Microbial fermentation production method of raspberry ketone

Abstract

The invention provides a microbial fermentation production method of raspberry ketone, which takes agricultural production waste as a raw material of a fermentation culture medium, adopts a one-step fermentation method of natural saccharomycetes from raspberry fruits to produce raspberry ketone, and solves the problems of complex operation, long fermentation period, high raw material price and the like. The method comprises the following operation steps: S1, activation of a fermentation strain, S2, preparation of a fermentation seed solution, S3, preparation of a sterilized raspberry leaf extracting solution, S4, preparation of a sterilized bamboo shoot skin extracting solution, S5, preparation of a fermentation culture medium, S6, production inoculation and fermentation of raspberry ketone, and S7, obtaining of a raspberry ketone product. According to the invention, resources are effectively utilized, and environmental pollution caused by discarded agricultural wastes is reduced; the fermentation culture medium is green and environment-friendly, the cost is low, the produced raspberry ketone has very high economic value, and waste is turned into wealth; the fermentation conditions are mild, the time is short, the efficiency is high, the yield is high, equipment required by industrial production is conventional fermentation equipment, the operation is simple, and the feasibility is high.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation synthesis in the field of biosynthesis, and specifically relates to a microbial fermentation production method of raspberry ketone. Background Art

[0002] Raspberry ketone, also known as Raspberry ketone (RK), has a chemical name of 4-(4-hydroxyphenyl)-2-butanone. It is mainly found in raspberries and is widely used in perfumes and cosmetics. It can also be used to flavor food and beverages. Raspberry ketone has good biological activity functions, such as regulating fatty liver, protecting and improving liver function, protecting the heart, regulating blood lipids and reducing weight and lipids, regulating blood sugar and anti-diabetic functions, antioxidant functions, anti-inflammatory functions, etc. It also has broad application prospects in the medical field.

[0003] The production of raspberry ketone mainly includes extraction method, chemical method and biological method. The extraction method generally uses natural plant raw materials such as raspberry fruit, and uses solvent extraction method or other auxiliary means to extract. However, due to the low content of raspberry ketone in natural plant raw materials, the extraction yield is low, and the extraction process is complicated, resulting in high price of naturally extracted raspberry ketone, which is not suitable for large-scale industrial production. The chemical synthesis of raspberry ketone has the advantages of high yield and low price. Chemical synthesis can be synthesized using natural raw materials, such as using natural anisaldehyde and natural acetone to catalyze aldol condensation to produce precursor anisole acetone; then, anisole acetone is converted into anisyl acetone by nickel or palladium / carbon catalytic hydrogenation; finally, raspberry ketone is obtained by demethylation reaction of anisyl acetone; however, this method of synthesis is limited by factors such as raw material prices and the use of catalysts. Other chemical synthesis methods, such as the use of phenol-butanone alcohol, phenol-methyl vinyl ketone, p-hydroxybenzaldehyde-acetone, methoxybenzyl chloride-ethyl acetoacetate and other raw materials, will produce problems such as toxic chemical reagent residues, heavy metal catalyst residues, acid and alkali corrosion, by-products that are difficult to remove, and products with bad odors, which affect the application of raspberry ketone synthesized by chemical methods in the fields of medicine, food, cosmetics, etc.; in addition, the environmental pollution caused by chemical synthesis cannot be ignored.

[0004] Compared with chemical synthesis, biosynthesis, which uses enzymes and active substances produced by the life activities of microorganisms to produce target compounds, has gradually attracted attention due to its mild and safe preparation conditions and green and environmentally friendly production process. The application of biosynthesis in the synthesis of raspberry ketone has also attracted attention.

[0005] Raspberry ketone is a derivative of phenylalanine produced by a complex and tightly linked enzyme and regulatory system. Raspberry ketone is produced through 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, dihydroxyacetone phosphate synthase condenses erythrose-4-phosphate derived from the pentose phosphate pathway and phosphoenolpyruvate derived from the glycolysis pathway, and further enzymatic steps produce the final product of the pathway, chorismate. Choristate mutase 1 then converts chorismate into prephenate, and finally prephenate may be biosynthesized into phenylalanine in plants through the pyruvic acid pathway. Phenylalanine may also be produced in the cytoplasm through the phenylpyruvate pathway under the action of chorismate mutase 2.

[0006] p-Coumaryl-CoA is one of the substrates for the synthesis of raspberry ketones in plants. It is produced through the phenylpropanoid biosynthesis pathway. Phenylalanine ammonia lyase catalyzes the deamination of phenylalanine to produce cinnamic acid, cinnamic acid hydroxylase catalyzes cinnamic acid to produce p-coumaric acid, and p-coumaryl-CoA ligase catalyzes p-coumaric acid to produce p-coumaryl-CoA. Due to the low price of p-coumaric acid, it is usually added exogenously during the synthesis process to increase the yield of raspberry ketones.

