CN105624244A - Method for extracting cyclo(glycinyl-alaninyl)dipeptide from bacillus coagulans - Google Patents
Method for extracting cyclo(glycinyl-alaninyl)dipeptide from bacillus coagulans Download PDFInfo
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- C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
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Abstract
The invention discloses a method for extracting cyclo(glycinyl-alaninyl)dipeptide from bacillus coagulans. The method mainly comprises the following steps: performing bacterial activation and culture to obtain a crude extract, coarsely separating the crude extract through silica gel column chromatography, purifying through recrystallization, performing TLC (thin layer chromatography) tracking and detection to obtain a compound, and performing nuclear magnetic resonance detection and document comparison to identify the compound as the cyclo(glycinyl-alaninyl)dipeptide. The bacillus coagulans used by the method as a raw material is a probiotic, is safe and harmless; the method is simple in extracting process and low in cost; the obtained compound is high in purity.
Description
Technical field
The invention belongs to the preparing technical field of ring (sweet-the third) dipeptides, it is specifically related to a kind of method extracting ring (sweet-the third) dipeptides from Bacillus coagulans.
Background technology
Bacillus coagulans (bacilluscoagulansLL1103), also known as lactic acid bacteria, had both had the characteristic of milk-acid bacteria lactic acid producing, had again abundant enzyme system and strong stress resistance, high temperature resistant, bile tolerance, the easy characteristic stored of genus bacillus. As the up-and-coming youngster of probiotic bacterium family, be made into probiotics oneself be widely used in aquaculture, herding watch the industries such as material, food and medicine. Bacillus coagulans is except, except the health-care effect maintained in intestinal microecology balance, common intestinal tract disease as chronic colitis, diarrhoea and constipation etc. have obvious curative effects, is also had the effect regulating blood pressure and blood lipoid and improving immunizing power by it.
Research shows to have the polypeptides matter bacteriocin suppressing spoilage organism and pathogenic bacterium in Bacillus coagulans meta-bolites, also has the report producing anti-plant pathogenic fungi about Bacillus coagulans in recent years. But Bacillus coagulans meta-bolites but has no the report of small-molecule substance CYCLIC DIPEPTIDES compounds; Cyclic dipeptides is a kind of diketopiperazine class material with stable six-membered ring structure, shows multiple biological activity and the pharmacologically actives such as antibacterium, antimycotic, antiviral, antitumor, immunosuppression, neuroprotective, hyperglycemia, anti-malarial.
Summary of the invention
It is an object of the invention to provide the method extracting ring (sweet-the third) dipeptides from Bacillus coagulans that a kind of preparation technology ring (sweet-the third) dipeptides purity simple, obtained is high.
For achieving the above object, the technical solution used in the present invention is, a kind of method extracting ring (sweet-the third) dipeptides from Bacillus coagulans, and the structural formula of ring (sweet-the third) dipeptides is as follows:
;
Described method comprises the following steps:
(1) actication of culture: be inoculated on plate culture medium by Bacillus coagulans, cultivates 24 hours under 37 DEG C of conditions, obtains activated spawn;
(2) elementary cultivation: the activated spawn obtained in step (1) is inoculated in MRS liquid nutrient medium, at 150r/min, under 37 DEG C of conditions, shaking table is cultivated 12��16 hours, obtains seed liquor;
(3) secondary cultivation: the seed liquor that step (2) obtains is inoculated in MRS liquid nutrient medium, the volume of seed liquor is the 4��6% of MRS liquid nutrient medium volume, and at 150r/min, under 37 DEG C of conditions, fermented liquid cultivated 48��72 hours to obtain by shaking table;
(4) crude extract preparation: centrifugal after fermented liquid concentrated by rotary evaporation prepared by step (3) to the 1/4 of original fermented solution volume, get supernatant liquor alcohol settling, filter and continue to concentrate filtrate 1/4 to former filtrate volume, then it is extracted with ethyl acetate, aqueous phase propyl carbinol is extracted again, butanol extraction liquid concentrating under reduced pressure is obtained crude extract;
(5) silica gel column chromatography rough segmentation: crude extract dissolve with methanol prepared by step (4), then post is filled after silica gel mixed sample, stationary phase is 80-100 object silica gel, with methylene dichloride: methyl alcohol volume ratio=50:1��1:1 gradient elution, silica gel thin-layer chromatography detects, and the volume ratio of combined dichloromethane-methyl alcohol is stream part of 1:1;
(6) stream part step (5) obtained adopts recrystallization method purifying, specifically being operating as of recrystallization method purifying: stream part concentrated by rotary evaporation step (5) obtained removes methylene dichloride and methyl alcohol, dry, methylene dichloride is added: the methylene chloride-methanol dissolution with solvents of methyl alcohol (volume ratio)=1:1 is complete at 50 DEG C, then condensation 12 ~ 24h at 4 DEG C, filter, dry, by methanol wash, solid after methanol wash is added methylene dichloride: the methylene chloride-methanol of methyl alcohol (volume ratio)=1:1 dissolves completely, then condensation 12 ~ 24h at 4 DEG C, filter, dry, obtain ring (sweet-the third) dipeptides.
