CN104605358A - Preparation method for alpha-glucosidase inhibitor as well as product and usage thereof - Google Patents
Preparation method for alpha-glucosidase inhibitor as well as product and usage thereof Download PDFInfo
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- CN104605358A CN104605358A CN201510080317.5A CN201510080317A CN104605358A CN 104605358 A CN104605358 A CN 104605358A CN 201510080317 A CN201510080317 A CN 201510080317A CN 104605358 A CN104605358 A CN 104605358A
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- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 title abstract 6
- 239000003888 alpha glucosidase inhibitor Substances 0.000 title abstract 6
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- Organic Chemistry (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Medicines Containing Plant Substances (AREA)
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Abstract
The invention discloses a preparation method for an alpha-glucosidase inhibitor as well as a product and a usage thereof. The preparation method comprises the following steps: centrifugalizing fermented soy bean milk and taking supernatant, extracting the obtained supernatant by using ethyl acetate, depressurizing and evaporating the obtained ethyl acetate-phase to be dry, adding ethyl acetate to prepare an alpha-glucosidase inhibitor crude extraction liquid, separating and eluting the obtained crude extraction liquid by using a silica gel column, testing inhibition of the obtained eluant to alpha-glucosidase, combining and collecting the eluants of component peak according to inhibition effects and freeze-drying to obtain the alpha-glucosidase inhibitor. The invention provides an active ingredient which is separated from a soy bean fermentation product and has obvious inhibition effect to alpha-glucosidase, and offers new ideas for industrial preparation and usage of soy bean fermentation-originated alpha-glucosidase inhibitor. According to the invention, the obtained alpha-glucosidase inhibitor can be directly used in production of products for controlling absorption of carbohydrate in intestinal tract, so as to achieve the purpose of blood glucose fluctuation control.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of alpha-glucosidase restrainer and product thereof and application.
Background technology
In recent years, diabetes are widely current in the whole world, have become the chronic disease of the third-largest class serious harm human health after tumour, angiocardiopathy at present.Along with China's economy, social developing rapidly, diet structure and living-pattern preservation, the quickening of pace of population aging, the illness rate of diabetes also presents the trend increased fast.Diabetes prevalence is zooming while, the awareness of China's diabetes, treatment rate and control rate are obviously on the low side.Diabetes have become the public health problem of a serious harm population of China health, produce more and more serious impact to socio-economic development.
Different according to pathogenesis, diabetes are divided into type i diabetes (insulin-dependent) and type ii diabetes (non-insulin-depending type), and the latter accounts for more than 85% of diabetes sum.Up to now, the medicine for the treatment of type ii diabetes is mainly divided into according to therapy mechanism difference: (1) Drugs Promoting Insulin Secretion: as sulfonylurea; (2) insulin sensitizer: as tetrahydrothiazole derivates such as guitar ancestors (troglitazone) in the wrong; (3) alpha-glucosidase restrainer (α-GI): as acarbose etc.Because α-GI has action temperature and lasting, the little even nontoxic advantage of toxic and side effect, therefore obtain the favor of more and more domestic and international researcher.
Research shows, alpha-glucosidase restrainer (α-GI) can reduce the postprandial hyperglycemia of diabetes patient effectively, as the one of OHA, its place of having an effect is positioned at small intestine, the mode of action suppresses the alpha-glucosidase activity in small intestine body, thus reach the object reducing postprandial hyperglycemia.If carbohydrate accounts for more than 50% in user's diet, then hypoglycemic effect is more obvious, and therefore it is applicable to take carbohydrate as the crowd of staple food, especially middle and elderly diabetic patient.Though the α-GI found at present can overcome some shortcomings of traditional antidiabetic drug, kind is relatively less, and people are still constantly devoted to development of new α-GI, especially the research of natural products.In recent years, from natural products, the α-GI of separation and extraction is mainly from herbaceous plant and actinomyces, only has minority about the hypoglycemic report of lactic acid bacteria product.
H.Fujita (J.Nutri.131:1211-1213,2001) mode adopting hot-water extraction from the flavouring fermented soya bean (Touchi) of Chinese tradition is reported, obtain having the active extract of Inhibiting α-glucosidase (rat cane sugar hydrolytic enzyme), this extract has hypoglycemic activity in rat and hyperglycemia population; It is mixed with the alpha-amylase inhibitor phaseolamin coming from navy bean (kidneybean) that US Patent No. 7354606B2 discloses a kind of fermented soya bean extract that adopts, and is used for the treatment of the method for hyperglycaemia.A.Momose (J.J.Soc.Food Tech.Sci, Feb, 2011) report the effect that the alpha-glucosidase restrainer be separated to from meso (a kind of fermented bean products used as flavoring of the natural fermented method production of employing of japanese traditional) has adjustment post-prandial glucose excursions.These researchs all rest on the crude extract aspect of soybean fermented product, rarely have about wherein inhibitor active ingredient separation with report qualitatively, therefore, deficient theoretical foundation greatly hinders the extensive preparation and application of alpha-glucosidase restrainer in bean product.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly only rest on crude extract aspect for the research that existing soybean fermented product has an alpha-glucosidase inhibition, the present situation rarely having separation method and qualitative analysis about wherein concrete active ingredient to report, provides a kind of preparation method of alpha-glucosidase restrainer and product thereof and application.
