CN109112169A - Polylysine glyceride and preparation method thereof, purposes and the method for preparing polylysine - Google Patents
Polylysine glyceride and preparation method thereof, purposes and the method for preparing polylysine Download PDFInfo
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Abstract
The invention proposes the method for preparing polylysine glyceride, polylysine glyceride in the purposes prepared in preservative and the method for preparing polylysine.The method for preparing polylysine glyceride includes: to carry out fermentation process as substrate using glycerol, obtains tunning；And processing is extracted to the tunning, to obtain polylysine glyceride.The method for preparing polylysine is to carry out fermentation process as substrate using glycerol, obtains polylysine glyceride；And the polylysine glyceride is hydrolyzed, to obtain polylysine.A large amount of polylysine glyceride can be obtained using the method for preparing polylysine glyceride of the invention, it is easy to operate.Moreover, the structure of polylysine glyceride is different from existing polylysine glyceride, there is preferable anti-corrosion effect.In addition, the polylysine of high yield can be obtained using the method for preparing polylysine of the invention.
Description
Technical field
The present invention relates to biological fields.In particular it relates to polylysine glyceride and its preparation method, purposes
And the method for preparing polylysine.
Background technique
Japanese scholars S.Shima and H.Sakai in 1977 from microorganism screen Dragendo~Positive (write a Chinese character in simplified form
During DP) substance, it was found that one kind contains the homotype monomer-polymer of 25-30 lysine residues, referred to as ε-poly
Lysine (ε-PL).
However, the derivative of ε-poly-D-lysine still requires study at present.
Summary of the invention
The present invention is directed to solve at least one the technical problems existing in the prior art at least to a certain extent.
It should be noted that the present invention is the following discovery based on inventor and completes:
Inventor's discovery when analyzing the polylysine fermentation liquid using glycerol as sole carbon source removes in fermentation liquid
There is also a kind of possible polylysine derivative for polylysine, and carries out polylysine fermentation by sole carbon source of glucose
When, then the derivative of polylysine is not found.The derivative is by being accredited as polylysine glyceride, the polylysine glyceride
Structure be different from existing polylysine glyceride, and have preferable anti-corrosion effect.But inventor is in the course of the research
It was found that the polylysine glyceride is affected in separation and Extraction by pH.In turn, inventor obtains more excellent by many experiments
Separation and Extraction condition, the yield of polylysine glyceride is higher with this condition.
For this purpose, in one aspect of the invention, the invention proposes a kind of methods for preparing polylysine glyceride.According to
The embodiment of the present invention, which comprises fermentation process is carried out as substrate using glycerol, obtains tunning；And it is right
The tunning extracts processing, to obtain polylysine glyceride.Preparation side according to an embodiment of the present invention as a result,
Method can obtain a large amount of polylysine glyceride, and easy to operate.
According to an embodiment of the invention, the above-mentioned method for preparing polylysine glyceride can also have following supplementary technology
Feature:
According to an embodiment of the invention, the extraction process carries out in acid condition.As a result, further to obtain
Largely and the higher polylysine glyceride of purity.
According to an embodiment of the invention, the extraction process includes: to be separated by solid-liquid separation the tunning, the is collected
One supernatant；The pH value of first supernatant is adjusted to alkalinity, centrifugation adjusts the pH value of the supernatant of collection to acidity, obtains
To the second supernatant；Second supernatant is filtered, filtrate is collected；The filtrate is concentrated, concentrate is obtained；
The concentrate is subjected to ethanol precipitation, precipitating is collected in centrifugation；The precipitating is carried out to heavy molten, centrifugation, collection third supernatant
Liquid；The pH value of the third supernatant is adjusted to acidity, obtains the 4th supernatant；And the 4th supernatant is subjected to ethyl alcohol
Precipitating, centrifugation collect precipitating, to obtain the polylysine glyceride.As a result, so as to further obtain a large amount of and purity compared with
High polylysine glyceride.
