CN105624244B - A method of extracting ring (sweet-the third) dipeptides from bacillus coagulans - Google Patents
A method of extracting ring (sweet-the third) dipeptides 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 method that the invention discloses one kind extracting ring (sweet-the third) dipeptides from bacillus coagulans.It includes actication of culture and culture to prepare ring (sweet-the third) dipeptides method mainly, the acquisition of crude extract, by crude extract through silica gel column chromatography rough segmentation, it is purified using recrystallization method, TLC tracing detections obtain compound and are detected through nuclear-magnetism and be accredited as ring (sweet-the third) dipeptides with document comparison.The raw materials used in the present invention bacillus coagulans are a kind of probiotics, and safe and harmless, extraction process is simple, at low cost；Compound purity obtained is high.
Description
Technical field
The invention belongs to the preparing technical fields of ring (sweet-the third) dipeptides, and in particular to one kind is carried from bacillus coagulans
The method for taking ring (sweet-the third) dipeptides.
Background technology
Bacillus coagulans（bacillus coagulans LL1103）, also known as lactic acid bacteria both possessed lactic acid bacteria production
The characteristic of lactic acid, and there is the strong abundant enzyme system and resistance of bacillus, high temperature resistant, bile tolerance, the characteristic easily stored.
As the up-and-coming youngster of probiotics family, be made into probiotics oneself be widely used in aquaculture, material is watched in herding,
The industries such as food and medicine.Bacillus coagulans are other than the health-care effect in maintaining intestinal microecology balance, to common
Intestines problem such as chronic colitis, diarrhea and constipation etc. have obvious curative effects, also have and adjust blood pressure and blood lipoid and improve immunity
Effect.
Research shows that having the polypeptides matter bacterium for inhibiting spoilage organisms and pathogenic bacteria in bacillus coagulans metabolite
Element generates the report of anti-plant pathogenic fungi also on bacillus coagulans in recent years.But bacillus coagulans metabolism production
But have no that the report of small-molecule substance CYCLIC DIPEPTIDES compounds, Cyclic dipeptides are a kind of diketone for having and stablizing six-membered ring structure in object
Piperazine substance shows antibacterium, antimycotic, antiviral and antitumor, immunosupress, neuroprotection, antihyperglycemic, antimalarial
The multiple biological activities such as disease and pharmacological activity.
Invention content
The object of the present invention is to provide a kind of preparation processes, and ring (sweet-the third) dipeptides purity simple, obtained is high from condensation
The method that ring (sweet-the third) dipeptides is extracted in bacillus.
To achieve the above object, the technical solution adopted by the present invention is, one kind extracted from bacillus coagulans ring (it is sweet-
The third) structural formula of the method for dipeptides, ring (sweet-the third) dipeptides is as follows：
；
It the described method comprises the following steps：
（1）Actication of culture：Bacillus coagulans are inoculated on plating medium, cultivates 24 hours, obtains under the conditions of 37 DEG C
Obtain activated spawn；
（2）Primary culture：By step（1）In obtained activated spawn be inoculated into MRS fluid nutrient mediums, in 150r/
Min, under the conditions of 37 DEG C, shaking table culture 12~16 hours obtains seed liquor；
（3）Secondary culture：By step（2）Obtained seed liquor is inoculated into MRS fluid nutrient mediums, and the volume of seed liquor is
The 4~6% of MRS fluid nutrient medium volumes, under the conditions of 150r/min, 37 DEG C, shaking table culture obtains zymotic fluid in 48~72 hours；
（4）It is prepared by crude extract：By step（3）It is centrifuged after the zymotic fluid concentrated by rotary evaporation of preparation to the 1/4 of original fermentation liquor volume,
Supernatant ethanol precipitation is taken, filter and continues to concentrate the filtrate to the 1/4 of former filtrate volume, is then extracted with ethyl acetate,
Water phase is extracted again with n-butanol, butanol extraction liquid is concentrated under reduced pressure and obtains crude extract；
（5）Silica gel column chromatography rough segmentation：By step（4）The crude extract of preparation is dissolved with methanol, is then filled after silica gel mixed sample
Column, stationary phase is the silica gel of 80-100 mesh, with dichloromethane：Methanol volume ratio=50:1~1:1 gradient elution, silica gel thin-layer layer
The volume ratio of analysis detection, combined dichloromethane-methanol is 1:1 fraction；
（6）By step（5）Obtained fraction is purified using recrystallization method, and the concrete operations of recrystallization method purifying are：It will step
Suddenly（5）Obtained fraction concentrated by rotary evaporation removes dichloromethane and methanol, dry, and dichloromethane is added at 50 DEG C：Methanol（Volume
Than）=1:1 methylene chloride-methanol solvent dissolving is complete, then condensation 12 at 4 DEG C ~ for 24 hours, filters, dry, is washed with methanol,
Dichloromethane is added in solid after methanol is washed：Methanol（Volume ratio）=1:1 methylene chloride-methanol dissolving is complete, and then 4
Condensation 12 at DEG C ~ for 24 hours, it filters, it is dry, obtain ring (sweet-the third) dipeptides.
