CN106520608B - A kind of alpha-glucosidase restrainer and preparation method thereof - Google Patents
A kind of alpha-glucosidase restrainer and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, specifically disclose a kind of preparation method of alpha-glucosidase restrainer, it ferments the following steps are included: ox series bacillus (Paenibacillus bovis) CGMCC No.8333 is inoculated in skimmed milk by (a), obtains acidified milk；(b) acidified milk for obtaining step (a) sterilizes, and centrifuging and taking supernatant, ultrafiltration takes low molecule part, is freeze-dried, obtains alpha-glucosidase restrainer.Preparation method in above-mentioned technical proposal, ox series bacillus (Paenibacillus bovis) CGMCC No.8333 is used for the first time, it ferments using skimmed milk as culture medium, and by sterilizing, centrifugation, ultrafiltration, freeze-drying, alpha-glucosidase restrainer is prepared, disclosing ox series bacillus CGMCC No.8333, there is fermentation skimmed milk to generate the new application of alpha-glucosidase restrainer, widen the source of alpha-glucosidase restrainer.Meanwhile the activity of alpha-glucosidase restrainer prepared by ox series bacillus CGMCC No.8333 fermentation skimmed milk is significant, stability is good.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of preparation method of alpha-glucosidase restrainer.In addition,
The invention further relates to a kind of alpha-glucosidase restrainers.
Background technique
In recent years, diabetes are widely current in the whole world, have become the third major class after tumour, cardiovascular disease at present
Seriously endanger the chronic disease of human health.With China's economy, the rapid development of society, diet structure and life style change
Become, the trend quicklyd increase is also presented in the quickening of pace of population aging, the illness rate of diabetes.It is quick in diabetes prevalence
While rising, the awarenesses of China's diabetes, treatment rate and control rate are obviously relatively low.Diabetes have become a serious harm
The public health problem of population of China health, increasingly severe influence is generated to socio-economic development.
According to pathogenesis difference, diabetes are divided into type-1 diabetes mellitus (insulin-dependent) and type-2 diabetes mellitus (non-pancreas islet
Plain dependent form), the latter accounts for about 85% or more of diabetes sum.So far, the drug of type-2 diabetes mellitus is treated according to treatment
Mechanism difference is broadly divided into: (1) Drugs Promoting Insulin Secretion；(2) insulin sensitizer；(3) alpha-glucosidase restrainer (α-
GI).Due to α-GI have the advantages that action temperature and persistently, toxic side effect it is small or even nontoxic, obtain it is more and more both at home and abroad
The favor of researcher.
Studies have shown that alpha-glucosidase restrainer (α-GI) can be effectively reduced the postprandial hyperglycemia of diabetes patient, make
For one kind of oral hypoglycemic agents, the place having an effect is located at small intestine, and the mode of action is to inhibit the intracorporal phlorose of small intestine
Glycosides enzymatic activity, to achieve the purpose that reduce postprandial hyperglycemia.If carbohydrate accounts for 50% or more in user's diet,
Hypoglycemic effect becomes apparent, therefore it is suitable for using carbohydrate as the crowd of staple food, especially middle and elderly diabetic patient.
Though α-the GI found at present can overcome some disadvantages of traditional antidiabetic drug, its source relative narrowness, main to collect
In in actinomyces, cause alpha-glucosidase restrainer source insufficient, this is those skilled in the art's urgent problem to be solved.
Summary of the invention
Based on above-mentioned technical problem, the present invention provides a kind of new methods of alpha-glucosidase restrainer preparation, with ox
Series bacillus CGMCC No.8333 is fermentation strain, using skimmed milk as culture medium, ferments and alpha-glucosidase inhibition is made
Agent.
Specifically, on the one hand, provide a kind of preparation method of alpha-glucosidase restrainer, comprising the following steps: (a)
Ox series bacillus (Paenibacillus bovis) CGMCC No.8333 is inoculated in skimmed milk and is fermented, sent out
Kefir milk；(b) acidified milk for obtaining step (a) sterilizes, centrifuging and taking supernatant, the portion that ultrafiltration takes molecular weight to be not more than 10,000Da
Point, freeze-drying obtains alpha-glucosidase restrainer.