According to an embodiment of the invention, the extraction process include: by the tunning under the revolving speed of 5000rpm from
Heart 20min collects the first supernatant；The pH value of first fermentation liquid is adjusted to 8.5, in 5000rpm's with 5M NaOH solution
Be centrifuged 10min at a temperature of revolving speed and 4 DEG C, and adjust collection supernatant pH value to 3.2, obtain the second supernatant；By institute
It states ultrafiltration membrance filter of second supernatant through 10KD, is centrifuged at a temperature of the revolving speed of 3000rpm and 4 DEG C, collects filtrate；With rotation
The filtrate is concentrated 5 times under the conditions of 60 DEG C, obtains concentrate by evaporimeter；4 times of volume pre-coolings are added into the concentrate
Dehydrated alcohol, stirring is centrifuged 10min at a temperature of the revolving speed of 5000rpm and 4 DEG C, collects precipitating；The precipitating is used into pH
7.0 phosphate buffers weight is molten, and 10min is centrifuged at a temperature of the revolving speed of 5000rpm and 4 DEG C, collects third supernatant；By described
The pH value of three supernatants is adjusted to 3.2, adds the dehydrated alcohol of 4 times of volumes pre-cooling, the revolving speed of 5000rpm and 4 DEG C of temperature
Lower centrifugation 10min collects precipitating, to obtain the polylysine glyceride.As a result, further to obtain a large amount of and purity
Higher polylysine glyceride.
According to an embodiment of the invention, the culture medium of the fermentation process includes: the (NH of the glycerol of 60g/L, 10g/L4)2SO4, 0.8g/L KH2PO4·3H2O, the MgSO of 0.5g/L4·7H2O, the KH of 1.36g/L2PO4, 0.04g/L ZnSO4·
7H2O, the FeSO of 0.03g/L4·7H2The yeast extract of O and 5g/L, the pH value of the culture medium are 6.8.As a result, so as to
Further obtain a large amount of and higher polylysine glyceride of purity.
According to an embodiment of the invention, the fermentation process condition is as follows: fermentation temperature is 30 DEG C, revolving speed 200rpm,
Time is 72 hours, and fermenting microbe is Streptomyces albulus.It is higher further to obtain a large amount of and purity as a result,
Polylysine glyceride.
In another aspect of this invention, the invention proposes a kind of polylysine glyceride.According to an embodiment of the invention,
The polylysine glyceride has structure shown in following formula, wherein n is 21~34, and the polylysine glyceride is to pass through
What the method noted earlier for preparing polylysine glyceride obtained.
In still another aspect of the invention, the invention proposes polylysine glyceride noted earlier in preparing preservative
Purposes.
In still another aspect of the invention, the invention proposes a kind of methods for preparing polylysine.Reality according to the present invention
Apply example, which comprises fermentation process is carried out as substrate using glycerol, obtains polylysine glyceride；And to described
Polylysine glyceride is hydrolyzed, to obtain polylysine.Since production bacterial strain is more tolerant to polylysine glyceride,
Therefore it uses and first obtains a large amount of polylysine glyceride, then it is hydrolyzed to obtain the method for polylysine, utilize this kind of side
Formula can obtain the polylysine of higher yield.
According to an embodiment of the invention, the hydrolysis process is carried out in microbial body.Thereby, it is possible to further obtain
Obtain high yield polylysine.
According to an embodiment of the invention, the hydrolysis process is carried out outside microbial body, the hydrolysis process includes:
The polylysine glyceride is dissolved in the citrate buffer solution that pH value is 3.2, and adjusts the pH value of mixed liquor to 7.0,
To obtain polylysine.Thereby, it is possible to further obtain high yield polylysine.
