A method for producing diosgenin with high green yield by using microorganisms

Abstract

The present invention belongs to the field of biotechnology and discloses a method for green and high-yield production of diosgenin using microorganisms. Specifically, after homogenizing Dioscorea zingiberensis, it is treated with bacterial strain A to release saponins, and then treated with double enzymes to saccharify starch. The filtrate and filter cake are obtained by plate and frame filtration. The filter cake is mixed with water and filtered again, and the filtrates are combined to obtain a Dioscorea zingiberensis sugar solution containing saponins. If the raw material is dry Dioscorea zingiberensis, a complex enzyme is added to the filtrate for treatment, and Dioscorea zingiberensis sugar solution and a saponin-containing precipitate are obtained by centrifugation. If the raw material is fresh ginger, the filtrate is subjected to membrane separation to obtain Dioscorea zingiberensis sugar solution and a saponin concentrate, and the supernatant saponin solution and a saponin-containing precipitate are obtained by centrifugation. The saponin-containing precipitate is extracted with ethanol to obtain a saponin extract. A culture medium is prepared with the supernatant saponin solution and the saponin extract, and treated with bacterial strain B, and a diosgenin-containing precipitate is obtained by centrifugation. The precipitate is dried and refined by solvent extraction to obtain a diosgenin product. The present invention improves the design of the overall process flow, can obtain two types of saponins, has a high saponin extraction rate, and greatly improves the diosgenin yield, which is 1.3 to 1.6 times that of the acid hydrolysis process.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method for green and high-yield production of diosgenin from Dioscorea zingiberensis by using microorganisms. Background Art

[0002] Dioscorea zingiberensis C.H.Wright, scientifically named Dioscorea zingiberensis, also known as Huotougen, is a perennial twining herbaceous plant, which is a unique wild plant resource in China and is mainly distributed in provinces such as Hunan, Shaanxi, Hubei, Sichuan, Guizhou and Yunnan. The rhizome of Dioscorea zingiberensis contains steroidal saponin components such as dioscin, about 45% starch, 40% cellulose, as well as some chemical components such as steroidal glycosides, alkaloids, flavonoid glycosides, cardiac glycosides, tannins, pigments, etc. Dioscorea zingiberensis is an important medicinal plant source and contains rich diosgenin, also known as diosgenin (hereinafter all referred to as diosgenin). Diosgenin is the aglycone of dioscin and exists in Dioscorea zingiberensis in the form of dioscin. Diosgenin from Dioscorea zingiberensis can be used to synthesize hundreds of drugs necessary for national economy and people's livelihood, mainly including steroid hormone intermediates, corticosteroids, sex hormones, anabolic hormones, etc., and is known as "medicinal gold". Steroid hormone drugs have become the second largest category of chemical drugs after antibiotics.

[0003] Components such as lignocellulose, starch, protein, pectin, etc. in Dioscorea zingiberensis plants have a certain encapsulation effect on steroidal saponins, and steroidal saponins are also connected to other components through C3-position glycosides. Therefore, the simple acid hydrolysis method has a low yield and it is difficult to extract diosgenin from plants completely, and only about 1 / 4 of it can be extracted.

[0004] At present, the production of diosgenin still adopts the separation methods of first extraction and then hydrolysis represented by Marker and Wall and the method of first hydrolysis and then extraction proposed by Rothrok (direct acid hydrolysis process). The traditional diosgenin production process has a large discharge of wastewater and waste residue, the wastewater has a high acid content and strong pigment concentration, serious pollution, high treatment difficulty and high cost.

[0005] Patent CN1515585A discloses a method for producing diosgenin in an environmentally friendly and pollution-free manner. However, during the sieving and separation of 40% of the cellulose portion, some saponins bound to cellulose are carried away, thereby reducing the yield of diosgenin. Moreover, in the actual industrial process, membrane filtration has problems such as low flux and easy clogging (the membrane filtration it uses directly filters the sieved mixed solution with a very small pore size ultrafiltration membrane, which contains a large amount of starch and other large particle substances and is extremely easy to clog). Patent CN1970785A discloses a method for the clean production and comprehensive utilization of diosgenin. After the raw ginger raw material is pulverized, it is directly subjected to solvent extraction, and the bound saponins covalently coupled with the lignocellulose structure of ginger cannot be obtained, and there is room for improvement in the diosgenin yield. Moreover, other components of ginger are not utilized in a high-value manner. Patent CN1821380A discloses a highly efficient composite microbial flora for treating ginger. The microbial flora it uses covers various microorganisms such as bacteria, molds, and yeasts. However, the growth time of fungi is relatively long, and there is an antagonistic phenomenon between bacteria and fungi during the growth process. Most microorganisms have a staged growth situation during the treatment of ginger lignocellulose, and there is uncontrollability like the natural fermentation process. Moreover, some microorganisms may degrade and transform the sapogenin nucleus, thereby reducing the diosgenin yield. Patent CN103060416A discloses a method for the clean production of ginger diosgenin using microbial technology. The method of using a single microorganism combined with steam explosion to treat dry or fresh ginger materials has obtained a good yield and greatly reduced the usage of acid, water, and organic solvents. However, there are still areas that need improvement: the ability of a single microorganism to treat ginger is limited, and pretreatment of ginger is required by combining means such as steam explosion. Processes such as steam explosion have noise, high energy consumption, and a small single-batch processing capacity. At the same time, some harmful decomposition products are generated. In addition, a small amount of acid is still used in the process of producing diosgenin, and green production throughout the process cannot be achieved. Patent CN106834407B discloses a method for the green production of ginger diosgenin by biological methods. It uses a mixture of multiple microorganisms to treat ginger materials, effectively promoting the release of saponins, and completely replacing chemical acid hydrolysis with the microbial treatment method. The diosgenin yield is relatively high, realizing the green manufacturing of diosgenin, and the usage of water and organic solvents has decreased significantly. However, there are still areas that need improvement: the proportions of water-soluble and fat-soluble saponins in dried ginger and fresh ginger are very different. Using the same process will result in excessive cost investment and low returns. A large amount of organic solvents are required to extract the small amount of residual saponins in the fiber residue during the treatment process, which will consume a large amount of organic solvents and increase costs. In addition, the membrane concentrate of saponins contains a large amount of cellulose-like macromolecular substances, which will inhibit the biological transformation of saponins by microorganisms, thereby reducing the diosgenin yield. At the same time, the amount of inhibitors in each batch of saponin concentrate will affect the conversion rate of saponins, making the diosgenin yield unstable. Therefore, there are still areas that need improvement.

[0006] Using different or general process technologies to process wild yam with different saponin compositions at the lowest cost, maximizing the yield of diosgenin from wild yam, ensuring the stability of each batch of products, and at the same time abandoning the traditional acid hydrolysis method, significantly reducing the amount of organic solvents used, thus significantly reducing the production cost and pollution level of diosgenin has become the core issue. It is urgently necessary to carry out scientific and technological innovation, develop new green production processes and apply them industrially. Summary of the Invention

[0007] Aiming at the above defects or improvement requirements of the prior art, the purpose of the present invention is to provide a method for producing diosgenin with high yield and green by using microorganisms. By improving the overall process flow design and adopting a series of microbial communities, including highly efficient microbial community A that can secrete lignocellulose-degrading enzyme systems, glycosyltransferases and surfactants, and microbial strain B that can secrete glycosidases, promoting saponin release, saponin enrichment and saponin conversion respectively, it can effectively solve the prominent problems in the production process of diosgenin, such as low release rate of wild yam saponins, low yield of diosgenin, high energy consumption, serious pollution, and insufficient utilization of resources, significantly reducing the production cost of diosgenin and improving the market competitiveness of diosgenin. The present invention uses microbial community A and microbial strain B to produce diosgenin green and with high yield by biological methods, without using acid at all. The yield of diosgenin is 1.3 - 1.6 times that of the traditional acid hydrolysis process, with less organic solvent used, less pollution, full utilization of resources, and easy industrial amplification, and has great popularization value in the diosgenin industry.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided a method for producing diosgenin with high yield and green by using microorganisms, characterized by comprising the following steps:

[0009] (1) Pretreatment of wild yam fermentation: Using fresh wild yam as raw material, making a uniform slurry by pulverizing and homogenizing according to the mass ratio of fresh wild yam to water of 1:1 - 1:3; then, directly inoculating 5 - 20 vol% of microbial community A that can secrete lignocellulose-degrading enzyme systems, glycosyltransferases and surfactants into the slurry, and fermenting and culturing for 2 - 8 hours to obtain the corresponding fermentation broth;

[0010] (2) Double enzyme treatment: Heating the fermentation broth obtained in step (1) to 90 - 100 °C, keeping warm for 20 - 40 minutes to completely gelatinize the starch, then adjusting the pH to 5.0 - 8.0, and adding high-temperature amylase to liquefy the starch;

[0011] Or, heating the fermentation broth obtained in step (1) to 90 - 100 °C, keeping warm for 20 - 40 minutes to completely gelatinize the starch, then cooling the temperature to 60 - 80 °C, and then adjusting the pH to 5.5 - 7.5, and adding medium-temperature amylase to liquefy the starch;

[0012] Next, after the starch liquefaction is complete, control the temperature to 50 - 65 °C, adjust the pH to 4.0 - 5.0, and then add glucoamylase and treat for 3 - 5 hours to completely convert the starch into glucose, obtaining a saccharified mash;

[0013] (3) Filter and separate saponins: Filter the saccharified mash obtained in step (2) once by plate - frame pressing filtration to obtain the first filtrate and the filter cake; then, repeat the plate - frame filtration at least 1 time. Specifically, according to the corresponding relationship of 1 - 3 mL of water corresponding to every 1 g of filter cake, add water to the filter cake, soak and mix well, and perform plate - frame filtration again to obtain the filter cake and the filtrate; then combine the filtrates obtained from the repeated plate - frame filtration with the first filtrate to obtain a saponin - containing yellow ginger saccharified liquid;

[0014] (4) Concentrate and enrich saponins: Subject the saponin - containing yellow ginger saccharified liquid obtained in step (3) to three - stage membrane filtration to obtain a retentate and a permeate. The retentate is a saponin concentrate, and the permeate is a yellow ginger sugar solution; then, separate the saponin concentrate by a centrifuge to obtain a supernatant saponin solution and a saponin - containing precipitate;

[0015] Then, extract the saponin - containing precipitate 3 - 5 times with a 40 - 90 vol% ethanol solution, combine the extracts, and separate the ethanol to obtain a saponin extract;

[0016] (5) Prepare a saponin conversion culture medium: Dilute the supernatant saponin solution or the saponin extract obtained in step (4) so that the total saponin concentration does not exceed 300 g / L; then, add other culture medium components to prepare a saponin conversion culture medium;

[0017] Or, mix and dilute the supernatant saponin solution and the saponin extract obtained in step (4) so that the total saponin concentration does not exceed 300 g / L, and then add other culture medium components to prepare a saponin conversion culture medium;

[0018] (6) Saponin bioconversion: After sterilizing and cooling the saponin conversion culture medium in step (5), add 0.05 - 0.1 vol% Tween 80, inoculate 5 - 20 vol% of microorganism B that can secrete a variety of glycosidases with different functions, and then carry out fermentation conversion; after fermentation is completed, centrifuge and filter the fermentation product to obtain a diosgenin - containing precipitate; among them, the microorganism B can secrete a variety of glycosidases with different functions, and these glycosidases can hydrolyze different saponins;

[0019] (7) Preparation of diosgenin product: Sun - dry or dry the diosgenin - containing precipitate obtained in step (6), and then extract and refine it with an organic solvent to obtain a diosgenin product.