[0007] Malonyl-CoA is another substrate for the synthesis of raspberry ketones. First, acetyl-CoA is catalyzed by acetyl-CoA carboxylase to generate malonyl-CoA. Under the catalysis of benzylideneacetone synthase, one molecule of malonyl-CoA and one molecule of p-coumaryl-CoA undergo decarboxylation condensation to generate benzylideneacetone, and finally, under the catalysis of benzylideneacetone reductase, they are reduced to generate the target compound raspberry ketones.

[0008] The above is the pathway for synthesizing raspberry ketones in plants. The method of using microorganisms to biosynthesize raspberry ketones is based on this pathway and the key enzymes in the pathway. Through genetic engineering means, the genes of the relevant enzymes are edited in the microorganisms, and the genes of the relevant enzymes are transferred into the microorganisms to obtain transgenic microorganisms that can express enzymes related to the raspberry ketone synthesis pathway, such as Escherichia coli, and then the transgenic microorganisms are used to ferment and produce raspberry ketones.

[0009] The biosynthesis methods that have been published so far mainly use the fermentation method of genetically engineered bacteria obtained in previous studies. For example, in 2022, researchers reported the synthesis of raspberry ketones in Escherichia coli by adding a simple carbon source; the reporter constructed a microbial platform for producing raspberry ketones from glucose; through genetic engineering and phenylalanine ammonia lyase optimization, glucose produced 19.3g / L and 1.9g / L of tyrosine and p-coumaric acid, respectively; the strain producing p-coumaric acid contains a plasmid to express p-coumaric acid ligase and benzoyl acetone synthase to produce raspberry ketones from glucose; the content of malonyl-CoA in the cells was increased by genetic and chemical manipulation, which increased the yield of raspberry ketones; finally, under optimal conditions, fed-batch culture was used to ferment glucose as a carbon source to produce 62mg / L of raspberry ketones. For another example, Wang Chengcheng et al. reported that they introduced p-coumaryl-CoA ligase, benzylideneacetone synthase and benzylideneacetone reductase genes from plants into Escherichia coli W3110 and successfully constructed the recombinant strain WCC-1; by optimizing the initial induction time, the concentration of the inducer isopropylthiogalactoside, and the induction temperature, the strain produced raspberry ketones at a mass concentration of 125.86 mg / L by shake flask fermentation; fed-batch fermentation was carried out in a 3L fermenter, and the mass concentration of raspberry ketones was increased to 178.13 mg / L. For another example, in 2020, it was reported that a heterologous pathway for producing raspberry ketones from p-coumaric acid was successfully constructed in Corymebacterium glutamicum, and the resulting strain accumulated up to 99.80 mg / L of raspberry ketones.

[0010] In the patent application published by the Patent Office of the State Intellectual Property Office, application number 202011316680X (application publication number: CN112391418A) discloses an industrial fermentation production method for raspberry ketone, which uses three kinds of microorganisms to obtain raspberry ketone through three stages of fermentation. In the first stage, actinomycetes sp.OMK-74 is used to ferment and produce p-hydroxybenzaldehyde with p-coumaric acid as the main raw material; in the second stage, Bacillus sp.OMK-75 is used as the fermentation strain, and p-hydroxybenzaldehyde and acetone synthesized and purified in the first stage are used as the main raw materials to synthesize 4-hydroxybenzylideneacetone; in the third stage, hydroxybenzylideneacetone synthesized and purified in the second stage is used as the main raw material, and yeast SaccharomycesOMK-75 is used as the fermentation strain, fermented and purified to obtain raspberry ketone. This method uses three different strains, undergoes three stages of fermentation and cultivation, and undergoes three separation and purification to obtain the final product raspberry ketone; excluding the seed liquid preparation and activation time, the fermentation and cultivation time for raspberry ketone production is about 300 hours, or 12.5 days (see its specific implementation method), and the intermediate product needs to be purified twice before subsequent reactions can be carried out. Another patent application published by the Patent Office of the State Intellectual Property Office (patent application number: 2018106524422, application publication number: CN108753852A) uses genetic engineering to transfer 4-coumaroyl-CoA ligase, benzyl acetone synthase and benzyl acetone reductase from plants parsley, palm rhubarb and raspberry into Escherichia coli for expression, and then obtains 70.62 mg / L of raspberry ketone under shake flask fermentation conditions. The above methods all achieve the microbial fermentation production of raspberry ketone, but the current microbial fermentation method still has disadvantages such as complex early genetic engineering bacteria construction operations, high raw material prices, stability of genetic engineering bacteria, or long-term fermentation pollution risks, which limits its industrial application. 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 fermentation medium raw materials and adopts a one-step fermentation method of natural yeast isolated from raspberry fruit to produce raspberry ketone, thereby solving the problems of complex operation, long fermentation cycle, high raw material price, etc.

[0012] The present invention is implemented by the following technical scheme: 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, and S7: obtaining raspberry ketone products.

[0013] 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 uniform mixing, the pH is adjusted to 4.5-6.5 under sterile conditions;

[0014] 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 the deposit number: CGMCC No.18712.

[0015] The specific operation details are as follows.

[0016] S1: Activation of fermentation bacteria.

