Debaryomyces and application thereof in preparation of paeoniflorin
By using the GT16 fermentation technology of Debali yeast GT16, the conversion of precursor substances in peony bark into peony glycoside was solved, and the problem of low extraction rate of peony glycoside in the existing technology was achieved, and the effective extraction effect of peony glycoside was achieved.
Patent Information
- Application Number
- CN202411482408.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to effectively improve the extraction rate of peony glycoside in peony peel, which limits the efficiency of peony glycoside extraction and production.
Debaryomyces prosopidis GT16 fermentation technology is used to ferment the precursor substances in the peony bark to promote their conversion to peony glycoside, thereby improving the content and extraction rate of peony glycoside.
Through the fermentation technology of Debali yeast, the content of peony glycoside has been significantly improved, making it reaching more than 10%, and the extraction rate reaches more than 0.4%, solving the problem of low extraction rate in the prior art.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a Debaryomyces yeast and application thereof in the preparation of paeoniflorin. Background Art
[0002] Peony is a woody plant of the genus Paeonia in the family Paeoniaceae, mainly produced in Anhui, Chongqing, Gansu, Shandong, Henan and other provinces in my country. In addition to being an ornamental plant, peony also has high medicinal value. The dried root bark of peony has been used as medicine in my country for more than 2,000 years. The medicinal dried root bark is called moutan bark. Moutan bark tastes bitter and pungent, is slightly cold in nature, and enters the heart, liver, and kidney meridians. It is mostly used to treat symptoms such as fever and rashes, no sweating and bone steaming, amenorrhea and dysmenorrhea, and bloating and sores.
[0003] A large number of modern studies have shown that peony bark contains a variety of biologically active secondary metabolites, including monoterpenes, monoterpenoid glycosides, polyphenols and their glycosides, paeonols, etc. Among them, paeoniflorin is a monoterpenoid glycoside compound in peony bark extract, and its structure is a β-D-pyranose glucopyranosyl group and a benzoyl group connected to the monoterpenoid mother nucleus. Paeoniflorin has pharmacological effects such as anti-tumor, anti-inflammatory, anti-allergic, immunomodulatory, treatment of cardiovascular diseases, treatment of osteoporosis, improvement of depression, protection of cerebral ischemia, protection of cerebral hypoxia, analgesia, liver and kidney protection. It has been made into a prescription drug for clinical treatment as an effective ingredient, and is also often used as an important raw material for the production of functional cosmetics. At present, various extraction technologies are mainly used to directly extract paeoniflorin from peony bark. With the continuous development of extraction technology, the inherent paeoniflorin content of peony bark has gradually become a limiting factor for the extraction rate. The use of microbial fermentation technology to promote the conversion of precursor substances in peony bark into paeoniflorin, thereby improving the extraction rate of paeoniflorin, is of great significance to the extraction and production of paeoniflorin from peony bark. Therefore, it is urgent to obtain microorganisms that can effectively convert precursor substances in Paeonia suffruticosa into paeoniflorin. Summary of the invention
[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Debaryomyces cerevisiae.
[0005] Another object of the present invention is to provide the use of the above-mentioned Debaryomyces in the preparation of paeoniflorin.
[0006] Another object of the present invention is to provide a method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa Andrews and its application.
[0007] The object of the present invention is achieved by the following technical scheme: a strain of Debaryomyces cerevisiae, named Debaryomyces cerevisiae ( Debaryomyces prosopidis) GT16, with the deposit number GDMCC No: 65196, was deposited on September 24, 2024 at the Guangdong Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.
[0008] The fermentation liquid of the Debaryomyces mellifera has high UDP-glycosyltransferase activity, which can promote the conversion of paeoniflorin and other precursor substances in the peony bark into paeoniflorin during the fermentation of the peony bark, thereby increasing the content of paeoniflorin. Therefore, the Debaryomyces mellifera is particularly suitable for use in the preparation of paeoniflorin.
