CN106978466A - A kind of preparation method of active ginseng saponin(e - Google Patents
A kind of preparation method of active ginseng saponin(e Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of active ginseng saponin(e.Including handling ginseng by vapours, then squeezed using ethanol, ginsenoside extracted, to prepare active ginseng saponin constituent.Utilize two kinds of temperature steps, two kinds of bacterial strains (Mo Hawei bacillus KJS 3 and poly- ferment bacillus KJS 2) of (45 DEG C and 55 DEG C) processing successively during the fermentation.Centrifugation gained saponin(e solution, separates and retains supernatant.Thing is staticly settled again using ethanol, is then filtered.Filtrate is concentrated, and freezes dried and obtains active ginseng saponin(e.The inventive method can prepare the CK containing compound of high yield active saponin.The saponin(e produced by fermenting can be used among food.
Description
Technical field
The present invention relates to a kind of preparation method of active ginseng saponin(e.It is contemplated that batch produces active ginseng.Activity
Ginseng has higher absorbability in human body.The present invention is also related to the method for food production.
Background technology
Ginseng means Korean ginseng, wild ginseng, domestication wild ginseng, Chinese ginseng (pseudo-ginseng ginseng), the West in the present invention
Ginseng, one kind in sun-dried ginseng, two or more ginseng combination.
Ginsenoside and non-saponin(e material include panacen, polysaccharide, amino acid derivativges and phenolic compound.The material has
There is good pharmacological activity.To being played an important role in terms of removing free radical, antitumor, blood pressure control, lipid-loweringing and anti-hepatic lesion.
Ginsenoside is divided into two classes：PPD (protopanoxadiol) and PPT (protopanaxatriol).PPD type saponin(es are in basic structure
There are a variety of different substituents groups on PPD, typically there is Rb1, Rb2, Rc, Rd, F2, Rg3 and ginsenoside compound K etc..PPT types
Saponin(e includes Re, Rf, Rg1 and Rh1 using PPT as basic structure.
In general, many in nature to contain saccharide compound, the product (aglycone) after glycolysis shows enhancing
Bioactivity (Med.Pharm.Soc.1992,9：1-13).If ginsenoside containing carbohydrate side chains (ginsenoside Rb1, Rb2,
Rb and Re) occur to hydrolyze and form ginseng sapoglycoside Rg 3, Rh1, Rh2, F2, CY and CK, they can produce more preferably effect in vivo
(Ginseng Res.2003,27：129-134).
Ginsenoside is referred to as inactive saponin(e, is a kind of carbohydrate complexes, only minimal amount of inactive saponin(e
Absorbed in small intestine and enter internal.Among the enteric microorganism extracted in human feces, the water on its ginsenoside Rb1
Solution ability test result indicates that, 21% enteric microorganism does not have hydrolysis ability.And it is existing it has proven convenient that 70% has certain journey
The enteric microorganism of the hydrolysis of degree, has very big difference (Planta in the ability for decomposing ginsenoside
Medica.1998,64：696-700).
General Ginseng Products are made up of the ginsenoside of highly-water-soluble (nonactive saponin(e component), but this height is water-soluble
The composition of property and low intestinal absorption is unhelpful to human body.But, if active component is changed into water insoluble, its absorbability and have
Effect property is also enhanced.
The saponin(e of the present invention or the property of processing of Panax ginseng product mainly have：
By making carbohydrate be removed from saponin(e, the active component in the present invention improves the oral absorption in human body (with day
Right ginseng is compared).Ginsenoside Rb1, Rb2, Rd and Re are coupled with the monose of three or more than three.The present invention passes through carbohydrate
Hydrolysis, generation ginseng sapoglycoside Rg 3, Rh1, Rh2, F2, CY and CK.Rg3, Rh1, Rh2, F2, CY and CK are that typically have
The saponin(e of higher level effect, i.e., the active ginseng saponin(e for having more preferable absorbability or physiologically active in human body.
Red ginseng is the Typical Representative for activating ginseng, refers to process the people of six hours in Modern Significance under the conditions of 90 DEG C
Ginseng.Some active components (especially compound CK) are that (compound CK is not deposit in nature by the generation of some enteric bacteria
).And compound CK is difficult to synthesize.However, the method for producing compound CK using strain fermentation is still not mature enough.Typically
For, compared with enzyme process is changed, the output of strain fermentation is very low.But, fermenting and producing is more likely produced than enzyme process conversion
Go out various active saponins.
