conditions are as follows: temperature 26°C, rotation speed 220 rpm, liquid loading volume ratio 50%, after fermentation for 20 h, carry out fed-batch fermentation, add 30 parts by weight of the sterilized raspberry leaf extract and 20 parts by weight of the sterilized bamboo shoot skin extract to the fermentation mixture, and continue to ferment for another 24 h after adding, to obtain a raspberry ketone fermentation broth. After detection, the concentration of raspberry ketone in the fermentation broth is 1291 mg / L.

[0138] S7: Obtaining of raspberry ketone product.

[0139] After the raspberry ketone fermentation step is completed, carry out extraction, separation and purification of raspberry ketone to obtain a raspberry ketone product.

[0140] Example 7

[0141] The determination method of raspberry ketone is carried out according to the following operations.

[0142] Analysis is carried out using an Agilent 1260 Infinity II high performance liquid chromatograph. Chromatographic column: Agilent ZORBAX Extend-C 18 (250 mm × 4.6 mm); mobile phase: A is methanol, B is 0.02% formic acid solution; gradient elution program: 0 - 5 min, 0 - 10% A, 100% - 90% B; 5 - 10 min, 10% - 20% A, 90% - 80% B; 10 - 20 min, 20% - 35% A, 80% - 65% B; 20 - 35 min, 35% - 40% A, 65% - 60% B; 35 - 40 min, 40% - 75% A, 60% - 25% B; 40 - 45 min, 75% - 10% A, 25% - 90% B; flow rate: 0.8 mL / min, injection volume: 10 μL, column temperature: 35°C, detection wavelength: 280 nm. The standard sample series and the sample to be measured are measured in sequence. Draw a standard curve with the standard sample concentration against the peak area, and calculate the content of raspberry ketone according to the standard curve and the peak area of the sample to be measured.

[0143] Standard curve: The raspberry ketone standard was dissolved in methanol to prepare standard solutions with different mass concentrations. After filtration through a 0.22 μm microporous membrane, HPLC analysis was performed to obtain the regression equation and correlation coefficient of the peak area (x) and the mass concentration of the active ingredient (y).

[0144] Treatment of the sample to be measured: Take a certain volume of the sample to be measured. After appropriate dilution, centrifuge at 8000 rpm for 5 min, aspirate the supernatant, filter it through a 0.22 μm membrane, and measure it according to the determination method of raspberry ketone described above. Calculate the result based on the standard curve.

[0145] As described above, only some embodiments of the present invention are provided and are not intended to limit the present invention. The scope of patent protection of the present invention is subject to the claims. Any equivalent structural changes made by using the content of the specification of the present invention should similarly be included in the protection scope of the present invention.

Claims

1. A method for producing raspberry ketone by microbial fermentation, characterized in that: The operation steps include: S1: activation of fermentation strains, S2: preparation of fermentation seed liquid, S3: preparation of sterilized raspberry leaf extract, S4: preparation of sterilized bamboo shoot peel extract, S5: preparation of fermentation medium, S6: production inoculation and fermentation of raspberry ketone, S7: obtaining raspberry ketone products; The fermentation strain is Issatchenkia terricola WJL-G4, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2019, with a deposit number of CGMCC No.18712, a proposed classification name of Issatchenkia terricola, and a test result of survival. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; The formula of the fermentation medium is composed of the following components by weight: 250-450 parts of sterilized raspberry leaf extract, 200-450 parts of sterilized bamboo shoot peel extract, 0.2-2 parts of sterilized nicotinic acid mother solution, 1-10 parts of sterilized methionine mother solution, and the balance is supplemented by sterilized water to a total weight of 1000 parts of the fermentation medium. After uniformly mixing, adjust the pH to 4.5-6.5 under sterile conditions.

2. The method for producing raspberry ketone by microbial fermentation according to claim 1, characterized in that: The raspberry leaves are fresh leaves of raspberry, a plant of the genus Rubus in the Rosaceae family.