Further, in above-mentioned steps (a), the inoculum concentration of ox series bacillus CGMCC No.8333 is 1.25x106~
1x107cfu/mL。
Further, in above-mentioned steps (a), skimmed milk includes skimmed milk powder and water, and skimmed milk powder accounts for the quality of skimmed milk
Percentage is 6~12%.
Further, it in above-mentioned steps (a), ferments for shaken cultivation, hunting speed is 100~300rpm.
Further, in above-mentioned steps (a), the temperature of fermentation is 20 DEG C~35 DEG C.
Further, in above-mentioned steps (a), the time of fermentation is 3~12h.
Further, in above-mentioned steps (b), the speed of centrifugation is 8,000~12,000g, and the time of centrifugation is 5~10 points
Clock.
On the other hand, a kind of alpha-glucosidase restrainer is additionally provided, is made by any of the above-described kind of preparation method.
Further, above-mentioned alpha-glucosidase restrainer to the rate concentration IC50 half-suppressed of alpha-glucosidase≤
55mg/mL。
Preparation method in above-mentioned technical proposal uses ox series bacillus (Paenibacillus bovis) for the first time
CGMCC No.8333, ferments using skimmed milk as culture medium, and by sterilizing, is centrifuged, ultrafiltration, and freeze-drying is prepared into
To alpha-glucosidase restrainer, disclosing ox series bacillus CGMCC No.8333, there is fermentation skimmed milk to generate α-grape
The new application of glycosidase inhibitor has widened the source of alpha-glucosidase restrainer.Meanwhile ox series bacillus CGMCC
The activity of alpha-glucosidase restrainer prepared by No.8333 fermentation skimmed milk is significant, and stability is good.
Specific embodiment
Technical solution of the present invention is illustrated to be clearer, below in conjunction with specific embodiment to skill of the invention
Art scheme is further elaborated:
In a specific embodiment, a kind of preparation method of alpha-glucosidase restrainer is provided, including with
Lower step: (a) ox series bacillus (Paenibacillus bovis) CGMCC No.8333 is inoculated in skimmed milk and is carried out
Fermentation, obtains acidified milk；(b) acidified milk for obtaining step (a) sterilizes, centrifuging and taking supernatant, and ultrafiltration takes molecular weight no more than 10,
The part of 000Da, freeze-drying, obtains alpha-glucosidase restrainer.
Preparation method in above-mentioned technical proposal is used ox series bacillus CGMCC No.8333 for the first time, is made with skimmed milk
It ferments for culture medium, and by sterilizing, is centrifuged, ultrafiltration, freeze-drying is prepared alpha-glucosidase restrainer, drapes over one's shoulders
Having revealed ox series bacillus CGMCC No.8333, there is fermentation skimmed milk to generate the new application of alpha-glucosidase restrainer, open up
The wide source of alpha-glucosidase restrainer.Meanwhile prepared by ox series bacillus CGMCC No.8333 fermentation skimmed milk
The activity of alpha-glucosidase restrainer is significant, and stability is good.
In addition, the preparation method in above-mentioned technical proposal greatlies simplify production stage, avoiding traditional complicated technology can
It can bring pollution problem；Secondly, whole preparation process is not related to any organic solvent, therefore fundamentally prevent organic molten
The problem of agent remains.Fermentation medium safety from a wealth of sources, low in cost, natural used by preparing avoids closing using chemistry
At culture medium；In addition fermenting microbe uses a kind of this single culture of ox series bacillus, the combination of above-mentioned raw material, strain has
Conducive to the standardization of product quality and the cost control of industrial mass production.
Further, in step (a), the inoculum concentration of ox series bacillus CGMCC No.8333 is 1.25x106~
1x107cfu/mL；Preferably 2.5x106~7.5x106Cfu/mL is more preferably 5x106cfu/mL。
Further, in above-mentioned steps (a), skimmed milk can be fresh skimmed milk, be also possible to be formulated de-
Rouge cream.The skimmed milk preferably prepared, the skimmed milk of preparation include skimmed milk powder and water, and skimmed milk powder accounts for the quality of skimmed milk
Percentage is 6~12%.Preparation method may comprise steps of: skimmed milk powder being added in distilled water, mixes sufficiently dissolution
Afterwards, it sterilizes 5~20 minutes for 95~125 DEG C, cooling to obtain the final product.
Further, in above-mentioned steps (a), preferred fermentation temperature is 20 DEG C~35 DEG C；Preferably 25 DEG C~35 DEG C；
It is more preferably 30 DEG C.
