CN102653778A - Enzymolysis preparation method of phosphatidylinositol - Google Patents
Enzymolysis preparation method of phosphatidylinositol Download PDFInfo
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- CN102653778A CN102653778A CN2012101410979A CN201210141097A CN102653778A CN 102653778 A CN102653778 A CN 102653778A CN 2012101410979 A CN2012101410979 A CN 2012101410979A CN 201210141097 A CN201210141097 A CN 201210141097A CN 102653778 A CN102653778 A CN 102653778A
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Abstract
The invention discloses an enzymolysis preparation method of phosphatidylinositol. The enzymolysis preparation method comprises the following steps of: dissolving pretreated soybean phosphatide in solvent, and adding phospholipase D in the solvent, wherein the addition of the phospholipase D is 50 to 150 units per 100mg the pretreated soybean phosphatide; stirring for 3 to 12 hours at 36 DEG C to 40 DEG C and pH value ranging from 7.0 to 9.0, thus obtaining an enzymolysis product; and performing extraction on the enzymolysis product by virtue of normal hexane, concentrating and drying extraction liquid, washing by virtue of ethanol or ethanol aqueous solution, and drying to obtain the phosphatidylinositol product. The pretreated soybean phosphatide is prepared by the following steps of: dissolving soybean phosphatide in chloroform, settling by virtue of methanol, collecting sediments, repeating the steps for several times, and drying the final obtained sediment to obtain the pretreated soybean phosphatide. The enzymolysis preparation method is simple and relatively low in cost; and the purity of the prepared phosphatidylinositol is high, and thus the phosphatidylinositol can serve as a chromogenic substrate to be applied to a chromogenic medium for quickly detecting LM (listeria monocytogenes).
Description
Technical field:
The invention belongs to biological technical field, be specifically related to a kind of enzymolysis preparation of PI.
Background technology:
(Listeria monocytogenes LM) is a kind of important food-borne pathogens to listeria monocytogenes.To the distinctive inositol monophosphate Phospholipid hydrolase of LM (Cphosphatidylinositol phospholipase C, PI-PLC) and β-D-glucuroide (method that the design of β-D-glucosidase) chromogenic substrate carries out rapid detection receives much concern in recent years.The PI-PLC enzyme can specificly act on PI (phosphatidylinositol; PI) structure; Decomposite the free fat hydrocarbon chain, because the hydrophobic interaction of macromole fat hydrocarbon chain, when being applied to color developing culture medium; Form the opaque and white haloing in the LM periphery of bacterial colonies, thereby LM and other food-borne pathogens and the non-listeria spp that singly increases are made a distinction.
At present domestic to separate the correlative study of purifying about PI less, and the main preparation method who adopts is confined to utilize solvent method and the column chromatography method of component polarity difference etc.Though but the simple and effective productive rate of column chromatography separated PI is lower, poor repeatability; Solvent extraction process purifying PI consumes a large amount of organic solvents, and cost is higher to be unfavorable for production application; The report that adopts HPLC and thin layer chromatography preparation is also arranged in addition, but since treatment capacity very little, and cost is high-leveled and difficult to use.Domestic preparation about PI has only Qiqihar University and Wuhan engineering college to do correlative study recent years; But because yield issues does not all obtain the application aspect the LM rapid detection, therefore seeking feasible high efficiency separation means becomes present problem demanding prompt solution.
The research that domestic LM chromogenic substrate prepares the aspect is backward relatively, has directly restricted its application in aspects such as LM detection color developing culture medium.Along with the development of research and deeply, the combined method means that adopt two or more separation methods to combine improve separation purity, and perhaps enzymolysis process prepares PI and all has the certain development potentiality.Along with increase, develop the developing direction that a kind of simple and feasible process for preparing high-purity will become future studies to the PI demand.
The preparation method of traditional P I mainly utilizes polar difference between its component and separates, and solvent extration, column chromatography, thin layer chromatography, HPLC (HPLC) etc. are arranged.But only rely on separation method to be difficult to obtain single phosphatide, and solvent-oil ratio is big, cost is higher, is difficult to widespread use.In conjunction with microbial process; Find that common four kinds of Phospholipid hydrolases (phospholipase A1, A2, C and D) have specific effect to the soybean lipositol; Its action site is suc as formula shown in 1, particularly the ester bond of Phospholipase D hydrolytic phosphatide acid specifically and base (choline etc.); And have base transfer characteristics (transphosphatidylation reaction), promptly under given conditions, can be attached on the base of phosphatide by the various substrates that contain brothers position hydroxyl of catalysis.