[0020] According to another aspect of the present invention, the present invention provides a method for producing diosgenin from yellow ginger with high yield using microorganisms in a green way, which is characterized by including the following steps:

[0021] (1) Pre-fermentation treatment of Dioscorea zingiberensis: Using sun-dried Dioscorea zingiberensis as raw material, crushing and homogenizing it into a uniform slurry according to the mass ratio of dried ginger to water of 1:3 - 1:10; then, directly inoculating 5 - 20 vol% of microbial flora A capable of secreting lignocellulose-degrading enzyme system, glycosyltransferase and surface active substances into the slurry, and fermenting and culturing for 2 - 8 hours to obtain the corresponding fermentation broth;

[0022] (2) Double enzyme treatment: Heating the fermentation broth obtained in step (1) to 90 - 100 °C, holding for 20 - 40 minutes to completely gelatinize the starch, then adjusting the pH to 5.0 - 8.0, and adding high-temperature amylase to liquefy the starch;

[0023] Or, heating the fermentation broth obtained in step (1) to 90 - 100 °C, holding for 20 - 40 minutes to completely gelatinize the starch, then cooling the temperature to 60 - 80 °C, adjusting the pH to 5.5 - 7.5, and adding medium-temperature amylase to liquefy the starch;

[0024] Then, after the starch liquefaction is complete, controlling the temperature at 50 - 65 °C, adjusting the pH to 4.0 - 5.0, and adding saccharifying enzyme to treat for 3 - 5 hours to completely convert the starch into glucose, obtaining saccharified mash;

[0025] (3) Filtering and separating saponins: Filtering the saccharified mash obtained in step (2) once with a plate and frame press to obtain the first filtrate and filter cake; then, repeating the plate and frame filtration at least 1 time. Specifically, according to the corresponding relationship of 1 - 3 mL of water corresponding to 1 g of filter cake, adding water to the filter cake, soaking and mixing evenly, and performing plate and frame filtration again to obtain filter cake and filtrate; then combining the filtrates obtained by repeating the plate and frame filtration with the first filtrate to obtain Dioscorea zingiberensis saccharified liquid containing saponins;

[0026] (4) Concentrating and enriching saponins: Adding cellulase and xylanase to the Dioscorea zingiberensis saccharified liquid containing saponins obtained in step (3), and the final specific activity of each enzyme in the system is 100 - 300 U / g of dry weight of Dioscorea zingiberensis, performing constant temperature treatment at 40 - 60 °C for 12 - 60 hours, centrifuging to obtain Dioscorea zingiberensis sugar solution and saponin-containing precipitate;

[0027] Then, extracting the saponin-containing precipitate 3 - 5 times with 40 - 90 vol% ethanol solution, combining the extraction liquids, and separating ethanol to obtain saponin extract;

[0028] (5) Preparing saponin conversion medium: Diluting the saponin extract obtained in step (4) to make the total saponin concentration not exceed 300 g / L; then, adding other medium components to make saponin conversion medium;

[0029] (6) Saponin biotransformation: After the saponin transformation medium in step (5) is sterilized and cooled, add 0.05 - 0.1 vol% Tween 80, inoculate 5 - 20 vol% of microorganism B that can secrete a variety of glycosidases with different functions, and then carry out fermentation transformation; after fermentation is completed, the fermentation product is centrifuged and filtered to obtain a saponin precipitate; wherein, the microorganism B can secrete a variety of glycosidases with different functions, and these glycosidases can hydrolyze different saponins.

[0030] (7) Preparation of saponin product: The saponin precipitate obtained in step (6) is dried by sunning or drying, and then refined by extraction with an organic solvent to obtain a saponin product.

[0031] As a further preference of the present invention, in the step (1), the surface active substance includes surfactin or lichenysin; the fermentation culture is carried out at 30 - 40 °C, a rotation speed of 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm for 2 - 8 h.

[0032] As a further preference of the present invention, in the step (1), the microbial flora A includes four kinds of bacteria: Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena.

[0033] Among them, Bacillus sp. is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M2016791; Bacillus licheniformis is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M2010113.

[0034] As a further preference of the present invention, the bacterial solution of the microbial flora A is prepared by a preparation method including the following steps:

[0035] (I) Respectively take single colonies of the strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena and inoculate them into LB medium, and culture them at 30 - 40 °C and 100 - 300 r / min for 8 - 16 h to obtain the first-stage seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena respectively.

[0036] (II) Respectively inoculate the primary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained in step (I) into LB medium at an inoculation amount of 1 - 5 vol%, and culture at 30 - 40 °C, 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm for 2 - 8 h to obtain the secondary seed solutions of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena respectively;

[0037] (III) Respectively inoculate the secondary seed solutions of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained in step (II) into a bacterial medium at an inoculation amount of 1 - 5 vol%, and culture at an aeration rate of 0.1 - 1 vvm, a stirring speed of 100 - 300 r / min, and a temperature of 30 - 40 °C for 2 - 8 h to obtain the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena respectively;

[0038] (IV) Mix the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained in step (III) according to a preset volume ratio to prepare a bacterial solution of microbial flora A; Preferably, the preset volume ratio is specifically an equal volume ratio;

[0039] Preferably, in step (III), the bacterial medium is a cheap bacterial medium, and its formula is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%.

[0040] As a further preference of the present invention, in step (6), the microbial bacteria B is specifically Aspergillus tubingensis, which is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC M2016389.

[0041] As a further preference of the present invention, the bacterial liquid of the microbial bacteria B is prepared by a preparation method including the following steps:

[0042] (S1) Take the slant-preserved strain Aspergillus tubingensis and inoculate it into a fungal medium, and culture it at 25-30 °C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 10-18 h to obtain the first-stage seeds of Aspergillus tubingensis;

[0043] (S2) Inoculate the first-stage seeds of Aspergillus tubingensis obtained in step (S1) into the fungal medium at an inoculation amount of 1-5 vol%, and culture it at 25-30 °C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 16-32 h to obtain the second-stage seeds of Aspergillus tubingensis;

[0044] (S3) Inoculate the second-stage seeds of Aspergillus tubingensis obtained in step (S2) into the fungal induction medium at an inoculation amount of 1-5 vol%, and culture it at 30-40 °C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 16-32 h to obtain the third-stage seeds of Aspergillus tubingensis;

[0045] Preferably, in the above step (S1) and step (S2), the formula of the fungal medium is as follows: peptone 5-10 g / L, yeast powder 2-5 g / L, glucose 5-10 g / L;

[0046] In the above step (S3), the fungal induction medium is an inexpensive fungal induction medium, and its formula is as follows: soybean cake powder 1-5 g / L, yeast powder 2-5 g / L, total saponins of Dioscorea zingiberensis alcohol extract 2-30 g / L; wherein, the total saponins of Dioscorea zingiberensis alcohol extract is preferably obtained by the following preparation method: take the dried Dioscorea zingiberensis, powder it, add 3 L of 70 vol% ethanol to every 1000 g of Dioscorea zingiberensis dry powder, stir and perform ultrasonic-assisted extraction for 30 minutes, after centrifugation, take the supernatant; continue to add 3 L of 70 vol% ethanol to the precipitate, repeat the above operation 2 times or more; combine the supernatants, separate the ethanol, and freeze-dry the extract to obtain the total saponins of Dioscorea zingiberensis alcohol extract.

[0047] As a further preference of the present invention, in the above step (5), the other medium components include inorganic salts and nitrogen sources; preferably include: sodium chloride 0.5-2 g / L, potassium nitrate 0.5-2 g / L, potassium dihydrogen phosphate 0.5-5 g / L, magnesium sulfate 0.5-2 g / L;

[0048] Alternatively, it preferably comprises: 0.5 - 2 g / L of sodium chloride, 1 - 5 g / L of soybean cake powder, 0.5 - 5 g / L of potassium dihydrogen phosphate, and 0.5 - 2 g / L of magnesium sulfate.

[0049] As a further preference of the present invention, in the step (6), the fermentation conversion is carried out under the conditions of 30 - 40 °C, a rotation speed of 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm for 24 - 144 h;

[0050] Among them, for a certain saponin conversion culture medium:

[0051] When the saponin conversion culture medium is prepared using the supernatant saponin solution and without using saponin extract, the fermentation conversion time is 24 - 48 h;

[0052] When the saponin conversion culture medium is prepared using saponin extract and without using the supernatant saponin solution, the fermentation conversion time is 96 - 144 h;

[0053] When the saponin conversion culture medium is prepared using both the supernatant saponin solution and saponin extract, the fermentation conversion time is 96 - 144 h.

[0054] As a further preference of the present invention, in the step (7), the organic solvent is selected from methanol, ethanol, gasoline, petroleum ether, and chloroform.

[0055] Through the above technical solutions conceived by the present invention, compared with the prior art, the following beneficial effects can be achieved:

[0056] 1. According to the changes of total saponins and other active ingredients during the harvesting and storage of Dioscorea zingiberensis, and the conversion effects of different types of saponins by microorganisms, the present invention divides the production process of diosgenin into fresh ginger process and dried ginger process to achieve high yield and efficient production of diosgenin.

[0057] 2. In addition, in the double - enzyme treatment stage, the present invention provides two schemes (i.e., high - temperature amylase + glucoamylase, or medium - temperature amylase + glucoamylase). In actual application, it can be combined with the existing double - enzyme market and different methods can be adopted according to different equipment, technology, cost and other requirements.

