CN109762866B - Method for improving rare saponin content in total ginsenoside by utilizing acetobacter whole-cell catalysis and application - Google Patents
Method for improving rare saponin content in total ginsenoside by utilizing acetobacter whole-cell catalysis and application Download PDFInfo
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Abstract
The invention discloses a method for improving the content of rare saponin in total ginsenoside by utilizing acetobacter whole-cell catalysis and application thereof. The method comprises the following steps: (1) crushing and sieving white ginseng, adding a solvent for extraction and concentration to obtain a ginseng extracting solution; (2) purifying the ginseng extract by using macroporous resin, and then concentrating to obtain a ginseng total saponin extract; (3) fermenting Acetobacter by high density fermentation method to obtain fermentation liquid with thallus concentration of 1.5 × 109More than cfu/mL, and then filtering and concentrating by a tangential flow microfiltration system to obtain concentrated fermentation liquor; (4) adding the ginseng total saponin extracting solution into the concentrated fermentation liquor for biotransformation to obtain the ginseng total saponin aqueous solution containing rare saponin. The method has mild conditions, can greatly improve the content of rare saponin in the ginseng total saponin extracting solution, and has higher application value.
Description
Technical Field
The invention belongs to the field of biotransformation, and particularly relates to a method for improving the content of rare saponins in total saponins of ginseng by utilizing whole-cell catalysis of acetobacter and application of the method.
Background
Ginsenoside is the main active component of ginseng, and has physiological effects of improving memory, improving blood circulation, resisting tumor, and enhancing immunity. Rare ginsenoside has a very small content in ginseng, but the pharmacological action is stronger than that of a major component, and the effects of the rare ginsenoside on the aspects of antibiosis, antiphlogosis and antitumor are increasingly concerned. Ginsenoside is mainly composed of terpenoid as mother nucleus and oligosaccharide as ligand, and is absorbed by organism, because of its large molecular weight, strong polarity and weak mucous membrane penetrability, it is poor to absorb in gastrointestinal tract, and its bioavailability is low, and its blood concentration can hardly reach the concentration required for fully exerting pharmacological activity, and most of the medicine is discharged out of body in original form; ginsenoside is metabolized by the internal environment of human body, sugar chains of ginsenoside ligands are degraded step by step to obtain rare saponin and sapogenin, the more complete the sugar chains are degraded, the lower the polarity of the product is, the greater the absorption rate of the product by the organism is, so the rare saponin and the sapogenin are main substances for the ginseng to exert the drug effect, but the content of natural rare saponin is often lower.
The existing patents mostly increase the content of certain rare saponin in ginseng extract. Because the concentration of the substrate is low, the conversion efficiency is not high, and the content of rare saponin in the obtained ginseng extract is low. The patent CN101244104B provides a method for increasing the content of rare saponins in a total ginsenoside extract, which comprises the steps of degreasing by using an organic solvent chloroform, extracting by using n-butyl alcohol saturated water through ultrasonic waves, steaming and converting at high temperature (120-130 ℃), and finally concentrating and extracting.
At present, the main preparation methods of rare saponins comprise acid and alkali hydrolysis and biotransformation. Because acid and alkali hydrolysis methods produce a large amount of waste acid and waste alkali, and cause great harm to the environment, a pollution-free production way is imperative to be found. Therefore, the microbial fermentation is adopted, the sugar chain structure of the ginsenoside is changed by using biotransformation, the rapid and effective transformation of the ginsenoside is realized, and the method has great research significance for improving the yield of rare active saponins.
However, previous researches show that the acetobacter is a high-yield strain of the beta-glucosidase, and the produced enzyme is an intracellular insoluble membrane-bound enzyme, namely, only whole cells show high enzyme activity, and the enzyme activity of both fermentation broth supernatant and broken cells is low.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for improving the content of rare saponins in total saponins of ginseng by utilizing the whole-cell catalysis of acetobacter.
The invention also aims to provide the application of the method for improving the content of rare saponin in the total ginsenoside by utilizing the acetobacter whole-cell catalysis.
The purpose of the invention is realized by the following technical scheme: a method for improving the content of rare saponins in total saponins of ginseng by utilizing the catalysis of acetobacter whole cells comprises the following steps:
(1) extracting the total saponins of ginseng: crushing and sieving white ginseng, and then adding a solvent for extraction to obtain an extracting solution; concentrating the extractive solution to obtain concentrated Ginseng radix extractive solution;
(2) and (3) macroporous resin purification: adding the concentrated ginseng extract obtained in the step (1) into macroporous resin, washing off impurities, performing gradient elution, collecting eluent, and concentrating the eluent to obtain a ginseng total saponin extract;
(3) preparing a high-concentration acetobacter whole-cell catalytic system: fermenting Acetobacter by high density fermentation method to obtain fermentation liquid with thallus concentration of 1.5 × 109Filtering and concentrating the obtained fermentation liquor by a tangential flow microfiltration system to obtain concentrated fermentation liquor, wherein the cfu/mL is more than the cfu/mL;
(4) and (3) biotransformation: and (3) adding the ginseng total saponin extracting solution obtained in the step (2) into the fermentation liquor concentrated in the step (3) for biotransformation to obtain a ginseng total saponin aqueous solution containing rare saponin.