[0017] Take the Issatchenkia terricola WJL-G4 strain stored in the ampoule bottle, inoculate it into YPD liquid medium, culture it in a 250mL conical flask with a liquid volume of 30mL at 25-28℃, 120-250rpm for 24h, and then dilute it to 10 -6 , draw 100 μL, spread it on YPD solid culture medium and culture it at 25-28℃ for 24 hours, pick a single colony and inoculate it into YPD slant culture medium, culture it at 25-28℃ for 24-36 hours to obtain activated bacteria.

[0018] S2: Preparation of fermentation seed solution.

[0019] Pick a ring of activated bacteria obtained from S1 and inoculate it into a new YPD liquid medium. After culturing for 24 hours at 25-28°C, 120-250rpm, and a liquid volume ratio of 10%-25%, add sterile glass beads and shake for 20 minutes to adjust the bacterial concentration to 1.0×10 7 -5.0×10 7 CFU / mL, and obtain the fermentation seed liquid.

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

[0021] The raspberry leaves are fresh leaves of the plant raspberry 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 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 cut and ground to obtain raspberry leaf grinding pulp. After adding distilled water according to a weight ratio of 1:1-2 between the raspberry leaf grinding pulp and distilled water, the leaves are beaten with a wall breaking machine for 1-3 minutes to obtain beaten raspberry leaves. Cellulase is added to the beaten raspberry leaves, and the weight of the cellulase added is 1:1. The method comprises the following steps: adding 0.01-0.05% of the weight of raspberry leaves to the mixture, 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 mixture in an ultrasonic extractor, turning on the ultrasonic extractor for simultaneous enzymolysis and extraction for 0.2-0.5 hours, and setting conditions of the ultrasonic extractor as follows: input power of 200-350W and ultrasonic frequency of 20-30KHz. After the simultaneous enzymolysis and extraction are completed, the mixture 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.

[0022] Preferably, the weight ratio of the raspberry leaf grinding slurry 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 pulped raspberry leaves, the enzymolysis time is 0.8-1.2h, and the temperature is 42-46°C.

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

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

[0026] The bamboo shoot skin is the fresh outer skin of fresh tender bamboo shoots of the perennial plant bamboo of the genus Bambusa of the Poaceae family, and the outermost layer that is in direct contact with the air and has become fibrotic is removed to obtain the inner layer of fresh bamboo shoot skin; the inner layer of fresh bamboo shoot skin is washed with distilled water to remove impurities such as soil residues, and the surface water is drained to obtain the washed bamboo shoot skin; the washed bamboo shoot skin is quickly frozen and stored at -15 to -20°C to obtain frozen bamboo shoot skin; the washed bamboo shoot skin or the frozen bamboo shoot skin thawed at 25-45°C for 30 minutes is taken, cut into pieces and ground to obtain bamboo shoot skin grinding slurry; distilled water is added according to a weight ratio of 1:1-2 between the bamboo shoot skin grinding slurry and distilled water, and the mixture is beaten with a wall breaking machine for 1-3 minutes to obtain bamboo shoot skin pulp; cellulase is added to the bamboo shoot skin pulp The weight of cellulase added is 0.01-0.05% of the weight of bamboo shoot peel pulp. After mixing evenly, a layer of plastic wrap is covered on the mouth of the container, the temperature is kept constant at 35-52°C, enzymolysis is carried out for 0.5-1.5h, and then placed in an ultrasonic extractor for enzymolysis and simultaneous extraction; the conditions for enzymolysis and simultaneous extraction are that the ultrasonic extractor is set at an input power of 200-400w, an ultrasonic frequency of 15-20KHz, a constant temperature of 35-52°C, and the enzymolysis and simultaneous extraction time is 0.1-0.5h, and then the temperature is quickly raised to 90°C to inactivate the enzyme for 5 minutes, followed by filtering with 8 layers of gauze, and the filtrate is sterilized by pasteurization, and then cooled to 22-28°C to obtain a sterilized bamboo shoot peel extract.

[0027] Preferably, the weight ratio of the bamboo shoot skin grinding slurry 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 peel pulp, the enzymolysis time is 0.8-1.2h, and the temperature is 42-46°C.

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

[0030] S5: Preparation of fermentation medium

[0031] First, a sterilized nicotinic acid mother solution and a sterilized methionine mother solution are prepared. Nicotinic acid is weighed and mixed in a weight ratio of 1:20 to obtain a nicotinic acid mother solution; methionine is weighed and mixed in a weight ratio of 1:20 to obtain a methionine mother solution; both mother solutions are sterilized at 121° C. for 15 minutes to obtain the sterilized nicotinic acid mother solution and the sterilized methionine mother solution, respectively.

[0032] The components are measured and mixed according to the formula weight and composition ratio of the fermentation medium:

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

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

[0035] 0.2-2 parts of sterilized nicotinic acid stock solution;

[0036] 1-10 parts of sterilized methionine stock solution;

[0037] The balance is supplemented with sterile water to make up to 1000 parts by weight of the fermentation medium;

[0038] After uniform mixing, the pH is aseptically adjusted to 4.5-6.5 to obtain the fermentation medium.

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

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

[0041] Preferably, the weight portion of the sterilized nicotinic acid mother solution is 1.0-1.2 parts.