The formula of the plating medium is：Peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, grape
Sugared 20.0g/L, sodium acetate 5.0g/L, lemon acid diamine 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganese sulfate
0.25g/L, agar 15.0g/L, Tween-80 1.0mL/L, pH 6.0.
The formula of the MRS fluid nutrient mediums is：Peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, Portugal
Grape sugar 20.0g/L, sodium acetate 5.0g/L, lemon acid diamine 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, sulfuric acid
Manganese 0.25g/L, Tween-80 1.0mL/L, pH 6.0.
The step（4）The process conditions centrifuged after middle zymotic fluid concentrated by rotary evaporation are：Centrifugal condition is 4 DEG C of temperature,
Rotating speed is 5000rpm, centrifugation time 20min.
The step（4）The concrete operations of middle supernatant ethanol precipitation are：It is added in equal volume into supernatant
The ethyl alcohol of 95vol%.
The step（4）The concrete operations that filtrate is extracted with ethyl acetate after middle concentration are：Filtrate and acetic acid after concentrating
1 ︰ 1 is mixed ethyl ester by volume, is poured into separatory funnel, and shaking flask extracts 2 ~ 3 times, stands 20~30min every time, obtains water phase；
The concrete operations of the water phase that ethyl acetate is obtained by extraction extracting n-butyl alcohol are：1 ︰ 1 is mixed water phase by volume with n-butanol,
Enter in separatory funnel, shaking flask extracts 2 ~ 3 times, stands 20~30min every time, obtains butanol extraction liquid.
The beneficial effect comprise that：The present invention carries out ring (sweet-the third) dipeptides by raw material of bacillus coagulans
Extraction, bacillus coagulans are a kind of probiotics, and metabolite has natural sex safety, and extracting method is simple, cost
It is low, have sustainability, ring (sweet-the third) dipeptides purity obtained high（Up to 99%）, to promoting ring (sweet-the third) dipeptides pharmacology
It further studies and is of great significance as lead compound development and utilization.
Description of the drawings
Fig. 1 is ring (sweet-the third) dipeptides prepared by embodiment 11H NMR figures；
Fig. 2 is ring (sweet-the third) dipeptides prepared by embodiment 113C NMR figures.
Specific implementation mode
The present invention is further explained in the light of specific embodiments, and but the scope of the present invention is not limited thereto.With
Bacillus coagulans used in lower embodiment derive from China typical culture collection center（Deposit number：CCTCC NO：
M2013193）.