The white ginseng in the step (1) is preferably sun-dried ginseng.
The sieving in the step (1) is to sieve the mixture through a sieve of 40-80 meshes.
The solvent used in step (1) is preferably water.
The extraction conditions in the step (1) are as follows: the ratio of the material to the liquid is 1: 5-20, the extraction temperature is 95 ℃, the extraction time is 1-3.0 h, and the extraction times are 2-6; preferably: the ratio of the material to the liquid is 1: 8-15, the extraction temperature is 95 ℃, the extraction time is 1.5-2.5 h, and the extraction times are 3-5; more preferably: the ratio of material to liquid is 1:10, the extraction time is 1.5h, and the extraction times are 4 times.
The concentration in the step (1) is carried out until the volume of the extracting solution is 10-20%.
The macroporous resin in the step (2) is preferably macroporous resin AB-8.
The step (2) of washing away impurities is to wash away impurities by using distilled water and ethanol solution in sequence; preferably, the impurities are washed off by using distilled water, a 10% (w/w) ethanol solution and a 20% (w/w) ethanol solution.
The gradient elution in the step (2) is gradient elution by using 30-95% (w/w) ethanol solution.
Concentrating the eluent in the step (2) to 10-35% of the volume of the eluent; preferably, the concentration is carried out to 15-25% of the volume of the eluent.
The Acetobacter in the step (3) is preferably Acetobacter aceti (Acetobacter pasteurianus) GIM 1.67.
The concentration of the cells in the fermentation broth in step (3) is preferably 1.6X 109～1.9×109cfu/mL; more preferably 1.63X 109～1.88×109cfu/mL。
The high-density fermentation method in the step (3) comprises the steps of strain activation, shake flask culture, seed tank propagation, fermentation in a fermentation tank and the like, and is preferably realized by the following steps: activating and expanding culture of acetobacter to obtain seed liquid; then inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid; more preferably by the following steps:
(I) inoculating acetobacter aceti to a solid agar culture medium for activation culture by streaking to obtain activated acetobacter aceti;
(II) inoculating the activated acetobacter into a culture medium in a shake flask for shake culture to obtain a seed solution A;
(III) inoculating the seed solution A to a culture medium in a seed tank for culture, and controlling the pH value of a culture system to be 4.0-5.5 to obtain a seed solution B;
(IV) inoculating the seed liquid B to a culture medium in a fermentation tank for fermentation culture, controlling the pH value of the fermentation liquid to be 4.0-5.5 in the fermentation process, and simultaneously feeding concentrated apple juice to obtain the fermentation liquid.
The conditions for the activation culture described in step (I) are preferably: culturing at 30 deg.C for 24 h.
The solid agar culture medium in the step (I) consists of the following components in percentage by mass: 1% of yeast extract, 2% of glucose, 2% of agar, 4% of ethanol and the balance of water, and the pH value is 5.5.
The conditions for the shaking culture described in step (II) are preferably: culturing at 100rpm and 30 deg.C for 16 h.
The amount of the activated acetobacter aceti inoculated in the step ((II) was 5% by volume of the medium.
The culture medium in the step (II) consists of the following components in percentage by mass: 1% of yeast extract, 1% of glucose and KH2P040.3 percent and 2 percent of ethanol.
The inoculation amount of the seed liquid A in the step (III) is 5 percent of the volume of the culture medium.
The conditions for the culture described in step (III) are preferably: the stirring speed is 150rpm, the aeration rate is 0.6vvm, the temperature is 30 ℃, and the culture time is 10 h.
The culture medium in the steps (III) and (IV) consists of the following components in percentage by mass: 2% of yeast extract, 2% of glucose and KH2P040.3 percent and 2 percent of ethanol.
Controlling the pH value of the culture system in the steps (III) and (IV) to be 4.0-5.5 by feeding sodium hydroxide; preferably, the control is carried out by feeding 4-8M of sodium hydroxide solution.
The dosage of the concentrated apple juice in the step (IV) is calculated according to the proportion of 10-25 g (preferably 10-20 g) of concentrated apple juice per liter of fermentation liquor, and the concentrated apple juice is added after 8-20 hours (preferably 8-16 hours) of fermentation is started.
The culture conditions in step (IV) are as follows: stirring at the rotation speed of 150rpm, ventilation volume of 0.6vvm, temperature of 30 ℃ and culture time of 12-28 h; preferably: the stirring speed is 150rpm, the ventilation volume is 0.6vvm, the temperature is 30 ℃, and the culture time is 16-24 h.
And (4) the tangential flow microfiltration system in the step (3) is a PALL tangential flow microfiltration system, and the tangential flow microfiltration system is adopted to filter and remove the fermentation liquor so as to achieve the purpose of concentrating the fermentation liquor and obtaining high-concentration thallus.
The flow rate of the tangential flow microfiltration system in the step (3) is 1.0L-2L/min.
