CN101724015A - Alpha-glucosidase inhibitor and preparation method thereof - Google Patents
Alpha-glucosidase inhibitor and preparation method thereof Download PDFInfo
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- CN101724015A CN101724015A CN200910114494A CN200910114494A CN101724015A CN 101724015 A CN101724015 A CN 101724015A CN 200910114494 A CN200910114494 A CN 200910114494A CN 200910114494 A CN200910114494 A CN 200910114494A CN 101724015 A CN101724015 A CN 101724015A
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- glucosidase inhibitor
- glucosidase
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Abstract
The invention relates to an alpha-glucosidase inhibitor and a preparation method thereof. The preparation method comprises the following steps: culturing and activating a strain; culturing seeds; inoculating the stain into a shake flask for fermentation culture; collecting fermentation liquor; and performing separation and purification to obtain the alpha-glucosidase inhibitor. In the invention, the alpha-glucosidase inhibitor is obtained by microbial fermentation and can be used for developing hypoglycemic medicaments and provide a new tool for further research on active site of enzymes such as the alpha-glucosidase.
Description
Technical field
The present invention relates to a kind of alpha-glucosidase inhibitor and microbial fermentation preparation method thereof.
Background technology
The very high and continuation increase of diabetes, obesity and the hyperlipidaemia frequency of disease development in world wide population.In general crowd, diabetes are one of the chronicest diseases, and it takes place along with fat and increasing of age.Though be useful on the medicine that the treatment diabetic syndrome is developed, the target that has challenge today in the treatment to diabetic most remains and makes glucose level as much as possible near normal.In addition, postprandial hyperglycemia disease and hyperinsulinemia are the independent hazard factors that diabetes great vessels complication takes place.Report in " diabetic medicine ", alpha-glucosidase inhibitor is present in the alpha-glucosidase of intestinal microvillus, thereby rising rapidly and its insulin concentration that causes of suppressing after the feed rise, because of suppressing the human and human sugar of animal in addition, the particularly oligosaccharides of starch source, Sucrose Metabolism, demonstrate the effect that blood sugar rises that suppresses, to improving various diseases such as obesity due to hyperglycemia symptom and the hyperglycemia, diabetes of great use.In addition, add the food of alpha-glucosidase inhibitor, be applicable to the edible for patients of metabolic disturbance, be applicable to that also healthy people prevents food as metabolic disturbance.
Existing at present three kinds of alpha-glucosidase inhibitors are applied to clinical, are respectively acarbose (Acarbose), miglitol (Miglitol), voglibose (Viglibose), but have been found that there is abdominal discomfort in these medicines, flatulence, gurgling sound, digestive tract reactions such as exhaust, idol has side effects such as diarrhoea, its application is restricted (Uchida, R, Nasu, Aet.al Chem..Pharm.Bull 47,187-193,1999).Because these restrictions, it is extremely urgent to seek novel alpha-glucosidase inhibitor.
At present, producing alpha-glucosidase inhibitor both at home and abroad mostly is to extract from Chinese medicine, and traditional Chinese medicine extraction efficient is lower, and the cost height is difficult to industrialization.
Summary of the invention
The purpose of this invention is to provide a kind of alpha-glucosidase inhibitor and preparation method thereof, it is low that this method can solve traditional Chinese medicine extraction efficient, is difficult to realize the problem of industrialization.
Purpose of the present invention is achieved through the following technical solutions: a kind of alpha-glucosidase inhibitor, described inhibitor are that molecular weight is less than 1000 polypeptide.
The microbial strains for preparing described alpha-glucosidase inhibitor is a rose Streptomyces fulvissimus.
The application of described alpha-glucosidase inhibitor in preparation treatment diabetes medicament.
Prepare this alpha-glucosidase inhibitor and obtain by microbial fermentation, its method steps is as follows:
(1) actication of culture
Preparation adopts before the seed the synthetic solid medium of Gao Shi to carry out actication of culture, and the activatory bacterial classification changed over to cultivates the accumulation spore on the slant medium and prepare usefulness for seed,
Substratum weight consists of: Zulkovsky starch 1.0～3.0%, KNO
30.01～0.1%, NaCl 0.001～0.05%, K
2HPO
40.01～0.05%, MgSO
47H
2O 0.01～0.05%, FeSO
40.001～0.005%, agar powder 1.2～2.2%, surplus is a water, pH 7.0～10.0,
Culture condition is pH7.0～10.0 on the described slant medium, 28～35 ℃ of temperature temperature, and incubation time is 5～7d,
(2) seed culture
Then bacterial classification is cultivated by described culture condition at following substratum:
Substratum weight consists of: Semen Maydis powder 1.0～3.0%, Zulkovsky starch 1.0～3.0%, KNO
30.1～1.0%, NaCl 0.01～0.05%, K
2HPO
40.01～0.05%, MgSO
47H
2O 0.01～0.05%, FeSO
40.001～0.005%, surplus is a water,
The seed culture condition is pH7.0～10.0, and liquid amount is 25～50ml/250ml, 28～35 ℃ of temperature temperature, and incubation time is 1～2d.