The formula of described plate culture medium is: peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid diamines 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, agar 15.0g/L, tween-80 1.0mL/L, pH6.0.
The formula of described MRS liquid nutrient medium is: peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid diamines 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween-80 1.0mL/L, pH6.0.
In described step (4), after fermented liquid concentrated by rotary evaporation, the processing condition of centrifugation are: centrifugal condition is temperature 4 DEG C, and rotating speed is 5000rpm, centrifugation time 20min.
Specifically being operating as of supernatant liquor alcohol settling in described step (4): the ethanol adding isopyknic 95vol% in supernatant liquor.
In described step (4) concentrated after the concrete operation that is extracted with ethyl acetate of filtrate be: after will concentrate, filtrate and ethyl acetate 11 mix by volume, pour in separating funnel, and shaking flask extracts 2 ~ 3 times, and standing 20��30min, obtains aqueous phase every time; The concrete operation of the aqueous phase n-butanol extraction that extraction into ethyl acetate obtains is: aqueous phase and propyl carbinol 11 mix by volume, pour in separating funnel, shaking flask extraction 2 ~ 3 times, leave standstill 20��30min every time, obtain butanol extraction liquid.
The useful effect that the present invention produces is: the present invention take Bacillus coagulans as the extraction that raw material carries out ring (sweet-the third) dipeptides, Bacillus coagulans is a kind of probiotic bacterium, its meta-bolites has natural sex security, and extracting method is simple, cost is low, there is sustainability, obtained ring (sweet-the third) dipeptides purity height (can reach 99%), studies further to promotion ring (sweet-the third) dipeptides pharmacology and significant as lead compound development and utilization.
Accompanying drawing explanation
Fig. 1 is ring (sweet-the third) dipeptides of embodiment 1 preparation1HNMR schemes;
Fig. 2 is ring (sweet-the third) dipeptides of embodiment 1 preparation13CNMR schemes.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but protection scope of the present invention is not limited to this. Bacillus coagulans used in following examples derives from China typical culture collection center (deposit number: CCTCCNO:M2013193).