[0042] Preferably, the weight portion of the sterilized methionine mother solution is 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] The fermentation seed liquid obtained in step S2 is taken, and production inoculation is performed according to a weight ratio of the fermentation seed liquid to the fermentation medium of 1-10:100 to obtain a fermentation mixed liquid, and then raspberry ketone is fermented and cultured under the conditions of temperature 22-28°C, rotation speed 120-250rpm, liquid volume ratio 20%-55%, fermentation time 20-48h, to obtain raspberry ketone fermentation liquid.

[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 volume ratio is 35-40%, and the fermentation time is 24-46 hours.

[0048] Optionally, the fermentation of raspberry ketone adopts a batch fed fermentation method. When the fermentation is carried out for 18-24 hours after inoculation, the sterilized raspberry leaf extract and the sterilized bamboo shoot peel extract are respectively added to the fermenting fermentation mixture, 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.

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

[0050] S7: Raspberry ketone product acquisition.

[0051] After the fermentation step of raspberry ketone is completed, the raspberry ketone is separated, extracted and purified. This step is carried out according to a known method, such as separation and purification using preparative high performance liquid chromatography to obtain a raspberry ketone product.

[0052] Advantages of the invention

[0053] The present invention makes full use of agricultural waste such as raspberry leaves and bamboo shoot skins, which effectively utilizes resources, reduces waste, and reduces the pollution of the environment by discarded agricultural waste; at the same time, the cost of the fermentation medium is very low, and the produced raspberry ketone has a high economic value, achieving the effect of turning waste into treasure.

[0054] The invention only needs one fermentation step to complete the fermentation production of raspberry ketone. The fermented bacteria are natural strains 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 environmentally friendly, the equipment required for industrial production is conventional fermentation equipment, the operation is simple, and the feasibility is strong.

[0055] Biological deposit information

[0056] The terrestrial Issatchenkia yeast (Issatchenkia terricola) WJL-G4 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, and a proposed classification name of: terrestrial Issatchenkia yeast Issatchenkia terricola. The test result was that it was alive. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0058] Example 1

[0059] A method for producing raspberry ketone by microbial fermentation, the specific implementation scheme is as follows:

[0060] S1: Activation of fermentation bacteria.

[0061] The Issatchenkia terricola WJL-G4 strain stored in the ampoule was inoculated into YPD liquid medium and cultured in a 250 mL conical flask with a liquid volume of 30 mL at 28°C and 160 rpm for 24 h. The mixture was then graded and diluted to 10 -6 , draw 100 μL, spread it on YPD solid culture medium and culture it at 28°C for 24 h, pick a single colony and inoculate it into YPD slant culture medium, culture it at 28°C for 24 h to obtain activated bacteria.

[0062] S2: Preparation of fermentation seed solution.

[0063] Pick one ring of activated bacteria from S1 and inoculate it into new YPD liquid medium. After culturing for 24 h in a 250 mL conical flask at 28 °C, 160 rpm and a liquid volume of 30 mL, add sterile glass beads and shake for 20 min to adjust the bacterial concentration to 1.0 × 10 7 CFU / mL, and obtain the fermentation seed liquid.

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

[0065] The raspberry leaves are fresh leaves of the plant raspberry 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 quickly frozen and stored at -20°C to obtain frozen raspberry leaves. The frozen raspberry leaves thawed at 25°C for 30 minutes are cut and ground to obtain raspberry leaf grinding slurry. Distilled water is added according to a weight ratio of 1:2 between the raspberry leaf grinding slurry and distilled water, and the leaves are beaten with a wall breaking machine for 2 minutes to obtain beaten raspberry leaves. Cellulase is added at a weight of 0.02% of the weight of the pulped raspberry leaves. After stirring evenly, a layer of plastic wrap is covered on the mouth of the container. The temperature is kept constant at 40°C for enzymolysis for 1.5 hours. Then, it is placed in an ultrasonic extractor with a set input power of 300w and an ultrasonic frequency of 30KHz. After enzymolysis and simultaneous extraction for 0.5 hours, the temperature is quickly raised to 90°C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is sterilized by pasteurization at 80°C for 15 minutes, and then cooled to 28°C to obtain a sterilized raspberry leaf extract.

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

[0067] The bamboo shoot skin is fresh bamboo shoot skin obtained from new tender bamboo shoots of the perennial plant bamboo of the genus Bambusa of the Poaceae family, and the outermost layer that is in direct contact with the air and has become fibrotic is removed to obtain an inner layer of fresh bamboo shoot skin, the inner layer of fresh bamboo shoot skin is washed with distilled water to remove impurities such as soil residues, the surface water is drained and then quickly frozen and stored at -20°C to obtain frozen bamboo shoot skin; the frozen bamboo shoot skin that has been thawed at 25°C for 30 minutes is cut into pieces and ground to obtain bamboo shoot skin grinding slurry, distilled water is added according to a weight ratio of 1:2 between the bamboo shoot skin grinding slurry and distilled water, and the mixture is beaten with a wall breaking machine for 3 minutes to obtain bamboo shoot skin pulp, and the bamboo shoot skin pulp is added with distilled water. Add cellulase with a weight of 0.02% of the weight of the bamboo shoot peel pulp. After mixing evenly, cover the container with a layer of plastic wrap, keep the temperature constant at 50°C for enzymatic hydrolysis for 1.5 hours, and then place it in an ultrasonic extractor for enzymatic hydrolysis and simultaneous extraction. The conditions are: the ultrasonic extractor is set to have an input power of 200w and an ultrasonic frequency of 20KHz, and the temperature is constant at 50°C. After enzymatic hydrolysis and simultaneous extraction for 0.5 hours, the temperature is rapidly raised to 90°C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is sterilized by pasteurization at 75°C for 20 minutes, and then cooled to 28°C to obtain a sterilized bamboo shoot peel extract.