According to an embodiment of the invention, the culture medium and fermentation process condition of the fermentation process are to prepare as previously described
Defined in the method for polylysine glyceride.Thereby, it is possible to further obtain high yield polylysine.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows the flow diagram according to an embodiment of the invention for preparing polylysine glyceride method；
Fig. 2 show zymotic fluid group according to an embodiment of the invention at chromatogram；
Fig. 3 shows the NMR chromatogram of polylysine glyceride according to an embodiment of the invention；
Fig. 4 show control group zymotic fluid group according to an embodiment of the invention at chromatogram, wherein ★ is indicated poly-
Lysine；
Fig. 5 show experimental group zymotic fluid group according to an embodiment of the invention at chromatogram, wherein ★ is indicated poly-
Lysine, ☆ are polylysine glyceride；
Fig. 6 shows that different carbon source according to an embodiment of the invention illustrates the analysis that polylysine glyceride influences
Figure；
Fig. 7 shows influence of the different temperatures according to an embodiment of the invention to polylysine glyceride stability
Analyze schematic diagram；
Fig. 8 shows the differential responses time according to an embodiment of the invention to the shadow of polylysine glyceride stability
Loud analysis schematic diagram；And
Fig. 9 shows influence of the different pH value according to an embodiment of the invention to polylysine glyceride stability
Analyze schematic diagram.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Explicitly or implicitly include one or more of the features.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is two or more.
The invention proposes a kind of method for preparing polylysine glyceride, polylysine glyceride and application thereof and systems
The method of standby polylysine, will be described in greater detail respectively below.
The method for preparing polylysine glyceride
In another aspect of this invention, the invention proposes a kind of sides for preparing previously described polylysine glyceride
Method.According to an embodiment of the invention, referring to Fig. 1, this method comprises: S100 fermentation process and S200 extraction process.Root as a result,
A large amount of polylysine glyceride can be obtained according to the preparation method of the embodiment of the present invention, and easy to operate.It is described more fully below
The preparation method.
According to an embodiment of the invention, referring to Fig. 1, this method comprises:
S100 fermentation process
In this step, fermentation process is carried out as substrate using glycerol, obtains tunning.Currently, with biofermentation
When method obtains polylysine glyceride, glycerol, glucose etc. are generallyd use as carbon source.Inventors have found that using glycerol as
When sole carbon source, there is only polylysines in tunning, and there is also polylysine glyceride of the invention.However, with Portugal
When grape sugar is as carbon source, do not find to generate polylysine glyceride.In turn, inventor uses the training using glycerol as sole carbon source
It supports base and carries out fermentation process, and separate and extract polylysine glyceride from obtained tunning.
According to an embodiment of the invention, the culture medium of fermentation process includes: the (NH of the glycerol of 60g/L, 10g/L4)2SO4、
The KH of 0.8g/L2PO4·3H2O, the MgSO of 0.5g/L4·7H2O, the KH of 1.36g/L2PO4, 0.04g/L ZnSO4·7H2O、
The FeSO of 0.03g/L4·7H2The yeast extract of O and 5g/L, the pH value of culture medium are 6.8.Inventors have found that culture medium group
Above-mentioned preferable culture medium composition is obtained at the yield that will affect polylysine glyceride, and then by many experiments, at this
The yield of polylysine glyceride can be further increased under part.
According to an embodiment of the invention, fermentation process condition is as follows: fermentation temperature is 30 DEG C, revolving speed 200rpm, the time
It is 72 hours, fermenting microbe is Streptomyces albulus.Inventors have found that fermentation process condition and fermenting microbe can shadows
The yield of polylysine glyceride is rung, and then obtains above-mentioned preferable fermentation process condition and fermenting microbe by many experiments,
The yield of polylysine glyceride can be further increased with this condition.
According to an embodiment of the invention, extraction process carries out in acid condition.Inventors have found that by tunning
PH value maintain 7.0 under conditions of, after 12 hours, polylysine glyceride is by complete hydrolysis at polylysine.In turn, it invents
People's discovery, guarantees that extraction process is to carry out in acid condition, can be reduced as far as polylysine glycerol ester hydrolysis, thus
Obtain a large amount of polylysine glyceride.