The present inventor finds that Lactobacillus plantarum ST-III (CGMCC No.0847) ferments the supernatant of gained soya-bean milk zymotic fluid after the technique separation and purification such as extraction, silicagel column, can obtain and there is strong alpha-glucosaccharase enzyme inhibition activity component, thus complete the present invention.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of preparation method of alpha-glucosidase restrainer, and described preparation method comprises the following steps:
(1) lactobacillus plantarum ST-III is inoculated in fermentation in aseptic soya-bean milk and obtains fermented soybean milk;
(2) fermented soybean milk of step (1) gained is heated, centrifuging and taking supernatant after cooling, gained supernatant uses the ethyl acetate of 2 ~ 5 times of volumes to extract, and gained ethyl acetate is added ethyl acetate after evaporated under reduced pressure and is mixed with crude extract;
(3) get step (2) gained crude extract and cross silicagel column, with ethyl acetate and n-hexane mixed liquor, gradient elution is carried out to silicagel column, the eluent of fraction collection alpha-glucosaccharase enzyme inhibition rate >=10%;
(4) dry after the eluent of alpha-glucosaccharase enzyme inhibition rate >=10% of step (3) gained being removed organic solvent, obtain alpha-glucosidase restrainer.
Wherein step (1) obtains fermented soybean milk for lactobacillus plantarum ST-III being inoculated in fermentation in aseptic soya-bean milk.The temperature of wherein said fermentation is preferably 25 ~ 40 DEG C, and the time of described fermentation is preferably 24 ~ 120 hours.Wherein said soya-bean milk is standby by the bean as main food kind, as being selected from soybean, red bean, mung bean, broad bean, big white kidney bean and small white kidney bean, and most preferably soybean.The preparation method of described soya-bean milk is conventional, as being prepared into soya-bean milk in soy bean milk making machine.Namely soya-bean milk obtains aseptic soya-bean milk after sterilizing, and sterilizing methods is conventional, as thermophilic digestion sterilizing, 118 DEG C 15 minutes.The described preferred solid content 3%-9% of sterilizing soya-bean milk (w/w), most preferably soymilk concentration is 5-6% (w/w).The preferred 0.5%-5% of the inoculum concentration of lactobacillus plantarum ST-III in aseptic soya-bean milk (v/v), most preferably 2% (v/v).The preferred 35-37 DEG C of fermentation temperature, it is still further preferred that 37 DEG C.Fermentation time is 24-30 hour more preferably.Lactobacillus plantarum ST-III is inoculated in aseptic soya-bean milk and ferments, and as unique fermented bacterium, also can ferment with other mushrooms simultaneously, and preferred plant lactobacillus ST-III of the present invention is as unique fermented bacterium.
Wherein step (2) is heated by the fermented soybean milk of step (1) gained, centrifuging and taking supernatant after cooling, gained supernatant uses the ethyl acetate of 2 ~ 5 times of volumes to extract, and gained ethyl acetate is added ethyl acetate after evaporated under reduced pressure and is mixed with crude extract.Wherein said evaporated under reduced pressure is the evaporation drying technology of this area routine, is preferably rotary evaporation drying, parallel evaporation drying etc.The temperature of wherein said fermented soybean milk heating is preferably 95 ~ 100 DEG C, and be more preferably 98 DEG C, the time of described heating is preferably 10 ~ 30 minutes, and be more preferably 20 minutes, gained supernatant preferably uses the ethyl acetate of 3 ~ 4 times of volumes to extract.
Wherein step (3) crosses silicagel column for getting step (2) gained crude extract, carries out gradient elution, the eluent of fraction collection alpha-glucosaccharase enzyme inhibition rate >=10% with ethyl acetate and n-hexane mixed liquor to silicagel column.The primary condition of wherein said silicagel column is preferably for the volume ratio of ethyl acetate and n-hexane is 2:3, in described ethyl acetate and n-hexane mixed liquor, the volume ratio of ethyl acetate and n-hexane is preferably 2:3 ~ 4:1, the volume ratio being more preferably 2:3, ethyl acetate and n-hexane for the volume ratio of ethyl acetate and n-hexane is the volume ratio of 3:2 and/or ethyl acetate and n-hexane is 4:1, and the flow velocity of described gradient elution is preferably 0.5 ~ 1.5mL/min.