[0068] S5: Preparation of fermentation medium

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

[0070] The components are measured and mixed according to the following weight portions and proportions: 450 parts of sterilized raspberry leaf extract; 450 parts of sterilized bamboo shoot peel extract; 2 parts of sterilized niacin mother solution; 10 parts of sterilized methionine mother solution; supplement with sterile water to a total weight of 1000 parts, 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] The fermentation seed liquid obtained in step S2 is taken, and production inoculation is performed according to a weight ratio of the fermentation seed liquid to the fermentation medium of 10:100 to obtain a fermentation mixture, and then raspberry ketone is fermented and cultured under the conditions of temperature 28°C, rotation speed 250rpm, liquid volume ratio 50%, fermentation for 48h, and raspberry ketone fermentation liquid is obtained. After testing, the concentration of raspberry ketone in the fermentation liquid is 1054mg / L.

[0073] S7: Raspberry ketone product acquisition.

[0074] After the raspberry ketone fermentation step is completed, the raspberry ketone is separated, extracted and purified. This step is carried out according to a known method to obtain a raspberry ketone product.

[0075] Example 2

[0076] The microbial fermentation production method of raspberry ketone has the following specific operation steps.

[0077] S1: Activation of fermentation bacteria.

[0078] The Issatchenkia terricola WJL-G4 strain stored in the ampoule was inoculated into YPD liquid medium and cultured in a 250 mL conical flask with a liquid volume of 30 mL at 25°C and 160 rpm for 24 h. The mixture was then graded and diluted to 10 -6 , draw 100 μL, spread it on YPD solid culture medium and culture it at 25°C for 24 h, pick a single colony and inoculate it into YPD slant culture medium, culture it at 25°C for 36 h to obtain activated bacteria.

[0079] S2: Preparation of fermentation seed solution.

[0080] Pick one ring of activated bacteria obtained from S1 and inoculate it into new YPD liquid medium. After culturing for 24 h at 26 °C, 160 rpm, and a liquid volume of 35 mL in a 250 mL conical flask, add sterile glass beads and shake for 20 min to adjust the bacterial concentration to 5.0 × 10 7 CFU / mL, and obtain the fermentation seed liquid.

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

[0082] The raspberry leaves are fresh leaves of the plant raspberry of 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 taken, chopped and ground to obtain raspberry leaf grinding slurry. After adding distilled water according to a weight ratio of 1:1 between the raspberry leaf grinding slurry and distilled water, a wall breaking machine is used to beat for 2 minutes to obtain beaten raspberry leaves. Cellulase is added to the beaten raspberry leaves, and the added weight is 0.02% of the weight of the beaten raspberry leaves. After stirring evenly, Cover the container with a layer of plastic wrap, keep the temperature constant at 45°C, and after enzymatic hydrolysis for 1.0 hour, place it in an ultrasonic extractor for enzymatic hydrolysis and extraction. The conditions are: the ultrasonic extractor is set at an input power of 300w and an ultrasonic frequency of 25KHz, and the temperature is kept constant at 45°C. After enzymatic hydrolysis and extraction for 0.2 hour, the temperature is rapidly raised to 90°C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is pasteurized at 80°C for 15 minutes, and then cooled to 26°C to obtain a sterilized raspberry leaf extract.

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

[0084] The bamboo shoot skin is the fresh outer skin of new tender bamboo shoots of the perennial plant bamboo of the genus Bambusa of the Poaceae 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 fibrotic, the inner layer of fresh bamboo shoot skin is taken and washed with distilled water to remove impurities such as soil residues, and the surface water is drained to obtain the washed bamboo shoot skin; the washed bamboo shoot skin is taken, cut into pieces and ground to obtain bamboo shoot skin grinding slurry, distilled water is added according to a weight ratio of 1:1 between the bamboo shoot skin grinding slurry and distilled water, and the mixture is beaten with a wall breaking machine for 2 minutes to obtain bamboo shoot skin pulp, and cellulase is added to the bamboo shoot skin pulp with a weight of The invention discloses a method for preparing the bamboo shoot skin pulp of claim 19, wherein the mixture is mixed with 0.02% of the weight of the bamboo shoot skin pulp, and then covered with a layer of plastic wrap. The temperature is kept constant at 45°C. After enzymolysis for 1.5 hours, the mixture is placed in an ultrasonic extractor for simultaneous enzymolysis and extraction. The conditions are as follows: the ultrasonic extractor is set at an input power of 250w and an ultrasonic frequency of 20KHz. The temperature is kept constant at 45°C. After enzymolysis and extraction for 0.5 hours, the temperature is rapidly raised to 90°C to inactivate the enzyme for 5 minutes. The mixture is then filtered through 8 layers of gauze. The filtrate is pasteurized at 80°C for 15 minutes, and then cooled to 26°C to obtain a sterilized bamboo shoot skin extract.