It should be noted that term " acidity " used in the present invention refers to pH value less than 7.0.It is according to the present invention preferred
Embodiment guarantees that extraction process is carried out under conditions of pH value is 3.2, can farthest reduce polylysine glyceride
Hydrolysis, to obtain a large amount of polylysine glyceride.
According to an embodiment of the invention, extraction process includes: to be separated by solid-liquid separation tunning, the first supernatant is collected
Liquid；The pH value of the first supernatant is adjusted to alkalinity, centrifugation adjusts the pH value of the supernatant of collection to acidity, obtains the second supernatant
Liquid；Second supernatant is filtered, filtrate is collected；Filtrate is concentrated, concentrate is obtained；Concentrate progress ethyl alcohol is sunk
It forms sediment, precipitating is collected in centrifugation；Precipitating is carried out to heavy molten, centrifugation, collection third supernatant；The pH value of third supernatant is adjusted to acid
Property, obtain the 4th supernatant；And the 4th supernatant is subjected to ethanol precipitation, centrifugation collects precipitating, to obtain polylysine
Glyceride.Inventor obtains above-mentioned preferable extraction process condition by many experiments, can obtain with this condition a large amount of poly-
Lysine glyceride, and purity is higher.
According to a particular embodiment of the invention, extraction process includes:
1) tunning is centrifuged under the revolving speed of 5000rpm 20min, collects the first supernatant.
2) pH value for being adjusted to the first fermentation liquid with 5M NaOH is centrifuged at a temperature of the revolving speed of 5000rpm and 4 DEG C to 8.5
10min, and adjust collection supernatant pH value to 3.2, obtain the second supernatant.As a result, to remove indissoluble in fermentation liquid
Impurity, this step is completed in 15min as far as possible, to avoid product degradation.
3) ultrafiltration membrance filter by the second supernatant through 10KD is centrifuged at a temperature of the revolving speed of 3000rpm and 4 DEG C, collects
Filtrate.
4) Rotary Evaporators are used, filtrate is concentrated 5 times under the conditions of 60 DEG C, obtains concentrate.
5) it is added the dehydrated alcohol of 4 times of volumes pre-cooling into concentrate, stirring, at a temperature of the revolving speed of 5000rpm and 4 DEG C
It is centrifuged 10min, collects precipitating.As a result, so as to make polylysine glyceride precipitate.
6) will precipitating it is molten with 7.0 phosphate buffer of pH weight, be centrifuged 10min at a temperature of the revolving speed of 5000rpm and 4 DEG C, receive
Collect third supernatant.This step is completed in 15min as far as possible, to avoid product degradation.
7) pH value of third supernatant is adjusted to 3.2, adds the dehydrated alcohol of 4 times of volumes pre-cooling, 5000rpm's turns
10min is centrifuged at a temperature of speed and 4 DEG C, precipitating is collected, to obtain polylysine glyceride.By the way that the pH value of system is adjusted
To 3.2, to avoid the degradation of polylysine glyceride.Further, use dehydrated alcohol to precipitate polylysine glyceride, thus
Obtain the higher polylysine glyceride of a large amount of purity.
Inventor pass through many experiments, in conjunction with polylysine glyceride characteristic and obtain above-mentioned preferable extraction process item
Part obtains a large amount of and higher polylysine glyceride of purity so as to polylysine glyceride degradation as less as possible.
It should be noted that, although the pH value of system is adjusted to 8.5 (alkalinity), but is centrifuged and receives in step 2)
Time used in collection supernatant is shorter, subsequent that the pH value of supernatant is adjusted to 3.2 (acidity) immediately, ensure that extraction process exists on the whole
It is carried out under acid condition, to ensure that the yield of polylysine glyceride.Similarly, in being used in step 6), in the short time
Property buffer salt weight molten precipitating, centrifugation and collect supernatant, ensure that extraction process carries out in acid condition on the whole, it is same to guarantee
The yield of polylysine glyceride.