3. The method for producing raspberry ketone by microbial fermentation according to claim 1, characterized in that: The preparation method of the sterilized raspberry leaf extract comprises the following steps: after fresh raspberry leaves are picked, they are washed with distilled water to remove surface impurities and drained to obtain washed raspberry leaves, the washed raspberry leaves are quickly frozen and stored at -15°C to -20°C to obtain frozen raspberry leaves; the washed raspberry leaves or frozen raspberry leaves thawed at 25-45°C for 30 minutes are taken, chopped and ground to obtain raspberry leaf grinding pulp, distilled water is added according to a weight ratio of 1:1-2 between the raspberry leaf grinding pulp and distilled water, and the mixture is beaten with a wall breaking machine for 1-3 minutes to obtain beaten raspberry leaves, and cellulase is added to the beaten raspberry leaves, wherein the weight of the cellulase added is 1:1 / 2 of the beaten raspberry leaves. The method comprises the following steps: adding 0.01-0.05% of the weight of the leaves to the container, stirring evenly, covering the container with a layer of preservative film, performing enzymolysis at a constant temperature of 35-55 DEG C for 0.5-1.5 hours, placing the container in an ultrasonic extractor, turning on the ultrasonic extractor for simultaneous enzymolysis and extraction for 0.2-0.5 hours, and setting the ultrasonic extractor to have an input power of 200-350W and an ultrasonic frequency of 20-30KHz. After the simultaneous enzymolysis and extraction are completed, the container is rapidly heated to 90 DEG C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is sterilized by pasteurization and then cooled to 22-28 DEG C to obtain a sterilized raspberry leaf extract.

4. The method for producing raspberry ketone by microbial fermentation according to claim 1, characterized in that: The bamboo shoot skin is the fresh outer skin of fresh young bamboo shoots of the perennial plant bamboo of the genus Bambusa of the Gramineae family, and the inner layer of fresh bamboo shoot skin is obtained by removing the outermost layer that is in direct contact with the air and has become fibrous.

5. The method for producing raspberry ketone by microbial fermentation according to claim 1 or 4, characterized in that: The preparation method of the sterilized bamboo shoot peel extract comprises the following steps: washing the inner layer of fresh bamboo shoot peel with distilled water to remove soil residues and other impurities, draining the surface water to obtain washed bamboo shoot peel, and quickly freezing the washed bamboo shoot peel and storing it at -15 to -20°C to obtain frozen bamboo shoot peel; taking the washed bamboo shoot peel or the frozen bamboo shoot peel thawed at 25-45°C for 30 minutes, cutting and grinding to obtain bamboo shoot peel grinding slurry, adding distilled water according to a weight ratio of 1:1-2 between the bamboo shoot peel grinding slurry and distilled water, and beating with a wall breaking machine for 1-3 minutes to obtain bamboo shoot peel pulp, and adding cellulase to the bamboo shoot peel pulp, wherein the added weight of the cellulase is 0.01% of the weight of the bamboo shoot peel pulp. -0.05%, after mixing evenly, cover the container with a layer of plastic wrap, keep the temperature constant at 35-52°C, perform enzymolysis for 0.5-1.5h, then place in an ultrasonic extractor for further enzymolysis and extraction; the conditions for the enzymolysis and extraction are that the ultrasonic extractor is set at an input power of 200-400w, an ultrasonic frequency of 15-20KHz, and a constant temperature of 35-52°C, the enzymolysis and extraction time is 0.1-0.5h, then the temperature is rapidly raised to 90°C to inactivate the enzyme for 5 minutes, then filtered through 8 layers of gauze, the filtrate is sterilized by pasteurization, and then cooled to 22-28°C to obtain a sterilized bamboo shoot peel extract.

6. The method for producing raspberry ketone by microbial fermentation according to claim 1, characterized in that: The production inoculation and raspberry ketone fermentation conditions are as follows: the production inoculation is carried out according to a weight ratio of the fermentation seed liquid to the fermentation medium of 1-10:100 to obtain a fermentation mixture, and then the raspberry ketone fermentation culture is carried out under the conditions of a temperature of 22-28°C, a rotation speed of 120-250rpm, a liquid volume ratio of 20%-55%, and fermentation for 20-48h to obtain a raspberry ketone fermentation liquid.

7. The method for producing raspberry ketone by microbial fermentation according to claim 1 or 6, characterized in that: A fed-batch fermentation method was adopted.

8. The method for producing raspberry ketone by microbial fermentation according to claim 7, characterized in that: The method of batch fed fermentation is that when the fermentation of raspberry ketone is carried out for 18-24 hours after production inoculation, the sterilized raspberry leaf extract and the sterilized bamboo shoot peel extract are respectively added to the fermentation mixture being fermented, and the weight parts of the sterilized raspberry leaf extract added are 30-50 parts, and the weight parts of the sterilized bamboo shoot peel extract added are 20-50 parts.