[0085] S5: Preparation of fermentation medium

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

[0087] The components are measured and mixed according to the following weight parts and proportions: 450 parts of sterilized raspberry leaf extract; 400 parts of sterilized bamboo shoot peel extract; 2 parts of sterilized niacin mother solution; 9 parts of sterilized methionine mother solution; supplement with sterile water to a total weight of 1000 parts, mix evenly, and aseptically adjust the pH to 5.5 to obtain the fermentation medium.

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

[0089] The fermentation seed liquid obtained in step S2 is taken, and production inoculation is carried out according to a weight ratio of the fermentation seed liquid to the fermentation medium of 5:100 to obtain a fermentation mixture, and then raspberry ketone fermentation culture is carried out under the conditions of temperature 26°C, rotation speed 180rpm, and liquid volume ratio 35%. After fermentation for 24 hours, fed-batch fermentation is carried out, and 45 parts by weight of the sterilized raspberry leaf extract and 35 parts by weight of the sterilized bamboo shoot peel extract are added to the fermentation mixture. After adding, fermentation is continued for 24 hours to obtain raspberry ketone fermentation liquid. After testing, the concentration of raspberry ketone in the fermentation liquid is 1695 mg / L.

[0090] S7: Raspberry ketone product acquisition.

[0091] After the raspberry ketone fermentation step is completed, the raspberry ketone is extracted, separated and purified to obtain a raspberry ketone product.

[0092] Example 3

[0093] The microbial fermentation production method of raspberry ketone has the following specific implementation steps.

[0094] S1: Activation of fermentation bacteria.

[0095] The Issatchenkia terricola WJL-G4 strain stored in the ampoule was inoculated into YPD liquid medium and cultured in a 250 mL conical flask with a liquid volume of 30 mL at 28°C and 180 rpm for 24 h. The mixture was then graded and diluted to 10 -6 , draw 100 μL, spread it on YPD solid culture medium and culture it at 28°C for 24 h, pick a single colony and inoculate it into YPD slant culture medium, culture it at 28°C for 24 h to obtain activated bacteria.

[0096] S2: Preparation of fermentation seed solution.

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

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

[0099] The raspberry leaves are fresh leaves of the plant raspberry 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 raspberry leaf grinding slurry. After adding distilled water according to a weight ratio of the raspberry leaf grinding slurry to distilled water of 1:1.5, the leaves are beaten with a wall breaking machine for 1.5 minutes to obtain beaten raspberry leaves. Cellulase is added to the beaten raspberry leaves, and the added weight is 0.04% of the weight of the beaten raspberry leaves, and the mixture is stirred evenly. Then, a layer of plastic wrap was covered on the mouth of the container, the temperature was kept constant at 40°C, and after enzymatic hydrolysis for 1 hour, it was placed in an ultrasonic extractor for enzymatic hydrolysis and extraction. The conditions were that the ultrasonic extractor was set at an input power of 350w and an ultrasonic frequency of 25KHz, the temperature was kept constant at 40°C, and after enzymatic hydrolysis and extraction for 0.2h, the temperature was rapidly raised to 90°C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate was pasteurized at 75°C for 25 minutes, and then cooled to 26°C to obtain a sterilized raspberry leaf extract.

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

[0101] The bamboo shoot skin is the fresh outer skin of new tender bamboo shoots of the perennial plant bamboo of the genus Bambusa of the Poaceae 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 fibrotic, the inner layer of fresh bamboo shoot skin is taken, washed with distilled water to remove impurities such as soil residues, the surface water is drained, and then quickly frozen and stored at -18°C to obtain frozen bamboo shoot skin; the frozen bamboo shoot skin that has been thawed at 35°C for 30 minutes is taken, cut into pieces and ground to obtain bamboo shoot skin grinding slurry, distilled water is added according to a weight ratio of 1:1.5 between the bamboo shoot skin grinding slurry and distilled water, and the mixture is beaten with a wall breaking machine for 1.5 minutes to obtain bamboo shoot skin slurry, and the bamboo shoot skin slurry is added with distilled water. Add cellulase in an amount of 0.03% of the weight of the bamboo shoot peel pulp, mix well, cover the container with a layer of plastic wrap, keep the temperature constant at 45°C, and perform enzymatic hydrolysis and extraction for 1.5 hours. Then, place the container in an ultrasonic extractor for enzymatic hydrolysis and extraction under the following conditions: the ultrasonic extractor is set at an input power of 250W and an ultrasonic frequency of 20KHz, and the temperature is kept constant at 45°C. After enzymatic hydrolysis and extraction for 0.2 hours, the temperature is rapidly raised to 90°C to inactivate the enzyme for 5 minutes, and then filtered through 8 layers of gauze. The filtrate is pasteurized at 80°C for 15 minutes, and then cooled to 26°C to obtain a sterilized bamboo shoot peel extract.