Wherein, the eluent of fraction collection alpha-glucosaccharase enzyme inhibition rate >=10% can operate by this area routine, be preferably when use ethyl acetate and n-hexane mixed liquor carry out gradient elution, be in charge of collection eluent, the alpha-glucosaccharase enzyme inhibition rate of each pipe eluent is tested, select each pipe eluent of wherein alpha-glucosaccharase enzyme inhibition rate >=10% to carry out merging by component peaks branch to collect, as in the present invention, two eluting peaks can be produced, then be in charge of the eluent of alpha-glucosaccharase enzyme inhibition rate >=10% in collection two eluting peaks, gained eluent is merged.The gathering speed of wherein said eluent is this area routine techniques, and the gathering speed of described eluent is preferably that every 3 ~ 8min/ manages.In elution process, if when in eluent, the volume ratio of different solvents is outside limited range of the present invention, can because the effect that inappropriate elution requirement causes two kinds of component wash-outs to be separated becomes very poor, its result is very likely that two components are eluted simultaneously, instead of sequentially eluting.In order to eliminate false positive results that experimental implementation error causes to the impact of this experiment, alpha-glucosaccharase enzyme inhibition rate is more preferably >=15%.
Wherein step (4) is dry after the eluent of alpha-glucosaccharase enzyme inhibition rate >=10% of step (3) gained being removed organic solvent, obtains alpha-glucosidase restrainer.Wherein said dry technology is preferably Freeze Drying Technique, and described Freeze Drying Technique is the Freeze Drying Technique of this area routine, as long as by removal of solvents, can obtain described alpha-glucosidase restrainer.
A preferred embodiments of the present invention is as follows:
(1) be inoculated in by lactobacillus plantarum ST-III in aseptic soya-bean milk, 25 ~ 40 DEG C ferment 24 ~ 120 hours, obtain fermented soybean milk;
(2) by the fermented soybean milk 95 ~ 100 DEG C of step (1) gained heating 10 ~ 30 minutes, centrifuging and taking supernatant after being cooled to 15 ~ 25 DEG C, gained supernatant uses the ethyl acetate of 2 ~ 5 times of volumes to extract, after the ethyl acetate phase rotary evaporation of gained is dry, add the crude extract that ethyl acetate is mixed with alpha-glucosidase restrainer;
(3) getting step (2) gained crude extract, to be splined on primary condition be ethyl acetate: the volume ratio of n-hexane is on the silicagel column of 2:3, by ethyl acetate: the volume ratio of n-hexane is 2:3, ethyl acetate: the volume ratio of n-hexane is 3:2, ethyl acetate: the volume ratio of n-hexane is 4:1, with the flow velocity of 0.5 ~ 1.5mL/min, gradient elution is carried out to silicagel column, wherein each gradient elution column volume, eluent collects eluent by the speed of every 3 ~ 8min/ pipe, and described ratio is volume ratio;
(4) be dissolved in water after each pipe eluent of step (3) gained being removed organic solvent, test gained solution to the inhibitory action of alpha-glucosidase, freeze-drying and get final product after merging according to inhibition the eluent collecting component peaks.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: the alpha-glucosidase restrainer of preparation method's gained as described herein.
Gained alpha-glucosidase restrainer of the present invention has higher alpha-glucosaccharase enzyme inhibition activity, can meet the demand of suitability for industrialized production completely.The molecular weight of alpha-glucosidase restrainer of the present invention is preferably 272.1Da, and its molecular weight is more preferably 256.1Da.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: alpha-glucosidase restrainer of the present invention is preparing the purposes in dietary supplements, antihypelipidemic foodstuff, health products or medicine.Antihypelipidemic foodstuff, dietary supplement, health products or medicine that wherein said purposes is preferably oral, reduce the postprandial hyperglycemia of diabetes patient, regulates post-prandial glucose excursions.
For solving the problems of the technologies described above, four of the technical scheme that the present invention takes is: a kind of extract of soybean with alpha-glucosaccharase enzyme inhibition activity, and the preparation method of described extract of soybean comprises the following steps:
(1) lactobacillus plantarum ST-III is inoculated in fermentation in aseptic soya-bean milk and obtains fermented soybean milk;
(2) heated by the fermented soybean milk of step (1) gained, centrifuging and taking supernatant after cooling, gained supernatant uses the ethyl acetate of 2 ~ 5 times of volumes to extract, by after the ethyl acetate of gained mutually drying and get final product.
The content of the preferred technique scheme of wherein said step (1) and step (2) as mentioned before.