And (4) concentrating the fermentation liquor in the step (3) until the volume of the fermentation liquor is 10-20%.
The adding amount of the total ginsenoside extracting solution in the step (4) is calculated according to 35-60% of the mass of the concentrated fermentation liquor; preferably 45 to 55 percent of the mass of the concentrated fermentation liquor.
The biotransformation conditions in the step (4) are as follows: reacting for 2-10 days under the conditions of pH5.5-6.5 and reaction temperature of 35-55 ℃; more preferably: reacting for 3-7 days under the conditions of pH5.5-6.5 and the reaction temperature of 40-50 ℃.
The rare saponin in the ginseng total saponin aqueous solution in the step (4) is one or more of Rg3, CK, F2 and Rh 2; preferably the rare saponins Rg3, CK, F2 and Rh 2.
The method for improving the content of rare saponin in the total ginsenoside by utilizing the whole-cell catalysis of acetobacter comprises the step of removing macromolecules such as thalli, enzyme and the like after the step (4); the method specifically comprises the following steps: and (3) removing macromolecules such as thalli, enzyme and the like from the total ginsenoside aqueous solution containing rare saponin obtained in the step (4) by adopting a tangential flow ultrafiltration system, wherein the tangential flow ultrafiltration system adopts a circulating ultrafiltration process and uses a microfiltration bag to control the flow rate in stages: in the first stage, the flow rate is 0.5-0.8L/min, and the ultrafiltration time is calculated according to the total reaction liquid volume (L)/0.3min (namely ultrafiltration time (min) ═ total reaction liquid volume (L) ÷ 0.3 (L/min)); the flow rate of the second stage is 0.3-0.5L/min, and the time is the total volume (L)/0.5min of the reaction solution.
The tangential flow ultrafiltration system is a PALL tangential flow ultrafiltration system.
The method for improving the content of rare saponin in the total ginsenoside by utilizing the catalysis of acetobacter whole cells is applied to the improvement of the content of rare saponin in the total ginsenoside.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention aims to overcome the defects and shortcomings of the prior art and the method, reduce the production cost, provide a method for improving the content of rare ginsenoside through whole-cell catalysis, and particularly relates to the method for improving the content of rare saponin through whole-cell transformation of total ginsenoside by high-concentration acetobacter aceti. Firstly, decocting and extracting a ginseng water extracting solution by an extraction tank, primarily purifying by macroporous resin, and removing macromolecular polysaccharide and strong-polarity micromolecular compounds to obtain a ginseng total saponin extracting solution; secondly, obtaining thallus whole cells through high-density fermentation (controlling feeding and pH double-flow feeding fermentation), removing most of fermentation liquor enriched thallus by using a fermentation tank coupled tangential flow microfiltration system, refluxing the enriched thallus to the fermentation tank, adding a total saponin extract into the fermentation tank to obtain a high-concentration whole cell catalytic system, and performing biotransformation under certain conditions; after the conversion is finished, macromolecules such as thallus, enzyme and the like are removed through a coupled tangential flow ultrafiltration system, and the content of rare saponin in the obtained total ginsenoside is greatly improved. The invention is characterized in that the concentration and enrichment of the bacteria in the fermentation liquor and the removal of the macromolecules such as the bacteria and the enzyme after the biotransformation are finished by the operation of a filtering system, the filtering is divided into microfiltration and ultrafiltration according to the size of the aperture, and the content of rare saponin in the total ginsenoside extracting solution can be greatly improved by a fermentation tank or an extraction tank and a tangential flow filtering (including microfiltration and ultrafiltration) system. Has great value for meeting the market demands of wide food, medical treatment and health care.
2. The content of 4 rare saponins in the total ginsenoside water solution prepared by the method is higher, wherein the content of Rg3 and CK is at least improved by 268 and 182 times, and F2 and Rh2 are rare saponins not contained in the original extracting solution. In the prior art, the content of rare saponin can be greatly improved through fermentation, biotransformation and some concentration steps: for example, in patent CN102366373A, the content of some rare saponins is increased by performing biotransformation on the ginseng extract by using the filtered supernatant (enzyme solution) of lactobacillus bulgaricus fermentation broth, wherein the content of rare saponins Rg3, Rh2 and CK is only increased by 16.89, 4.59 and 26.17 times, which is far lower than that of the invention.
3. The method has the advantages of simple raw material source and treatment, mild conditions, direct fermentation of the whole-cell catalyst, no need of further purification, coupling of the fermentation, concentration and purification processes, shortening of the preparation time, greatly improved content of 4 rare saponins in the total saponins of panax ginseng, and capability of meeting the application of different requirements of the total saponins of panax ginseng. The enzyme activity of the effective enzyme is improved and the conversion efficiency is improved by concentrating and enriching the leavening agent and the conversion substrate.