Lactic acid bacteria is difficult to ferment under the conditions of 37 DEG C or so, and its fermentation temperature should be not less than 42 DEG C.High temperature changes bacterial strain
The tertiary structure of producing enzyme, therefore the enzyme can not play a role.When temperature raises 10 DEG C, the ability of enzyme is also improved twice therewith.
But high temperature is also unfavorable for bacterial strain existence.Therefore present inventor uses high temperature resistant, can produce the bacterial strain of a variety of enzymes.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of side of producing of the active ginseng saponin(e with more preferable effect
Method, the saponin(e in vivo has more preferable absorbability or physiologically active.
The fermentation temperature that the present invention is used is higher than 42 DEG C.The temperature is unfavorable for the growth of general bacterium.The present invention is from ginseng
Additional sugar is isolated in saponin(e, the active component of the forms such as compound CK, Rd, Rg3 is then obtained using fermentation process.
The present invention realizes that the technical scheme of above-mentioned target comprises the following steps：
1) steam heating ginseng is used；
2) crush, extract ginseng using ethanol, obtain ginsenoside extract solution；
3) Mo Hawei bacillus KJS-3 (Moja-3) nutrient solutions and poly- ferment bacillus KJS-2 (Bp-2) are cultivated
Liquid, which is added in ginsenoside extract solution, to ferment；
4) the ginsenoside solution after centrifugation fermentation, separates and retains supernatant；Sediment is reclaimed, is redissolved using ethanol,
Filtering, collects filtrate；
5) after filtrate is concentrated, freeze-drying obtains active ginseng saponin(e；Supernatant continues after cultivating, and concentration, freezing is dry
It is dry, also obtain active ginseng saponin(e.
In such scheme, step 3) fermentation temperature in the range of 42 DEG C -50 DEG C；As a preferred embodiment, fermentation temperature is
45℃。
Step 5) in, supernatant liquor fermentation temperature is at 45~60 DEG C.As a preferred embodiment, fermentation temperature is 55 DEG C.
Step 1) in, processing of Panax ginseng temperature is 110 DEG C, and steam pressure is 1.1kg/cm2, the duration is 10 minutes.Weight
It is multiple three times.
Step 2) in, the ginseng chopping after heat treatment is put to high speed agitator and stirred, 80vol% alcohol is then statically placed in
In, extract saponin(e component.
The present invention has the following effects that.
Active saponin composition is prepared by heat treatment.Extract after saponin(e, during the fermentation, using two kinds of bacterial strains, successively
Using two kinds of Temperature Treatments, the CK containing compound of high yield active saponin can be prepared.Because active material is by fermenting
Produce, can be used in food.
Embodiment
Way to solve the problem of the present invention will be described in detail in applicant.The present invention will not be discussed in detail related to known technology
Details, in order to avoid obscure present subject matter.
In general, under conditions of the secondary structure for ensureing enzyme is not destroyed, production of enzyme of the bacterial strain when increasing by 10 DEG C
It is original twice.
Bacterial strain of the present invention, which is found under the optimum growth temperature higher than common bacterium, to be grown very well.By a large amount of
Research find, Mo Hawei bacillus KJS-3 (Moja-3, August is preserved in Korean Culture Center on 22nd within 2008,
Deposit number is KCCM 10961P.The growth conditions of the bacterium is：20~50 DEG C, pH=6~7.5).In 42-45 DEG C of temperature range
Inside show maximum growth amount.We, which also select, growth temperature is likely to be breached 60 DEG C of bp-2 (2006.08.16 is preserved in Korea Spro
State's Organism Depositary, deposit number is KCCM10769P).Bp-2 most suitable growth temperature is 45 DEG C to 55 DEG C, and fermentation is most
High-temperature is 55 DEG C.Under the temperature conditionss, most of bacterium can not grow, so as to prevent the midway suppression caused by living contaminants
System.
The present invention hereinafter will be described in detail according to embodiment.
In the heat treatment process of the present invention, specifically, processing of Panax ginseng temperature is 110 DEG C, and steam pressure is 1.1kg/
cm2, the duration is 10 minutes.In triplicate.
Then chopping is put to high speed agitator, is then statically placed in 80vol% alcohol, extracts saponin(e component.Protoplast's ginseng two
Than protopanaxatriol, ((PPT) is easier to extract in 80% ethanol solution to obtain alcohol (PPD).Concentrate after saponin extract solution, will
It is multiple soluble in water.This uses the raw material as active PPD.
The fermentation liquid of a certain amount of Moja-3, Bp-2 bacterial strain is injected into PPD suspension using peristaltic pump.The hair
Zymotic fluid is used for the carbohydrate side chains for decomposing saponin(e, then generates the activation PPD solution of the CK containing compound.