Lactobacillus plantarum ST-III described in the present invention is prior art, and its deposit number is CGMCCNo.0847.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can be combined, obtain the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
1, the present invention has broken the present situation that research that relevant soybean fermented product at present has an alpha-glucosidase inhibition only rests on crude extract aspect, provides separation from soybean fermented product, and clearly has an active component of alpha-glucosidase inhibition.
2, the invention provides a kind of method preparing alpha-glucosidase restrainer from soybean fermented product, for the extensive preparation and application of the alpha-glucosidase restrainer in soybean fermented product source provide new approaches.
3, the alpha-glucosidase restrainer adopting the present invention to prepare can be directly used in have and control the production of carbohydrate at the product of intestinal absorption effect, thus reaches control blood glucose fluctuation object.
Accompanying drawing explanation
Fig. 1 is the silicagel column gradient elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.
Fig. 2 is the silicagel column gradient elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.
Fig. 3 is the silicagel column gradient elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.
Fig. 4 is the HPLC-UV detection of the alpha-glucosidase restrainer F1-B that CGMCC No.0847 produces.
Fig. 5 is the HPLC-UV detection of the alpha-glucosidase restrainer F2-B that CGMCC No.0847 produces.
Fig. 6 is the mass spectrogram of the alpha-glucosidase restrainer F1-B that CGMCC No.0847 produces.
Fig. 7 is the mass spectrogram of the alpha-glucosidase restrainer F2-B that CGMCC No.0847 produces.
Fig. 8 is the silicagel column ethyl acetate isocratic elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.
Fig. 9 is the silicagel column normal hexane isocratic elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.
Detailed description of the invention
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue." room temperature " described in the present invention refers to the temperature of carrying out the operation room tested, and is generally 25 DEG C.
Due to the one that maltose is in alpha-glucosaccharase enzyme family, therefore in the present invention, sample is to the inhibiting rate of alpha-glucosidase by embodying the inhibiting rate of maltose, and concrete assay method is as follows:
Get 100 μ L testing samples to manage in 1.5mL EP, then 50 μ L maltose (100mU/mL are added, sigma, the U.S.), mixing, 37 DEG C of incubation 15min, then the PNPG (sigma, the U.S.) of 80 μ L 2mM is added, mixing, 37 DEG C of reaction 15min, finally add the Na of 80 μ L 0.2M
2cO
3cessation reaction, then 4 DEG C, 10,000rpm centrifugal 2min, get 200 μ L supernatants in 96 hole microwell plate (greiner bio-one, Germany), under 405nm wavelength, measure absorbance with the multi-functional ELIASA of Spectra Max M5 (Molecular Devices, the U.S.).
Testing sample is to the inhibit activities of alpha-glucosidase (maltose) as shown by the following formula:
Wherein: negative control group is that the phosphate buffer (PBS) of 0.1M, pH=6.8 substitutes testing sample; Negative blank group substitutes the maltose in negative control group for PBS; Sample blank group is the maltose that PBS substitutes in testing sample group.Two kinds of alpha-glucosidase restrainers are prepared in the examples below, by its called after F1 and F2, according to the difference of elution time and elution requirement in different embodiment, divide into groups alpha-glucosidase restrainer F1 called after F1-A, F1-B and F1-C, by alpha-glucosidase restrainer F2 divide into groups called after be F2-A, F2-B and F2-C.
The preparation of embodiment 1 two kinds of alpha-glucosidase restrainers
Take deposit number as the lactobacillus plantarum ST-III of CGMCC No.0847 be fermented bacterium 25 DEG C fermentation 24 hours fermentation aseptic soybean milk 1L, the zymotic fluid of gained is at 100 DEG C of heating 10min, centrifuging and taking supernatant after being cooled to 25 DEG C, the centrifugal supernatant getting supernatant gained for 10 minutes of 8000rpm uses the ethyl acetate of 2 times of volumes to extract, after the ethyl acetate phase rotary evaporation of gained is dry, add 20mL ethyl acetate and be mixed with alpha-glucosidase restrainer crude extract, getting this crude extract 1.5mL, to be splined on primary condition be ethyl acetate: the volume ratio of n-hexane is on the silicagel column of 2:3 (v/v), by ethyl acetate: the volume ratio of n-hexane is 2:3 (v/v), ethyl acetate: the volume ratio of n-hexane is 3:2 (v/v), ethyl acetate: the volume ratio of n-hexane be 4:1 (v/v) sequentially, with the flow velocity of 0.5mL/min, gradient elution is carried out to sample, wherein each gradient elution column volume, eluent is collected by the speed of every 8min/ pipe, each pipe eluent of gained is removed after organic solvent through pressure, often pipe adds the dissolving of 0.5ml ultra-pure water, above-mentioned detection method is utilized to test its inhibitory action to alpha-glucosidase, result as shown in Figure 1, Fig. 1 is the silicagel column gradient elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.