[0009] A method for preparing a paeoniflorin-rich extract by fermenting peony bark, comprising the following steps: (1) Microbial fermentation of peony bark: inoculating the above-mentioned Debaryomyces or Debaryomyces culture solution into a sterilized fermentation medium containing peony bark, and fermenting to obtain a fermentation material; (2) Reflux extraction and slag removal: subjecting the fermented material obtained in step (1) to reflux extraction and centrifugal separation to obtain an extraction supernatant; (3) impurity removal and drying: the extraction supernatant obtained in step (2) is concentrated, anhydrous ethanol is added to the obtained concentrated solution, the mixture is stirred evenly, allowed to stand, and the solid and liquid are separated to obtain an impurity-removed supernatant; the ethanol is then distilled to recover the ethanol, and the impurity-removed and ethanol-removed concentrated solution is spray-dried to obtain a powdered extract.
[0010] In step (1): The fermentation medium consists of the following ingredients: peony bark powder, glucose, sodium citrate, peptone, yeast extract, potassium dihydrogen phosphate, magnesium sulfate and water.
[0011] The peony bark powder is obtained by crushing and sieving the dried peony bark; preferably the peony bark powder has a size of less than 100 meshes.
[0012] The water is preferably purified water.
[0013] The dosage of the peony bark powder is preferably calculated based on a ratio of 100 to 110 g / L with water.
[0014] The amount of glucose used is preferably calculated based on a ratio of glucose to water of 160-180 g / L.
[0015] The dosage of the sodium citrate is preferably calculated based on a ratio of 3.0 to 3.4 g / L with water.
[0016] The amount of peptone used is preferably calculated based on a ratio of 6 to 10 g / L of peptone to water.
[0017] The amount of yeast extract used is preferably calculated based on a ratio of 6 to 10 g / L of yeast extract to water.
[0018] The dosage of the potassium dihydrogen phosphate is preferably calculated based on a ratio of 2.4 to 2.8 g / L with water.
[0019] The dosage of the magnesium sulfate is preferably 0.22 to 0.26 g / L according to the ratio of magnesium sulfate to water.
[0020] The pH of the fermentation medium is preferably 6.3-6.5.
[0021] The sterilization condition is preferably sterilized at 121° C. for 15 to 20 min.
[0022] The Debaryomyces cerevisiae culture solution is prepared by the following steps: inoculating the preserved strain of Debaryomyces cerevisiae into a liquid culture medium, and performing shaking culture to obtain the Debaryomyces cerevisiae culture solution.
[0023] The liquid culture medium is composed of 80-100 g / L glucose, 8-10 g / L peptone, 9-11 g / L yeast extract, 2.4-2.8 g / L potassium dihydrogen phosphate, and 0.22-0.26 g / L magnesium sulfate, with pH adjusted to 6.3-6.5, and the solvent is water.
[0024] The inoculation amount of the Debaryomyces yeast preserved strain is preferably 2 loops of bacterial moss per 100 mL to 200 mL of liquid culture medium.
[0025] The shaking culture conditions are preferably 27-29° C. and 180-220 r / min for 18-22 h.
[0026] The inoculation amount of the Debaryomyces cerevisiae culture solution is preferably calculated as 0.4% to 1.2% of the volume of the water used in the fermentation medium.
[0027] The fermentation conditions are preferably as follows: temperature 27-29° C., stirring speed 220-260 r / min, ventilation ratio controlled at 0.24-0.36 vvm for 0-16 h of fermentation, ventilation ratio controlled at 0.36-0.48 vvm for 17-32 h of fermentation, ventilation ratio controlled at 0.32-0.36 vvm for 33-60 h of fermentation, ventilation ratio controlled at 0.28-0.32 vvm for 61 h and thereafter, and fermentation period 72-84 h.
[0028] In step (2): The temperature of the reflux extraction is preferably 90-100°C.
[0029] The reflux extraction time is preferably 40 to 60 min.