[0102] S5: Preparation of fermentation medium

[0103] The preparation method of the sterilized nicotinic acid mother solution and the sterilized methionine mother solution is the same as that in Example 1.

[0104] The components are measured and mixed according to the following weight portions and proportions: 250 parts of sterilized raspberry leaf extract; 200 parts of sterilized bamboo shoot peel extract; 0.2 parts of sterilized niacin mother solution; 2 parts of sterilized methionine mother solution; add sterile water to a total weight of 1000 parts, mix well, and aseptically adjust the pH to 4.5 to obtain the fermentation medium.

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

[0106] The fermentation seed liquid obtained in step S2 is taken, and production inoculation is performed according to a weight ratio of the fermentation seed liquid to the fermentation medium of 2:100 to obtain a fermentation mixed liquid, and then raspberry ketone is fermented and cultured under the conditions of temperature 26°C, rotation speed 150rpm, and liquid volume ratio 45%. After fermentation for 24 hours, raspberry ketone fermentation liquid is obtained; after testing, the concentration of raspberry ketone in the fermentation liquid is 916 mg / L.

[0107] S7: Raspberry ketone product acquisition.

[0108] After the raspberry ketone fermentation step is completed, the raspberry ketone is extracted, separated and purified to obtain a raspberry ketone product.

[0109] Example 4

[0110] A method for producing raspberry ketone by microbial fermentation, the specific implementation scheme is as follows.

[0111] S1: Activation of fermentation bacteria.

[0112] The Issatchenkia terricola WJL-G4 strain stored in the ampoule was inoculated into YPD liquid medium and cultured in a 250 mL conical flask with a liquid volume of 30 mL at 28°C and 160 rpm for 24 h. The mixture was then graded and diluted to 10 -6 , draw 100 μL, spread it on YPD solid culture medium and culture it at 28°C for 24 h, pick a single colony and inoculate it into YPD slant culture medium, culture it at 28°C for 24 h to obtain activated bacteria.

[0113] S2: Preparation of fermentation seed solution.

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

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

[0116] The raspberry leaves are fresh leaves of the plant raspberry 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 quickly frozen and stored at -20°C to obtain frozen raspberry leaves. The frozen raspberry leaves thawed at 25°C for 30 minutes are cut and ground to obtain raspberry leaf grinding slurry. After adding distilled water according to a weight ratio of 1:2 between the raspberry leaf grinding slurry and distilled water, the leaves are beaten with a wall breaking machine for 1.8 minutes to obtain beaten raspberry leaves. Cellulase is added to the beaten raspberry leaves with a weight of 1:2. The invention discloses a method of preparing a raspberry leaf extractor comprising: adding 0.03% of the weight of raspberry leaves to the mixture, stirring the mixture evenly, and then covering the container with a layer of plastic wrap, keeping the temperature constant at 50°C, performing enzymolysis for 1.4 hours, and then placing the extract in an ultrasonic extractor for simultaneous enzymolysis and extraction. The conditions are as follows: the ultrasonic extractor is set at an input power of 350W and an ultrasonic frequency of 20KHz, and the temperature is kept constant at 50°C. After the enzymolysis and extraction are performed for 0.2 hours, the temperature is rapidly raised to 90°C for inactivating the enzyme for 5 minutes, and then the extract is filtered through 8 layers of gauze. The filtrate is pasteurized at 80°C for 15 minutes, and then cooled to 26°C to obtain a sterilized raspberry leaf extract.

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

[0118] The bamboo shoot skin is the fresh outer skin of new tender bamboo shoots of the perennial plant bamboo of the genus Bambusa of the Poaceae family. The bamboo shoot skin is peeled from the fresh tender bamboo shoots, 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 fibrotic. The inner layer of fresh bamboo shoot skin is washed with distilled water to remove impurities such as soil residues, and the surface water is drained to obtain washed bamboo shoot skin. The washed bamboo shoot skin is cut into pieces and ground to obtain bamboo shoot skin grinding slurry. Distilled water is added according to a weight ratio of 1:2 between the bamboo shoot skin grinding slurry and distilled water, and the mixture is beaten with a wall breaking machine for 1.5 minutes to obtain bamboo shoot skin slurry. Cellulase is added to the bamboo shoot skin slurry. The added weight is 0.05% of the weight of the bamboo shoot skin pulp. After mixing evenly, cover the container with a layer of plastic wrap, keep the temperature constant at 45°C, and carry out enzymatic hydrolysis and extraction for 1.5 hours. Then, place it in an ultrasonic extractor for enzymatic hydrolysis and extraction. The conditions are that the ultrasonic extractor is set at an input power of 280w and an ultrasonic frequency of 19KHz, and the temperature is kept constant at 45°C. After enzymatic hydrolysis and extraction for 0.3 hours, the temperature is rapidly raised to 90°C to inactivate the enzyme for 5 minutes, and then filtered with 8 layers of gauze. The filtrate is pasteurized at 80°C for 15 minutes, and then cooled to 26°C to obtain a sterilized bamboo shoot skin extract.