49 ~ 57 pipes, 71 ~ 78 pipes as can be seen from the results, the eluate in these two regions is significantly higher than the inhibiting rate of other pipes to alpha-glucosaccharase enzyme inhibition rate, therefore the eluate in these two regions is collected respectively, after freeze-drying, obtain two kinds of alpha-glucosidase restrainers, respectively called after F1-A, F2-A.
The preparation of embodiment 2 two kinds of alpha-glucosidase restrainers
Take deposit number as the lactobacillus plantarum ST-III of CGMCC No.0847 be fermented bacterium 40 DEG C fermentation 120 hours fermentation aseptic soybean milk 1L, the zymotic fluid of gained is at 98 DEG C of heating 20min, after being cooled to 20 DEG C, 9000rpm gets supernatant in centrifugal 15 minutes, the supernatant of gained uses the ethyl acetate of 4 times of volumes to extract, after the ethyl acetate phase rotary evaporation of gained is dry, add 20mL ethyl acetate and be mixed with alpha-glucosidase restrainer crude extract, getting this crude extract 1.5mL, to be splined on primary condition be ethyl acetate: the volume ratio of n-hexane is on the silicagel column of 2:3 (v/v), by ethyl acetate: the volume ratio of n-hexane is 2:3 (v/v), ethyl acetate: the volume ratio of n-hexane is 3:2 (v/v), ethyl acetate: the volume ratio of n-hexane be 4:1 (v/v) sequentially, with the flow velocity of 1mL/min, gradient elution is carried out to sample, wherein each gradient elution column volume, eluent is collected by the speed of every 6min/ pipe, each pipe eluent of gained is removed after organic solvent through pressure, often pipe adds the dissolving of 0.5ml ultra-pure water, its inhibitory action to alpha-glucosidase is tested with above-mentioned detection method, result as shown in Figure 2, Fig. 2 is the silicagel column gradient elution curve map of the alpha-glucosidase restrainer crude extract that CGMCCNo.0847 fermentation soybean soya-bean milk obtains.
33 ~ 38 pipes, 48 ~ 52 pipes can be found out, the eluate in these two regions is significantly higher than the inhibiting rate of other pipes to alpha-glucosaccharase enzyme inhibition rate, therefore collected respectively, after freeze-drying by the eluate in these two regions, obtain two kinds of alpha-glucosidase restrainers, respectively called after F1-B, F2-B.
The preparation of embodiment 3 two kinds of alpha-glucosidase restrainers
Take deposit number as the lactobacillus plantarum ST-III of CGMCC No.0847 be fermented bacterium 37 DEG C fermentation 72 hours fermentation aseptic soybean milk 1L, the zymotic fluid of gained is at 95 DEG C of heating 30min, after being cooled to 15 DEG C, 8000rpm gets supernatant in centrifugal 10 minutes, the supernatant of gained uses the ethyl acetate of 5 times of volumes to extract, after the ethyl acetate phase rotary evaporation of gained is dry, add 20mL ethyl acetate and be mixed with alpha-glucosidase restrainer crude extract, getting this crude extract 1.5mL, to be splined on primary condition be ethyl acetate: the volume ratio of n-hexane is on the silicagel column of 2:3 (v/v), by ethyl acetate: the volume ratio of n-hexane is 2:3 (v/v), ethyl acetate: the volume ratio of n-hexane is 3:2 (v/v), ethyl acetate: the volume ratio of n-hexane be 4:1 (v/v) sequentially, with the flow velocity of 1.5mL/min, gradient elution is carried out to sample, wherein each gradient elution column volume, eluent is collected by the speed of every 3min/ pipe, each pipe eluent of gained is removed after organic solvent through pressure, often pipe adds the dissolving of 0.5ml ultra-pure water, its inhibitory action to alpha-glucosidase is tested with above-mentioned detection method, result as shown in Figure 3, Fig. 3 is the silicagel column gradient elution curve map of the alpha-glucosidase restrainer crude extract that CGMCCNo.0847 fermentation soybean soya-bean milk obtains.
44 ~ 51 pipes, 64 ~ 69 pipes can be found out, the eluate in these two regions is significantly higher than the inhibiting rate of other pipes to alpha-glucosaccharase enzyme inhibition rate, therefore collected respectively, after freeze-drying by the eluate in these two regions, obtain two kinds of alpha-glucosidase restrainers, respectively called after F1-C, F2-C.