Polylysine glyceride
In one aspect of the invention, the invention proposes a kind of polylysine glyceride.According to an embodiment of the invention,
The polylysine glyceride has structure shown in following formula, wherein n is 21~34, and polylysine glyceride is by front institute
What the method that description prepares polylysine glyceride obtained.In existing polylysine glyceride structure, the carboxyl of polylysine
Esterification has occurred in end and 1,3 hydroxyls of glycerol.However in polylysine glyceride of the invention, the carboxyl of polylysine
Esterification has occurred in end and 2 hydroxyls of glycerol, and the two structure is not identical.In addition, inventor is it was unexpectedly observed that the present invention
Polylysine glyceride have preferable anti-corrosion effect.
Purposes
In still another aspect of the invention, the invention proposes polylysine glyceride described above to prepare preservative
In purposes.Inventor is it was unexpectedly observed that polylysine glyceride of the invention can be used for preparing with preferable anti-corrosion effect
Preservative.
The method for preparing polylysine
In still another aspect of the invention, the invention proposes a kind of methods for preparing polylysine.Reality according to the present invention
Example is applied, this method comprises: carrying out fermentation process as substrate using glycerol, obtains polylysine glyceride；And to poly- bad ammonia
Acid glyceride is hydrolyzed, to obtain polylysine.Inventors have found that since production bacterial strain is sweet more tolerant to polylysine
Grease, therefore it is hydrolyzed to obtain the method for polylysine again using a large amount of polylysine glyceride are first obtained, utilize this
Kind mode can obtain the polylysine of higher yield.
It should be noted that not making considered critical for hydrolysis process condition, can both be carried out in microbial body, it can also
To be carried out outside microbial body.According to a particular embodiment of the invention, hydrolysis process is carried out outside microbial body, at hydrolysis
Reason include: polylysine glyceride is dissolved in the citrate buffer solution that pH value is 3.2, and adjust the pH value of mixed liquor to
7.0, to obtain polylysine.Inventors have found that polylysine glyceride can be degraded to poly- rely under conditions of pH value 7.0
Propylhomoserin, and the yield of polylysine is higher.As a result, to obtain high yield polylysine.
According to an embodiment of the invention, the culture medium and fermentation process condition of fermentation process are to prepare as previously described
Defined in the method for polylysine glyceride.Since production bacterial strain is more tolerant to polylysine glyceride, uses and first obtain
It obtains a large amount of polylysine glyceride it to be hydrolyzed to obtain the method for polylysine again, fermenting in the way of this kind can obtain
The polylysine of higher yield.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
In this embodiment, polylysine glyceride is obtained in following manner:
1, fermentation process
Streptomyces albulus NBRC 14147 is inoculated in fermentation medium and carries out shake flask fermentation, 30 DEG C,
200rpm is cultivated 72 hours, obtains fermentation liquid.
Wherein, fermentation medium is as follows:
Glycerol 60g/L；(NH4)2SO410g/L；KH2PO4·3H2O 0.8g/L；MgSO4·7H2O 0.5g/L；KH2PO4
1.36；ZnSO4·7H2O 0.04g/L；FeSO4·7H2O 0.03g/L；Yeast extract 5g/L, pH 6.8.
2, extraction process
Scheme is as follows: fermentation liquid, which is separated by solid-liquid separation, --- -- adjusts pH to alkalinity and --- -- adjusts pH to carry out ultrafiltration to acidity --- -- to go
Except macromolecular foreign protein --- -- ethanol precipitation --- -- the molten removing insoluble protein of weight under neutrallty condition ----adjust pH to acidity ----
Ethyl alcohol precipitates obtain product again.