(3) the seed liquid of fermentative production alpha-glucosidase inhibitor be inoculated in the fermention medium ferment,
Described fermention medium weight consists of: Semen Maydis powder 1.0～4.0%, analysis for soybean powder 0.5～2.0%, Zulkovsky starch 0.1～1.0%, KNO
30.5～2.0%, NaCl 0.01～0.05%, K
2HPO
40.01～0.05%, MgSO
47H
2O 0.01～0.05%, FeSO
40.001～0.005%, CaCO
30.1～0.35%, surplus is a water,
The condition of fermentation culture is pH7.0～10.0, and liquid amount is 25～50ml/250ml, 28～35 ℃ of temperature, and incubation time 4～6d,
(4) adopt conventional method then, separation and purification obtains alpha-glucosidase inhibitor from the fermentation culture product.
Outstanding advantage of the present invention is:
1, adopt the high yield alpha-glucosidase that screening obtains from soil to suppress active wild strain as producing bacterial strain, with Semen Maydis powder, corn steep liquor as the fermentation base-material, can guarantee that streptomycete produces breeding soon, the alpha-glucosidase inhibitor of producing is nontoxic, harmless, and containing has very strong inhibiting rate to alpha-glucosidase, α-Dian Fenmei, α-Pu Taotang amylase.
2, by the microorganisms producing enzyme inhibitors, raw material sources are simple, and production cost is low, and is pollution-free, and operation is implemented convenient, can enhance productivity greatly.The present invention is fermented, and to produce secondary metabolite be a kind of novel alpha-glucosidase inhibitor, and the appearance of novel alpha-glucosidase inhibitor also provides new instrument for enzyme active sites such as further research alpha-glucosidases.
Embodiment
Below further specify the present invention by specific examples.
Embodiment 1
(1) actication of culture: and the activatory bacterial classification changed on the slant medium cultivate a large amount of spores of accumulation and prepare usefulness in a large number for seeds.
Substratum weight consists of: Zulkovsky starch 2.0%, KNO
30.1%, NaCl 0.01%, K
2HPO
40.01%, MgSO
47H
2O 0.01%, FeSO
40.001%, agar powder 1.8%, surplus is a water, pH 7.0,28 ℃ of culture temperature, incubation time is 7d.
(2) seed culture
Spore on the inclined-plane is received in the seed culture medium with transfering loop picking one ring spore, after the inoculation, will be shaken bottle respectively under 26 ℃, 28 ℃, 30 ℃, 32 ℃, the 34 ℃ temperature, 160r/min cultivates, 24h, and each temperature is provided with three parallel laboratory tests.
Substratum weight consists of: Semen Maydis powder 2%, Zulkovsky starch 1.0%, KNO
30.5%, NaCl 0.05%, K
2HPO
40.05%, MgSO
47H
20.05%, FeSO
40.001%, surplus is a water.
PH 7.2, and liquid amount is 50ml/250ml.
(3) fermentation culture
Fermentation shake flask is 250ml, the fermention medium 50ml that packs into is after the sterilization, during switching, at first three parallel seeds being shaken bottle mixes, then transfer in the fermentation shake flask with the seed of 5ml liquid-transfering gun absorption 5ml, cultivation is respectively inoculated 9 from 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃ seed and is shaken bottle, places corresponding temperature condition mutually down, 160r/min cultivates, 5d, 6d, 7d get three shake the bottle detect.
Substratum weight consists of: Semen Maydis powder 2.0%, analysis for soybean powder 0.8%, Zulkovsky starch 0.5%, KNO
31.2%, NaCl 0.05%, K
2HPO
40.05%, MgSO
47H
2O 0.05%, FeSO
40.001%, CaCO
30.25%, surplus is a water, pH8.0.
(4) adopt conventional method, the novel alpha-glucosidase inhibitor of separation and purification from the fermentation culture product then.
Behind the centrifugal removal thalline of fermented liquid, measure alpha-glucosidase by aforesaid method and suppress active.It the results are shown in Table 1
Under the different incubation times of table 1 differing temps to the inhibition activity of alpha-glucosidase
Interpretation:
Temperature is in the biosynthesizing that is fit to alpha-glucosidase inhibitor more than 28 ℃, and wherein inhibiting rate reaches the highest 30 ℃ the time, and when incubation time was 5d, secondary metabolite reached the highest to the inhibition activity of alpha-glucosidase.
Embodiment 2
(1) actication of culture
With embodiment 1.
(2) seed culture
Prepare seed culture medium, the initial pH of each seed culture medium is respectively 6.0,7.0,8.0,9.0,10.0, after the sterilization cooling.Spore on the inclined-plane is received in the seed culture medium with transfering loop picking one ring spore, will be shaken bottle respectively at 30 ℃, 160r/min cultivates, 24h, and each pH value is provided with three parallel laboratory tests.
Substratum weight is formed with embodiment 1.