[0058] 3. The present invention uses the microbial flora A capable of secreting lignocellulose - degrading enzyme system, glycosyltransferase, and surface - active substances to pretreat the Dioscorea zingiberensis raw materials, making the lignocellulose structure loose, effectively breaking the glycosidic bond, releasing the bound saponins, and making the saponins wrapped by lignocellulose, pectin, starch, etc. in the Dioscorea zingiberensis tissue cells fully free; then through transglycosylation and surface - active substances, the solubility of free saponins in water is increased, and a small amount of saponins in the solid matter are dissolved in the aqueous phase.

[0059] The cellulase system of a single strain is incomplete and the enzyme activity is very low. Due to the encapsulation of hemicellulose and lignin, the microorganism fails to contact cellulose, so almost no cellulase is secreted. The xylanase, mannanase and pectinase enzyme activity levels secreted by a single strain are very low, affecting the overall degradation effect, and the effect of promoting the release of dioscin is very limited. The microbial flora A of the present invention is a mixed microbial flora capable of secreting lignocellulose-degrading enzyme systems, glycosyltransferases and surfactants, with very high enzyme activity. They can cooperate with each other, avoiding the competition of different microorganisms for limited substrates, improving the degradation efficiency of different substrates, secreting various enzymes with high enzyme activity at the same time, and the synergistic effect between various enzymes is more obvious, quickly releasing the encapsulated and covalently bound dioscin. The microbial flora A in the present invention can preferably adopt Bacillus sp. (more preferably Bacillus sp. corresponding to the preservation number CCTCC M 2016791), Bacillus licheniformis (more preferably Bacillus licheniformis corresponding to the preservation number CCTCC M 2010113), Pectobacterium carotovorum (unrestricted) and Cellulomonas flavigena (unrestricted). Among them, Bacillus sp. can produce amylase, glycosyltransferase and lipopeptide biosurfactants, Bacillus licheniformis can produce amylase and protease, Pectobacterium carotovorum can produce pectinase, and Cellulomonas flavigena can produce cellulase. The activities of these bacteria can efficiently destroy the tissue structure of Dioscorea zingiberensis, so that dioscin is fully released and freed into the aqueous phase.

[0060] 4. Through the treatment with the microbial flora A and multiple plate-and-frame filtration (such as plate-and-frame filtration again after water immersion), the amount of saponins contained in the Dioscorea zingiberensis fiber residue can be ignored, which is less than 2% of the total saponins in Dioscorea zingiberensis. There is no need to extract the saponins in the Dioscorea zingiberensis fiber residue with organic solvents, avoiding the large use and loss of organic solvents, greatly saving the production cost. After the Dioscorea zingiberensis fiber residue is dried, it can be directly used as raw materials for biological activated carbon, boards, organic fertilizers, etc.

[0061] 5. The membrane concentrate of dioscin contains 25 - 40% solids. Among the solid dry matter, 40 - 65% is cellulose-like macromolecular substances, which can effectively inhibit the biotransformation of saponins. The inhibitors can be effectively removed by multiple extractions with 40 - 90% ethanol. Compared with directly extracting saponins from Dioscorea zingiberensis or its fiber residue with organic solvents, extracting saponins from the membrane concentrate precipitate of Dioscorea zingiberensis saponins with organic solvents not only avoids the large consumption and loss of organic solvents, but also greatly saves production costs. At the same time, it relieves the inhibition of macromolecular substances on the biotransformation of saponins, improving the transformation efficiency of saponins and the yield of diosgenin. In addition, the membrane separation in the method of the present invention adopts three-stage membrane treatment, that is, filtration separation with three membranes of different pore sizes, which can effectively solve the problems of low flux and easy blockage in the treatment with a single pore-size membrane.

[0062] 6. The supernatant saponin solution and saponin extract obtained in the present invention can be further extracted and purified respectively to obtain water-soluble saponins and fat-soluble saponins, which are used to prepare various saponin products and drugs, such as different kinds of saponin reference standards, Dioscorea saponin tablets, etc.

[0063] 7. Using microorganism B that can secrete a series of highly active glycosidases to directly transform total saponins into diosgenin in a transformation medium prepared from aqueous-phase saponins or a saponin mixture, the obtained diosgenin product has high purity and high yield, and there are no by-products generated compared with the acid hydrolysis process. Microorganism B in the present invention can preferably be Aspergillus tubingensis (deposit number CCTCC M2016389). It can grow and reproduce using dioscin as a carbon source, and produce various glycosidases that can fully hydrolyze different dioscin saponins. It has good hydrolysis ability for both aqueous-phase saponins and non-aqueous-phase saponins, and will not degrade sapogenins, enabling the accumulation of sapogenins. The saponin transformation medium can particularly preferably adopt a formula including: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L (or soybean cake powder 1 - 5 g / L), potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L, and 0.05 - 0.1 vol% Tween 80, which can provide essential growth factors, nitrogen sources, and inorganic salts for the growth and reproduction of Aspergillus tubingensis. For example, magnesium ions can effectively improve the activity of glycosidases produced by Aspergillus tubingensis. Adding Tween 80 before inoculation after the medium is sterilized can effectively improve the solubility of non-aqueous-phase saponins in water and improve the enzymatic reaction efficiency.

[0064] 8. A method for green and high-yield production of diosgenin from Dioscorea zingiberensis using microorganisms in the present invention has the advantages of being green, environmentally friendly, highly adaptable, pollution-free, low-cost, capable of producing multiple products, high in product added value, and having broad industrialization prospects.

[0065] In summary, in the present invention, different single microbial strains or microbial communities and enzymes are added in stages to treat the turmeric material, so that the lignocellulose structure becomes loose, the glycosidic bond is effectively broken, and the bound dioscin is released. Then, through the action of glycosyltransferase, the free reducing sugar is linked to the saponin, increasing its solubility in water. At the same time, the biosurfactant secreted by the strain makes the free water-insoluble saponin dissolve in the aqueous phase; the saponin in the solid matter has a high degree of dissociation and a low residue; the saponin in the aqueous phase and the non-aqueous phase saponin from which inhibitors are removed are all converted into diosgenin by the highly efficient glycosidase produced by the microbial strain, completely replacing acid hydrolysis.

[0066] Compared with the direct acid hydrolysis method, the method of the present invention does not use acid at all, has no wastewater discharge, the turmeric resources can be fully utilized, and compared with the traditional acid hydrolysis method, the diosgenin yield can be increased by 31.3 - 58.7%. In addition, the new process has separated and enriched the water-soluble saponin and the fat-soluble saponin, and the fat-soluble saponin can also be directly subjected to acid hydrolysis treatment. At this time, a large amount of strong acid does not need to be consumed, the cost is low, and the pollution is easy to handle. Compared with the patent CN106834407B, the method of the present invention has a wider adaptability, and the saponin conversion is more efficient, solving the problems of the influence of inhibitors and the instability of diosgenin yield. At the same time, the water-phase saponin can be efficiently utilized to produce diosgenin and saponin products. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 is a process flow chart of the green production of diosgenin from fresh turmeric by the microbial method based on the present invention.

[0068] Figure 2 is a process flow chart of the green production of diosgenin from dried turmeric by the microbial method based on the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0069] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0070] Based on the present invention, taking fresh turmeric as an example, as Figure 1 shown, the method for the green and high-yield production of diosgenin from turmeric by using microorganisms may include the following steps:

[0071] (1) Pretreatment of turmeric material

[0072] Take an appropriate amount of fresh turmeric, wash away the soil and remove the whiskers, and crush and homogenize it according to the ratio of fresh turmeric weight to water of 1:1 - 1:3 (w / w) to prepare a turmeric slurry.

[0073] (2) Preparation of microbial flora

[0074] The preparation method of microbial flora A includes the following steps:

[0075] 1) Inoculate a single colony of the strain Bacillus sp. into a 250 mL shake flask containing 100 mL of LB medium, and culture it at 30 - 40 °C and 100 - 300 r / min for 8 - 16 h to obtain the first-stage seed of Bacillus sp.

[0076] 2) Inoculate the first-stage seed of Bacillus sp. into a 10 L seed tank containing LB medium, with an inoculation amount of 1 vol%, a tank temperature of 30 - 40 °C, 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm, and culture it for 2 - 8 h to obtain the second-stage seed of Bacillus sp.

[0077] 3) Inoculate the second-stage seed of Bacillus sp. into a 1 m 3 fermentation tank containing a cheap bacterial medium, with an aeration rate of 0.1 - 1 vvm, a stirring speed of 100 - 300 r / min, a tank temperature of 30 - 40 °C, and culture it for 2 - 8 h to obtain the third-stage seed of Bacillus sp.

[0078] Use the same method as above to obtain the third-stage seeds of Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena respectively.

[0079] 4) Mix the third-stage seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena in equal proportions to prepare microbial flora A.

[0080] The formula of the cheap bacterial medium is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%;

[0081] The preparation method of microbial bacteria B includes the following steps:

[0082] 1) Inoculate the spore suspension of the slant-preserved strain Aspergillus tubingensis into a 250 mL shake flask containing 100 mL of fungal medium, and culture it at 25 - 30 °C and 100 - 300 r / min for 10 - 18 h to obtain the first-stage seeds of Aspergillus tubingensis.

[0083] 2) Inoculate the first-stage seeds of Aspergillus tubingensis into a 7 L seed tank filled with fungal medium, with an inoculation amount of 1 - 5 vol%, a tank temperature of 25 - 30 °C, 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm, and culture for 16 - 32 h to obtain the second-stage seeds of Aspergillus tubingensis.

[0084] 3) Inoculate the second-stage seeds of Aspergillus tubingensis into a 1 m 3 fermenter filled with a cheap fungal induction medium, with an inoculation amount of 1 - 5 vol%, an aeration rate of 0.1 - 1 vvm, a stirring speed of 100 - 300 r / min, and a tank temperature of 30 - 40 °C, and culture for 16 - 32 h. After centrifugation, obtain the third-stage seeds of Aspergillus tubingensis.

[0085] (3) Microbial fermentation to promote saponin dissociation

[0086] Inoculate 5 - 20 vol% of the microbial flora A into the Dioscorea zingiberensis slurry obtained in step (1), and ferment and culture it at 30 - 40 °C, a rotation speed of 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm for 2 - 8 h to obtain the corresponding fermentation broth.