Further, in above-mentioned steps (a), preferred fermentation time is 3~12h；Preferably 6~12 hours；More preferably
Ground is 6 hours.
Further, in above-mentioned steps (a), preferred fermentation method be shaken cultivation, hunting speed be 100~
300rpm；Preferably 150~250rpm；It is more preferably 200rpm.
In conjunction with the embodiments with comparative example 1 also it is found that when except the range of preferred fermentation parameter, ox series bacillus
CGMCC No.8333 fermentation skimmed milk, presses down using the alpha-glucosidase obtained after sterilizing, centrifugation, ultrafiltration and freeze-drying
The inhibitory effect of preparation is decreased obviously.And within preferred scope, inoculum concentration, skimmed milk concentration, fermentation temperature and time are mutual
It influences, so that the activity for the alpha-glucosidase restrainer that ox series bacillus CGMCC No.8333 fermentation generates is more preferably.Example
Such as, when inoculum concentration is very few, culture propagation speed is slow, so that the activity of the alpha-glucosidase restrainer finally obtained is lower than excellent
The inoculum concentration range of choosing；When inoculum concentration is excessive, strain individual is dead due to the limitation of living space and nutrition after a certain period of time
Or growth arrest, it is unfavorable for the accumulation of metabolin, but also the activity of the alpha-glucosidase restrainer finally obtained is lower than excellent
The inoculum concentration range of choosing.In another example strain metabolism slowly, will lead to phlorose when fermentation temperature is lower than preferred scope value
The activity of glycosides enzyme inhibitor decreases.Further for example, when the hunting speed of fermentation is lower than preferred scope, the dissolved oxygen amount of strain fermentation
Not enough, production and metabolism are restricted, and the activity of alpha-glucosidase restrainer is caused to reduce.
Further, in above-mentioned steps (b), the molecular cut off of ultrafiltration is 1,000~10,000Da；Preferably 1,
000~5,000Da are more preferably 1,000Da.
In conjunction with the embodiments and comparative example 1 is also it is found that when except preferred molecular cut off, alpha-glucosidase restrainer
Inhibitory effect be decreased obviously.
Further, in above-mentioned steps (b), the temperature of sterilizing is preferably 95 DEG C~125 DEG C, is more preferably 98~110
DEG C, it is optimally 100 DEG C；The time of sterilizing is 5~20 minutes, is more preferably 10~18 minutes, is optimally 15 minutes.
Further, in above-mentioned steps (b), the speed of centrifugation is preferably 8, and 000~12,000g are more preferably 10000
~11000g；The time of centrifugation is preferably 5~10 minutes, is more preferably 6~8 minutes.
Further, in above-mentioned steps (b), above-mentioned freeze-drying is vacuum freeze drying, vacuum freeze drying condition
Preferably: plate layer limiting temperature≤- 60 DEG C, -70 DEG C of cold-trap limiting temperature, plate layer charging 0.5~2.0mm of thickness, vacuum degree
10~30Pa.
In another particular embodiment of the invention, a kind of alpha-glucosidase restrainer is provided, by above-mentioned any
Preparation method is made.
Above-mentioned preparation method has above-mentioned beneficial effect, therefore, the alpha-glucosidase as obtained by above-mentioned preparation method
Inhibitor also has corresponding beneficial effect, and details are not described herein again.
Further, above-mentioned alpha-glucosidase restrainer to the rate concentration IC50 half-suppressed of alpha-glucosidase≤
55mg/mL.In conjunction with the embodiments also it is found that the inhibitory activity of above-mentioned alpha-glucosidase restrainer is significantly higher than other conventional degreasings
Milk fermentation bacterial strain.
Above-mentioned specific embodiment is further illustrated below by embodiment, but is not therefore limited the present invention to described
Scope of embodiments among.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or presses
It is selected according to product manual.It is analytical reagents if reagent used in embodiment does not add explanation, buys from traditional Chinese medicines collection
Group.Other test apparatuses, reagent, strain are not illustrated such as, can directly be bought by commercial sources.