[0030] The centrifugal separation conditions are preferably: centrifugal speed 6000-7000 r / min, centrifugal time 18-22 min.
[0031] In step (3): The concentration is preferably carried out using an ultrafiltration membrane; more preferably, it is carried out using a 360 Da ultrafiltration membrane.
[0032] The degree of concentration is preferably such that the volume of the concentrated liquid is 10% to 20% of the volume of the extracted supernatant.
[0033] The amount of anhydrous ethanol added is preferably 8.5 to 9.5 times the volume of the concentrated solution.
[0034] The standing time is 60 to 120 minutes, more preferably 60 to 90 minutes.
[0035] The solid-liquid separation method is preferably centrifugation.
[0036] The centrifugal conditions are preferably: centrifugal speed 7500-8500 r / min, separation time 18-22 min.
[0037] The distillation temperature is preferably 90-100°C.
[0038] The volume of the concentrated liquid is 8% to 10% of the volume of the impurity-removed supernatant.
[0039] The spray drying conditions are preferably: inlet air temperature 160-170°C, outlet air temperature 40-50°C.
[0040] The method for preparing a paeoniflorin-rich extract by fermenting peony bark is used in preparing paeoniflorin.
[0041] A powdery extract of paeoniflorin from peony bark is obtained by the method, wherein the paeoniflorin content is more than 10%, and the extraction rate of paeoniflorin from peony bark is more than 0.4%.
[0042] Compared with the prior art, the present invention has the following advantages and effects: (1) The Debaryomyces GT16 screened by the present invention can be used to increase the content of paeoniflorin in Paeonia suffruticosa root by fermentation.
[0043] (2) There are no research reports or production precedents on using microbial fermentation to increase the content of paeoniflorin in peony bark. The present invention uses Debaryomyces suffruticosa to ferment peony bark, which promotes the conversion of other flavonoid compounds in peony bark into paeoniflorin, thereby significantly increasing the content of paeoniflorin in the extract; compared with the unfermented control, the content of paeoniflorin is more than 1.2 times that of the control. The obtained extract can be used as a raw material in the fields of medicine, cosmetics, etc., which is conducive to promoting the high-value-added development and utilization of peony bark. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 These are scanning electron microscope photos of strain GT16; the magnification of A is 5000, and the magnification of B is 10000. DETAILED DESCRIPTION
[0045] The present invention is described in further detail below, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention can be obtained commercially.
[0046] Example 1 (1) Enrichment culture Enrichment medium: 100 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 2.5 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate. Dissolve various materials in water, adjust pH to 7.0, and prepare the medium. Prepare 100 mL of enrichment medium, put it into a 500 mL conical flask, sterilize it at 121°C for 20 minutes, cool it, inoculate 10 g of soybean paste (Foshan Guanggu Condiment Co., Ltd.), and place it at 30°C and 200 r / min for 16 hours to obtain enrichment culture fluid.
[0047] (2) Plate separation Plate screening culture medium: 20 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 2.0 g / L potassium dihydrogen phosphate, 20 g / L agar. Dissolve various materials in water, adjust pH to 7.0, and prepare the culture medium.
[0048] The plate culture medium was sterilized at 121℃ for 20 min, poured into a culture dish, and made into a plate. The enriched culture solution was diluted with sterile water, spread on the plate culture medium, and cultured at 30℃ for 48 h. 48 colonies with larger diameters were selected and inoculated into the slant culture medium (the composition of the slant culture medium is the same as that of the plate culture medium), cultured at 30℃ for 36 h, and stored in a 4℃ refrigerator for later use.