The purity detecting of embodiment 4 two kinds of alpha-glucosidase restrainers
Alpha-glucosidase restrainer F1-A, F1-B, F1-C, F2-A, F2-B and F2-C ultra-pure water prepared by embodiment 1 ~ 3 is dissolved, after being made into the 0.5mg/mL aqueous solution, carries out the analysis of high-efficient liquid phase chromatogram HPLC method.The Waters 2690 high-efficient liquid phase system adopted, is equipped with Waters 2410 differential refraction detector; Select TSK SW2000 gel chromatographic columns (TOSOH company, Japan).Mobile phase: ultra-pure water; Column temperature: 45 DEG C; Flow velocity: 1mL/min; Sample size: 50 μ L.
Result shows, F1-A, F1-B, F1-C, F2-A, F2-B and F2-C six samples all only present simple spike, show that alpha-glucosidase restrainer prepared by the present invention has higher degree.Wherein, the chromatographic results of sample F 1-B and F2-B as shown in Figure 4, Figure 5.Wherein Fig. 4 is the HPLC-UV detection of the alpha-glucosidase restrainer F1-B that CGMCC No.0847 produces, Fig. 5 is the HPLC-UV detection of the alpha-glucosidase restrainer F2-B that CGMCC No.0847 produces, wherein abscissa is retention time, and ordinate is signal value.
The molecular weight determination of embodiment 5 two kinds of alpha-glucosidase restrainers
Alpha-glucosidase restrainer F1-B and F2-B of AB4800MALDI-TOF-TOF (AB Sciex, the U.S.) mass spectrograph to preparation in embodiment 2 is adopted to carry out Mass Spectrometer Method.As shown in Figure 6 and Figure 7, wherein Fig. 6 is the mass spectrogram of the alpha-glucosidase restrainer F1-B that CGMCC No.0847 produces to result, and Fig. 7 is the mass spectrogram of the alpha-glucosidase restrainer F2-B that CGMCC No.0847 produces.Acquired results shows that the molecular weight of alpha-glucosidase restrainer F1-B and F2-B prepared by the present invention is respectively 272.1 and 256.1Da.
The effectiveness comparison of comparative example 1 and existing alpha-glucosidase restrainer
Alpha-glucosidase restrainer F1-A, F1-B, F1-C, F2-A, F2-B, F2-C that embodiment 1 ~ 3 is prepared and acarbose (sigma, the U.S.) with ultra-pure water be made into 0.2,0.4,0.6,0.8, the 1.0mg/mL aqueous solution, utilize above-mentioned detection method to measure the sample of variable concentrations to the inhibiting rate of alpha-glucosidase, the linear relationship that concentration and alpha-glucosaccharase enzyme inhibition rate are formed per sample calculates the half inhibiting rate concentration (IC of sample to alpha-glucosidase
50).
The different sample of table 1 is to half inhibiting rate concentration (IC of alpha-glucosidase
50)
Each sample is to half inhibiting rate concentration (IC of alpha-glucosidase
50) as shown in table 1, F1-A, F1-B and F1-C are suitable with traditional inhibitors acarbose to the inhibition of alpha-glucosidase, and the effect of F2-A, F2-B and F2-C is a little less than acarbose, therefore, the alternative existing alpha-glucosidase restrainer of alpha-glucosidase restrainer that prepared by the present invention is applied to association area.
Effectiveness comparison before comparative example 2 and separation and purification
Lactobacillus plantarum ST-III soya-bean milk zymotic fluid centrifuged supernatant prepared by embodiment 1 ~ 3 is carried out freeze drying, obtain mixture M-A, M-B and M-C respectively, compare said mixture and its active principle F1-A, F1-B, F1-C, F2-A, F2-B, F2-C to half inhibiting rate concentration (IC of alpha-glucosidase
50).
The different sample of table 2 is to half inhibiting rate concentration (IC of alpha-glucosidase
50)
Each sample is to half inhibiting rate concentration (IC of alpha-glucosidase
50) as shown in table 2, component F1 and F2 is to the inhibition of alpha-glucosidase and be significantly better than mixture, shows that active ingredient reaches significant concentration effect in preparation process.
The preparation of comparative example 3 two kinds of alpha-glucosidase restrainers
With reference to method described in embodiment 3, ethyl acetate is used respectively to the eluent of sample instead and n-hexane carries out isocratic elution, detect the separating effect of alpha-glucosidase under two kinds of elution process, result is as Fig. 8 and Fig. 9, wherein Fig. 8 is the silicagel column ethyl acetate isocratic elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains, and Fig. 9 is the silicagel column normal hexane isocratic elution curve map of the alpha-glucosidase restrainer crude extract that CGMCC No.0847 fermentation soybean soya-bean milk obtains.Experimentally result, Fig. 8 shows ethyl acetate isocratic elution curve map, and because selected eluent polarity is excessive, active principle F1 and F2 does not separate completely and is just eluted, so this elution requirement is not suitable for the separation of active ingredient of the present invention.