If not a small amount of raw material is added dropwise, reaction can be prevented from occurring very well, to cause yield relatively low.Therefore, originally
Invention is the zymotic fluid that the reaction raw materials of scheduled volume are injected into Moja3, Bp2 bacterial strain by peristaltic pump, after fermentation, is prepared containing work
The PPD solution of property component, inclusion compounds CK (i.e. CKS solution).
After the completion of CKS solution centrifugals, thing is staticly settled again in ethanol, filter, filtrate centralized recovery CKS solution.
The activity PPD's of embodiment 1 produces
The porous shelf that ginseng is loaded in reative cell.By automatic temperature-adjusting and humidity conditioner, it is passed through to reative cell
Steam, excludes air, is directly heat-treated by steam, steam pressure 1.1kg/cm2.121 DEG C of keeping temperature, 10 points of time
Clock, removes the unnecessary moisture of ginseng.
Heat treated ginseng is cooled to less than 90 DEG C, air bleeding valve injection air is opened, continues to repeat operation two above
It is secondary.
Heat treated ginseng is milled to the particle that particle diameter is no more than 1 millimeter, 80vol% ethanol is added after drying, plus
It is hot fully to extract ginsenoside.More than 500 microns of solid particle is filtered out by filter paper, the ethanol through filter paper is collected molten
Liquid, is referred to as heat-treated saponin(e ethanol solution.
Use Rotary Evaporators concentration heat treatment saponin(e ethanol solution.Heat treated concentration soap is dissolved with a small amount of ethanol
Glycosides, adds sterile purified water and prepares suspension for preserving.Concentration process can prevent harmful bacteria from growing, and be because causing
The miscellaneous bacteria of pollution is difficult survival more than 50 DEG C.
(45 DEG C) generation active saponins of the Moja-3 strain fermentations of embodiment 2
The PPD of isoreactivity containing Rg3 prepared in Example 1 ginsenoside, after drying, takes 24 grams, for following experiment.
First, Mojia-3 bacterial strains are cultivated with Tryptose soy meat soup (TSB), temperature 45 C, the time is not less than 16 hours.
100 milliliters of nutrient solutions are taken to be cultivated 60 minutes under the conditions of adding to Secondary Culture in 1000 milliliters of fresh TBS, 45 DEG C.After the completion of culture,
100 milliliter of 10% (w/v) activity PPD aaerosol solution is added, flexible pipe pump rate is 20 ml/mins.Under the conditions of 45 DEG C again
Culture 6 hours, obtains CKS solution.
CKS solution centrifugals obtain supernatant, referred to as solution A.Sediment after centrifugation is concentrated, and is again dissolved in a small amount of ethanol
In.Filtering, takes filtrate, is concentrated to give the higher CKS solution of concentration.CKS solution is freeze-dried, 0.054 gram of CKS is obtained (primary
React CKS).
Continue culture solution A under the conditions of 45 DEG C, the time is 6 hours.Identical with primary CKS preparation methods, concentration, freezing are dry
It is dry to obtain 0.18 gram of CKS (secondary reaction CKS).Primary and secondary reaction CKS total amounts are 0.234g.
The Bp-2 of embodiment 3, which ferments (55 DEG C), generates active saponins
The PPD of isoreactivity containing Rg3 prepared in Example 1 ginsenoside, after drying, takes 24 grams, for following experiment.
First, Bp-2 bacterial strains are cultivated with Tryptose soy meat soup (TSB), temperature 45 C, the time is not less than 16 hours.Take
100 milliliters of nutrient solutions are cultivated 60 minutes under the conditions of adding to Secondary Culture in 1000 milliliters of fresh TBS, 55 DEG C.After the completion of culture, plus
Enter 100 milliliter 10% (w/v) activity PPD aaerosol solutions, flexible pipe pump rate is 20 ml/mins.Trained again under the conditions of 55 DEG C
Support 6 hours, obtain CKS solution.
CKS solution centrifugals obtain supernatant, referred to as solution B.Sediment is dissolved with a small amount of ethanol, filtering takes filtrate, concentrated
Obtain the higher CKS solution of concentration.CKS solution is freeze-dried, 0.0645g CKS (CKS primary reactions) are obtained.
Continue culture solution B under the conditions of 55 DEG C, the time is 6 hours.Identical with primary CKS preparation methods, concentration, freezing are dry
It is dry to obtain 0.139 gram of CKS (secondary reaction CKS).Main and secondary reaction CKS total amounts are 0.2035 gram.