4. The method can be used for preparing the total ginsenoside with high content of rare saponin in a large scale with high efficiency and environmental protection. In the method, edible safety fungi are adopted, and a whole-cell catalyst is obtained through high-density fermentation in a full-automatic fermentation tank, so that the water extract of the total ginsenoside is converted, and the content of rare saponin is improved.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
In the following examples, the filtration system used is a PALL tangential flow filtration system, which can be assembled with different filtration elements (microfiltration and ultrafiltration elements) to perform different functions, and is divided into microfiltration and ultrafiltration (i.e. PALL tangential flow microfiltration system and PALL tangential flow ultrafiltration system) according to the pore size, wherein the microfiltration column used is a PALL hollow fiber membrane column UMP-1147R membrane column, and the ultrafiltration bag is a PALL T series membrane OS030T 12.
In the following examples, sun-dried ginseng was purchased from Jilin plus Earth products Ltd; the macroporous adsorption resin AB-8 is purchased from Shanghai leaf Biotech limited; concentrated apple juice was purchased from Qin' an great wall juice beverage, Inc.
The method has universality on Acetobacter, and the Acetobacter (Acetobacter pasteurianus) used in the examples is purchased from Guangdong province microorganism strain collection center with the collection number of GIM 1.67.
HPLC-UV detection is adopted for the four rare ginsenoside standards, and the peak emergence time of the 4 rare saponin standards is shown in Table 1. The 4 rare ginsenoside standard substances are purchased from Shanghai leaf Biotech limited. The following invention will be described in detail with reference to the following examples, which are not intended to be limiting, and the examples are generally conducted under the conventional conditions or under the conditions recommended by the manufacturers. Proportions and percentages are by weight unless otherwise indicated.
TABLE 1 time to peak of four saponin standards
| Serial number | Rare ginsenoside | Time to peak |
| 1 | F2 | 43.200 |
| 2 | Rg3 | 46.811 |
| 3 | CK | 59.825 |
| 4 | Rh2 | 65.179 |
Example 1
1. The preparation method of the total ginsenoside comprises the following steps:
(1) the extraction method of the total ginsenoside comprises the following steps: crushing sun-dried ginseng, sieving with a 80-mesh sieve, decocting and extracting in an extraction tank, wherein the mass ratio of ginseng powder to water is 1:8, the extraction temperature is 95 ℃, the extraction time is 1h, the extraction times are 5 times, combining the extracting solutions, and concentrating under reduced pressure to 10% of the volume of the stock solution to obtain the ginseng extracting solution.
(2) Primary purification of the ginseng extract by macroporous resin: loading the mixture into a macroporous adsorption resin AB-8 with the volume of 60% of that of a chromatographic column with the thickness of 45mm by 80cm, loading the sample by a wet method, washing impurities by distilled water, 10% (w/w) ethanol solution and 20% (w/w) ethanol solution in sequence, carrying out gradient elution by 30-95% (w/w) ethanol solution, and collecting eluent; concentrating the eluate to 20% (v/v) to obtain Ginseng radix total saponin extract.
2. High-density feed fermentation of acetobacter:
(1) and (3) activation of thalli: streaking and inoculating the acetobacter strains preserved on the inclined plane to a solid agar culture medium for culturing, and culturing at 30 ℃ for 24h to realize thallus activation; wherein the agar culture medium comprises the following components: 1% of yeast extract, 2% of glucose, 2% of agar, 4% of ethanol and 5.5% of pHs, wherein the percentages are mass percentages, and the balance is water;
(2) and (3) shake flask culture: selecting a single colony on the solid culture medium in the step (1) to be inoculated on the culture medium in a shake flask for shake culture, and obtaining a seed solution; the culture conditions are as follows: shaking at 100rpm at 30 deg.C for 16 h; the formula (mass percent) of the culture medium of the shake flask is as follows: 1% of yeast extract, 1% of glucose and KH2P040.3 percent of ethanol and 2 percent of ethanol;
(3) expanding culture in a seeding tank: inoculating the seed liquid obtained in the step (2) into a culture medium in a seed tank according to the inoculation amount of 5% (v/v) for culture; the culture conditions are as follows: stirring at 150rpm, ventilating amount of 0.6vvm, temperature of 30 deg.C, adding 8M sodium hydroxide solution, controlling pH of the culture solution at 5.5, and culturing for 10 hr; the culture medium of the seeding tank comprises the following components in percentage by mass: 2% of yeast extract, 2% of glucose and KH2P040.3 percent of ethanol and 2 percent of ethanol; the volume of the seeding tank is 10L, and the liquid filling amount is 70%;
(4) fermentation in a fermentation tank: inoculating the seed solution obtained in step (3) into a culture medium in a fermentation tank according to the inoculation amount of 5% (v/v), culturing, adding 10g/L concentrated apple juice (mass of the concentrated apple juice/volume of the fermentation liquid in the fermentation tank) in the fermentation process, adding 10 hours after the fermentation is started, culturing for 16 hours, and obtaining the fermentation liquid with the thallus density of 1.73 × 109(cfu/mL)。
3. And (3) thallus enrichment: and (3) concentrating the fermentation liquor obtained in the fermentation tank in the step (4) to 20% (v/v) by using a PALL tangential flow microfiltration system, wherein the flow rate is 1.0L/min. The fermentation liquor in the microfiltration process is sterile through pipelines. The thalli are enriched and then flow back to a seeding tank.