(3) fermentation culture
Fermentation shake flask is 250ml, fermention medium 50ml packs into, after the initial pH value of fermention medium is respectively 6.0,7.0,8.0,9.0,10.0 sterilizations, during switching, at first three parallel seeds are shaken bottle and mix, then transfer in the fermentation shake flask with the seed of 5ml liquid-transfering gun absorption 5ml, cultivation is respectively inoculated 3 from pH6.0,7.0,8.0,9.0,10.0 seed and is shaken bottle, place 30 ℃, cultivate under the condition of 160r/min, the 5d taking-up is shaken bottle and is detected.
Substratum weight is formed with embodiment 1.
(4) adopt conventional method, the novel alpha-glucosidase inhibitor of separation and purification from the fermentation culture product then.
Behind the centrifugal removal thalline of fermented liquid, press preceding method and measure alpha-glucosidase inhibition activity.It the results are shown in Table 1
The different initial pH bottom fermentation liquid of table 1 are to the inhibition activity of alpha-glucosidase
Interpretation:
The initial pH of substratum is in the biosynthesizing that is fit to alpha-glucosidase inhibitor more than 8.0, and wherein 9.0 o'clock inhibiting rates reach the highest, and secondary metabolite reaches the highest to the inhibition activity of alpha-glucosidase.
Embodiment 3
(1) actication of culture:
With embodiment 1.
(2) seed culture
Prepare seed culture medium, initial pH value is 9.0, the liquid amount 50ml/250ml of seed culture medium, the spore on the inclined-plane is received in the seed culture medium with transfering loop picking one ring spore, to shake bottle respectively at 30 ℃, 160r/min cultivates, and 24h is provided with three parallel laboratory tests.
Substratum weight is formed with embodiment 1.
(3) fermentation culture
Prepare fermention medium, initial pH is 9.0, liquid amount is respectively 20ml/250ml, 30ml/250ml, 40ml/250ml, 50ml/250ml, 60ml/250ml, 70ml/250ml, 80ml/250ml, 90ml/250ml, 100ml/250ml, after the sterilization, during switching, at first three parallel seeds being shaken bottle mixes, then transfer in the fermentation shake flask with the seed of 5ml liquid-transfering gun absorption 5ml, each is inoculated 3 and shakes bottle, place 30 ℃, cultivate under the condition of 160r/min, the 5d taking-up is shaken bottle and is detected.
Substratum weight is formed with embodiment 1.
(4) adopt conventional method, the novel alpha-glucosidase inhibitor of separation and purification from the fermentation culture product then.
Behind the centrifugal removal thalline of fermented liquid, measure alpha-glucosidase by aforesaid method and suppress active.It the results are shown in Table 1
The different liquid amount bottom fermentation of table 1 liquid is to the inhibition activity of alpha-glucosidase
Interpretation:
Experimental result shows, under the moderate aeration condition, as 40ml/250ml, helps the biosynthesizing of alpha-glucosidase inhibitor, and too high or low excessively aeration condition all will be unfavorable for the biosynthesizing of alpha-glucosidase inhibitor.
Claims (3)
1. an alpha-glucosidase inhibitor is characterized in that, described inhibitor is that molecular weight is less than 1000 polypeptide.
2. the microbial strains of the described alpha-glucosidase inhibitor of preparation claim 1 is a rose Streptomyces fulvissimus.
3. the application of the described alpha-glucosidase inhibitor of claim 1 in preparation treatment diabetes medicament.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910114494A CN101724015A (en) | 2009-10-30 | 2009-10-30 | Alpha-glucosidase inhibitor and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910114494A CN101724015A (en) | 2009-10-30 | 2009-10-30 | Alpha-glucosidase inhibitor and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114099A (en) * | 2013-02-07 | 2013-05-22 | 广西大学 | Beta-glucosaccharase gene for coding glycosyl hydrolase family 1 and application thereof |
| CN109021076A (en) * | 2018-08-31 | 2018-12-18 | 华南理工大学 | A kind of hypoglycemic heptapeptide |
| CN109021079A (en) * | 2018-08-31 | 2018-12-18 | 华南理工大学 | A kind of hypoglycemic ten hexapeptide |
| CN109021075A (en) * | 2018-08-31 | 2018-12-18 | 华南理工大学 | A kind of hypoglycemic decapeptide |
-
2009
- 2009-10-30 CN CN200910114494A patent/CN101724015A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114099A (en) * | 2013-02-07 | 2013-05-22 | 广西大学 | Beta-glucosaccharase gene for coding glycosyl hydrolase family 1 and application thereof |
| CN103114099B (en) * | 2013-02-07 | 2014-06-11 | 广西大学 | Beta-glucosaccharase gene for coding glycosyl hydrolase family 1 and application thereof |
| CN109021076A (en) * | 2018-08-31 | 2018-12-18 | 华南理工大学 | A kind of hypoglycemic heptapeptide |
| CN109021079A (en) * | 2018-08-31 | 2018-12-18 | 华南理工大学 | A kind of hypoglycemic ten hexapeptide |
| CN109021075A (en) * | 2018-08-31 | 2018-12-18 | 华南理工大学 | A kind of hypoglycemic decapeptide |
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Open date: 20100609 |