Fig. 9 then shows normal hexane isocratic elution curve map, and the active ingredient in sample is all adsorbed on silica gel, is not eluted, and therefore, this elution requirement is not suitable for the separation of active ingredient in the present invention equally.
Application Example 1 have alpha-glucosaccharase enzyme inhibition activity dietary supplements preparation
By lactobacillus plantarum ST-III, with inoculum concentration 2% (v/v) aseptic inoculation, in aseptic tomato juice culture medium, (mature tomato cleans, peeling, juice extractor squeezes, 100 order filtered through gauze get juice, boil 5min, 8000g is centrifugal, and 10min gets supernatant, 121 DEG C of sterilizing 20min, be cooled to room temperature and get final product), cultivate 8 hours for 37 DEG C, obtain zymotic fluid, regulate pH to 7.0, add the F1-B of embodiment 2 preparation simultaneously with 0.02% (w/v) to zymotic fluid, fully after mixing, after freeze drying, namely obtain the dietary supplements with alpha-glucosaccharase enzyme inhibition activity.
Add the phosphate buffer (PBS) of 0.1M, pH=6.8 to this dietary supplements, the volume before making it be reduced to freeze-drying, recording it to the inhibitory action of alpha-glucosidase is 22%.
Application Example 2 have alpha-glucosaccharase enzyme inhibition activity dietary supplements preparation
By lactobacillus plantarum ST-III, with inoculum concentration 2% (v/v) aseptic inoculation, in aseptic tomato juice culture medium, (mature tomato cleans, peeling, juice extractor squeezes, 100 order filtered through gauze get juice, boil 5min, 8000g is centrifugal, and 10min gets supernatant, 121 DEG C of sterilizing 20min, be cooled to room temperature and get final product), cultivate 8 hours for 37 DEG C, obtain zymotic fluid, regulate pH to 7.0, add the F2-B of embodiment 2 preparation simultaneously with 0.04 (w/v) to zymotic fluid, fully after mixing, after freeze drying, namely obtain the dietary supplements with alpha-glucosaccharase enzyme inhibition activity.
Add the phosphate buffer (PBS) of 0.1M, pH=6.8 to this dietary supplements, the volume before making it be reduced to freeze-drying, recording it to the inhibitory action of alpha-glucosidase is 27%.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a preparation method for alpha-glucosidase restrainer, is characterized in that, described preparation method comprises the following steps:
(1) lactobacillus plantarum ST-III is inoculated in fermentation in aseptic soya-bean milk and obtains fermented soybean milk;
(2) fermented soybean milk of step (1) gained is heated, centrifuging and taking supernatant after cooling, gained supernatant uses the ethyl acetate of 2 ~ 5 times of volumes to extract, and gained ethyl acetate is added ethyl acetate after evaporated under reduced pressure and is mixed with crude extract;
(3) get step (2) gained crude extract and cross silicagel column, with ethyl acetate and n-hexane mixed liquor, gradient elution is carried out to silicagel column, the eluent of fraction collection alpha-glucosaccharase enzyme inhibition rate >=10%;
(4) dry after the eluent of alpha-glucosaccharase enzyme inhibition rate >=10% of step (3) gained being removed organic solvent, obtain alpha-glucosidase restrainer.
2. preparation method as claimed in claim 1, it is characterized in that, step (1) described soya-bean milk is soybean milk, and the temperature of described fermentation is 25 ~ 40 DEG C, and the time of described fermentation is 24 ~ 120 hours.
3. preparation method as claimed in claim 1, it is characterized in that, the temperature of step (2) described heating is 95 ~ 100 DEG C, and the time of heating is 10 ~ 30 minutes.
4. preparation method as claimed in claim 1, it is characterized in that, the primary condition of step (3) described silicagel column is the volume ratio of ethyl acetate and n-hexane is 2:3, in described ethyl acetate and n-hexane mixed liquor, the volume ratio of ethyl acetate and n-hexane is 2:3 ~ 4:1, and the flow velocity of wash-out is 0.5 ~ 1.5mL/min.
5. the alpha-glucosidase restrainer of preparation method's gained as described in any one of Claims 1 to 4.
6. alpha-glucosidase restrainer as claimed in claim 5, it is characterized in that, the molecular weight of described alpha-glucosidase restrainer is 272.1Da.
7. alpha-glucosidase restrainer as claimed in claim 6, it is characterized in that, the molecular weight of described alpha-glucosidase restrainer is 256.1Da.
8. an alpha-glucosidase restrainer as claimed in claim 7 is preparing the purposes in dietary supplements, antihypelipidemic foodstuff, health products or medicine.