[0087] (4) Double enzyme treatment

[0088] Heat the fermentation broth obtained in step (3) to 90 - 100 °C and keep it warm for 20 - 40 minutes to completely gelatinize the starch. Then add high-temperature α-amylase according to the volume of the Dioscorea zingiberensis slurry to make the enzyme activity unit reach 300 U / mL or more, and liquefy it at pH 6.2 and 95 °C for 20 min (of course, the pH value and temperature conditions can also be set according to the other high-temperature amylase setting values in the prior art); then add glucoamylase according to the volume of the Dioscorea zingiberensis slurry to make the enzyme activity unit reach 300 U / mL or more, and saccharify it at pH 4.2 and a temperature of 60 °C for 3 - 6 h to completely convert the starch into glucose and obtain the saccharified mash.

[0089] Of course, in addition to high-temperature amylase, medium-temperature amylase can also be used (at this time, the pH value and temperature conditions can also be set according to the known medium-temperature amylase setting values in the prior art), and with subsequent glucoamylase treatment, the saccharified mash can also be obtained.

[0090] (5) Filter and separate saponins

[0091] Press and filter the saccharified mash obtained in step (4) once with a plate and frame filter press to obtain a filtrate and a filter cake. Then, add water to the filter cake according to a mass-to-volume ratio of 1:1 to 1:3, soak and mix evenly, and perform a secondary plate and frame filtration to obtain a second filter cake and a filtrate. Then, combine the two filtrates to obtain a saponin-containing yellow ginger saccharified liquid (containing solids), and the filter cake is yellow ginger fiber residue.

[0092] (6) Concentrate and enrich saponins

[0093] The saponin-containing yellow ginger saccharified liquid obtained in step (5) is successively passed through microfiltration, ultrafiltration, and nanofiltration to obtain a retentate (containing solids) and a permeate. The retentate is a saponin concentrate, and the permeate is a yellow ginger starch saccharified liquid. Centrifuge the retentate to obtain a supernatant saponin solution and a saponin-containing precipitate. The saponin-containing precipitate is extracted 3 to 5 times with a 40 to 90 vol% ethanol solution, and then the extraction liquids are combined and ethanol is recovered to obtain a saponin extract.

[0094] (7) Prepare a saponin transformation medium

[0095] Mix the supernatant saponin solution and the saponin extract obtained in step (6) so that the total saponin concentration does not exceed 300 g / L, and then add conventional medium components such as inorganic salts to prepare a saponin transformation medium. For example, it may include: sodium chloride 0.5 to 2 g / L, potassium nitrate 0.5 to 2 g / L, potassium dihydrogen phosphate 0.5 to 5 g / L, and magnesium sulfate 0.5 to 2 g / L.

[0096] Or it may include: sodium chloride 0.5 to 2 g / L, soybean cake powder 1 to 5 g / L, potassium dihydrogen phosphate 0.5 to 5 g / L, and magnesium sulfate 0.5 to 2 g / L.

[0097] (8) Biotransformation of saponins

[0098] After sterilizing and cooling the saponin transformation medium obtained in step (7), add 0.05 to 0.1 vol% Tween 80, and inoculate with microorganism B that can secrete a variety of glycosidases with different functions at an inoculation volume ratio of 5 to 20 vol%, and ferment and culture for 2 to 6 days to obtain the corresponding fermentation product. Then, centrifuge and filter to obtain a saponin-containing precipitate.

[0099] (9) Preparation of diosgenin product

[0100] The precipitate obtained in step (8) can be dried and then extracted with anhydrous ethanol for 4 h. The organic solvent is recovered and recycled, and the extract is crystallized and dried to obtain a white diosgenin product.

[0101] The preferred range of the inoculation amount of microbial flora A in step (3) is 5 to 20 vol%. The gradual increase of microbial flora A helps to improve its working efficiency. The enzyme secreted by flora A makes the saponin free and dissolved in the aqueous phase during the pretreatment reaction time. Considering that if the amount of microbial flora A added is too large, the sugar group on the free saponin will be further degraded, thereby reducing the solubility of the saponin and causing it to precipitate, affecting the solubility of the saponin, the inoculation ratio is controlled to 5 to 20 vol%.

[0102] The high temperature amylase, saccharifying enzyme, etc. used in step (4), including the cellulase and xylanase used in the following text, are not limited, and enzymes of the same type and having the same function known in the prior art can participate. These enzymes used in the present invention are all existing commercial products. The high temperature α-amylase and saccharifying enzyme used in the following examples are purchased from Su Kehan Biology, and the cellulase and xylanase are purchased from Aladdin.

[0103] The filter cake obtained in step (5) is turmeric fiber residue, which can be directly used as raw material for biological activated carbon, board material, organic fertilizer, etc. after drying.

[0104] The turmeric sugar solution obtained in step (6) has a glucose concentration of 5 to 9 g / L and can be recycled as water for at least 10 times. The sugar concentration has no obvious inhibitory effect on the activities of amylase and saccharifying enzyme during the double enzyme treatment and has no effect on the release of saponins.

[0105] The supernatant saponin solution and saponin extract obtained in step (6) can be further extracted and purified to obtain water-soluble saponins and fat-soluble saponins, which are used to prepare various saponin products and drugs, such as different types of saponin standard products, diosgenin tablets, etc. Among them, the saponin extract is mainly fat-soluble saponins in the non-aqueous phase, and the bioconversion efficiency is relatively low. This part of saponins can also be treated by traditional acid hydrolysis process, which uses less strong acid, less wastewater, and has very low COD and BOD of wastewater, which is easy to treat and low in cost.

[0106] The preferred range of microorganism B in step (8) is 5 to 20 vol%. Gradually increasing the amount of microorganism B helps to improve its working efficiency, greatly shorten the adjustment period of microorganism B, and accelerate enzyme production. Considering that further increasing its addition amount will not significantly improve the saponin conversion efficiency and will increase costs, the inoculation ratio is controlled to 5 to 20 vol%.

[0107] The fermentation waste liquid obtained in step (8) can be recycled at least 10 times without adding inorganic salts. Water needs to be added for each recycling, and a small amount of nitrogen source, such as soybean cake powder, potassium nitrate, etc., needs to be added when recycled for more than 10 times.

[0108] The cellulase system of a single strain is incomplete and its enzyme activity is very low. Due to the encapsulation of hemicellulose and lignin, the microorganism fails to contact cellulose, so it hardly secretes cellulase. The xylanase, mannanase and pectinase activities secreted by a single strain are very low, affecting the overall degradation effect, and the effect of promoting the release of dioscin by fermentation is very limited. However, the microbial flora A of the present invention is a mixed microbial flora capable of secreting lignocellulose-degrading enzyme systems, glycosyltransferases and surfactants, which can cooperate with each other, avoiding the competition of different microorganisms for limited substrates, improving the degradation efficiency of different substrates, secreting various enzymes with high enzyme activity at the same time, and the synergistic effect between various enzymes is more obvious, quickly releasing the dioscin encapsulated and covalently bound. The microbial strain B of the present invention can secrete a variety of highly active glycosidases. These rich glycosidase enzyme systems cooperate with each other synergistically. At the same time, it does not require harsh environment and culture conditions. Only by removing the inhibitors can it ensure high-efficiency and low-cost conversion of saponins.

[0109] In the present invention, different microbial single strains or flora and enzymes are added in stages to treat the dioscorea material, making the lignocellulose structure loose, effectively breaking the glycosidic bond, releasing the bound dioscin, and then connecting the free reducing sugar to the saponin through the action of glycosyltransferase, increasing its solubility in water. At the same time, the biosurfactant secreted by the strain makes the free water-insoluble saponin dissolve in the aqueous phase; the degree of free saponin in the solid is high and the residue is low; the saponin in the aqueous phase and the non-aqueous phase saponin from which the inhibitor is removed are all converted into sapogenin by the highly efficient glycosidase produced by the microbial strain, completely replacing acid hydrolysis. The present invention has a high yield of dioscorea sapogenin, does not use acid at all in the whole production process, uses less organic solvent, has no wastewater discharge, less waste residue, product diversification, high availability of dioscorea resources, and the multi-process treatment is applicable to all fresh and dried plants containing saponins.

[0110] The microbial flora A includes but is not limited to Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena; the microbial strain B is Aspergillustubingensis.

[0111] The Bacillus sp. was deposited at the China Center for Type Culture Collection in Wuhan, China on December 30, 2016, and its deposit number is CCTCC M 2016791.

[0112] The Bacillus licheniformis was deposited at the China Center for Type Culture Collection in Wuhan, China on May 12, 2010, and its deposit number is CCTCC M 2010113.

[0113] The Pectobacterium carotovorum has no specific strain limitation.

[0114] The Cellulomonas flavigena has no specific strain limitation.

[0115] The Aspergillus tubingensis was deposited on July 11, 2016 at the China Center for Type Culture Collection in Wuhan, China, with the deposit number CCTCC M2016389.

[0116] In addition, due to the seasonal variation characteristics of diosgenin production in diosgenin production enterprises, large-scale production of diosgenin only starts during the harvesting period of Dioscorea zingiberensis. Therefore, enterprises mostly purchase fresh Dioscorea zingiberensis, and some are dried Dioscorea zingiberensis due to storage reasons. Therefore, in the following examples, fresh Dioscorea zingiberensis and dried Dioscorea zingiberensis will be used as examples respectively to illustrate the method of the present invention in detail (when using dried Dioscorea zingiberensis as the raw material, the process schematic diagram based on the method of the present invention is as Figure 2 shown).

[0117] The following are specific examples (wherein, Examples 1-3 use fresh Dioscorea zingiberensis as the raw material, and Examples 4-5 use dried Dioscorea zingiberensis as the raw material):

[0118] Example 1

[0119] A method for producing diosgenin from Dioscorea zingiberensis with high green yield by using microorganisms, comprising the following steps:

[0120] (1) Pretreatment of Dioscorea zingiberensis materials

[0121] Take 8850 kg of fresh Dioscorea zingiberensis, wash away the soil and whiskers, and pulverize and homogenize it according to the ratio of fresh weight of Dioscorea zingiberensis to water of 1:1 (w / w) to prepare a Dioscorea zingiberensis slurry. This Dioscorea zingiberensis slurry does not require sterilization treatment, and no additional inorganic salts, nitrogen sources, carbon sources, growth factors and other substances need to be added to the homogeneous slurry (that is to say, no additional nutrient elements or treatments are required for the homogeneous slurry).

[0122] (2) Preparation of microbial flora

[0123] The preparation method of microbial flora A is as follows:

[0124] Take single colonies of the strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena and inoculate them into 250 mL shake flasks containing 100 mL of LB medium, one bottle for each strain, and culture them at 30 °C and 100 r / min for 12 h to obtain primary seeds.