Embodiment 1
A kind of method extracting ring (sweet-the third) dipeptides from Bacillus coagulans, comprises the following steps:
(1) actication of culture: be inoculated on plate culture medium by Bacillus coagulans, cultivates 24 hours under 37 DEG C of conditions, obtains activated spawn;
(2) elementary cultivation: the activated spawn obtained in step (1) is inoculated in MRS liquid nutrient medium, at 150r/min, under 37 DEG C of conditions, shaking table cultivates 12 hours, obtains seed liquor 1L;
(3) secondary cultivation: the seed liquor that step (2) obtains is inoculated in MRS liquid nutrient medium, the volume of seed liquor is the 4% of MRS liquid nutrient medium volume, and at 150r/min, under 37 DEG C of conditions, fermented liquid 20L cultivated 48 hours to obtain by shaking table;
(4) crude extract preparation: centrifugal after fermented liquid concentrated by rotary evaporation prepared by step (3) to the 1/4 of original volume, get supernatant liquor alcohol settling, filter and continue to concentrate filtrate 1/4 to original volume, then it is extracted with ethyl acetate, aqueous phase propyl carbinol is extracted again, butanol extraction liquid concentrating under reduced pressure is obtained crude extract 20g;
(5) silica gel column chromatography rough segmentation: then crude extract dissolve with methanol step (4) prepared fills post after silica gel mixed sample, stationary phase is 80-100 object silica gel, taking methylene dichloride: methyl alcohol volume ratio=50:1��1:1 gradient elution (methylene dichloride: methyl alcohol volume ratio is as 50:1,40:1,30:1,20:1,10:1,1:1), silica gel thin-layer chromatography detects, and the volume ratio collecting methylene chloride-methanol is stream part of 1:1;
(6) stream part step (5) obtained adopts recrystallization method purifying, specifically being operating as of recrystallization method purifying: stream part concentrated by rotary evaporation step (5) obtained removes methylene dichloride and methyl alcohol, dry, methylene dichloride is added: the methylene chloride-methanol dissolution with solvents of methyl alcohol=1:1 is complete at 50 DEG C, then condensation 12 ~ 24h at 4 DEG C, filter, dry, by methanol wash, solid after methanol wash is added methylene dichloride: the methylene chloride-methanol dissolution with solvents of methyl alcohol=1:1 is complete, then condensation 12 ~ 24h at 4 DEG C, filter, dry, obtain ring (sweet-the third) dipeptides (940mg, purity 99.1%).
The formula of described plate culture medium is: peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid diamines 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, agar 15.0g/L, tween-80 1.0mL/L, pH6.0.
The formula of described MRS liquid nutrient medium is: peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid diamines 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween-80 1.0mL/L, pH6.0.
In described step (4), after fermented liquid concentrated by rotary evaporation, the processing condition of centrifugation are: centrifugal condition is temperature 4 DEG C, and rotating speed is 5000rpm, centrifugation time 20min.
Specifically being operating as of described step (4) supernatant liquor alcohol settling: add the ethanol of isopyknic 95% in supernatant liquor.
In described step (4) concentrated after the concrete operation that is extracted with ethyl acetate of filtrate be: will concentrate filtrate and ethyl acetate afterwards and 11 mix by volume, and pour in separating funnel, shaking flask extracts 2 times, leaves standstill 30min every time, obtains aqueous phase; Extraction into ethyl acetate obtains the concrete operation of aqueous phase n-butanol extraction: aqueous phase and propyl carbinol 11 mix by volume, pour in separating funnel, and shaking flask extracts 2 times, leaves standstill 30min every time, obtains butanol extraction liquid.
By Fig. 1-2 it will be seen that warp1HNMR��13CNMR analyzes, comparison document (Guo Qiong, Wang Jian, Yao Junhua, etc. strain South Sea coral bacterium L-4 anti-tumor activity secondary metabolite research [J]. Zhongshan University's journal, 2013,52 (3): 77-82.) compound, confirming to extract is ring (sweet-the third) dipeptides��
Above-described embodiment is the preferred embodiment of the present invention, but embodiments of the present invention are not restricted to the described embodiments, and the change that other any the present invention of not deviating from does all should be the substitute mode of equivalence, is included within protection scope of the present invention.