Embodiment 1
A method of it extracting ring (sweet-the third) dipeptides from bacillus coagulans, includes the following steps：
（1）Actication of culture：Bacillus coagulans are inoculated on plating medium, cultivates 24 hours, obtains under the conditions of 37 DEG C
Obtain activated spawn；
（2）Primary culture：By step（1）In obtained activated spawn be inoculated into MRS fluid nutrient mediums, in 150r/
Min, under the conditions of 37 DEG C, shaking table culture 12 hours obtains seed liquor 1L；
（3）Secondary culture：By step（2）Obtained seed liquor is inoculated into MRS fluid nutrient mediums, and the volume of seed liquor is
The 4% of MRS fluid nutrient medium volumes, under the conditions of 150r/min, 37 DEG C, shaking table culture obtains zymotic fluid 20L in 48 hours；
（4）It is prepared by crude extract：By step（3）It centrifuges, takes after the zymotic fluid concentrated by rotary evaporation to the 1/4 of original volume of preparation
Clear liquid ethanol precipitation filters and continues to concentrate the filtrate to the 1/4 of original volume, is then extracted with ethyl acetate, by water phase
It is extracted again with n-butanol, butanol extraction liquid is concentrated under reduced pressure and obtains crude extract 20g；
（5）Silica gel column chromatography rough segmentation：By step（4）The crude extract of preparation fills column after dissolving then silica gel mixed sample with methanol,
Stationary phase is the silica gel of 80-100 mesh, with dichloromethane：Methanol volume ratio=50:1~1:1 gradient elution（Dichloromethane：Methanol
Volume ratio is 50:Isosorbide-5-Nitrae 0:1,30:1,20:1,10:1,1:1）, silica gel thin-layer chromatography detection, collect the body of methylene chloride-methanol
Product is than being 1:1 fraction；
（6）By step（5）Obtained fraction is purified using recrystallization method, and the concrete operations of recrystallization method purifying are：It will step
Suddenly（5）Obtained fraction concentrated by rotary evaporation removes dichloromethane and methanol, dry, and dichloromethane is added at 50 DEG C：Methanol=1:1
The dissolving of methylene chloride-methanol solvent it is complete, then condensation 12 at 4 DEG C ~ for 24 hours is filtered, dry, is washed with methanol, methanol is washed
Dichloromethane is added in solid after washing：Methanol=1:1 methylene chloride-methanol solvent dissolving is complete, then condensation 12 at 4 DEG C ~
For 24 hours, it filters, it is dry, obtain ring (sweet-the third) dipeptides（940mg, purity 99.1%）.
The formula of the plating medium is：Peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, grape
Sugared 20.0g/L, sodium acetate 5.0g/L, lemon acid diamine 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganese sulfate
0.25g/L, agar 15.0g/L, Tween-80 1.0mL/L, pH 6.0.
The formula of the MRS fluid nutrient mediums is：Peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, Portugal
Grape sugar 20.0g/L, sodium acetate 5.0g/L, lemon acid diamine 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, sulfuric acid
Manganese 0.25g/L, Tween-80 1.0mL/L, pH 6.0.
The step（4）The process conditions centrifuged after middle zymotic fluid concentrated by rotary evaporation are：Centrifugal condition is 4 DEG C of temperature,
Rotating speed is 5000rpm, centrifugation time 20min.
The step（4）The concrete operations of supernatant ethanol precipitation are：95% isometric second is added into supernatant
Alcohol.
The step（4）The concrete operations that filtrate is extracted with ethyl acetate after middle concentration are：Filtrate and acetic acid after concentrating
1 ︰ 1 is mixed ethyl ester by volume, is poured into separatory funnel, shaking flask extracts 2 times, stands 30min every time, obtains water phase；Acetic acid second
The concrete operations that water phase extracting n-butyl alcohol is obtained by extraction in ester are：1 ︰ 1 is mixed water phase by volume with n-butanol, pours into liquid separation leakage
In bucket, shaking flask extracts 2 times, stands 30min every time, obtains butanol extraction liquid.
By Fig. 1-2 it is found that through1H NMR、13C NMR analyses, compare document（Guo Qiong, Wang Jian, Yao Junhua wait mono-
Strain South Sea coral bacterium L-4 antitumor activity secondary metabolites research [J] Zhongshan University journal, 2013,52 (3):
77-82.）, confirm that the compound extracted 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 by above-described embodiment
Limitation, it is other any without departing from the substitute mode that should be equivalent is changed made by the present invention, it is included in the guarantor of the present invention
Within the scope of shield.