Embodiment 1
1, materials and methods
(a) preparation of seed (fermenting microbe): by ox series bacillus CGMCC No.8333, (source of the bacterial strain please join
See the Chinese patent of Publication No. CN 103740618A) freeze-dried powder dissolved with a small amount of sterile distilled water, take a ring with oese
TYC solid medium (buying from OXOID Co., Britain) is lined, 30 DEG C of aerobic culture 48h take out, with oese picking list
Bacterium colony is put into 10mL TYC fluid nutrient medium (buying from OXOID Co., Britain), with turbula shaker that bacterium colony is evenly dispersed
In in fluid nutrient medium, 30 DEG C, 180rpm shaken cultivation take out for 24 hours, TYC fluid nutrient medium is inoculated in 2% (v/v) inoculum concentration
(purchase from OXOID Co., Britain), 30 DEG C, 180rpm shaken cultivation for 24 hours after, culture 15,000rpm is centrifuged 10 minutes, is abandoned
After going supernatant, thallus to wash 2 times with sterile distilled water, is suspended with the sterile distilled water of former volume of culture, obtain the kind of fermentation
Son, the bacteria concentration of seed liquor are 2.5x108cfu/mL。
(b) preparation of skimmed milk: the skimmed milk powder that mass percent is 10% is mixed with distilled water, after completely dissolution,
Sterilize 5min at 125 DEG C, is cooled to room temperature to get the skimmed milk of required concentration.
(c) prepared by sample to be tested: alpha-glucosidase restrainer is dissolved in sterile water, pH is adjusted to 6.80, then 4
DEG C, 10,000rpm be centrifuged 10min, take supernatant, pH is adjusted to 6.80,4 DEG C again, 10,000rpm be centrifuged 10min, acquirement it is upper
It can be used to the detection of alpha-glucosaccharase enzyme inhibition activity clearly.
The detection of alpha-glucosaccharase enzyme inhibition activity: it takes 100 μ L samples to be tested to manage in 1.5mL EP (Eppendorf), connects
50 μ L alpha-glucosidases (100mU/mL, sigma, the U.S.) are added, mix, then 80 μ L are added in 37 DEG C of incubation 15min
The PNPG (sigma, the U.S.) of 2mM, mixes, and 37 DEG C of reaction 15min are eventually adding the Na of 80 μ L 0.2M2CO3Terminate reaction, then 4
DEG C, 10,000rpm be centrifuged 2min, take 200 μ L supernatants in 96 hole microwell plates (Greinerbio-one, Germany), in 405nm wavelength
It is lower to measure absorbance value with Spectra Max M5 multi-function microplate reader (MolecularDevices, the U.S.).Sample to be tested pair
The inhibitory activity of alpha-glucosidase is as shown by the following formula:
Wherein: negative control group is that blank skimmed milk substitutes sample to be tested；
Negative blank group is that the phosphate buffer (PBS) of 0.1M, pH=6.8 substitute the phlorose in negative control group
Glycosides enzyme；
Sample blank group is the alpha-glucosidase in PBS substitution sample to be tested group.
The measurement of alpha-glucosidase rate concentration IC50 half-suppressed: testing sample solution is carried out in sesquialter diluted method
Dilution, obtains the sample to be tested group of various concentration, using the sample to be tested of above-mentioned detection method measurement various concentration to α-grape
Sample is calculated to α-grape according to the linear relationship that concentration and alpha-glucosaccharase enzyme inhibition rate are constituted in the inhibiting rate of glycosidase
The rate concentration (IC50) half-suppressed of glycosidase.
2, the preparation of alpha-glucosidase restrainer
Ox series bacillus CGMCC No.8333 seed is de- in 10% (w/w) with (v/v) aseptic inoculation of inoculum concentration 2%
Rouge cream, 30 DEG C, 200rpm cultivate 6 hours acidified milk.
Acidified milk 95 DEG C of sterilizings 20min, 8,000g centrifugation 10min are taken into supernatant, ultrafiltration (Mwco=1,000Da) (ultrafiltration
Equipment Pellicon 0.1M2And ultrafiltration membrane is all bought from Millipore Corp., similarly hereinafter) low molecular weight part is taken, it is freeze-dried to obtain the final product
Alpha-glucosidase restrainer product A.
3, the measurement of alpha-glucosaccharase enzyme inhibition activity
Alpha-glucosidase restrainer product A is in the above way prepared as to measure it to alpha-glucosaccharase after sample to be tested
Enzyme inhibition activity, the results showed that half-suppressed rate concentration IC50=42mg/mL of the product A to alpha-glucosidase.