[0049] (3) Shake flask fermentation screening Shake flask medium: 100 g / L peony bark powder, 150 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 30 g / L sodium citrate, 2.5 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate, adjust pH7.0. The medium was divided into 500 mL conical flasks, each containing 100 mL of liquid, and sterilized at 121°C for 20 min. After cooling, each bottle was inoculated with the strain picked from the plate, and each bottle was inoculated with 2 rings of bacterial lawn, and cultured at 28°C and 200 r / min for 48 h. Then, each bottle of culture solution was centrifuged at 4°C and 8000 r / min for 20 min, and the supernatant was collected as a crude enzyme solution for UDP-glycosyltransferase activity detection. According to the enzyme activity test results, the optimal UDP-glycosyltransferase producing bacteria were screened.
[0050] Before the enzyme activity test, 2.5, 12.5, 25, 50, and 200 μg / mL steviol standard solutions were prepared and detected by high performance liquid chromatography (reference: Hu Lin, Kou Yanjie, Weng Yongjing, et al. Determination of steviol content in steviol glycosides by high performance liquid chromatography [J]. Natural Product Research and Development, 2014, 26: 1644-1646.), a steviol standard curve was drawn, and the regression equation of the standard curve was established.
[0051] Improve the method described in the literature (Chen Meiqi. Construction of glycosyltransferase cascade reaction system and its synthesis in steviol glycosides [D]. Guangzhou: South China University of Technology, 2023.), and determine the UDP-glycosyltransferase activity in the supernatant. Take two test tubes, according to the reaction system shown in Table 1, add 100 mmol phosphate buffer (pH7.0), 2.5 mmol steviol solution, 16 mmol diphosphate glucose solution, 80 mmol magnesium chloride solution, crude enzyme solution, water to the two test tubes respectively, shake well, and place in a 45℃ water bath for 20 min. Then, place in a 100℃ water bath to inactivate the enzyme for 10 min, transfer the solutions in the two test tubes to volumetric flasks respectively, cool, add methanol, dilute to 10 mL, shake well, filter through a 0.22 μm membrane, and analyze the steviol content in the filtrate by liquid chromatography. Calculate the amount of steviol converted after the enzyme reaction, conversion amount = steviol mass in the control tube - steviol mass in the reaction tube. Activity of UDP-glycosyltransferase: Under the above reaction conditions, the amount of enzyme required to convert 1 nmol of steviol in 1 min is 1 activity unit. The results of enzyme activity determination are shown in Table 2. Among them, the UDP-glycosyltransferase activity in the fermentation broth of strain GT16 is the highest, so strain GT16 is preferred as the test strain of the present invention. Under the microscope, strain GT16 is oval in shape, and the bacterial morphology under the scanning electron microscope is as follows Figure 1 shown.
[0052] (4) Identification of bacterial species The strain GT16 was sent to a gene sequencing company for ITS region DNA detection. The detected sequence was retrieved and compared with the NCBI data platform, and the strain was identified as Debaryomyces ( Debaryomyces prosopidis The strain was named Debaryomyces ( Debaryomyces prosopidis ) GT16 was deposited on September 24, 2024 in the Guangdong Provincial Microbiological Culture Collection Center, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC NO:65196.
[0053] Debaryomyces Debaryomyces prosopidis ) The ITS region sequence of GT16 is as follows: .
[0054] Example 2 (1) Cultivation of Debaryomyces yeast Prepare 180 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, adjust pH to 6.4, solvent is water), put it into a 1000 mL conical flask, sterilize it at 121°C for 20 min, after cooling, inoculate 2 rings of Debaryomyces GT16 slant strain, and culture it at 28°C and 200 r / min for 20 h.
[0055] (2) Peony bark fermentation Crushed peony bark, passed through a 100-mesh sieve, weighed 2.31 kg of peony bark powder, mixed with 22 L of pure water, put into a 30L fermentation tank, added 3.74 kg of glucose, 70.4 g of sodium citrate, 176 g of peptone, 176 g of yeast extract, 57.2 g of potassium dihydrogen phosphate, 5.28 g of magnesium sulfate, stirred evenly, adjusted pH 6.4, and sterilized at 121°C for 18 min. After cooling, the above 180 mL of Debaryomyces cerevisiae culture solution was added, stirring was started, the stirring speed was controlled at 240 r / min, and the fermentation temperature was controlled at 28°C. During the fermentation process, the ventilation ratio was controlled at 0.24-0.36 vvm for 0-16 h of fermentation, 0.36-0.48 vvm for 17-32 h of fermentation, 0.32-0.36 vvm for 33-60 h of fermentation, 0.28-0.32 vvm for 61 h and thereafter, and fermentation for 78 h.