4. And (3) biotransformation: adding the obtained Ginseng radix total saponin extractive solution into concentrated fermentation broth, wherein the addition amount of Ginseng radix total saponin extractive solution is 55% of the fermentation broth, pH is controlled at 5.5, reaction temperature is 50 deg.C, and reaction time is 7 days (intermittent stirring can be adopted for reaction).
5. Removing macromolecules such as thalli and enzyme: after the conversion is finished, macromolecules such as thalli and enzyme are removed by using a PALL tangential flow ultrafiltration system, the ultrafiltration system is a circulating ultrafiltration process, and the flow rate is controlled by using a microfiltration bag in stages: the flow rate of the first stage is 0.8L/min, the ultrafiltration time is total reaction liquid volume (L)/0.3min, and the flow rate of the second stage is 0.5L/min, and the time is total reaction liquid volume (L)/0.5 min.
6. Separation and detection of rare saponins: detecting the content of rare saponin in the total ginsenoside water solution after transforming and removing macromolecules such as thallus and enzyme by HPLC-UV (high performance liquid chromatography-ultraviolet detection), specifically:
taking 5mL of the concentrated solution obtained in the step 5, extracting the ginseng fermentation liquor for 4 times by using a water saturated n-butyl alcohol solution with the volume being 3 times that of the concentrated solution, combining organic phases, carrying out reduced pressure rotary evaporation to obtain a crude product of ginsenoside, dissolving the crude product of ginsenoside by using 2mL of chromatographic pure methanol, and detecting the concentration of rare saponin by using HPLC-UV; meanwhile, the total ginsenoside extract (crude extract) obtained in step 1 of this example was used as a control, and the rest of the steps were the same. The results are shown in Table 2.
TABLE 2 rare saponin content and fold increase (μ g/mL)
The content of 4 rare saponins in the total ginsenoside water solution prepared by the embodiment is higher, wherein the content of Rg3 and CK is increased by 275 and 182 times, and F2 and Rh2 are rare saponins not contained in the original extracting solution.
Example 2
1. The preparation method of the total ginsenoside comprises the following steps:
(1) the extraction method of the total ginsenoside comprises the following steps: crushing sun-dried ginseng, sieving with a 60-mesh sieve, extracting in an extraction tank at the extraction temperature of 95 ℃ for 4 times at the material-liquid ratio of 1:10 for 1.5h, combining the extracting solutions, and concentrating under reduced pressure to 15% of the volume of the original solution to obtain the ginseng extracting solution.
(2) Primary purification of the ginseng extract by macroporous resin:
loading the mixture into a macroporous adsorption resin AB-8 with the volume of 60% of that of a chromatographic column with the thickness of 45mm by 80cm, loading the sample by a wet method, washing impurities by distilled water, 10% (w/w) ethanol solution and 20% (w/w) ethanol solution in sequence, carrying out gradient elution by 30-95% (w/w) ethanol solution, and collecting eluent; concentrating the eluate to 15% (v/v) to obtain Ginseng radix total saponin extract.
2. High-density feed fermentation of acetobacter:
(1) the procedures of thallus activation and shake flask culture are the same as those in example 1;
(2) expanding culture in a seeding tank: inoculating the fermentation liquor obtained by shake flask culture into a seeding tank according to the inoculation amount of 5% (v/v) for culture; the culture conditions are as follows: adding 6M sodium hydroxide solution, controlling pH of the culture solution to 5.0, and culturing under the same conditions as in example 1; the medium composition of the seeding tank was the same as in example 1;
(3) fermentation in a fermentation tank: inoculating the seed liquid obtained by expanding culture of the seed tank into a culture medium in a fermentation tank according to the inoculation amount of 5% (v/v) for culture, feeding 15g/L of concentrated apple juice (the mass of the concentrated apple juice/the volume of fermentation liquid in the fermentation tank) in the fermentation process, finishing feeding 16 hours after the start of fermentation, culturing for 20 hours, and expanding culture of other culture media and culture conditions with the seed tank; the obtained fermentation liquid has a cell density of 1.88 × 109(cfu/mL)。
3. And (3) thallus enrichment: the fermentation broth from the fermentor was concentrated to 15% (v/v) using a PALL tangential flow microfiltration system at a flow rate of 1.5L/min. The fermentation liquor in the microfiltration process is sterile through pipelines. The thalli are enriched and then flow back to a seeding tank.
4. And (3) biotransformation: adding the obtained Ginseng radix total saponin extractive solution into concentrated fermentation broth, wherein the addition amount of Ginseng radix total saponin extractive solution is 50% of the mass of the fermentation broth, pH is controlled at 6.0, reaction temperature is 45 deg.C, and reaction time is 5 days (intermittent stirring can be adopted for reaction).