[0125] The primary seeds were separately inoculated into 10-L seed tanks containing 8 L of LB medium, and cultured at a tank temperature of 37 °C, 100 r / min, and an aeration rate of 0.1 vvm for 6 h to obtain secondary seeds.

[0126] The secondary seeds were separately inoculated into 1-m 3 fermenters containing 800 L of inexpensive bacterial medium, and cultured at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.1 vvm for 6 h to obtain tertiary seeds.

[0127] The obtained tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena were mixed in equal proportions to prepare microbial flora A.

[0128] The formula of the inexpensive bacterial medium is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%.

[0129] The preparation method of microbial flora B is as follows:

[0130] The slant-preserved strain Aspergillus tubingensis was inoculated into a 250-mL shake flask containing 100 mL of fungal medium, with 1 flask inoculated, and cultured at 28 °C and 100 r / min for 18 h to obtain primary seeds.

[0131] The primary seeds were inoculated into a 7-L seed tank containing 4 L of fungal medium, and cultured at a tank temperature of 28 °C, 200 r / min, and an aeration rate of 0.2 vvm for 24 h to obtain secondary seeds.

[0132] The secondary seeds were inoculated into 1-m 3 fermenters containing 400 L of inexpensive fungal induction medium, and cultured at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.2 vvm for 24 h to obtain tertiary seeds.

[0133] (3) Microbial fermentation to promote saponin dissociation

[0134] Microbial flora A was inoculated into the Dioscorea zingiberensis slurry obtained in step (1), and fermented and cultured at 37 °C, a rotation speed of 200 r / min, and an aeration of 0.1 vvm for 8 h to obtain the corresponding fermentation broth.

[0135] (4) Double enzyme treatment

[0136] Heat the fermentation broth obtained in step (3) to 90 - 100 °C, hold for 20 - 40 minutes to completely gelatinize the starch, then adjust the pH to 6.2, add high-temperature amylase to make the enzyme activity unit reach 300 U / mL or more, and carry out liquefaction at pH 6.2 and 95 °C for 20 min; then add saccharifying enzyme according to the volume of the yellow ginger slurry to make the enzyme activity unit reach 300 U / mL or more, and carry out saccharification at pH 4.2 and 60 °C for 6 h to completely convert the starch into glucose, obtaining saccharified mash.

[0137] (5) Filter and separate saponins

[0138] Filter the saccharified mash obtained in step (4) once with a plate and frame press to obtain a filtrate and a filter cake. Add water to the filter cake according to the mass - volume ratio of 1:1, soak and mix evenly, then carry out the second plate and frame filtration, and then combine the filtrates. The filtrate is the yellow ginger saccharified solution containing saponins (containing solids), and the filter cake is the yellow ginger fiber residue.

[0139] In addition, in this step, the number of times of repeated plate and frame filtration can be more, and the filtrates are also combined. There is no use of organic solvents in this process.

[0140] (6) Concentrate and enrich saponins

[0141] The yellow ginger saponin - containing saccharified solution obtained in step (5) is successively passed through microfiltration, ultrafiltration and nanofiltration to obtain a retentate (containing solids) and a permeate. The retentate is the saponin concentrate, and the permeate is the yellow ginger starch saccharified solution. Centrifuge the retentate to obtain a supernatant saponin solution and a saponin - containing precipitate. The saponin - containing precipitate is extracted 3 times with 70 vol% ethanol solution, and then the extraction liquids are combined and ethanol is recovered to obtain a saponin extract.

[0142] (7) Prepare a saponin conversion medium

[0143] Mix and dilute the supernatant saponin solution and the saponin extract obtained in step (6), and add inorganic salts to prepare a saponin conversion medium with a total volume of 1.7 m 3 .

[0144] The other medium components include inorganic salts and nitrogen sources, including: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L;

[0145] Or, including: sodium chloride 0.5 - 2 g / L, soybean cake powder 1 - 5 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L.

[0146] (8) Microbial conversion of saponins

[0147] Sterilize the saponin conversion medium obtained in step (7), and after cooling, add 0.05 vol% Tween 80, inoculate with 5 vol% of microorganism B that can secrete various glycosidases with different functions, and ferment and culture for 6 days to obtain the corresponding fermentation product. Then, centrifuge and filter to obtain a precipitate containing saponin.

[0148] (9) Preparation of saponin product

[0149] Dry the precipitate obtained in step (8), then extract with absolute ethanol for 4 h, recycle the organic solvent, and after the extract is crystallized and dried, obtain a white diosgenin product.

[0150] Compared with the direct acid hydrolysis method, the biological method does not use acid at all, is pollution-free, and obtains dioscorea starch sugar solution and dioscorea fiber residue that can be comprehensively utilized. The saponin yield is 133.43 mg / g of dry dioscorea weight, and the saponin yield is increased by 35.0% (the dry dioscorea weight in this application, similar to the conventional definition, refers to the weight of dry dioscorea / dried dioscorea obtained after fresh dioscorea is dried or sun-dried). Compared with patent CN106834407B, due to the removal of enzyme inhibitors and process optimization, the saponin yield is relatively stable, and the saponin yield is significantly increased.

[0151] Example 2

[0152] A method for producing diosgenin from dioscorea with high yield and green by using microorganisms, comprising the following steps:

[0153] (1) Pretreatment of dioscorea material

[0154] Take 5010 kg of fresh dioscorea, wash away the soil and remove the whiskers, and pulverize and homogenize it according to the ratio of fresh dioscorea weight to water of 1:1 (w / w) to prepare a dioscorea slurry. This dioscorea slurry does not require sterilization treatment, and no additional inorganic salts, nitrogen sources, carbon sources, growth factors and other substances are required in the homogeneous slurry.

[0155] (2) Preparation of microbial flora

[0156] The preparation method of microbial flora A is as follows:

[0157] Inoculate the single colonies of strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena into 250 mL shake flasks containing 100 mL of LB medium, one bottle for each strain, and culture at 30 °C and 100 r / min for 12 h to obtain first-stage seeds.

[0158] The primary seeds were separately inoculated into seed tanks containing 6 L of LB medium, and cultured at a tank temperature of 37 °C, 100 r / min, and an aeration rate of 0.1 vvm for 8 h to obtain secondary seeds.

[0159] The secondary seeds were separately inoculated into 1 m 3 fermenters containing 550 L of inexpensive bacterial medium, and cultured at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.1 vvm for 6 h to obtain tertiary seeds.

[0160] The obtained tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena were mixed in equal proportions to prepare microbial flora A.

[0161] The formula of the inexpensive bacterial medium is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%.

[0162] The preparation method of microbial bacteria B is as follows:

[0163] The slant-preserved strain Aspergillus tubingensis was inoculated into a 250 mL shake flask containing 100 mL of fungal medium, and 1 flask was inoculated and cultured at 28 °C and 100 r / min for 18 h to obtain primary seeds.

[0164] The primary seeds were inoculated into a 7 L seed tank containing 2.5 L of fungal medium, and cultured at a tank temperature of 28 °C, 200 r / min, and an aeration rate of 0.2 vvm for 24 h to obtain secondary seeds.

[0165] The secondary seeds were inoculated into 1 m 3 fermenters containing 250 L of inexpensive fungal induction medium, and cultured at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.2 vvm for 36 h to obtain tertiary seeds.

[0166] (3) Microbial fermentation to promote saponin dissociation

[0167] Microbial flora A was inoculated into the ginger tuber slurry obtained in step (1), and fermented and cultured at 37 °C, a rotation speed of 200 r / min, and an aeration of 0.1 vvm for 6 h to obtain the corresponding fermentation broth.

[0168] (4) Double enzyme treatment

[0169] Heat the fermentation broth obtained in step (3) to 90 - 100 °C and keep it warm for 20 - 40 minutes to completely gelatinize the starch. Then adjust the pH to 6.2, add high-temperature amylase to make the enzyme activity unit 300 U / mL or above, and liquefy it for 20 min at pH 6.2 and 95 °C. Then add glucoamylase according to the volume of the yellow ginger slurry to make the enzyme activity unit 300 U / mL or above, and saccharify it for 6 h at pH 4.2 and a temperature of 60 °C to completely convert the starch into glucose, obtaining a saccharified mash.

[0170] (5) Filter and separate saponins

[0171] Filter the saccharified mash obtained in step (4) once with a plate and frame press to obtain a filtrate and a filter cake. Then, add water to the filter cake according to the mass - volume ratio of 1:2, soak and mix well, and perform a secondary plate and frame filtration to obtain a second filter cake and a filtrate. Then combine the two filtrates, which is the yellow ginger saccharified liquid containing saponins (containing solids), and the filter cake is the yellow ginger fiber residue.

[0172] (6) Concentrate and enrich saponins

[0173] Pass the yellow ginger saponin - containing saccharified liquid obtained in step (5) through microfiltration, ultrafiltration, and nanofiltration successively to obtain a retentate (containing solids) and a permeate. The retentate is the saponin concentrate, and the permeate is the yellow ginger starch saccharified liquid. Centrifuge the retentate to obtain a supernatant saponin solution and a saponin - containing precipitate. The saponin - containing precipitate is extracted 4 times with a 70 vol% ethanol solution, then the extraction liquids are combined, and ethanol is recovered to obtain a saponin extract.

[0174] (7) Prepare a saponin conversion medium

[0175] Mix and dilute the supernatant saponin solution and the saponin extract obtained in step (6), and add inorganic salts to make a saponin conversion medium, with a total volume of 1.0 m 3 .

[0176] The other medium components include inorganic salts and nitrogen sources, including: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L;

[0177] Or, including: sodium chloride 0.5 - 2 g / L, soybean cake powder 1 - 5 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L.

[0178] (8) Microbial conversion of saponins

[0179] Sterilize the saponin conversion medium obtained in step (7). After cooling, add 0.1 vol% Tween 80, inoculate 10 vol% of microorganism B that can secrete various glycosidases with different functions, and ferment and culture for 6 days to obtain the corresponding fermentation product. Then, centrifuge and filter to obtain the saponin precipitate.

[0180] (9) Preparation of saponin product

[0181] Dry the precipitate obtained in step (8), then extract it with absolute ethanol for 4 h, recycle the organic solvent, and dry the extract by crystallization to obtain the white diosgenin product.