Claims (7)
1. extracting a method for ring (sweet-the third) dipeptides from Bacillus coagulans, the structural formula of ring (sweet-the third) dipeptides is as follows:
;
It is characterized in that, described method comprises the following steps:
(1) actication of culture: be inoculated on plate culture medium by Bacillus coagulans, cultivates 24 hours under 37 DEG C of conditions, obtains activated spawn;
(2) elementary cultivation: the activated spawn obtained in step (1) is inoculated in MRS liquid nutrient medium, at 150r/min, under 37 DEG C of conditions, shaking table is cultivated 12��16 hours, obtains seed liquor;
(3) secondary cultivation: the seed liquor that step (2) obtains is inoculated in MRS liquid nutrient medium, the volume of seed liquor is the 4��6% of MRS liquid nutrient medium volume, and at 150r/min, under 37 DEG C of conditions, fermented liquid cultivated 48��72 hours to obtain by shaking table;
(4) crude extract preparation: centrifugal after fermented liquid concentrated by rotary evaporation prepared by step (3) to the 1/4 of original volume, get supernatant liquor alcohol settling, filter and continue to concentrate filtrate 1/4 to original volume, then it is extracted with ethyl acetate, aqueous phase propyl carbinol is extracted again, butanol extraction liquid concentrating under reduced pressure is obtained crude extract;
(5) silica gel column chromatography rough segmentation: crude extract dissolve with methanol prepared by step (4), then post is filled after silica gel mixed sample, stationary phase is 80-100 object silica gel, with methylene dichloride: methyl alcohol volume ratio=50:1��1:1 gradient elution, silica gel thin-layer chromatography detects, and the volume ratio collecting methylene chloride-methanol is stream part of 1:1;
(6) stream part step (5) obtained adopts recrystallization method purifying to obtain ring (sweet-the third) dipeptides.
2. from Bacillus coagulans, extract the method for ring (sweet-the third) dipeptides as claimed in claim 1, it is characterized in that, the formula of described plate culture medium is: peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid diamines 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, agar 15.0g/L, tween-80 1.0mL/L, pH6.0.
3. from Bacillus coagulans, extract the method for ring (sweet-the third) dipeptides as claimed in claim 1, it is characterized in that, the formula of described MRS liquid nutrient medium is: peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid diamines 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween-80 1.0mL/L, pH6.0.
4. from Bacillus coagulans, extract the method for ring (sweet-the third) dipeptides as claimed in claim 1, it is characterized in that, processing condition centrifugal after fermented liquid concentrated by rotary evaporation in described step (4) are: centrifugal condition is temperature 4 DEG C, and rotating speed is 5000rpm, centrifugation time 20min.
5. from Bacillus coagulans, extract the method for ring (sweet-the third) dipeptides as claimed in claim 1, it is characterised in that, being specifically operating as of described step (4) supernatant liquor alcohol settling: add the ethanol of isopyknic 95% in supernatant liquor.
6. from Bacillus coagulans, extract the method for ring (sweet-the third) dipeptides as claimed in claim 1, it is characterized in that, in described step (4) concentrated after the concrete operation that is extracted with ethyl acetate of filtrate be: will concentrate filtrate and ethyl acetate afterwards and 11 mix by volume, pour in separating funnel, shaking flask extraction 2 ~ 3 times, leave standstill 20��30min every time, obtain aqueous phase; Extraction into ethyl acetate obtains the concrete operation of aqueous phase n-butanol extraction: aqueous phase and propyl carbinol 11 mix by volume, pour in separating funnel, shaking flask extraction 2 ~ 3 times, leave standstill 20��30min every time, obtain butanol extraction liquid.
7. from gemma lactobacillus, extract the method for ring (sweet-the third) dipeptides as claimed in claim 1, it is characterized in that, stream part step (5) obtained in described step (6) adopts specifically being operating as of recrystallization method purifying: stream part concentrated by rotary evaporation step (5) obtained removes methylene dichloride and methyl alcohol, dry, methylene dichloride is added: the methylene chloride-methanol dissolution with solvents of methyl alcohol=1:1 at 50 DEG C, then condensation 12 ~ 24h at 4 DEG C, filter, dry, by methanol wash, solid after methanol wash is added methylene dichloride: the methylene chloride-methanol of methyl alcohol=1:1 dissolves, then condensation 12 ~ 24h at 4 DEG C, filter, dry, obtain ring (dried meat-Gan) dipeptides.
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| CN201610181179.4A CN105624244B (en) | 2016-03-28 | 2016-03-28 | A method of extracting ring (sweet-the third) dipeptides from bacillus coagulans |
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Non-Patent Citations (2)
| Title |
|---|
| 姚遥等: "海洋细菌Bacillus sp.环二肽类代谢产物的研究", 《中国药物化学杂志》 * |
| 高昊等: "海洋细菌Bacillus sp.次生代谢产物的分离与鉴定", 《沈阳药科大学学报》 * |
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