[0119] S5: Preparation of fermentation medium

[0120] The preparation method of the sterilized nicotinic acid mother solution and the sterilized methionine mother solution is the same as that in Example 1.

[0121] The components are measured and mixed according to the following weight parts and proportions: 250 parts of sterilized raspberry leaf extract; 200 parts of sterilized bamboo shoot peel extract; 0.2 parts of sterilized niacin mother solution; 1 part of sterilized methionine mother solution; add sterile water to a total weight of 1000 parts, mix well, and aseptically adjust the pH to 6.5 to obtain the fermentation medium.

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

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

[0124] S7: Raspberry ketone product acquisition.

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

[0126] Example 5

[0127] A method for producing raspberry ketone by microbial fermentation, the specific implementation scheme 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] The fermentation seed liquid obtained in step S2 is taken, and production inoculation is performed according to a weight ratio of the fermentation seed liquid to the fermentation medium of 2:100 to obtain a fermentation mixture, and then raspberry ketone fermentation culture is performed under the conditions of temperature 26°C, rotation speed 150rpm, and liquid volume ratio 45%. After fermentation for 24 hours, fed-batch fermentation is performed, and 30 parts by weight of the sterilized raspberry leaf extract and 20 parts by weight of the sterilized bamboo shoot peel extract are added to the fermentation mixture. After adding, fermentation is continued for 24 hours to obtain raspberry ketone fermentation liquid. After testing, the concentration of raspberry ketone in the fermentation liquid is 1429 mg / L.

[0131] S7: Raspberry ketone product acquisition.

[0132] After the raspberry ketone fermentation step is completed, the raspberry ketone is extracted, separated and purified to obtain a raspberry ketone product.

[0133] Example 6

[0134] A method for producing raspberry ketone by microbial fermentation, the specific implementation scheme is as follows.

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

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

[0137] The fermentation seed liquid obtained in step S2 is taken, and production inoculation is performed according to a weight ratio of the fermentation seed liquid to the fermentation medium of 1:100 to obtain a fermentation mixture, and then raspberry ketone fermentation culture is performed under the conditions of temperature 26°C, rotation speed 220rpm, and liquid volume ratio 50%. After fermentation for 20 hours, fed-batch fermentation is performed, and 30 parts by weight of the sterilized raspberry leaf extract and 20 parts by weight of the sterilized bamboo shoot peel extract are added to the fermentation mixture. After adding, fermentation is continued for 24 hours to obtain raspberry ketone fermentation liquid. After testing, the concentration of raspberry ketone in the fermentation liquid is 1291 mg / L.

[0138] S7: Raspberry ketone product acquisition.

[0139] After the raspberry ketone fermentation step is completed, the raspberry ketone is extracted, separated and purified to obtain a raspberry ketone product.

[0140] Example 7

[0141] The determination method of raspberry ketone is carried out as follows.

[0142] Agilent 1260 Infinity II HPLC was used for analysis. Chromatographic column: Agilent ZORBAX Extend-C 18 (250mm×4.6mm); mobile phase: A is methanol, B is 0.02% formic acid solution; gradient elution program: 0-5min, 0-10% A, 100%-90% B; 5-10min, 10%-20% A, 90%-80% B; 10-20min, 20%-35% A, 80%-65% B; 20-35min, 35%-40% A, 65%-60% B; 35-40min, 40%-75% A, 60%-25% B; 40-45min, 75%-10% A, 25%-90% B; flow rate: 0.8mL / min, injection volume: 10μL, column temperature: 35℃, detection wavelength: 280nm. The standard sample series and the sample to be tested were measured in turn. A standard curve was drawn using the standard sample concentration versus the peak area, and the content of raspberry ketone was calculated based on the standard curve and the peak area of ​​the sample to be tested.

[0143] Standard curve: Raspberry ketone standard was dissolved in methanol to prepare standard solutions of different mass concentrations, filtered through a 0.22 μm microporous filter membrane, and then analyzed by HPLC 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 tested: Take a certain volume of the sample to be tested, dilute it appropriately, centrifuge it at 8000 rpm for 5 minutes, aspirate the supernatant, filter it through a 0.22 μm filter membrane, and then determine it according to the raspberry ketone determination method, and calculate the result according to the standard curve.

[0145] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. The patent protection scope of the present invention shall be based on the claims. All equivalent structural changes made using the contents of the specification of the present invention shall 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 and claim 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 and claim 7, characterized in that: Optionally, the raspberry ketone fermentation adopts a fed-batch fermentation method.

8. The method for producing raspberry ketone by microbial fermentation according to claim 8, characterized in that: The method of batch fed fermentation is as follows: when the fermentation of raspberry ketone after production inoculation reaches 18-24 hours, the sterilized raspberry leaf extract and the sterilized bamboo shoot peel extract are respectively added to the fermentation mixed liquid 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.