Specific steps:
1) fermentation liquid is separated by solid-liquid separation in 5000rpm, 20min；
2) being adjusted to pH value with 5M NaOH is 8.5；10min is centrifuged at a temperature of the revolving speed of 5000rpm and 4 DEG C, it is heavy to remove
It forms sediment, collects supernatant, and adjust supernatant pH value to 3.2；
3) ultrafiltration membrance filter by supernatant through 10KD, 3000rpm 4 DEG C, retain macromolecular foreign protein, collect filtrate；
4) Rotary Evaporators are used, filtrate is concentrated 5 times under the conditions of 60 DEG C, obtains concentrate；
5) it is added the dehydrated alcohol of 4 times of volumes pre-cooling into concentrate, stirring, at a temperature of the revolving speed of 5000rpm and 4 DEG C
It is centrifuged 10min, collects precipitating；
6) will precipitating it is molten with 7.0 phosphate buffer of pH weight, be centrifuged 10min at a temperature of the revolving speed of 5000rpm and 4 DEG C, remove
Remove the foreign protein of indissoluble；
7) supernatant is quickly adjusted to pH3.2, then plus the pre-cooling of 4 times of volumes dehydrated alcohol, the revolving speed of 5000rpm and 4 DEG C
At a temperature of be centrifuged 10min, precipitating is collected by centrifugation；
8) it is lyophilized, obtains pale yellow powder.
What entire extraction process process was substantially completed in acid condition.
The product of extraction is subjected to LC-MS analysis, as a result (Fig. 2) shows that the product in the product and fermentation liquid of extraction is several
It is the same, it does not degrade, product is single, belongs to glyceride.
The identification of 2 polylysine glyceride structure of embodiment
Qs glycerin ester is taken to be dissolved in heavy water (D2O in), 13C-Nuclear Magnetic Resonance (NMR) mirror is carried out
It is fixed.As a result as shown in figure 3, by spectrum analysis, determine that the structure of glyceride is the c-terminus of polylysine and 2 hydroxyls of glycerol
Esterification has occurred in base, obtains final product, 2- ε-poly (L-lysyl)-glycerol.
The Activity determination of 3 polylysine glyceride of embodiment
By Escherichia coli 8099 (3.7 × 109Cfu/ml), Escherichia coli O 157: H7 (1.05 × 109Cfu/ml) and
ATCC6538(1.9×108Cfu/ml) three plants of bacterium press 10 times of gradient dilution opportunistic pathogen suspensions, dilute 100 times altogether and bacteria suspension is made, so
4.5mL 1g/L polylysine glyceride is respectively drawn afterwards, the bacteria suspension that 0.5ml has diluted is added, be uniformly mixed (volume 9:1).It inhales
The above-mentioned mixed solution of 1ml is placed in plate, is poured into cool to 45 DEG C of LB culture medium, and pivotal plate keeps its full and uniform.It is solidifying
It is counted after being inverted culture 48h after Gu.Calculate bacteriostasis rate and sterilizing rate: X=(A-B)/A × 100% is (in formula: X-bacteriostasis rate, %；
A-control sample average colony number；B-subject sample average clump count)
As a result as shown in the table, it can be seen that polylysine glyceride has apparent bacteriostatic activity.
Embodiment 4
In this embodiment, influence of the research different carbon source to polylysine glyceride.
Experimental group: it is carried out according to the fermentation process of embodiment 1；
Control group: it is carried out according to the fermentation process of embodiment 1, difference is that fermentation medium is as follows: glucose 50g/L；
(NH4)2SO410g/L；K2HPO4·3H2O 0.8g/L；MgSO4·7H2O 0.5g/L；KH2PO41.36g/L；ZnSO4·7H2O
0.04g/L；FeSO4·7H2O 0.03g/L；Yeast extract 5g/L.
As a result as shown in figures 4-6.After fermented, with the product that Mass Spectrometer Method generates in control group be polymerization degree n=
The polylysine (Fig. 4) of 25-31, and the product in experimental group is the polylysine glyceride and n that the degree of polymerization is n=25-30
The polylysine (Fig. 5) of=25-31.In addition, yield is high (Fig. 6) in the yield increased group of polylysine in experimental group.As a result,
Fermentation process is carried out as sole carbon source using glycerol, polylysine glyceride can also be obtained while obtaining polylysine.