Formula 1
Phospholipid hydrolase has obtained effective application at the aspect such as synthetic of the directed modification of phosphatide, medicine.It is 70% PI that Nakazato in 1987 utilizes mixed phosphatide under the Phospholipase D effect, to obtain purity; Rakhimov in 1989 etc. prepare lipositol in the presence of pure and mild inositol; Juneja in 1989 etc. have reported that in the presence of L or D-Serine (phosphatidylcholine PC) is converted into phosphatidylserine (phosphatidylserine with phosphatidylcholine; PS); Transformation efficiency is respectively 98.5% and 100%, and the visible method that adopts Phospholipid hydrolase to prepare single phosphatide is feasible, but different phosphate lipase is under varying environment; When acting on different types of phosphatide, its decomposition condition is all inequality.
Summary of the invention:
The purpose of this invention is to provide a kind of with low cost, easy and simple to handle, enzymolysis preparation that can obtain highly purified PI.
The present invention utilizes the ester bond of Phospholipase D hydrolytic phosphatide acid specifically and base (choline etc.); And have phosphatide transfer characteristics (transphosphatidylation reaction), promptly under given conditions, can be attached on the base of phosphatide by the various substrates that contain brothers position hydroxyl of catalysis; And PI is unaffected; Utilize its decomposition, thereby purify highly purified PI, in order to realize enzyme digestion reaction other component in the mixed phosphatide; Explore Phospholipase D decomposing soybean phosphatide and obtain the condition of high purity PI, thereby realized the object of the invention.
The enzymolysis preparation of PI of the present invention is characterized in that may further comprise the steps:
Soybean phospholipid after the pre-treatment is dissolved in the solvent, adds Phospholipase D again, the add-on of Phospholipase D is added by the amount of soybean phospholipid after 50 ~ 150unints/100mg pre-treatment; Then at 36 ~ 40 ℃; The pH value is 7.0-9.0, and stirring reaction 3 ~ 12 hours obtains enzymolysis product; Enzymolysis product is used the normal hexane lixiviate, and washing also with ethanol or aqueous ethanolic solution again behind the vat liquor concentrate drying, drying obtains the PI product; Soybean phospholipid prepares through following method after the described pre-treatment: soybean phospholipid is dissolved in the chloroform, uses methanol extraction again, and the collecting precipitation thing, several times so repeatedly, the throw out that obtains at last is soybean phospholipid after obtaining pre-treatment after the drying.
Described solvent is preferably ether.
Further preferred, the add-on of described Phospholipase D press the amount interpolation of soybean phospholipid after the 100unints/100mg pre-treatment, and then at 37 ℃, the pH value is 8.0, stirring reaction 4 hours; It is 5% the acidic ethanol aqueous solution that described aqueous ethanolic solution is preferably volume(tric)fraction.Use this optimal way and can obtain highly purified PI product.
The enzymolysis preparation of PI of the present invention, method is simple, and is with low cost, and the purity of prepared PI can be applied in the color developing culture medium the LM rapid detection as chromogenic substrate than higher.Utilize the PI of the inventive method preparation because its purity is higher; Have certain commercialization and be worth, and this method is simple, easily operation; Reaction times is short; And environment friendly and pollution-free, be a kind of preparation method who has the PI of application prospect, for good basis has been laid in the extension preparation and the application of PI.
Description of drawings:
Fig. 1 be the PI product to different strains color developing effect figure, a, LM bacterial strain, b, listeria grayi strain;
Fig. 2 is different substrate color developing effect figure, a, PI standard substance, b, PI product.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
One, the preparation of PI product and mensuration
The pre-treatment of step 1, raw material
Take by weighing 5g powder soybean phospholipid, with sample dissolution, fully the dissolving back adds the 60mL cold methanol, stirs 1h with the cold trichloromethane of 50mL (4 ℃).Centrifugal 10min under 2 ℃, 5000r/min discards upper solution, collects solid with the cold chloroform of 25mL (4 ℃) dissolving, adds the lixiviate of 60mL alkaline methanol again, fully stirs centrifugal 10min under the same terms, collection solid.Aforesaid operations continues twice of repetition.Be soybean phospholipid after the pre-treatment after the insoluble solids drying that obtains at last.