Embodiment 2
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of skimmed milk: the skimmed milk powder that mass percent is 12% is mixed with distilled water, after completely dissolution,
Sterilize 20min at 95 DEG C, is cooled to room temperature to get the skimmed milk of required concentration.
(c) prepared by sample to be tested: with embodiment 1.
2, the preparation of alpha-glucosidase restrainer
By ox series bacillus CGMCC No.8333 seed with (v/v) aseptic inoculation of inoculum concentration 0.5% in 12% (w/w)
Skimmed milk, 35 DEG C, 100rpm cultivate 12 hours acidified milk.
Acidified milk 125 DEG C of sterilizings 5min, 10,000g centrifugation 8min are taken into supernatant, ultrafiltration (Mwco=5,000Da) takes low point
Son amount part, is freeze-dried up to alpha-glucosidase restrainer product B.
3, the measurement of alpha-glucosaccharase enzyme inhibition activity
Alpha-glucosidase restrainer product B is in the above way prepared as to measure it to alpha-glucosaccharase after sample to be tested
Enzyme inhibition activity, the results showed that half-suppressed rate concentration IC50=49mg/mL of the product B to alpha-glucosidase.
Embodiment 3
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of skimmed milk: the skimmed milk powder that mass percent is 6% is mixed with distilled water, after completely dissolution,
Sterilize 15min at 100 DEG C, is cooled to room temperature to get the skimmed milk of required concentration.
(c) prepared by sample to be tested: with embodiment 1.
2, the preparation of alpha-glucosidase restrainer
By ox series bacillus CGMCC No.8333 seed with (v/v) aseptic inoculation of inoculum concentration 4% in 6% (w/w) degreasing
Cream, 20 DEG C, 300rpm cultivate 3 hours acidified milk.
Acidified milk 120 DEG C of sterilizings 10min, 12,000g centrifugation 5min are taken into supernatant, ultrafiltration (Mwco=10,000Da) takes low
Molecular weight fractions are freeze-dried up to alpha-glucosidase restrainer products C.
3, the measurement of alpha-glucosaccharase enzyme inhibition activity
Alpha-glucosidase restrainer products C is in the above way prepared as to measure it to alpha-glucosaccharase after sample to be tested
Enzyme inhibition activity, the results showed that half-suppressed rate concentration IC50=55mg/mL of the products C to alpha-glucosidase.
Embodiment 4
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of skimmed milk: the skimmed milk powder that mass percent is 8% is mixed with distilled water, after completely dissolution,
Sterilize 10min at 120 DEG C, is cooled to room temperature to get the skimmed milk of required concentration.
(c) prepared by sample to be tested: with embodiment 1.
2, the preparation of alpha-glucosidase restrainer
By ox series bacillus CGMCC No.8333 seed with (v/v) aseptic inoculation of inoculum concentration 3% in 8% (w/w) degreasing
Cream, 25 DEG C, 250rpm cultivate 5 hours acidified milk.
Acidified milk 100 DEG C of sterilizings 15min, 10,000g centrifugation 6min are taken into supernatant, ultrafiltration (Mwco=1,000Da) takes low
Molecular weight fractions are freeze-dried up to alpha-glucosidase restrainer product D.
3, the measurement of alpha-glucosaccharase enzyme inhibition activity
Alpha-glucosidase restrainer product D is in the above way prepared as to measure it to alpha-glucosaccharase after sample to be tested
Enzyme inhibition activity, the results showed that half-suppressed rate concentration IC50=46mg/mL of the product D to alpha-glucosidase.
Embodiment 5
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of skimmed milk: the skimmed milk powder that mass percent is 9% is mixed with distilled water, after completely dissolution,
Sterilize 12min at 115 DEG C, is cooled to room temperature to get the skimmed milk of required concentration.
(c) prepared by sample to be tested: with embodiment 1.
2, the preparation of alpha-glucosidase restrainer
By ox series bacillus CGMCC No.8333 seed with (v/v) aseptic inoculation of inoculum concentration 1% in 9% (w/w) degreasing
Cream, 28 DEG C, 150rpm cultivate 9 hours acidified milk.
Acidified milk 115 DEG C of sterilizings 12min, 11,000g centrifugation 8min are taken into supernatant, ultrafiltration (Mwco=5,000Da) takes low
Molecular weight fractions are freeze-dried up to alpha-glucosidase restrainer product E.