Embodiment 5
1, influence of the research different temperatures to polylysine glyceride stability
Under the conditions of 7.0 pH, respectively take the obtained fermentation liquid of the step 1 of 1mL embodiment 1, respectively 25,30,35,40,
12h is placed under the conditions of 45 and 50 DEG C, LC-MS result (Fig. 7) shows that polylysine glyceride is more complete when temperature is higher than 40 DEG C
Ground is hydrolyzed to polylysine, so that polylysine glyceride can not be obtained.
2, influence of the research differential responses time to polylysine glyceride stability
Under the conditions of pH7.0,40 DEG C, the obtained fermentation liquid of the step 1 of 1mL embodiment 1 is respectively taken, reacts 1,5,7 respectively
And 12h, compare degree of hydrolysis with the situation of change of hydrolysis time.When LC-MS result (Fig. 8) shows that the reaction time reaches 5h, gathers and rely
Propylhomoserin glyceride is more fully hydrolyzed to polylysine, so that polylysine glyceride can not be obtained.
3, influence of the different pH value to polylysine glyceride stability is studied
The pH stable of the obtained fermentation liquid of the step 1 of embodiment 1 is being 3.2 or so, therefore 3.2 He of initial option pH
PH 7.0 is compared, and 12h, influence of the more different pH to polylysine glycerol ester hydrolysis are reacted under the conditions of 40 DEG C.LC-MS result
(Fig. 9) shows under the conditions of 3.2 pH, and after hydrolyzing 12h, polylysine glyceride is not hydrolyzed, and under conditions of pH 7.0, gathers
Lysine glyceride is more fully hydrolyzed to polylysine, so that polylysine glyceride can not be obtained.
As long as guaranteeing that the pH value of system in extraction process maintains in acid condition (preferably 3.2), to can get big as a result,
Measure polylysine glyceride.Also, under the conditions of guaranteeing acidic extraction, Extracting temperature and time are for polylysine glyceride
Yield influences not significant.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (10)
1. the method for preparing polylysine glyceride characterized by comprising
Fermentation process is carried out as substrate using glycerol, obtains tunning；And
Processing is extracted to the tunning, to obtain polylysine glyceride.
2. the method according to claim 1, wherein the extraction process carries out in acid condition.
3. the method according to claim 1, wherein the extraction process includes:
The tunning is separated by solid-liquid separation, the first supernatant is collected；
The pH value of first supernatant is adjusted to alkalinity, centrifugation adjusts the pH value of the supernatant of collection to acidity, obtains second
Supernatant；
Second supernatant is filtered, filtrate is collected；
The filtrate is concentrated, concentrate is obtained；
The concentrate is subjected to ethanol precipitation, precipitating is collected in centrifugation；
The precipitating is carried out to heavy molten, centrifugation, collection third supernatant；
The pH value of the third supernatant is adjusted to acidity, obtains the 4th supernatant；And
4th supernatant is subjected to ethanol precipitation, precipitating is collected in centrifugation, to obtain the polylysine glyceride,
Preferably, the extraction process includes:
The tunning is centrifuged 20min under the revolving speed of 5000rpm, collects the first supernatant；
Adjust the pH value of first fermentation liquid to 8.5 with 5M NaOH solution, at a temperature of the revolving speed of 5000rpm and 4 DEG C from
Heart 10min, and adjust collection supernatant pH value to 3.2, obtain the second supernatant；
By the ultrafiltration membrance filter of second supernatant through 10KD, it is centrifuged at a temperature of the revolving speed of 3000rpm and 4 DEG C, collects filter
Liquid；
With Rotary Evaporators, the filtrate is concentrated 5 times under the conditions of 60 DEG C, obtains concentrate；
Be added the dehydrated alcohol of 4 times of volumes pre-cooling into the concentrate, stirring, at a temperature of the revolving speed of 5000rpm and 4 DEG C from
Heart 10min collects precipitating；
The precipitating is molten with 7.0 phosphate buffer of pH weight, it is centrifuged 10min at a temperature of the revolving speed of 5000rpm and 4 DEG C, is collected
Third supernatant；
The pH value of the third supernatant is adjusted to 3.2, adds the dehydrated alcohol of 4 times of volumes pre-cooling, the revolving speed of 5000rpm
And 10min is centrifuged at a temperature of 4 DEG C, precipitating is collected, to obtain the polylysine glyceride.