[0056] (3) Reflux extraction and slag removal The fermented material was placed in a reflux extraction device and refluxed at 95°C for 50 min. Then, the extracted material was centrifuged at 6500 r / min for 20 min, and the supernatant was collected to obtain 17.9 L of supernatant. The paeoniflorin in the supernatant was detected by high performance liquid chromatography (reference: Deng Kaiying, Zhu Biyue, Zhu Zhaojing. Improved RP-HPLC method for simultaneous determination of paeoniflorin and paeonol in Paeonia suffruticosa root [J]. Chinese Pharmacy, 2013, 15: 1406-1408.), and the paeoniflorin concentration was 661.2 mg / L.
[0057] (4) Impurity removal and drying 17.9 L of the extracted supernatant was concentrated with a 360 Da ultrafiltration membrane to obtain 2.69 L of concentrated solution, 24.2 L of anhydrous ethanol was added, mixed evenly, allowed to stand for 90 min, centrifuged at 8000 r / min for 20 min, and the precipitate was removed to obtain 26.6 L of supernatant. The impurity-free supernatant was distilled at 95 ° C, ethanol was recovered, and 2.39 L of concentrated solution was obtained. Spray drying was performed, and the inlet air temperature was controlled at 160-170 ° C and the outlet air temperature was controlled at 40-50 ° C. 93.2 g of powder was collected. 0.25 g of powder was weighed and prepared into 50 mL of solution. The concentration of paeoniflorin in the solution was detected by high performance liquid chromatography. After conversion, the paeoniflorin content of the powder was 10.22%. Therefore, the extraction rate of paeoniflorin in the powder to the raw material of Paeonia suffruticosa moutan was 0.41%.
[0058] Example 3 (1) Cultivation of Debaryomyces yeast Prepare 88 mL of liquid culture medium (culture medium formula: 80 g / L glucose, 8 g / L peptone, 9 g / L yeast extract, 2.4 g / L potassium dihydrogen phosphate, 0.22 g / L magnesium sulfate, adjust pH to 6.3, solvent is water), put it into a 500 mL conical flask, sterilize it at 121°C for 20 min, and after cooling, inoculate 2 rings of Debaryomyces GT16 slant strains, and culture it at 27°C and 180 r / min for 22 h.
[0059] (2) Peony bark fermentation Crushed peony bark, passed through a 100-mesh sieve, weighed 2.2 kg of peony bark powder, mixed with 22 L of pure water, put into a 30L fermentation tank, added 3.52 kg of glucose, 66 g of sodium citrate, 132 g of peptone, 132 g of yeast extract, 52.8 g of potassium dihydrogen phosphate, 4.84 g of magnesium sulfate, stirred evenly, adjusted pH 6.3, and sterilized at 121°C for 16 min. After cooling, the above 88 mL of Debaryomyces cerevisiae culture solution was added, stirring was started, the stirring speed was controlled at 220 r / min, and the fermentation temperature was controlled at 27°C. During the fermentation process, the ventilation ratio was controlled at 0.24-0.36 vvm for 0-16 h of fermentation, 0.36-0.48 vvm for 17-32 h of fermentation, 0.32-0.36 vvm for 33-60 h of fermentation, 0.28-0.32 vvm for 61 h and thereafter, and fermentation lasted for 84 h.