The step 2 purification of products
With every 50mg enzymolysis product with 10-100mL normal hexane dissolution with solvents; At 200r/min, 4 ℃ are fully stirred 1h down; At 8000r/min; 4 ℃ centrifugal down, and with supernatant concentrate drying in vacuum drier, it is that 5% acidic ethanol (pH=6) embathes three times and drying obtains the PI product that dry thing uses volume(tric)fraction again.
Step 3, the experiment of Phospholipase D enzymolysis
One, enzyme concentration factor 50-150units
Embodiment 1:
Handle soybean phospholipid with reference to the method for step 1 raw materials pretreatment, obtain soybean phospholipid after the pre-treatment.Take by weighing soybean phospholipid 100mg after the pre-treatment, be dissolved in the 20mL ether, add and contain 0.001mol/LCa
2+Tris-HCl damping fluid 10mL, regulating pH is 8, adds Phospholipase D 50units, 37 ℃ of thermostat water baths; Rotating speed 800r/min is reaction 4h down, obtains enzymolysis product, with reference to step 2; Every 50mg enzymolysis product is with 100mL normal hexane dissolution with solvents, and at 200r/min, 4 ℃ are fully stirred 1h down;, at 8000r/min, 4 ℃ centrifugal down; Supernatant is placed the vacuum drier concentrate drying, and it is that 5% acidic ethanol (pH=6) embathes three times and drying obtains the PI product that dry thing uses volume(tric)fraction again, and the purity of PI is 80.18%.
Embodiment 2:
Present embodiment and embodiment 1 are basic identical, and the amount that just adds Phospholipase D is 150units, obtains the PI product, and the purity of its PI is 90.13%.
Two, pH factor (7-9)
Embodiment 3:
Handle soybean phospholipid with reference to the method for step 1 raw materials pretreatment, obtain soybean phospholipid after the pre-treatment.Take by weighing soybean phospholipid 100mg after the pre-treatment, be dissolved in the 20mL ether, add and contain 0.001mol/LCa
2+Tris-HCl damping fluid 10mL, regulating pH is 7.0, adds Phospholipase D 150units, 37 ℃ of thermostat water baths; Rotating speed 800r/min is reaction 4h down, obtains enzymolysis product, with reference to step 2; Every 50mg enzymolysis product is with 100mL normal hexane dissolution with solvents, and at 200r/min, 4 ℃ are fully stirred 1h down;, at 8000r/min, 4 ℃ centrifugal down; Supernatant is placed the vacuum drier concentrate drying, and it is that 5% acidic ethanol (pH=6) embathes three times and drying obtains the PI product that dry thing uses volume(tric)fraction again, and the purity of its PI is 87.68%.
Embodiment 4:
Present embodiment and embodiment 3 are basic identical, and just soybean phospholipid 100mg after taking by weighing pre-treatment is dissolved in the 20mL ether, add and contain 0.001mol/LCa
2+Tris-HCl damping fluid 10mL after, regulating pH is 9.0, other are identical with embodiment 3, obtain the PI product at last, the purity of its PI is 88.81%.
Three, temperature factor (36-40 ℃)
Embodiment 5:
Handle soybean phospholipid with reference to the method for step 1 raw materials pretreatment, obtain soybean phospholipid after the pre-treatment.Take by weighing soybean phospholipid 100mg after the pre-treatment, be dissolved in the 20mL ether, add and contain 0.001mol/LCa
2+Tris-HCl damping fluid 10mL, regulating pH is 8.0, adds Phospholipase D 150units, 36 ℃ of thermostat water baths; Rotating speed 800r/min is reaction 4h down, obtains enzymolysis product, with reference to step 2; Every 50mg enzymolysis product is with 100mL normal hexane dissolution with solvents, and at 200r/min, 4 ℃ are fully stirred 1h down;, at 8000r/min, 4 ℃ centrifugal down; Supernatant is placed on concentrate drying in the vacuum drier, and it is that 5% acidic ethanol (pH=6) embathes three times and drying obtains the PI product that dry thing uses volume(tric)fraction again, and the purity of its PI is 77.33%.
Embodiment 6:
Present embodiment and embodiment 5 are basic identical, and just the temperature of thermostat water bath is 40 ℃, obtains the PI product at last, and the purity of its PI is 72.17%.