[0182] Compared with the direct acid hydrolysis method, the biological method does not use acid at all, has less pollution, and obtains the comprehensively utilizable yellow ginger starch sugar solution and yellow ginger fiber residue. The saponin yield is 144.57 mg / g of dry yellow ginger weight, and the saponin yield is increased by 46.3%. Compared with patent CN106834407B, due to the removal of enzyme inhibitors and process optimization, the saponin yield is relatively stable, and the saponin yield is significantly increased.

[0183] Example 3

[0184] A method for producing diosgenin from yellow ginger with high green yield by using microorganisms, comprising the following steps:

[0185] (1) Pretreatment of yellow ginger material

[0186] Take 6500 kg of fresh yellow ginger, wash away the soil and remove the whiskers, and crush and homogenize it according to the ratio of fresh yellow ginger weight to water of 1:1 (w / w) to prepare a yellow ginger slurry. This yellow ginger slurry does not require sterilization treatment, and no additional inorganic salts, nitrogen sources, carbon sources, growth factors and other substances are required in the uniform slurry.

[0187] (2) Preparation of microbial flora

[0188] The preparation method of microbial flora A is as follows:

[0189] Inoculate the single colonies of strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena into 250 mL shake flasks containing 100 mL of LB medium, one bottle for each strain, and culture at 30 °C and 100 r / min for 12 h to obtain the first-stage seeds.

[0190] Respectively inoculate the first-stage seeds into a 10 L seed tank containing 7 L of LB medium, and culture at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.1 vvm for 6 h to obtain the second-stage seeds.

[0191] Inoculate the secondary seeds into a 1 m³ fermenter containing 700 L of a cheap bacterial medium, and culture at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.1 vvm for 6 h to obtain the tertiary seeds. 3

[0192] Mix the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained above in equal proportions to prepare a microbial flora A.

[0193] The formula of the cheap bacterial medium is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%.

[0194] The preparation method of microbial bacteria B is as follows:

[0195] Inoculate the slant-preserved strain Aspergillus tubingensis into a 250 mL shake flask containing 100 mL of a fungal medium, inoculate 1 flask, and culture at 28 °C and 100 r / min for 18 h to obtain the primary seeds.

[0196] Inoculate the primary seeds into a 7 L seed tank containing 3 L of a fungal medium, and culture at a tank temperature of 28 °C, 200 r / min, and an aeration rate of 0.2 vvm for 24 h to obtain the secondary seeds.

[0197] Inoculate the secondary seeds into a 1 m³ fermenter containing 300 L of a cheap fungal induction medium, 3 and culture at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.2 vvm for 48 h to obtain the tertiary seeds.

[0198] (3) Microbial fermentation promotes saponin dissociation

[0199] Inoculate the microbial flora A into the Dioscorea zingiberensis slurry obtained in step (1), and ferment and culture at 37 °C, a rotation speed of 200 r / min, and an aeration of 0.1 vvm for 4 h to obtain the corresponding fermentation broth.

[0200] (4) Double enzyme treatment

[0201] Heat the fermentation broth obtained in step (3) to 90 - 100 °C and keep it warm for 20 - 40 minutes to completely gelatinize the starch. Then adjust the pH to 6.2, add high-temperature amylase to make the enzyme activity unit reach 300 U / mL or more, and carry out liquefaction at pH 6.2 and 95 °C for 20 min. Then add glucoamylase according to the volume of the yellow ginger slurry to make the enzyme activity unit reach 300 U / mL or more, and carry out saccharification at pH 4.2 and a temperature of 60 °C for 6 h to completely convert the starch into glucose, obtaining a saccharified mash.

[0202] (5) Filter and separate saponins

[0203] Filter the saccharified mash obtained in step (4) once with a plate and frame press to obtain a filtrate and a filter cake. According to the mass-volume ratio of 1:3, add water to the filter cake, soak and mix well, then carry out the second plate and frame filtration, and then combine the filtrates. The filtrate is a yellow ginger saccharified solution containing saponins (containing solids).

[0204] (6) Concentrate and enrich saponins

[0205] Pass the yellow ginger saponin-containing saccharified solution obtained in step (5) through microfiltration, ultrafiltration, and nanofiltration membranes successively to obtain a permeate and a retentate (containing solids). The retentate is a saponin concentrate, and the permeate is a yellow ginger starch saccharified solution. Centrifuge the retentate to obtain a supernatant saponin solution and a saponin-containing precipitate. The saponin-containing precipitate is extracted 5 times with a 70 vol% ethanol solution, and then the extraction solutions are combined and ethanol is recovered to obtain a saponin extract.

[0206] (7) Prepare a saponin conversion medium

[0207] Mix and dilute the supernatant saponin solution and the saponin extract obtained in step (6), and add inorganic salts to prepare a saponin conversion medium with a total volume of 1.25 m 3 .

[0208] The other medium components include inorganic salts and nitrogen sources, including: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L;

[0209] Or, including: sodium chloride 0.5 - 2 g / L, soybean cake powder 1 - 5 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L.

[0210] (8) Microbial conversion of saponins

[0211] Sterilize the saponin conversion medium obtained in step (7), cool it, add 0.1 vol% Tween 80, inoculate 15 vol% of microorganism B that can secrete a variety of glycosidases with different functions, and carry out fermentation culture and conversion for 6 days to obtain the corresponding fermentation product. Then, obtain a saponin-containing precipitate through centrifugation and filtration;

[0212] (9) Preparation of saponin product

[0213] The precipitate obtained in step (8) is dried and then extracted with absolute ethanol for 4 h. The organic solvent is recovered for recycling. After the extract is crystallized and dried, a white dioscorea zingiberensis saponin product is obtained.

[0214] Compared with the direct acid hydrolysis method, the biological method does not use acid at all, has less pollution, and obtains dioscorea zingiberensis starch sugar solution and dioscorea zingiberensis fiber residue that can be comprehensively utilized. The saponin yield is 156.84 mg / g of dry dioscorea zingiberensis weight, and the saponin yield is increased by 58.7%. Compared with patent CN106834407B, due to the removal of enzyme inhibitors and process optimization, the saponin yield is relatively stable, and the saponin yield is significantly increased.

[0215] Example 4

[0216] A method for producing dioscorea zingiberensis saponin with high yield by using microorganisms greenly, comprising the following steps:

[0217] (1) Pretreatment of dioscorea zingiberensis materials

[0218] Take 2200 kg of sun-dried dioscorea zingiberensis, wash away the soil and whiskers, and pulverize and homogenize it according to the ratio of fresh dioscorea zingiberensis weight to water of 1:3 (w / w) to prepare a dioscorea zingiberensis slurry. This dioscorea zingiberensis slurry does not require sterilization treatment, and no additional inorganic salts, nitrogen sources, carbon sources, growth factors and other substances are needed in the homogeneous slurry.

[0219] (2) Preparation of microbial flora

[0220] The preparation method of microbial flora A is as follows:

[0221] Take the single colonies of strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena and inoculate them into 250 mL shake flasks containing 100 mL of LB medium, one bottle for each strain, and culture them at 30 °C and 100 r / min for 12 h to obtain primary seeds.

[0222] Respectively inoculate the primary seeds into a 10 L seed tank containing 6 L of LB medium, with the tank temperature at 37 °C, 200 r / min, and the ventilation rate of 0.1 vvm, and culture for 6 h to obtain secondary seeds.

[0223] Respectively inoculate the secondary seeds into a 1 m 3 Fermenter containing 500 L of cheap bacterial medium, with the tank temperature at 37 °C, 200 r / min, and the ventilation rate of 0.1 vvm, and culture for 6 h to obtain tertiary seeds.

[0224] Mix the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained above in equal proportions to prepare microbial flora A.

[0225] The formula of the inexpensive bacterial culture medium is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%.

[0226] The preparation method of microbial bacteria B is as follows:

[0227] Inoculate the slant-preserved strain Aspergillus tubingensis into a 100 mL shaking flask containing 30 mL of fungal culture medium, inoculate 1 flask, and culture at 28 °C and 100 r / min for 18 h to obtain primary seeds.

[0228] Inoculate the primary seeds into a 7 L seed tank containing 2 L of fungal culture medium, and culture at a tank temperature of 28 °C, 200 r / min, and an aeration rate of 0.2 vvm for 24 h to obtain secondary seeds.

[0229] Inoculate the secondary seeds into a 1 m 3 Fermenter containing 200 L of inexpensive fungal induction medium, and culture at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.2 vvm for 48 h to obtain tertiary seeds.

[0230] (3) Microbial fermentation promotes saponin dissociation

[0231] Inoculate microbial flora A into the Dioscorea zingiberensis slurry obtained in step (1), and ferment and culture at 37 °C, a rotation speed of 200 r / min, and an aeration of 0.1 vvm for 4 h to obtain the corresponding fermentation broth.

[0232] (4) Double enzyme treatment

[0233] Heat the fermentation broth obtained in step (3) to 90 - 100 °C, keep it warm for 20 - 40 minutes to completely gelatinize the starch, then adjust the pH to 6.2, and then add high-temperature amylase to make the enzyme activity unit 300 U / mL or more, and liquefy at pH 6.2 and 95 °C for 20 min; then add glucoamylase according to the volume of the Dioscorea zingiberensis slurry to make the enzyme activity unit 300 U / mL or more, and saccharify at pH 4.2 and a temperature of 60 °C for 6 h to completely convert the starch into glucose to obtain saccharified mash.

[0234] (5) Filter and separate saponins

[0235] The saccharified mash obtained in step (4) is filtered once by plate-and-frame pressing to obtain a filtrate and a filter cake. Water is added to the filter cake at a mass-to-volume ratio of 1:2, soaked and mixed evenly, and then subjected to a second plate-and-frame filtration. After that, the filtrates are combined, and the filtrate is a saponin-containing yellow ginger saccharified liquid (containing solids).

[0236] (6) Concentrate and enrich saponins

[0237] Cellulase and xylanase are added to the saponin-containing yellow ginger saccharified liquid obtained in step (5), and the final specific activity of each enzyme in the system is 300 U / g of dry yellow ginger weight. It is treated at 50 °C for 24 hours, and centrifuged to obtain yellow ginger sugar liquid and saponin-containing precipitate. The saponin-containing precipitate is extracted 3 times with 70 vol% ethanol solution, and the extraction liquids are combined, and ethanol is recovered to obtain a saponin extract paste.

[0238] (7) Prepare a saponin conversion culture medium

[0239] The saponin extract paste obtained in step (6) is diluted with tap water and inorganic salts are added to prepare a saponin conversion culture medium, with a total volume of 1 m 3 .