[0060] (3) Reflux extraction and slag removal The fermented material was placed in a reflux extraction device and refluxed at 90°C for 60 min. Then, the extracted material was centrifuged at 6000 r / min for 22 min, and the supernatant was collected to obtain 18.5 L of supernatant. The paeoniflorin in the supernatant was detected by high performance liquid chromatography (reference: Deng Kaiying, Zhu Biyue, Zhu Zhaojing. Improved RP-HPLC method for simultaneous determination of paeoniflorin and paeonol in Paeonia suffruticosa root [J]. Chinese Pharmacy, 2013, 15: 1406-1408.), and the paeoniflorin concentration was 620.7 mg / L.
[0061] (4) Impurity removal and drying 18.5 L of the extracted supernatant was concentrated using a 360 Da ultrafiltration membrane to obtain 1.85 L of concentrated solution, 15.7 L of anhydrous ethanol was added, mixed evenly, allowed to stand for 60 min, centrifuged at 7500 r / min for 22 min, and the precipitate was removed to obtain 17.3 L of supernatant. The impurity-free supernatant was distilled at 90 ° C, ethanol was recovered, and 1.73 L of concentrated solution was obtained. Spray drying was performed, and the inlet air temperature was controlled at 160-170 ° C and the outlet air temperature was controlled at 40-50 ° C. 88.5 g of powder was collected. 0.25 g of powder was weighed and prepared into 50 mL of solution. The concentration of paeoniflorin in the solution was detected by high performance liquid chromatography. After conversion, the paeoniflorin content of the powder was 10.37%. Therefore, the extraction rate of paeoniflorin in the powder to the raw material of Paeonia suffruticosa moutan was 0.42%.
[0062] Example 4 (1) Cultivation of Debaryomyces yeast Prepare 264 mL of liquid culture medium (culture medium formula: glucose 100 g / L, peptone 10 g / L, yeast extract 11 g / L, potassium dihydrogen phosphate 2.8 g / L, magnesium sulfate 0.26 g / L, adjust pH 6.4, solvent is water), divide it into two 500 mL conical flasks, sterilize it at 121°C for 20 min, and after cooling, inoculate two rings of Debaryomyces GT16 slant into each flask, and culture it at 29°C and 200 r / min for 18 h.
[0063] (2) Peony bark fermentation Crushed peony bark, passed through a 100-mesh sieve, weighed 2.42 kg of peony bark powder, mixed with 22 L of pure water, put into a 30L fermentation tank, added 3.96 kg of glucose, 74.8 g of sodium citrate, 220 g of peptone, 220 g of yeast extract, 61.6 g of potassium dihydrogen phosphate, 5.72 g of magnesium sulfate, stirred evenly, adjusted pH 6.5, and sterilized at 121°C for 22 min. After cooling, the above 264 mL of Debaryomyces cerevisiae culture solution was added, stirring was started, the stirring speed was controlled at 260 r / min, and the fermentation temperature was controlled at 29°C. During the fermentation process, the ventilation ratio was controlled at 0.24-0.36 vvm for 0-16 h of fermentation, 0.36-0.48 vvm for 17-32 h of fermentation, 0.32-0.36 vvm for 33-60 h of fermentation, 0.28-0.32 vvm for 61 h and thereafter, and 0.32 vvm for 72 h of fermentation.
[0064] (3) Reflux extraction and slag removal The fermented material was placed in a reflux extraction device and refluxed at 100°C for 40 min. Then, the extracted material was centrifuged at 7000 r / min for 18 min, and the supernatant was collected to obtain 16.9 L of supernatant. The paeoniflorin in the supernatant was detected by high performance liquid chromatography (reference: Deng Kaiying, Zhu Biyue, Zhu Zhaojing. Improved RP-HPLC method for simultaneous determination of paeoniflorin and paeonol in Paeonia suffruticosa root [J]. Chinese Pharmacy, 2013, 15: 1406-1408.), and the paeoniflorin concentration was 720.5 mg / L.