5. Removing macromolecules such as thalli and enzyme: after the conversion is finished, macromolecules such as thalli and enzyme are removed by using a PALL tangential flow ultrafiltration system, the ultrafiltration system is a circulating ultrafiltration process, and the flow rate is controlled by using a microfiltration bag in stages: the flow rate of the first stage is 0.6L/min, the ultrafiltration time is total reaction liquid volume (L)/0.3min, and the flow rate of the second stage is 0.4L/min, and the ultrafiltration time is total reaction liquid volume (L)/0.5 min.
2. The content of rare saponins in the total ginsenoside aqueous solution after conversion and removal of macromolecules such as thallus and enzyme was measured by HPLC-UV, and the total ginsenoside extract (crude extract) obtained in step 1 of this example was used as a control. The detection method is the same as that of step 6 of example 1. The results are shown in Table 3.
TABLE 3 rare saponin content and fold increase (μ g/mL)
The content of 4 rare saponins in the total ginsenoside water solution prepared by the embodiment is higher, wherein the content of Rg3 and CK is improved by 310 and 251 times, and F2 and Rh2 are rare saponins not contained in the original extracting solution.
Example 3
1. The preparation method of the total ginsenoside comprises the following steps:
(1) the extraction method of the total ginsenoside comprises the following steps: sun-dried ginseng is smashed and sieved by a 40-mesh sieve, and is extracted in an extraction tank, the material-liquid ratio is 1:15, the extraction temperature is 95 ℃, the extraction time is 1.5h, and the extraction times are 3 times. Mixing extractive solutions, and concentrating under reduced pressure to 20% of stock solution to obtain Ginseng radix extractive solution.
(2) Primary purification of the ginseng extract by macroporous resin: loading the mixture into a macroporous adsorption resin AB-8 with the volume of 60% of that of a chromatographic column with the thickness of 45mm by 80cm, loading the sample by a wet method, washing impurities by distilled water, 10% (w/w) ethanol solution and 20% (w/w) ethanol solution in sequence, carrying out gradient elution by 30-95% (w/w) ethanol solution, and collecting eluent; concentrating the eluate to 25% (v/v) to obtain Ginseng radix total saponin extract.
2. High-density feed fermentation of acetobacter:
(1) the procedures of thallus activation and shake flask culture are the same as those in example 1;
(2) expanding culture in a seeding tank: inoculating the fermentation liquor obtained by shake flask culture into a seeding tank according to the inoculation amount of 5% (v/v) for culture; the culture conditions are as follows: adding 4M sodium hydroxide solution, controlling pH of the culture solution to 4.0, and culturing under the same conditions as in example 1; the medium composition of the seeding tank was the same as in example 1;
(3) fermentation in a fermentation tank: inoculating the seed liquid obtained by expanding culture of the seed tank into a culture medium in a fermentation tank according to the inoculation amount of 5% (v/v) for culture, feeding 20g/L of concentrated apple juice (mass of the concentrated apple juice/volume of fermentation liquid in the fermentation tank) in the fermentation process, finishing feeding 20 hours after the start of fermentation, culturing for 24 hours, and expanding culture of other culture media and culture conditions with the seed tank; the obtained fermentation liquid has a cell density of 1.63 × 109(cfu/mL)。
3. And (3) thallus enrichment: the fermentation broth from the fermentor was concentrated to 10% (v/v) using a PALL tangential flow microfiltration system at a flow rate of 2.0L/min. The fermentation liquor in the microfiltration process is sterile through pipelines. The thalli are enriched and then flow back to a seeding tank.
4. And (3) biotransformation: adding the obtained Ginseng radix total saponin extractive solution into concentrated fermentation broth, controlling pH at 6.5 and 40 deg.C for 3 days (intermittent stirring can be used for reaction).
5. Removing macromolecules such as thalli and enzyme: after the conversion is finished, macromolecules such as thalli and enzyme are removed by using a PALL tangential flow ultrafiltration system, the ultrafiltration system is a circulating ultrafiltration process, and the flow rate is controlled by using a microfiltration bag in stages: the flow rate of the first stage is 0.5L/min, the ultrafiltration time is total reaction liquid volume (L)/0.3min, and the flow rate of the second stage is 0.3L/min, and the ultrafiltration time is total reaction liquid volume (L)/0.5 min.
3. The content of rare saponins in the aqueous solution of total saponins of panax ginseng after conversion and removal of macromolecules such as thallus and enzyme was measured by HPLC-UV, and the total saponins of panax ginseng extract (crude extract) obtained in step 1 of this example was used as a control. The detection method is the same as that of step 6 of example 1. The results are shown in Table 4.
TABLE 4 rare saponin content and fold increase (μ g/mL)
The content of 4 rare saponins in the total ginsenoside water solution prepared by the embodiment is higher, wherein the content of Rg3 and CK is increased by 268 and 208 times, and F2 and Rh2 are rare saponins not contained in the original extracting solution.