4. the method according to claim 1, wherein the culture medium of the fermentation process includes:
(the NH of the glycerol of 60g/L, 10g/L4)2SO4, 0.8g/L KH2PO4·3H2O, the MgSO of 0.5g/L4·7H2O、1.36g/
The KH of L2PO4, 0.04g/L ZnSO4·7H2O, the FeSO of 0.03g/L4·7H2The yeast extract of O and 5g/L, the training
The pH value for supporting base is 6.8,
Optionally, the fermentation process condition is as follows: fermentation temperature is 30 DEG C, revolving speed 200rpm, and the time is 72 hours, fermentation
Strain is Streptomyces albulus.
5. a kind of polylysine glyceride, which is characterized in that have structure shown in following formula:
Wherein, n is 21~34,
The polylysine glyceride is obtained by any one of Claims 1 to 4 method for preparing polylysine glyceride
?.
6. polylysine glyceride described in claim 5 is preparing the purposes in preservative.
7. a kind of method for preparing polylysine characterized by comprising
Fermentation process is carried out as substrate using glycerol, obtains polylysine glyceride；And
The polylysine glyceride is hydrolyzed, to obtain polylysine.
8. the method according to the description of claim 7 is characterized in that the hydrolysis process is carried out in microbial body.
9. the method according to the description of claim 7 is characterized in that the hydrolysis process is carried out outside microbial body, institute
Stating hydrolysis process includes:
The polylysine glyceride is dissolved in the citrate buffer solution that pH value is 3.2, and adjust the pH value of mixed liquor to
7.0, to obtain polylysine.
10. the method according to the description of claim 7 is characterized in that the culture medium and fermentation process condition of the fermentation process
It is to be prepared defined in the method for polylysine glyceride as claimed in claim 4.
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| PCT/CN2018/092487 WO2019001371A1 (en) | 2017-06-26 | 2018-06-22 | Polylysine glyceride, preparation method therefor and uses thereof, and polylysine preparation method |
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| JP2006028408A (en) * | 2004-07-20 | 2006-02-02 | Okayama Prefecture | Epsilon-poly-l-lysine-alcoholic ester and method for producing the same |
| CN102352385A (en) * | 2011-08-29 | 2012-02-15 | 江南大学 | Method for producing epsilon-polylysine through fermentation by using glycerin as single carbon source |
| CN102363797A (en) * | 2011-10-28 | 2012-02-29 | 天津科技大学 | Method for producing epsilon-poly-L-lysine |
| EP2543728A4 (en) * | 2010-03-03 | 2013-11-13 | Jnc Corp | Process for production of useful substance using genetically modified streptomyces ray fungus |
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| JP2006028408A (en) * | 2004-07-20 | 2006-02-02 | Okayama Prefecture | Epsilon-poly-l-lysine-alcoholic ester and method for producing the same |
| EP2543728A4 (en) * | 2010-03-03 | 2013-11-13 | Jnc Corp | Process for production of useful substance using genetically modified streptomyces ray fungus |
| CN102352385A (en) * | 2011-08-29 | 2012-02-15 | 江南大学 | Method for producing epsilon-polylysine through fermentation by using glycerin as single carbon source |
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