[0065] (4) Impurity removal and drying 16.9 L of the extracted supernatant was concentrated using a 360 Da ultrafiltration membrane to obtain 3.38 L of concentrated solution, 32.1 L of anhydrous ethanol was added, mixed evenly, allowed to stand for 90 min, centrifuged at 8500 r / min for 18 min, and the precipitate was removed to obtain 35.1 L of supernatant. The impurity-free supernatant was distilled at 100 ° C, ethanol was recovered, and 2.81 L of concentrated solution was obtained. It was spray-dried, and the inlet air temperature was controlled at 160-170 ° C and the outlet air temperature was controlled at 40-50 ° C. 96.9 g of powder was collected. 0.25 g of powder was weighed and prepared into 50 mL of solution. The concentration of paeoniflorin in the solution was detected by high performance liquid chromatography. After conversion, the paeoniflorin content of the powder was 10.02%. Therefore, the extraction rate of paeoniflorin in the powder to the raw material of Paeonia suffruticosa moutan was 0.40%.
[0066] Comparative Example (1) Reflux extraction and slag removal Crushed peony bark, passed through a 100-mesh sieve, weighed 2.31 kg of peony bark powder, mixed with 22 L of pure water, put into a 30L fermentation tank, added 3.74 kg of glucose, 70.4 g of sodium citrate, 176 g of peptone, 176 g of yeast extract, 57.2 g of potassium dihydrogen phosphate, 5.28 g of magnesium sulfate, stirred evenly, adjusted pH 6.4, and sterilized at 121°C for 18 min. After cooling, add the above 180 mL of pure water, start stirring, control the stirring speed to 240 r / min, and control the temperature to 28°C. During the stirring process, the ventilation ratio was controlled at 0.24-0.36 vvm for 0-16 h, 0.36-0.48 vvm for 17-32 h, 0.32-0.36 vvm for 33-60 h, and 0.28-0.32 vvm for 61 h and thereafter, with stirring for 78 h.
[0067] Then, the material was placed in a reflux extraction device and refluxed at 95°C for 50 min. Then, the extracted material was centrifuged at 6500 r / min for 20 min, and the supernatant was collected to obtain 18 L of supernatant. The paeoniflorin in the supernatant was detected by high performance liquid chromatography (reference: Deng Kaiying, Zhu Biyue, Zhu Zhaojing. Improved RP-HPLC method for simultaneous determination of paeoniflorin and paeonol in Paeonia suffruticosa root [J]. Chinese Pharmacy, 2013, 15: 1406-1408.), and the paeoniflorin concentration was 516.6 mg / L.
[0068] (2) Impurity removal and drying 18 L of the extracted supernatant was concentrated using a 360 Da ultrafiltration membrane to obtain 2.7 L of concentrated solution, 24.3 L of anhydrous ethanol was added, mixed evenly, allowed to stand for 90 min, centrifuged at 8000 r / min for 20 min, and the precipitate was removed to obtain 26.7 L of supernatant. The impurity-free supernatant was distilled at 95 ° C, ethanol was recovered, and 2.4 L of concentrated solution was obtained. Spray drying was performed, and the inlet air temperature was controlled at 160-170 ° C and the outlet air temperature was controlled at 40-50 ° C. 73.1 g of powder was collected. 0.25 g of powder was weighed and prepared into 50 mL of solution. The concentration of paeoniflorin in the solution was detected by high performance liquid chromatography. After conversion, the paeoniflorin content of the powder was 10.12%. Therefore, the extraction rate of paeoniflorin in the powder to the raw material of Paeonia suffruticosa moutan was 0.32%.
[0069] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A strain of Debaryomyces yeast, characterized in that: The name of the Debaryomyces cerevisiae is Debaryomyces cerevisiae ( Debaryomyces prosopidis ) GT16, with the deposit number GDMCC No: 65196, was deposited on September 24, 2024 at the Guangdong Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.