Comparative example 1
1. The preparation method of the ginseng total saponin extract is the same as that of example 1.
2. High-density feed fermentation of acetobacter:
(1) the steps of thallus activation, shake flask culture and seeding tank propagation are the same as example 1;
(2) fermentation in a fermentation tank: inoculating the seed liquid obtained by expanding culture in the seed tank into the culture medium in the fermentation tank according to the inoculation amount of 5% (v/v), culturing, adding 10g/L concentrated apple juice (mass of the concentrated apple juice/volume of the fermentation liquid in the fermentation tank) in the fermentation process, adding 10 hours after the fermentation is started, culturing for 16 hours, and obtaining the fermentation liquid with the thallus density of 1.69 multiplied by 10 by the culture condition in the same step (3)9(cfu/mL)。
3. And (3) thallus enrichment: and (3) discharging the fermentation liquor obtained in the step (2) from the fermentation tank, centrifuging to remove 80% (volume) of the fermentation liquor to obtain 20% (volume) of concentrated liquor containing the bacteria of the original fermentation liquor, and filling the concentrated fermentation liquor into a seed tank.
4. And (3) biotransformation: and (2) adding the ginseng total saponin extracting solution obtained in the step (1) into concentrated fermentation liquor, wherein the adding amount of the ginseng total saponin extracting solution accounts for 60% of the mass of the fermentation liquor, the pH value is controlled to be 5.5, the reaction temperature is 50 ℃, and the reaction time is 7 days (the reaction can be carried out by adopting an intermittent stirring mode).
5. Removing macromolecules such as thalli and enzyme: after the conversion is finished, macromolecules such as thalli and enzyme are removed by using a PALL tangential flow ultrafiltration system, the ultrafiltration system is a circulating ultrafiltration process, and the flow rate is controlled by using a microfiltration bag in stages: the flow rate of the first stage is 0.8L/min, the ultrafiltration time is total reaction liquid volume (L)/0.3min, and the flow rate of the second stage is 0.5L/min, and the time is total reaction liquid volume (L)/0.5 min.
6. The content of rare saponins in the aqueous solution of total saponins of panax ginseng after conversion and removal of macromolecules such as thallus and enzyme was measured by HPLC-UV, and the total saponins of panax ginseng extract (crude extract) obtained in step 1 of this example was used as a control. The detection method is the same as that of step 6 of example 1. The results are shown in Table 5.
TABLE 5 rare saponin content and fold increase (μ g/mL)
Comparative example 2
1. The preparation method of the ginseng total saponin extract is the same as that of example 3.
2. High-density feed fermentation and biotransformation of acetobacter:
(1) the steps of thallus activation, shake flask culture and seeding tank propagation are the same as example 1;
(2) fermentation in a fermentation tank and biotransformation: inoculating the seed liquid obtained by expanding culture of the seed tank into a fermentation tank according to the inoculation amount of 5% (v/v), adding 6% (v/v) of the total ginsenoside extracting solution prepared in the step 1 into the fermentation tank for fermentation culture, feeding 10g/L of concentrated apple juice (mass of the concentrated apple juice/volume of fermentation liquid in the fermentation tank) in the fermentation process, feeding the concentrated apple juice 10 hours after the start of fermentation, and culturing for 16 hours, wherein other culture mediums and culture conditions are the same as those in the example 3. The thallus density of the fermentation liquor is 1.52 multiplied by 10 at most in the fermentation process9(cfu/mL)。
3. Concentration and impurity removal:
(1) after the conversion is finished, macromolecules such as thalli and enzyme are removed by using a PALL tangential flow ultrafiltration system, the ultrafiltration system is a circulating ultrafiltration process, and the flow rate is controlled by using a microfiltration bag in stages: the flow rate of the first stage is 0.8L/min, the ultrafiltration time is total reaction liquid volume (L)/0.3min, and the flow rate of the second stage is 0.5L/min, and the time is total reaction liquid volume (L)/0.5 min.
(2) Concentrating the fermentation liquid without macromolecules such as thallus and enzyme to 15% of the original fermentation liquid volume to obtain the converted total ginsenoside water solution.
4. The content of rare saponins in the concentrated total ginsenoside aqueous solution was measured by HPLC-UV, and the total ginsenoside extract (crude extract) obtained in step 1 of this example was used as a control. The detection method is the same as that of step 6 of example 1. The results are shown in Table 6.