Claims (3)
1. a kind of method preparing ring (sweet-the third) dipeptides using microorganism, the structural formula of ring (sweet-the third) dipeptides are as follows：
；
It is characterized in that, the described method comprises the following steps：
（1）Actication of culture：Bacillus coagulans are inoculated on plating medium, cultivates 24 hours, is lived under the conditions of 37 DEG C
Change strain；The deposit number of the bacillus coagulans is：CCTCC NO：M2013193；
（2）Primary culture：By step（1）In obtained activated spawn be inoculated into MRS fluid nutrient mediums, in 150r/min, 37
Under the conditions of DEG C, shaking table culture 12~16 hours obtains seed liquor；
（3）Secondary culture：By step（2）Obtained seed liquor is inoculated into MRS fluid nutrient mediums, and the volume of seed liquor is MRS
The 4~6% of fluid nutrient medium volume, under the conditions of 150r/min, 37 DEG C, shaking table culture obtains zymotic fluid in 48~72 hours；
（4）It is prepared by crude extract：By step（3）It is centrifuged after the zymotic fluid concentrated by rotary evaporation to the 1/4 of original volume of preparation, takes supernatant
With ethanol precipitation, the 1/4 of original volume is filtered and continue to concentrate the filtrate to, is then extracted with ethyl acetate, by water phase with just
Butanol is extracted again, and butanol extraction liquid is concentrated under reduced pressure and obtains crude extract；
（5）Silica gel column chromatography rough segmentation：By step（4）The crude extract of preparation is dissolved with methanol, then fills column after silica gel mixed sample, Gu
Fixed is mutually the silica gel of 80-100 mesh, with dichloromethane：Methanol volume ratio=50:1~1:1 gradient elution, silica gel thin-layer chromatography inspection
It surveys, the volume ratio for collecting methylene chloride-methanol is 1:1 fraction；
（6）By step（5）Obtained fraction purifies to obtain ring (sweet-the third) dipeptides using recrystallization method；
The formula of the plating medium is：Peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, glucose
20.0g/L, sodium acetate 5.0g/L, lemon acid diamine 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganese sulfate
0.25g/L, agar 15.0g/L, Tween-80 1.0mL/L, pH 6.0；
The formula of the MRS fluid nutrient mediums is：Peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, glucose
20.0g/L, sodium acetate 5.0g/L, lemon acid diamine 2.0g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.58g/L, manganese sulfate
0.25g/L, Tween-80 1.0mL/L, pH 6.0；
The step（4）The concrete operations that filtrate is extracted with ethyl acetate after middle concentration are：Filtrate and ethyl acetate after concentrating
1 ︰ 1 is mixed by volume, is poured into separatory funnel, and shaking flask extracts 2 ~ 3 times, stands 20~30min every time, obtains water phase；Acetic acid
The concrete operations that water phase extracting n-butyl alcohol is obtained by extraction in ethyl ester are：1 ︰ 1 is mixed water phase by volume with n-butanol, pours into liquid separation
In funnel, shaking flask extracts 2 ~ 3 times, stands 20~30min every time, obtains butanol extraction liquid；
The step（6）It is middle by step（5）Obtained fraction use recrystallization method purify concrete operations for：By step（5）?
The fraction concentrated by rotary evaporation arrived removes dichloromethane and methanol, dry, and dichloromethane is added at 50 DEG C：Methanol=1:1 dichloromethane
Alkane-methanol solvate dissolving, then condensation 12 at 4 DEG C ~ for 24 hours, is filtered, dry, is washed with methanol, the solid after methanol is washed adds
Enter dichloromethane：Methanol=1:1 methylene chloride-methanol dissolving, then condensation 12 at 4 DEG C ~ for 24 hours, is filtered, dry, obtain ring (it is sweet-
The third) dipeptides.
2. the method for preparing ring (sweet-the third) dipeptides using microorganism as described in claim 1, which is characterized in that the step
（4）The process conditions centrifuged after middle zymotic fluid concentrated by rotary evaporation are：Centrifugal condition is 4 DEG C, rotating speed 5000rpm of temperature, when centrifugation
Between 20min.
3. the method for preparing ring (sweet-the third) dipeptides using microorganism as described in claim 1, which is characterized in that the step
（4）The concrete operations of supernatant ethanol precipitation are：95% isometric ethyl alcohol is added into supernatant.
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Non-Patent Citations (2)
| Title |
|---|
| 海洋细菌Bacillus sp.次生代谢产物的分离与鉴定;高昊等;《沈阳药科大学学报》;20100131;第27卷(第1期);摘要，第69页右栏第2-3段，第70页第1段、右栏第1段，第72页左栏第2段，图1 * |
| 海洋细菌Bacillus sp.环二肽类代谢产物的研究;姚遥等;《中国药物化学杂志》;20071031;第17卷(第5期);第311页左栏第4段-右栏第2段，第311页右栏第4段-第312页左栏第1段 * |
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