[0240] The other culture medium components include inorganic salts and nitrogen sources, including: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L;

[0241] Or, including: sodium chloride 0.5 - 2 g / L, soybean cake powder 1 - 5 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L.

[0242] (8) Microbial conversion of saponins

[0243] The saponin conversion culture medium obtained in step (7) is sterilized, cooled, 0.1 vol% Tween 80 is added, and 15 vol% of microorganism B capable of secreting a variety of glycosidases with different functions is inoculated, and fermented and cultured for 6 days to obtain the corresponding fermentation product. Then, it is centrifuged and filtered to obtain a diosgenin-containing precipitate;

[0244] (9) Preparation of diosgenin product

[0245] The precipitate obtained in step (8) is dried and then extracted with absolute ethanol for 4 h. The organic solvent is recovered and recycled. After the extract is crystallized and dried, a white yellow ginger diosgenin product is obtained.

[0246] Compared with the direct acid hydrolysis method, the biological method does not use acid at all, has less pollution, and obtains the comprehensively utilizable Dioscorea zingiberensis starch sugar solution and Dioscorea zingiberensis fiber residue. The saponin yield is 129.80 mg / g of dry Dioscorea zingiberensis weight, and the saponin yield has increased by 31.3% (since the dry Dioscorea zingiberensis weight used in this example is the weight of the sun-dried Dioscorea zingiberensis raw material). Compared with patent CN106834407B, due to the removal of enzyme inhibitors and process optimization, the saponin yield is relatively stable, and the saponin yield has increased significantly.

[0247] Example 5

[0248] A method for producing diosgenin from Dioscorea zingiberensis with high yield by using microorganisms, comprising the following steps:

[0249] (1) Pretreatment of Dioscorea zingiberensis materials

[0250] Take 2600 kg of sun-dried Dioscorea zingiberensis, wash away the soil and whiskers, and pulverize and homogenize it according to the ratio of fresh Dioscorea zingiberensis weight to water of 1:4 (w / w) to prepare a Dioscorea zingiberensis slurry. This Dioscorea zingiberensis slurry does not require sterilization treatment, and no additional inorganic salts, nitrogen sources, carbon sources, growth factors and other substances need to be added to the homogeneous slurry.

[0251] (2) Preparation of microbial flora

[0252] The preparation method of microbial flora A is as follows:

[0253] Take the single colonies of strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum and Cellulomonas flavigena and inoculate them into 250 mL shake flasks containing 100 mL of LB medium, one flask for each strain, and culture them at 30 °C and 100 r / min for 12 h to obtain primary seeds.

[0254] Respectively inoculate the primary seeds into 10 L seed tanks containing 7 L of LB medium, with the tank temperature at 37 °C, 200 r / min, and the ventilation rate at 0.1 vvm, and culture for 6 h to obtain secondary seeds.

[0255] Respectively inoculate the secondary seeds into a 1 m 3 Fermenter containing 700 L of cheap bacterial medium, with the tank temperature at 37 °C, 200 r / min, and the ventilation rate at 0.1 vvm, and culture for 6 h to obtain tertiary seeds.

[0256] Mix the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained above in equal proportions to prepare microbial flora A.

[0257] The formula of the cheap bacterial culture medium is as follows: soybean cake powder 1 - 5 g / L, peptone 2 - 5 g / L, yeast powder 1 - 2.5 g / L, sodium chloride 5 - 12 g / L, antifoaming agent 0.05 - 0.1 vol%.

[0258] The preparation method of microbial bacteria B is as follows:

[0259] Inoculate the slant-preserved strain Aspergillus tubingensis into a 100 mL shake flask containing 30 mL of fungal culture medium, inoculate 1 flask, and culture at 28 °C and 100 r / min for 18 h to obtain primary seeds.

[0260] Inoculate the primary seeds into a 7 L seed tank containing 3 L of fungal culture medium, and culture at a tank temperature of 28 °C, 200 r / min, and an aeration rate of 0.2 vvm for 24 h to obtain secondary seeds.

[0261] Inoculate the secondary seeds into a 1 m 3 Fermenter containing 300 L of cheap fungal induction medium, and culture at a tank temperature of 37 °C, 200 r / min, and an aeration rate of 0.2 vvm for 48 h to obtain tertiary seeds.

[0262] (3) Microbial fermentation promotes saponin dissociation

[0263] Inoculate microbial flora A into the Dioscorea zingiberensis slurry obtained in step (1), and ferment and culture at 37 °C, a rotation speed of 200 r / min, and an aeration of 0.1 vvm for 4 h to obtain the corresponding fermentation broth.

[0264] (4) Double enzyme treatment

[0265] Heat the fermentation broth obtained in step (3) to 90 - 100 °C and keep it warm for 20 - 40 minutes to completely gelatinize the starch. Then adjust the pH to 6.2, and add high-temperature amylase to make the enzyme activity unit 300 U / mL or more. Liquefy at pH 6.2 and 95 °C for 20 min; then add glucoamylase according to the volume of the Dioscorea zingiberensis slurry to make the enzyme activity unit 300 U / mL or more, and saccharify at pH 4.2 and a temperature of 60 °C for 6 h to completely convert the starch into glucose and obtain saccharified mash.

[0266] (5) Filter and separate saponins

[0267] The saccharified mash obtained in step (4) is subjected to plate-and-frame pressing filtration once to obtain a filtrate and a filter cake. Water is added to the filter cake according to a mass-to-volume ratio of 1:2, soaked and mixed evenly, and then subjected to a second plate-and-frame filtration. After that, the filtrates are combined. The filtrate is a saponin-containing yellow ginger saccharified solution (containing solids).

[0268] (6) Concentrate and enrich saponins

[0269] Cellulase and xylanase are added to the saponin-containing yellow ginger saccharified solution obtained in step (5), and the final specific activity of each enzyme in the system is 300 U / g of dry weight of yellow ginger. It is treated at 55 °C for 48 hours, and centrifuged to obtain yellow ginger sugar solution and saponin-containing precipitate. The saponin-containing precipitate is extracted 5 times with 70% ethanol solution, and the extraction solutions are combined, and ethanol is recovered to obtain a saponin extract paste.

[0270] (7) Prepare a saponin conversion medium

[0271] The saponin extract paste obtained in step (6) is diluted with tap water and inorganic salts are added to prepare a saponin conversion medium, with a total volume of 1.5 m 3 .

[0272] The other medium components include inorganic salts and nitrogen sources, including: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L;

[0273] Or, including: sodium chloride 0.5 - 2 g / L, soybean cake powder 1 - 5 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L.

[0274] (8) Microbial conversion of saponins

[0275] The saponin conversion medium obtained in step (7) is sterilized, cooled, 0.1 vol% Tween 80 is added, and 15 vol% of microbial strain B that can secrete a variety of glycosidases with different functions is inoculated, and fermented and cultured for 6 days to obtain the corresponding fermentation product. Then, it is centrifuged and filtered to obtain a saponin-containing precipitate;

[0276] (9) Preparation of diosgenin products

[0277] The precipitate obtained in step (8) is dried and then extracted with absolute ethanol for 4 h. The organic solvent is recovered and recycled. After the extract is crystallized and dried, a white diosgenin product is obtained.

[0278] Compared with the direct acid hydrolysis method, the biological method does not use acid at all, has less pollution, and obtains Dioscorea zingiberensis starch sugar solution and Dioscorea zingiberensis fiber residue that can be comprehensively utilized. The saponin yield is 134.42 mg / g of dry weight of Dioscorea zingiberensis, and the saponin yield is increased by 36.0%. Compared with Patent CN106834407B, due to the removal of enzyme inhibitors and process optimization, the saponin yield is relatively stable, and the saponin yield is significantly increased.

[0279] Bacillus sp. used in this application is preserved in the China Center for Type Culture Collection (CCTCC), and its microorganism preservation number is: CCTCC M 2016791; the taxonomic name is: Bacillus; the preservation time is: December 30, 2016; the preservation unit is: China Center for Type Culture Collection; the preservation address is: Preservation Center of Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province.

[0280] Bacillus licheniformis used in this application is preserved in the China Center for Type Culture Collection (CCTCC), and its microorganism preservation number is: CCTCC M 2010113; the taxonomic name is: Bacillus licheniformis; the preservation time is: May 12, 2010; the preservation unit is: China Center for Type Culture Collection; the preservation address is: Preservation Center of Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province.

[0281] Aspergillus tubingensis used in this application is preserved in the China Center for Type Culture Collection (CCTCC), and its microorganism preservation number is: CCTCC M2016389; the taxonomic name is: Aspergillus tubingensis; the preservation time is: July 11, 2016; the preservation unit is: China Center for Type Culture Collection; the preservation address is: Preservation Center of Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province.

[0282] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for the green and high-yield production of diosgenin using microorganisms, characterized in that, It includes the following steps: (1) Pre-fermentation treatment of Dioscorea zingiberensis: Crushing and homogenizing Dioscorea zingiberensis fresh ginger and water according to the mass ratio of 1:1 to 1:3 to make a uniform slurry; inoculating 5-20 vol% of microbial flora A in the slurry, and fermenting and culturing for 2-8 hours to obtain the corresponding fermentation broth; (2) Double enzyme treatment: Heating the fermentation broth obtained in step (1) to 90-100 °C, holding for 20-40 minutes to completely gelatinize the starch, then adjusting the pH to 5.0-8.0, and adding high-temperature amylase to liquefy the starch; Alternatively, heating the fermentation broth obtained in step (1) to 90-100 °C, holding for 20-40 minutes to completely gelatinize the starch, then cooling the temperature to 60-80 °C, and then adjusting the pH to 5.5-7.5, and adding medium-temperature amylase to liquefy the starch; After the starch liquefaction is complete, controlling the temperature at 50-65 °C, adjusting the pH to 4.0-5.0, and adding saccharifying enzyme to treat for 3-5 hours to completely convert the starch into glucose to obtain a saccharified mash; (3) Filtering and separating saponins: Filtering the saccharified mash obtained in step (2) once with a plate and frame press to obtain the first filtrate and filter cake; then, repeating the plate and frame filtration at least 1 time. Specifically, according to the corresponding relationship of 1-3 mL of water per 1 g of filter cake, adding water to the filter cake to soak and mix evenly, and performing plate and frame filtration again to obtain a filter cake and filtrate; then combining the filtrates obtained by repeating the plate and frame filtration with the first filtrate to obtain a Dioscorea zingiberensis saccharified liquid containing saponins; (4) Concentrating and enriching saponins: Passing the Dioscorea zingiberensis saccharified liquid containing saponins obtained in step (3) through three-stage membrane filtration to obtain a retentate and a permeate. The retentate is a saponin concentrate, and the permeate is a Dioscorea zingiberensis sugar solution; then, separating the saponin concentrate with a centrifuge to obtain a supernatant saponin solution and a saponin-containing precipitate; Then, extracting the saponin-containing precipitate 3-5 times with a 40-90 vol% ethanol solution, combining the extraction liquids, and separating ethanol to obtain a saponin extract; (5) Preparing a saponin conversion culture medium: Diluting the supernatant saponin solution and / or saponin extract obtained in step (4) so that the total saponin concentration does not exceed 300 g / L; then, adding other culture medium components to make a saponin conversion culture medium; (6) Saponin bioconversion: After sterilizing and cooling the saponin conversion culture medium in step (5), adding 0.05-0.1 vol% of Tween 80, inoculating 5-20 vol% of microbial strain B that can secrete a variety of glycosidases with different functions, and then performing fermentation conversion; after the fermentation is completed, centrifuging and filtering the fermentation product to obtain a diosgenin-containing precipitate; (7) Preparation of diosgenin product: Drying or baking the diosgenin-containing precipitate obtained in step (6), and then extracting and refining with an organic solvent to obtain a diosgenin product; In step (1), the microbial flora A is the four strains of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena; In step (6), the microorganism B is specifically Aspergillus tubingensis, with the preservation number of CCTCC M2016389.