(55 DEG C) fermentation generation active saponin compounds of the Moja-3 of embodiment 4 (45 DEG C) and Bp-2
The PPD of isoreactivity containing Rg3 prepared in Example 1 ginsenoside, after drying, takes 24 grams, for following experiment.
First, Bp-2 bacterial strains and Moja-3 bacterial strain temperature 45 Cs are cultivated with Tryptose soy meat soup (TSB), the time is not less than
16 hours.100 milliliters of nutrient solutions are taken to be cultivated 60 minutes under the conditions of adding to Secondary Culture in 1000 milliliters of fresh TBS, 45 DEG C.Culture
After the completion of, 100 milliliter of 10% (w/v) activity PPD aaerosol solution is added, flexible pipe pump rate is 20 ml/mins.In 45 DEG C of conditions
Under cultivate again 6 hours, obtain CKS solution.
CKS solution centrifugals obtain supernatant, referred to as solution C.Sediment is dissolved with a small amount of ethanol.Filtering, takes filtrate, concentrates
Obtain the higher CKS solution of concentration.CKS solution is freeze-dried, 1.04 grams of CKS (CKS primary reactions) are obtained.
Continue culture solution C under the conditions of 55 DEG C, the time is 6 hours.Identical with primary CKS preparation methods, concentration, freezing are dry
It is dry to obtain 0.2747 gram of CKS (secondary reaction CKS).Primary and secondary reaction CKS total amounts are 1.3147g.
Now it has proven convenient that utilizing two kinds of bacterial strain collective effects under the high temperature conditions, it is possible to produce a large amount of active saponins.
(55 DEG C) fermentation generation active saponin compounds of the Moja-3 of embodiment 5 (45 DEG C) and Bp-2
The PPD of isoreactivity containing Rg3 prepared in Example 1 ginsenoside, after drying, takes 24 grams, for following experiment.
First, Bp-2 bacterial strains and Moja-3 bacterial strain temperature 45 Cs are cultivated with Tryptose soy meat soup (TSB), the time is not less than
16 hours.100 milliliters of nutrient solutions are taken to be cultivated 60 minutes under the conditions of adding to Secondary Culture in 1000 milliliters of fresh TBS, 55 DEG C.Culture
After the completion of, 100 milliliters of 10w/v% activity PPD aaerosol solutions are added, flexible pipe pump rate is 20 ml/mins.Under the conditions of 45 DEG C
Cultivate 12 hours again, obtain CKS solution.CKS solution centrifugals obtain supernatant, referred to as solution D.Dissolved and precipitated with a small amount of ethanol
Thing.Filtering, takes filtrate, is concentrated to give the higher CKS solution of concentration.CKS solution is freeze-dried, 1.02 grams of CKS are obtained (at the beginning of CKS
Order reaction).
Continue culture solution D under the conditions of 55 DEG C, the time is 12 hours.It is identical with primary CKS preparation methods, concentration, freezing
It is dried to obtain 0.210 gram of CKS (secondary reaction CKS).Primary and secondary reaction CKS total amounts are 1.23 grams.
Now it has proven convenient that utilizing two kinds of bacterial strain collective effects under the high temperature conditions, it is possible to produce a large amount of active saponins.Fermentation
After time was more than 6 hours, CKS is no longer produced.
The saponin(e condition determination of embodiment 6
Chromatographic isolation, 35 DEG C of temperature are carried out using Kinetex C18, Detection wavelength is 203 nanometers.Water (DW) and acetonitrile
(ACN) as mobile phase, gradient elution comprises the following steps, and percentage is volume ratio.Chromatographic column is set to buffer again before operation flat
Weighing apparatus 20 minutes.Sampling volume is 20 microlitres.It is volume ratio that analysis condition details, which see the table below percentage in 1, mobile phase,.
Table 1, chromatographiccondition
Produce the result explaination of saponin：
The representative CKS saponin(es of embodiment 1 to embodiment 4 are analyzed.Using the electric mist of high performance liquid chromatography
Formula detector method (HPLC-CAD) carries out saponin(e analysis.Compound CK, Rg3, Rd, Rb1 and Rf composition in the typical saponin(e of analysis.