2. Use of the Debaryomyces yeast according to claim 1 in the preparation of paeoniflorin.
3. A method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan bark, characterized in that The steps include: (1) Microbial fermentation of peony bark: inoculating the Debaryomyces or Debaryomyces culture solution of claim 1 into a sterilized fermentation medium containing peony bark, and fermenting to obtain a fermentation material; (2) Reflux extraction and slag removal: subjecting the fermented material obtained in step (1) to reflux extraction and centrifugal separation to obtain an extraction supernatant; (3) impurity removal and drying: the extraction supernatant obtained in step (2) is concentrated, anhydrous ethanol is added to the obtained concentrated solution, the mixture is stirred evenly, allowed to stand, and the solid and liquid are separated to obtain an impurity-removed supernatant; the ethanol is then distilled to recover the ethanol, and the impurity-removed and ethanol-removed concentrated solution is spray-dried to obtain a powdered extract.
4. The method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan bark according to claim 3, characterized in that: The fermentation medium in step (1) is composed of the following ingredients: peony bark powder, glucose, sodium citrate, peptone, yeast extract, potassium dihydrogen phosphate, magnesium sulfate and water; The inoculation amount of the Debaryomyces cerevisiae culture solution in step (1) is calculated as 0.4% to 1.2% of the volume of the water used for the fermentation medium; The fermentation conditions described in step (1) are as follows: temperature 27-29°C, stirring speed 220-260 r / min, ventilation ratio controlled at 0.24-0.36 vvm for 0-16 h of fermentation, ventilation ratio controlled at 0.36-0.48 vvm for 17-32 h of fermentation, ventilation ratio controlled at 0.32-0.36 vvm for 33-60 h of fermentation, ventilation ratio controlled at 0.28-0.32 vvm for 61 h and thereafter, and fermentation period 72-84 h.
5. The method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan bark according to claim 4, characterized in that: The dosage of the peony bark powder is calculated based on a ratio of 100 to 110 g / L with water; The amount of glucose is calculated based on a ratio of 160 to 180 g / L of glucose to water; The dosage of the sodium citrate is calculated based on the ratio of sodium citrate to water of 3.0 to 3.4 g / L; The amount of peptone used is calculated based on a ratio of 6 to 10 g / L with water; The amount of yeast extract is calculated based on a ratio of 6 to 10 g / L to water; The dosage of potassium dihydrogen phosphate is calculated based on a ratio of 2.4 to 2.8 g / L with water; The dosage of the magnesium sulfate is 0.22-0.26 g / L according to the ratio of magnesium sulfate to water; The pH of the fermentation medium is 6.3-6.
5.
6. The method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan bark according to claim 3, characterized in that: The Debaryomyces cerevisiae culture solution is prepared by the following steps: inoculating the preserved strain of Debaryomyces cerevisiae into a liquid culture medium, and performing shaking culture to obtain the Debaryomyces cerevisiae culture solution.
7. The method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan bark according to claim 3, characterized in that: The temperature of the reflux extraction in step (2) is 90-100°C; The reflux extraction time in step (2) is 40 to 60 min.
8. The method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan bark according to claim 3, characterized in that: The degree of concentration described in step (3) is that the volume of the concentrate is 10% to 20% of the volume of the extraction supernatant; The amount of anhydrous ethanol added in step (3) is 8.5 to 9.5 times the volume of the concentrated solution; The standing time in step (3) is 60 to 120 minutes; The solid-liquid separation method in step (3) is centrifugation; The distillation temperature in step (3) is 90-100°C; The volume of the concentrated solution in step (3) is 8% to 10% of the volume of the impurity-removed supernatant; The spray drying conditions in step (3) are: inlet air temperature 160-170°C, outlet air temperature 40-50°C.
9. Use of the method for preparing a paeoniflorin-rich extract by fermenting Paeonia suffruticosa moutan cortex according to any one of claims 3 to 8 in preparing paeoniflorin.
10. A powdery extract of paeoniflorin from Paeonia suffruticosa moutan, characterized in that: Obtained by the method according to any one of claims 3 to 8.