Four. time factor 3-12h
Embodiment 7:
Handle soybean phospholipid with reference to the method for step 1 raw materials pretreatment, obtain soybean phospholipid after the pre-treatment.Take by weighing soybean phospholipid 100mg after the pre-treatment, be dissolved in the 20mL ether, add and contain 0.001mol/LCa
2+Tris-HCl damping fluid 10mL, regulating pH is 8.0, adds Phospholipase D 150units, 37 ℃ of thermostat water baths; Rotating speed 800r/min is reaction 3h down, obtains enzymolysis product, with reference to step 2; Every 50mg enzymolysis product is with 100mL normal hexane dissolution with solvents, and at 200r/min, 4 ℃ are fully stirred 1h down;, at 8000r/min, 4 ℃ centrifugal down; Supernatant is placed on concentrate drying in the vacuum drier, and it is that 5% acidic ethanol (pH=6) embathes three times and drying obtains the PI product that dry thing uses volume(tric)fraction again, and the purity of its PI is 85.21%.
Embodiment 8:
Present embodiment and embodiment 7 are basic identical, and just 37 ℃ of thermostat water baths, rotating speed 800r/min is reaction 12h down, obtains the PI product at last, and the purity of its PI is 90.04%.
Step 4 HPLC condition determination
4, performance liquid (HPLC) chromatographic determination condition
Agilent RTX-Si chromatographic column (4.6mm * 250mm * 5 μ m); 30 ℃ of column temperatures; Moving phase: V (normal hexane): V (Virahol): V (1% formic acid)=8: 8: 1-; Flow velocity 1mL/min; Sample size is 10 μ L; The DAD detector, the detection wavelength is 203nm; Adopt the isocratic elution method.
Step 5 colour developing treadmill test
5, colour developing culture condition
Take by weighing the PI product after above-mentioned steps is handled; According to prescription agar 15.0g/L; Peptone and YE 23.0g/L, β-D-glu 0.5-2g/L, PI product 1-8g/L prepare color developing culture medium, regulate pH=7.0 ± 0.2; High-temperature sterilization, and a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices prepares the color developing culture medium flat board.
6. orthogonal test
For further optimization experiment condition, on the basis of experiment of single factor, choose the principal element that influences enzyme digestion reaction; Be 4 of solvent temperature, enzyme concentration, pH, time as reaction factor, as raw material, be evaluation index with soybean phospholipid after the pre-treatment with the PI product P I purity after the aftertreatment; The design orthogonal test; Experiment condition is carried out orthogonal optimum seeking, and orthogonal test level of factor table is seen table 1, and orthogonal experiments is seen table 2.
Table 1 orthogonal experiment level of factor
Table 2 orthogonal experiments
Can know that by table 2 each factor is A to the influence of PI purity in proper order>C>D>B, promptly Influence of Temperature is maximum, secondly is the influence of enzyme concentration, moreover is the influence of pH, and the influence of time is minimum.Best of breed is A
2B
1C
3D
2Be 37 ℃ promptly in temperature, add 2.0 μ L/100mg Phospholipase Ds (100units/100mg), under pH=8.0 during reaction 4h, PI purity can reach maximum value, and verifies through further experiment; This condition gained PI purity is 90.13%, and the recovery is 54.82%.
The experiment of step 6 color developing effect
The color developing effect test
LM bacterial strain (a) and the listeria grayi (b) of 1, choosing recovery with transfering loop are respectively rule on the colour developing flat board; Place 37 ℃ to cultivate 24 ~ 48h down postvaccinal flat board; Observe colonial morphology; As shown in Figure 1: specific reaction can take place with the distinctive PI-PLC of LM in PI product of the present invention; LM is grown to the blue-greenish colour circular bacterium colony of rule and has opaque halo on every side on the colour developing flat board, but not the LM bacterium does not show tangible opaque halo on color developing culture medium, thereby can carry out specific detection to LM.
2, adopt identical color developing culture medium, prepare color developing culture medium with PI (Sigma) standard substance and the PI product of the present invention bought as chromogenic substrate respectively, cultivate 24 ~ 28h down at 37 ℃, the observation experiment result is as shown in Figure 2.LM all presents the blue-greenish colour bacterium colony on two kinds of colour developing flat boards, and is surrounded by opaque halo on every side.Two kinds of substrates have suitable color developing effect, and the PI product price of the present invention's preparation is lower, and the substitute that can be used as the import PI is applied in the LM color developing culture medium.