9. have an extract of soybean for alpha-glucosaccharase enzyme inhibition activity, it is characterized in that, the preparation method of described extract of soybean comprises the following steps:
(1) lactobacillus plantarum ST-III is inoculated in fermentation in aseptic soya-bean milk and obtains fermented soybean milk;
(2) heated by the fermented soybean milk of step (1) gained, centrifuging and taking supernatant after cooling, gained supernatant uses the ethyl acetate of 2 ~ 5 times of volumes to extract, by after the ethyl acetate of gained mutually drying and get final product.
10. extract of soybean as claimed in claim 9, it is characterized in that, preparation method's step (1) the described soya-bean milk of described extract of soybean is soybean milk, the temperature of described fermentation is 25 ~ 40 DEG C, the time of described fermentation is 24 ~ 120 hours, the temperature of step (2) described heating is 95 ~ 100 DEG C, and the time of heating is 10 ~ 30 minutes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132318A (en) * | 2015-09-01 | 2015-12-09 | 扬州大学 | Lactobacillus plantarum grx16 and application thereof |
| CN108004171A (en) * | 2017-12-18 | 2018-05-08 | 富乐顿生物工程科技（北京）有限公司 | A kind of spatial plant lactobacillus SS18-5 for reducing alpha-glucosidase activity and its application |
| CN108018236A (en) * | 2017-12-18 | 2018-05-11 | 富乐顿生物工程科技（北京）有限公司 | A kind of spatial plant lactobacillus SS18-37 for reducing alpha-glucosidase activity and its application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103169075A (en) * | 2011-12-21 | 2013-06-26 | 光明乳业股份有限公司 | Fermented bean product fermented by lactobacillus plantarum ST-III and alpha-glucosidase inhibitor |
| WO2013113966A1 (en) * | 2012-02-02 | 2013-08-08 | Consejo Superior De Investigaciones Científicas (Csic) | Lactic acid bacteria that grow in soy milk and active isoflaviones, product containing said bacteria and uses thereof |
| CN103610750A (en) * | 2013-11-07 | 2014-03-05 | 李刚 | Method for extracting, separating and purifying glucosidase inhibitor |
| JP2014172902A (en) * | 2013-03-05 | 2014-09-22 | Shonan Institute For Medical & Preventive Science | Diabetic conditions improving agent of sargassum horneri fermentation product using lactobacillus plantarum |
-
2015
- 2015-02-13 CN CN201510080317.5A patent/CN104605358B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103169075A (en) * | 2011-12-21 | 2013-06-26 | 光明乳业股份有限公司 | Fermented bean product fermented by lactobacillus plantarum ST-III and alpha-glucosidase inhibitor |
| WO2013113966A1 (en) * | 2012-02-02 | 2013-08-08 | Consejo Superior De Investigaciones Científicas (Csic) | Lactic acid bacteria that grow in soy milk and active isoflaviones, product containing said bacteria and uses thereof |
| JP2014172902A (en) * | 2013-03-05 | 2014-09-22 | Shonan Institute For Medical & Preventive Science | Diabetic conditions improving agent of sargassum horneri fermentation product using lactobacillus plantarum |
| CN103610750A (en) * | 2013-11-07 | 2014-03-05 | 李刚 | Method for extracting, separating and purifying glucosidase inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| 孙敏等: "α-糖苷酶抑制剂SH－9766的分离和鉴别", 《食品与生物技术学报》 * |
| 韩瑨等: "产 α-葡萄糖苷酶抑制剂乳酸菌的筛选及发酵条件优化", 《天然产物研究与开发》 * |
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| CN105132318A (en) * | 2015-09-01 | 2015-12-09 | 扬州大学 | Lactobacillus plantarum grx16 and application thereof |
| CN105132318B (en) * | 2015-09-01 | 2018-07-06 | 扬州大学 | Lactobacillus plantarum Grx16 and its application |
| CN108004171A (en) * | 2017-12-18 | 2018-05-08 | 富乐顿生物工程科技（北京）有限公司 | A kind of spatial plant lactobacillus SS18-5 for reducing alpha-glucosidase activity and its application |
| CN108018236A (en) * | 2017-12-18 | 2018-05-11 | 富乐顿生物工程科技（北京）有限公司 | A kind of spatial plant lactobacillus SS18-37 for reducing alpha-glucosidase activity and its application |
| CN108018236B (en) * | 2017-12-18 | 2021-02-12 | 富乐顿生物工程科技（北京）有限公司 | Space lactobacillus plantarum SS18-37 capable of reducing activity of alpha-glucosidase and application thereof |
| CN108004171B (en) * | 2017-12-18 | 2021-02-19 | 富乐顿生物工程科技（北京）有限公司 | Space lactobacillus plantarum SS18-5 capable of reducing activity of alpha-glucosidase and application thereof |
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