3, the measurement of alpha-glucosaccharase enzyme inhibition activity
Alpha-glucosidase restrainer product E is in the above way prepared as to measure it to alpha-glucosaccharase after sample to be tested
Enzyme inhibition activity, the results showed that half-suppressed rate concentration IC50=50mg/mL of the product E to alpha-glucosidase.
Effect example 1The stability of alpha-glucosidase restrainer inhibitory effect under refrigerated condition
After product A, B, C, D and E prepared by embodiment 1-5 are respectively charged into hermetic bag, it is placed in (8 DEG C) of refrigerated condition preservations
0, it is taken out after 10,20 and 30 days, measures each sample respectively to the rate concentration IC50 value half-suppressed of alpha-glucosidase, as a result such as table
Shown in 1.
The stability of alpha-glucosidase restrainer product inhibitory effect under 1 refrigerated condition of table
As shown in Table 1, the alpha-glucosidase restrainer product of all tests is after (8 DEG C) of refrigeration save 30 days, to α-
Glucosidase inhibitory active is stably held in same level, and stability is preferable.
Comparative example 1
By the inoculum concentration in embodiment 1, skimmed milk concentration, cultivation temperature, the speed and surpass that fermentation time, fermentation are vibrated
Filter molecular cut off is adjusted one by one, obtains the alpha-glucosidase restrainer product of following set of distinct methods preparation,
Alpha-glucosidase restrainer obtained by each group is as shown in table 2 to the inhibitory effect of alpha-glucosidase.
The inhibitory effect of 2 distinct methods of table preparation gained alpha-glucosidase restrainer
It can be concluded that, it will be connect in the preparation method of the alpha-glucosidase restrainer product from result shown in table 2
Kind amount, skimmed milk concentration, cultivation temperature, fermentation time, the speed of fermentation oscillation and ultrafiltration molecular cut off are adjusted to preferred
When except range, ox series bacillus CGMCC No.8333 can still ferment skimmed milk generate alpha-glucosidase suppression
Preparation, but the alpha-glucosidase inhibitory effect of product is decreased obviously.
Comparative example 2
1 the method for reference implementation example compares by ox series bacillus CGMCC No.8333, Lactobacillus casei
(L.casei) (purchase is from ATCC), lactobacillus bulgaricus (L.bulgaricus) LB340 (by Denis section public affairs by ATCC 393
Department provide), streptococcus thermophilus (S.thermophilus) ST-BODY-3 (by Hansen Corp., section provide) preparation phlorose
For glycosides enzyme inhibitor to the inhibitory effect of alpha-glucosidase, concrete operations are as follows:
1, materials and methods
(a) preparation of seed (fermenting microbe):
The preparation of Lactobacillus casei and lactobacillus bulgaricus seed: by Lactobacillus casei ATCC 393 and bulgarian milk
The freeze-dried powder of bacillus LB340 is dissolved with a small amount of sterile distilled water respectively, and each personal oese takes a ring to line MRS solid culture
On base (purchase is German from Merck Co.), 37 DEG C of Anaerobic culturels take out for 24 hours, are put into 1mL MRS liquid with oese picking single colonie
Bacterium colony, is dispersed in fluid nutrient medium by body (purchase is German from Merck Co.) with vortex oscillator, 37 DEG C of anaerobism trainings
It supports and takes out for 24 hours, 50mL MRS liquid is inoculated in 2% (v/v) inoculum concentration, after 37 DEG C of cultures for 24 hours, culture 9,000rpm centrifugation
It 10 minutes, discards supernatant, after thallus washs 2 times with sterile distilled water, is suspended with the sterile distilled water of former volume of culture, obtain phase
The seed for the fermentation answered.
The preparation of streptococcus thermophilus seed: the freeze-dried powder of streptococcus thermophilus ST-BODY-3 is molten with a small amount of sterile distilled water
Solution takes a ring to line on M17 solid medium (purchase is German from Merck Co.) with oese, and 40 DEG C of Anaerobic culturels take for 24 hours
Out, it is put into 1mL M17 liquid (purchase from Merck Co. Germany) with oese picking single colonie, with vortex oscillator by bacterium
It falls and is dispersed in fluid nutrient medium, 40 DEG C of Anaerobic culturels take out for 24 hours, are inoculated in 50mLM17 liquid with 2% (v/v) inoculum concentration
Body, after 40 DEG C of cultures for 24 hours, culture 9,000rpm is centrifuged 10 minutes, is discarded supernatant, after thallus washs 2 times with sterile distilled water,
It is suspended with the sterile distilled water of former volume of culture, obtains the seed of fermentation.