2. A method for producing diosgenin with high green yield by using microorganisms, characterized in that, It includes the following steps: (1) Pre-fermentation treatment of Dioscorea zingiberensis: Crushing and homogenizing to make a uniform slurry according to the mass ratio of dried ginger to water of 1:3 - 1:10; inoculating 5 - 20 vol% of the microbial flora A into the slurry, and fermenting and culturing for 2 - 8 hours to obtain the corresponding fermentation broth; (2) Double-enzyme treatment: Heating the fermentation broth obtained in step (1) to 90 - 100 °C, holding for 20 - 40 minutes to completely gelatinize the starch, then adjusting the pH to 5.0 - 8.0, and adding high-temperature amylase to liquefy the starch; Alternatively, heating the fermentation broth obtained in step (1) to 90 - 100 °C, holding for 20 - 40 minutes to completely gelatinize the starch, then cooling the temperature to 60 - 80 °C, adjusting the pH to 5.5 - 7.5, and adding medium-temperature amylase to liquefy the starch; After the starch liquefaction is complete, controlling the temperature at 50 - 65 °C, adjusting the pH to 4.0 - 5.0, and adding glucoamylase to treat for 3 - 5 hours to completely convert the starch into glucose to obtain saccharified mash; (3) Filtering and separating saponins: Filtering the saccharified mash obtained in step (2) once with a plate-and-frame press to obtain the first filtrate and the filter cake; then, repeating the plate-and-frame filtration at least 1 time. Specifically, according to the corresponding relationship of 1 - 3 mL of water corresponding to 1 g of the filter cake, adding water to the filter cake to soak and mix evenly, and performing plate-and-frame filtration again to obtain the filter cake and the filtrate; then combining the filtrates obtained by repeating the plate-and-frame filtration with the first filtrate to obtain the saponin-containing Dioscorea zingiberensis saccharified liquid; (4) Concentrating and enriching saponins: Adding cellulase and xylanase to the saponin-containing Dioscorea zingiberensis saccharified liquid obtained in step (3), and the final specific activity of each enzyme in the system is 100 - 300 U / g of dry weight of Dioscorea zingiberensis, carrying out constant-temperature treatment at 40 - 60 °C for 12 - 60 hours, and centrifuging to obtain Dioscorea zingiberensis sugar solution and saponin-containing precipitate; Then, extracting the saponin-containing precipitate 3 - 5 times with 40 - 90 vol% ethanol solution, combining the extraction liquids, and separating ethanol to obtain saponin extract; (5) Preparing saponin conversion medium: Diluting the saponin extract obtained in step (4) to make the total saponin concentration not exceed 300 g / L; then adding other medium components to make the saponin conversion medium; (6) Saponin bioconversion: After sterilizing and cooling the saponin conversion medium in step (5), adding 0.05 - 0.1 vol% of Tween 80, inoculating 5 - 20 vol% of the microorganism B capable of secreting various glycosidases with different functions, and then carrying out fermentation conversion; after the fermentation is completed, centrifuging and filtering the fermentation product to obtain saponin-containing precipitate; (7) Preparing sapogenin product: Drying the saponin-containing precipitate obtained in step (6) by sunning or drying, and then extracting and refining with an organic solvent to obtain the sapogenin product; In the step (1), the microbial flora A is composed of four kinds of bacteria: Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena. In the step (6), the microbial bacteria B is specifically Aspergillus tubingensis, and the preservation number is CCTCC M 2016389.

3. The method for producing diosgenin with high green yield by using microorganisms as claimed in claim 1 or 2, characterized in that In the step (1), the surfactant includes surfactin or lichenysin; the fermentation culture is carried out at 30-40°C, with a rotation speed of 100-300 r / min and an aeration rate of 0.1-1 vvm for 2-8 h.

4. The method for green and high-yield production of diosgenin using microorganisms as claimed in claim 1 or 2, characterized in that, Bacillus sp. is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M 2016791; Bacillus licheniformis is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M 2010113.

5. The method for green and high-yield production of diosgenin using microorganisms according to claim 4, characterized in that, The bacterial solution of the microbial flora A is prepared by a preparation method including the following steps: (I) Single colonies of the strains Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena are respectively inoculated into LB medium and cultured at 30-40°C and 100-300 r / min for 8-16 h to obtain the first-stage seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena respectively. (II) The first-stage seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained in the step (I) are respectively inoculated into LB medium at an inoculation amount of 1-5 vol% and cultured at 30-40°C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 2-8 h to obtain the second-stage seed solutions of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena respectively. (III) Respectively inoculate the secondary seed solutions of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained in step (II) into the bacterial medium at an inoculation amount of 1-5 vol%, and culture at an aeration rate of 0.1-1 vvm, a stirring speed of 100-300 r / min, and a temperature of 30-40 °C for 2-8 h to obtain the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena respectively; (IV) Mix the tertiary seeds of Bacillus sp., Bacillus licheniformis, Pectobacterium carotovorum, and Cellulomonas flavigena obtained in step (III) according to a preset volume ratio to prepare a bacterial solution of microbial flora A; the preset volume ratio is specifically an equal volume ratio.

6. The method for green and high-yield production of diosgenin using microorganisms according to claim 5, characterized in that, In step (III), the bacterial medium is an inexpensive bacterial medium, and its formula is as follows: soybean cake powder 1-5 g / L, peptone 2-5 g / L, yeast powder 1-2.5 g / L, sodium chloride 5-12 g / L, antifoaming agent 0.05-0.1 vol%.

7. The method for producing diosgenin with high green yield by using microorganisms as claimed in claim 1 or 2, characterized in that The bacterial solution of microbial bacteria B is prepared by a preparation method including the following steps: (S1) Take the slant-preserved strain Aspergillus tubingensis and inoculate it into the fungal medium, and culture at 25-30 °C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 10-18 h to obtain the primary seed of Aspergillus tubingensis; (S2) Inoculate the primary seed of Aspergillus tubingensis obtained in step (S1) into the fungal medium at an inoculation amount of 1-5 vol%, and culture at 25-30 °C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 16-32 h to obtain the secondary seed of Aspergillus tubingensis; (S3) Inoculate the secondary seed of Aspergillus tubingensis obtained in step (S2) into the fungal induction medium at an inoculation amount of 1-5 vol%, and culture at 30-40 °C, 100-300 r / min, and an aeration rate of 0.1-1 vvm for 16-32 h to obtain the tertiary seed of Aspergillus tubingensis; In the step (S3), the fungal induction medium is an inexpensive fungal induction medium, and its formula is as follows: soybean cake powder 1 - 5 g / L, yeast powder 2 - 5 g / L, total saponins of Dioscorea zingiberensis ethanol extract 2 - 30 g / L; wherein, the total saponins of Dioscorea zingiberensis ethanol extract is obtained by the following preparation method: take dried Dioscorea zingiberensis, powder it, add 3 L of 70 vol% ethanol to every 1000 g of Dioscorea zingiberensis dry powder, stir and perform ultrasonic-assisted extraction for 30 minutes, after centrifugation, take the supernatant; continue to add 3 L of 70 vol% ethanol to the precipitate, repeat the above operation 2 times or more; combine the supernatants, separate the ethanol, and then freeze-dry the extract to obtain the total saponins of Dioscorea zingiberensis ethanol extract.

8. The method for green and high-yield production of diosgenin using microorganisms as claimed in claim 7, characterized in that, In the step (S1) and the step (S2), the formula of the fungal medium is as follows: peptone 5 - 10 g / L, yeast powder 2 - 5 g / L, glucose 5 - 10 g / L.

9. The method for producing diosgenin with high green yield by using microorganisms according to claim 1 or 2, characterized in that, In the step (5), the other medium components include inorganic salts and nitrogen sources.

10. The method for producing diosgenin with high green yield by using microorganisms according to claim 9, characterized in that, In the step (5), the other medium components include: sodium chloride 0.5 - 2 g / L, potassium nitrate 0.5 - 2 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L; Or, include: sodium chloride 0.5 - 2 g / L, soybean cake powder 1 - 5 g / L, potassium dihydrogen phosphate 0.5 - 5 g / L, magnesium sulfate 0.5 - 2 g / L.

11. The method for green and high-yield production of diosgenin using microorganisms according to claim 1 or 2, characterized in that, In the step (6), the fermentation conversion is carried out under the conditions of 30 - 40 °C, a rotation speed of 100 - 300 r / min, and an aeration rate of 0.1 - 1 vvm for 24 - 144 h; Among them, for a certain saponin conversion medium: When the saponin conversion medium is prepared using the supernatant saponin solution and without using the saponin extract, the fermentation conversion time is 24 - 48 h; When the saponin conversion medium is prepared using the saponin extract and without using the supernatant saponin solution, the fermentation conversion time is 96 - 144 h; When the saponin conversion medium is prepared using both the supernatant saponin solution and the saponin extract, the fermentation conversion time is 96 - 144 h.

12. The method for green and high-yield production of diosgenin using microorganisms according to claim 1 or 2, characterized in that, In the step (7), the organic solvent is selected from methanol, ethanol, gasoline, petroleum ether, chloroform.

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