Because each embodiment takes 24 grams of sun-dried ginsengs to be fermented, as a result there is comparative sense.Every kind of saponin content is in absolute number form
Represent, as a result as shown in table 2, the CKS yield of Moja-3 and Bp2 fermentations is respectively 234 milligrams and 203 milligrams.But, Mixed Microbes
The CKS yield of strain (Moja3 and Bp2) fermentation reaches 1315 milligrams.Realize batch production CKS target.Rg3 is the weight of red ginseng
Want index.Moja3 generates 8.39 milligrams of Rg3, and Bp2 generates 12.48 milligrams of Rg3.But hybrid bacterial strain (Moja3 and Bp-2)
The Rg3 of fermenting and producing ferments higher than single strain.Its yield is twice original (32.64 milligrams).Non-existentization in nature
Compound CK, it is only generated by enteric bacteria or enzyme.The compound K yield of Moja3 and bp2 fermentations is respectively 0.04 milligram and 0.14
Milligram.And the compound CK of hybrid bacterial strain (Moja-3 and Bp-2) fermenting and producing is higher than single strain fermentation (0.26 milligram).
The chromatography result of table 2
Claims (8)
1. a kind of preparation method of active ginseng saponin(e, it is characterised in that comprise the following steps：
1) steam heating ginseng is used；
2) crush, extract ginseng using ethanol, obtain ginsenoside extract solution；
3) Mo Hawei bacillus KJS-3 nutrient solutions and poly- ferment bacillus KJS-2 nutrient solutions are carried added to ginsenoside
Take in liquid and ferment；
4) the ginsenoside solution after centrifugation fermentation, separates and retains supernatant；Sediment is reclaimed, is redissolved using ethanol, is filtered,
Collect filtrate；
5) after filtrate is concentrated, freeze-drying obtains active ginseng saponin(e；Supernatant continues after cultivating, and concentrates, freeze-drying,
Also active ginseng saponin(e is obtained.
2. preparation method according to claim 1, it is characterised in that step 3) fermentation temperature in 42 DEG C of -50 DEG C of scopes
It is interior.
3. preparation method according to claim 2, it is characterised in that fermentation temperature is 45 DEG C.
4. preparation method according to claim 1, it is characterised in that step 5) in, supernatant liquor fermentation temperature 45~
60℃。
5. preparation method according to claim 4, it is characterised in that fermentation temperature is 55 DEG C.
6. preparation method according to claim 1, it is characterised in that step 1) in, processing of Panax ginseng temperature is 110 DEG C, is steamed
Vapor pressure is 1.1kg/cm2, the duration is 10 minutes, in triplicate.
7. preparation method according to claim 1, it is characterised in that step 2) in, the ginseng chopping after heat treatment is put
To agitator stirring, then it is statically placed in 80vol% alcohol, extracts saponin(e component.
8. preparation method according to claim 1, it is characterised in that the ginseng is Korean ginseng, wild ginseng, cultivation people
It is more than one or both of ginseng, pseudo-ginseng ginseng, American Ginseng.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068490A (en) * | 2022-06-23 | 2022-09-20 | 四川大学 | Application of ginsenoside CK in preparation of bacteriostatic agent for mycobacterium abscessus or/and mycobacterium tuberculosis |
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| CN102618616A (en) * | 2012-04-13 | 2012-08-01 | 吉林农业大学 | Method of improving ginsenoside extraction rate by utilizing steam-explosion combined bio-composite enzyme technology |
| CN102657331A (en) * | 2012-05-02 | 2012-09-12 | 金光洙 | Fermented ginseng fermented by bacillus subtilis, fermented ginseng natto and application of extracts |
| CN104784230A (en) * | 2014-01-17 | 2015-07-22 | 百济红蔘株式会社 | Fermented red ginseng and manufacturing method thereof |
| CN103931659B (en) * | 2014-04-30 | 2016-08-24 | 武汉虹睿生物科技开发有限公司 | Mo Hawei bacillus KJS-3 is as the application of biological pesticide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102618616A (en) * | 2012-04-13 | 2012-08-01 | 吉林农业大学 | Method of improving ginsenoside extraction rate by utilizing steam-explosion combined bio-composite enzyme technology |
| CN102657331A (en) * | 2012-05-02 | 2012-09-12 | 金光洙 | Fermented ginseng fermented by bacillus subtilis, fermented ginseng natto and application of extracts |
| CN104784230A (en) * | 2014-01-17 | 2015-07-22 | 百济红蔘株式会社 | Fermented red ginseng and manufacturing method thereof |
| CN103931659B (en) * | 2014-04-30 | 2016-08-24 | 武汉虹睿生物科技开发有限公司 | Mo Hawei bacillus KJS-3 is as the application of biological pesticide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115068490A (en) * | 2022-06-23 | 2022-09-20 | 四川大学 | Application of ginsenoside CK in preparation of bacteriostatic agent for mycobacterium abscessus or/and mycobacterium tuberculosis |
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