(b) preparation of skimmed milk: with embodiment 1.
(c) prepared by sample to be tested: with embodiment 1.
2, the preparation of alpha-glucosidase restrainer product
By each bacterial strain with 2% (v/v) inoculum concentration aseptic inoculation in 10% (w/w) skimmed milk, (bulgarian milk is cultivated respectively
37 DEG C of Anaerobic culturels of bacillus and Lactobacillus casei, 40 DEG C of Anaerobic culturels of streptococcus thermophilus, 30 DEG C of ox series bacillus, 200rpm vibration
Swing culture) 6h, obtain corresponding acidified milk.
Above-mentioned acidified milk is sterilized according to method described in embodiment 1, is centrifuged, ultrafiltration, freeze-drying is prepared
Alpha-glucosidase restrainer.
3, the active measurement of alpha-glucosidase restrainer
The inhibitory effect of the alpha-glucosidase restrainer of above-mentioned different strains preparation is as shown in table 3:
The inhibitory effect of the alpha-glucosidase restrainer of 3 different strains of table preparation
As shown in Table 3, other normal fermentation bacterial strains do not have the energy that fermentation skimmed milk generates alpha-glucosidase restrainer
Power, the skimmed milk and ox series bacillus CGMCC No.8333 can ferment, finally obtains alpha-glucosidase restrainer, to α-
The inhibitory activity highly significant of glucuroide.
Alpha-glucosidase restrainer provided by the present invention and preparation method thereof is described in detail above.Herein
In apply that a specific example illustrates the principle and implementation of the invention, the explanation of above example is only intended to sides
Assistant solves method and its core concept of the invention.It should be pointed out that for those skilled in the art, not
, can be with several improvements and modifications are made to the present invention under the premise of being detached from the principle of the invention, these improvement and modification are also fallen into
In the protection scope of the claims in the present invention.
Claims (7)
1. a kind of preparation method of alpha-glucosidase restrainer, which comprises the following steps:
(a) ox series bacillus (Paenibacillus bovis) CGMCC No.8333 is inoculated in skimmed milk and is sent out
Ferment obtains acidified milk；
(b) acidified milk for obtaining step (a) sterilizes, centrifuging and taking supernatant, the part that ultrafiltration takes molecular weight to be not more than 10,000Da,
Freeze-drying, obtains alpha-glucosidase restrainer.
2. the preparation method of alpha-glucosidase restrainer according to claim 1, which is characterized in that in step (a), institute
The inoculum concentration for stating ox series bacillus CGMCC No.8333 is 1.25x106~1x107cfu/mL。
3. the preparation method of alpha-glucosidase restrainer according to claim 1, which is characterized in that in step (a), institute
Stating skimmed milk includes skimmed milk powder and water, and the mass percent that skimmed milk powder accounts for skimmed milk is 6~12%.
4. the preparation method of alpha-glucosidase restrainer according to claim 1, which is characterized in that in step (a), institute
Stating fermentation is shaken cultivation, and hunting speed is 100~300rpm.
5. the preparation method of alpha-glucosidase restrainer according to claim 1, which is characterized in that in step (a), institute
The temperature for stating fermentation is 20 DEG C~35 DEG C.
6. the preparation method of alpha-glucosidase restrainer according to claim 1, which is characterized in that in step (a), institute
The time for stating fermentation is 3~12h.
7. the preparation method of alpha-glucosidase restrainer according to claim 1, which is characterized in that in step (b), institute
The speed for stating centrifugation is 8, and 000~12,000g, the time of centrifugation are 5~10 minutes.
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| Natural Products as a-Amylase and a-Glucosidase Inhibitors and their Hypoglycaemic Potential in the Treatment of Diabetes: An Update;R.Tundis等;《 Mini-Reviews in Medicinal Chemistry》;20101031;315-331 |
| Production and Characterization of a New α-Glucosidase Inhibitory Peptide from Aspergillus oryzae N159-1;Min-Gu Kang等;《Mycobiology》;20130930;149-154 |
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