Claims (3)
1. the enzymolysis preparation of a PI is characterized in that, may further comprise the steps: soybean phospholipid after the pre-treatment is dissolved in the solvent; Add Phospholipase D again; The add-on of Phospholipase D is added by the amount of soybean phospholipid after 50 ~ 150unints/100mg pre-treatment, and then at 36 ~ 40 ℃, the pH value is 7.0 ~ 9.0; Stirring reaction 3 ~ 12 hours; Obtain enzymolysis product, enzymolysis product is used the normal hexane lixiviate, and washing also with ethanol or aqueous ethanolic solution again behind the vat liquor concentrate drying, drying obtains the PI product; Soybean phospholipid prepares through following method after the described pre-treatment: soybean phospholipid is dissolved in the chloroform, uses methanol extraction again, and the collecting precipitation thing, several times so repeatedly, the throw out that obtains at last is soybean phospholipid after obtaining pre-treatment after the drying.
2. enzymolysis preparation according to claim 1 is characterized in that, described solvent is an ether.
3. enzymolysis preparation according to claim 1 is characterized in that, the add-on of described Phospholipase D is pressed the amount of soybean phospholipid after the 100unints/100mg pre-treatment and added, and then at 37 ℃, the pH value is 8.0, stirring reaction 4 hours; Described aqueous ethanolic solution is that volume(tric)fraction is 5% the acidic ethanol aqueous solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315367A (en) * | 2018-03-19 | 2018-07-24 | 郑州四维健康管理有限公司 | Method for producing creatine phosphate by two-step enzymolysis method |
| CN110590833A (en) * | 2019-08-23 | 2019-12-20 | 翁源广业清怡食品科技有限公司 | Preparation method of phosphatidylinositol |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5100787A (en) * | 1989-08-30 | 1992-03-31 | The Nisshin Oil Mills, Ltd. | Method for preparing highly purified phosphatidylinositol |
| US20040235119A1 (en) * | 2001-08-28 | 2004-11-25 | Hans-Ulrich Hoppe | Method for the production of phospholipids |
| JP2008061546A (en) * | 2006-09-06 | 2008-03-21 | Shinko Boeki Kk | Method for producing phosphatidylinositol |
| CN101379187A (en) * | 2006-02-02 | 2009-03-04 | 国立大学法人名古屋大学 | Novel phospholipase D |
| JP2009072152A (en) * | 2007-09-21 | 2009-04-09 | Univ Nagoya | Method for producing natural type phosphatidylinositol and modified phospholipase d therefor |
-
2012
- 2012-05-08 CN CN2012101410979A patent/CN102653778A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5100787A (en) * | 1989-08-30 | 1992-03-31 | The Nisshin Oil Mills, Ltd. | Method for preparing highly purified phosphatidylinositol |
| US20040235119A1 (en) * | 2001-08-28 | 2004-11-25 | Hans-Ulrich Hoppe | Method for the production of phospholipids |
| CN101379187A (en) * | 2006-02-02 | 2009-03-04 | 国立大学法人名古屋大学 | Novel phospholipase D |
| JP2008061546A (en) * | 2006-09-06 | 2008-03-21 | Shinko Boeki Kk | Method for producing phosphatidylinositol |
| JP2009072152A (en) * | 2007-09-21 | 2009-04-09 | Univ Nagoya | Method for producing natural type phosphatidylinositol and modified phospholipase d therefor |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315367A (en) * | 2018-03-19 | 2018-07-24 | 郑州四维健康管理有限公司 | Method for producing creatine phosphate by two-step enzymolysis method |
| CN108315367B (en) * | 2018-03-19 | 2021-05-11 | 郑州四维健康管理有限公司 | Method for producing creatine phosphate by two-step enzymolysis method |
| CN110590833A (en) * | 2019-08-23 | 2019-12-20 | 翁源广业清怡食品科技有限公司 | Preparation method of phosphatidylinositol |
| CN110590833B (en) * | 2019-08-23 | 2022-06-21 | 翁源广业清怡食品科技有限公司 | Preparation method of phosphatidylinositol |
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Application publication date: 20120905 |