TABLE 6 rare saponin content and fold increase (μ g/mL)
In comparative examples 1 and 2, the conventional centrifugal concentrated thalli and the non-concentrated thalli are used for direct fermentation, and the obtained total ginsenoside aqueous solution has lower contents of 4 rare saponins and lower biotransformation efficiency compared with the total ginsenoside aqueous solution prepared in the embodiment of the invention.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (2)
1. A method for improving the content of rare saponin in total ginsenoside by utilizing the catalysis of acetobacter whole cells is characterized by comprising the following steps:
(1) extracting the total saponins of ginseng: crushing and sieving white ginseng, and then adding a solvent for extraction to obtain an extracting solution; concentrating the extractive solution to obtain concentrated Ginseng radix extractive solution;
(2) and (3) macroporous resin purification: adding the concentrated ginseng extract obtained in the step (1) into macroporous resin, washing off impurities, performing gradient elution, collecting eluent, and concentrating the eluent to obtain a ginseng total saponin extract;
(3) preparing a high-concentration acetobacter whole-cell catalytic system: fermenting Acetobacter by high density fermentation method to obtain fermentation liquid with thallus concentration of 1.5 × 109Filtering and concentrating the obtained fermentation liquor by a tangential flow microfiltration system to obtain concentrated fermentation liquor, wherein the cfu/mL is more than the cfu/mL;
(4) and (3) biotransformation: adding the ginseng total saponin extracting solution obtained in the step (2) into the fermentation liquor concentrated in the step (3) for biotransformation to obtain a ginseng total saponin aqueous solution containing rare saponins;
the white ginseng in the step (1) is sun-dried ginseng;
sieving in the step (1) is to pass through a sieve of 40-80 meshes;
the solvent in the step (1) is water;
concentrating the extract in the step (1) to 10-20% of the volume of the extract;
the extraction conditions in the step (1) are as follows: the ratio of the material to the liquid is 1: 5-20, the extraction temperature is 95 ℃, the extraction time is 1-3.0 h, and the extraction times are 2-6;
the macroporous resin in the step (2) is macroporous resin AB-8;
the step (2) of washing away impurities is to wash away impurities by using distilled water and ethanol solution in sequence;
the gradient elution in the step (2) is gradient elution by using 30-95% (w/w) ethanol solution;
concentrating the eluent in the step (2) to 10-35% of the volume of the eluent;
the Acetobacter in the step (3) is Acetobacter pasteurianus GIM 1.67;
concentrating in the step (3) to 10-20% of the volume of the fermentation liquor;
the tangential flow microfiltration system in the step (3) is a PALL tangential flow microfiltration system;
the flow rate of the tangential flow microfiltration system in the step (3) is 1.0L-2L/min;
the high-density fermentation method in the step (3) is realized by the following steps:
(I) inoculating acetobacter aceti to a solid agar culture medium for activation culture by streaking to obtain activated acetobacter aceti;
(II) inoculating the activated acetobacter into a culture medium in a shake flask for shake culture to obtain a seed solution A;
(III) inoculating the seed solution A to a culture medium in a seed tank for culture, and controlling the pH value of a culture system to be 4.0-5.5 to obtain a seed solution B;
(IV) inoculating the seed liquid B to a culture medium in a fermentation tank for fermentation culture, controlling the pH value of the fermentation liquid to be 4.0-5.5 in the fermentation process, and simultaneously feeding concentrated apple juice to obtain the fermentation liquid;
the solid agar culture medium in the step (I) consists of the following components in percentage by mass: 1% of yeast extract, 2% of glucose, 2% of agar, 4% of ethanol and the balance of water, wherein the pH value is 5.5;
the culture medium in the step (II) consists of the following components in percentage by mass: 1% of yeast extract, 1% of glucose and KH2P04 0.3 percent of ethanol and 2 percent of ethanol;
the culture conditions in step (III) are as follows: stirring at 150rpm, ventilation rate of 0.6vvm, temperature of 30 deg.C, and culture time of 10 h;
the culture medium in the steps (III) and (IV) consists of the following components in percentage by mass: 2% of yeast extract, 2% of glucose and KH2P040.3 percent of ethanol and 2 percent of ethanol;
controlling the pH value of the culture system in the steps (III) and (IV) to be 4.0-5.5 by feeding sodium hydroxide;
the dosage of the concentrated apple juice in the step (IV) is calculated according to the proportion of 10-25 g of concentrated apple juice per liter of fermentation liquor, and the concentrated apple juice is added after 8-20 hours after the fermentation is started;
the culture conditions in step (IV) are as follows: stirring at the rotating speed of 150rpm, ventilation volume of 0.6vvm, temperature of 30 ℃ and culture time of 12-24 h;
the biotransformation conditions in the step (4) are as follows: reacting for 2-10 days under the conditions of pH5.5-6.5 and reaction temperature of 35-55 ℃;
the adding amount of the total ginsenoside extracting solution in the step (4) is calculated according to 35-60% of the mass of the concentrated fermentation liquor;
the method for improving the content of rare saponin in the total ginsenoside by utilizing the whole-cell catalysis of acetobacter comprises the step of removing thallus and enzyme after the step (4); the method specifically comprises the following steps:
and (3) removing thalli and enzyme from the total ginsenoside aqueous solution containing rare saponin obtained in the step (4) by using a tangential flow ultrafiltration system, wherein the tangential flow ultrafiltration system adopts a circulating ultrafiltration process and uses a microfiltration bag to control the flow rate in stages: the flow rate of the first stage is 0.5-0.8L/min, and the ultrafiltration time is the total reaction liquid volume/0.3 min; the flow rate of the second stage is 0.3-0.5L/min, and the time is 0.5 min/volume of the total reaction liquid.
2. The use of the method for increasing the content of rare saponins in total saponins of panax ginseng using acetobacter whole-cell catalysis as claimed in claim 1 for increasing the content of rare saponins in total saponins of panax ginseng.
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