CN109112169B - Polylysine glyceride, preparation method and application thereof, and method for preparing polylysine - Google Patents
Polylysine glyceride, preparation method and application thereof, and method for preparing polylysine Download PDFInfo
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Abstract
The invention provides a method for preparing polylysine glyceride, application of polylysine glyceride in preparing a preservative and a method for preparing polylysine. The method for preparing the polylysine glyceride comprises the following steps: adopting glycerol as a substrate to carry out fermentation treatment to obtain a fermentation product; and carrying out extraction treatment on the fermentation product so as to obtain the polylysine glyceride. The method for preparing polylysine comprises the steps of performing fermentation treatment by using glycerol as a substrate to obtain polylysine glyceride; and hydrolyzing the polylysine glyceride to obtain polylysine. The method for preparing the polylysine glyceride can obtain a large amount of polylysine glyceride, and is simple and convenient to operate. And the structure of the polylysine glyceride is different from that of the existing polylysine glyceride, and the polylysine glyceride has a good anticorrosion effect. In addition, the method for preparing polylysine according to the present invention can provide polylysine in high yield.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to polylysine glyceride, a preparation method and application thereof, and a method for preparing polylysine.
Background
In the course of screening Dragendo-Positive (abbreviated as DP) substances from microorganisms by Japanese scholars S.Shima and H.Sakai in 1977, a homo-type monomer polymer containing 25 to 30 lysine residues, called epsilon-polylysine (epsilon-PL), was discovered.
However, derivatives of epsilon-polylysine are still under investigation.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.
It should be noted that the present invention has been completed based on the following findings of the inventors:
the inventor analyzes the polylysine fermentation liquor which takes glycerol as the only carbon source, and finds that the fermentation liquor has a possible polylysine derivative besides polylysine, and the polylysine derivative is not found when the polylysine fermentation is carried out by taking glucose as the only carbon source. The derivative is identified as polylysine glyceride, the structure of the polylysine glyceride is different from that of the existing polylysine glyceride, and the polylysine glyceride has a good antiseptic effect. However, the inventor finds that the polylysine glyceride is greatly influenced by pH in separation and extraction in the research process. Furthermore, the inventor obtains better separation and extraction conditions through a large number of experiments, and the yield of the polylysine glyceride is higher under the conditions.
To this end, in one aspect of the invention, the invention proposes a process for preparing polylysine glycerides. According to an embodiment of the invention, the method comprises: adopting glycerol as a substrate to carry out fermentation treatment to obtain a fermentation product; and carrying out extraction treatment on the fermentation product so as to obtain the polylysine glyceride. Therefore, the preparation method provided by the embodiment of the invention can obtain a large amount of polylysine glyceride and is simple and convenient to operate.
According to an embodiment of the present invention, the above method for preparing polylysine glyceride may further have the following additional technical features:
according to an embodiment of the invention, the extraction treatment is carried out under acidic conditions. Therefore, the polylysine glyceride with high quantity and purity can be further obtained.
According to an embodiment of the present invention, the extraction process includes: carrying out solid-liquid separation on the fermentation product, and collecting a first supernatant; adjusting the pH value of the first supernatant to be alkaline, centrifuging, and adjusting the pH value of the collected supernatant to be acidic to obtain a second supernatant; filtering the second supernatant, and collecting filtrate; concentrating the filtrate to obtain a concentrated solution; carrying out ethanol precipitation on the concentrated solution, centrifuging, and collecting precipitate; re-dissolving the precipitate, centrifuging, and collecting a third supernatant; adjusting the pH value of the third supernatant to be acidic to obtain a fourth supernatant; and subjecting the fourth supernatant to ethanol precipitation, centrifuging and collecting the precipitate so as to obtain the polylysine glyceride. Therefore, the polylysine glyceride with high quantity and purity can be further obtained.
According to an embodiment of the present invention, the extraction process includes: centrifuging the fermentation product at the rotating speed of 5000rpm for 20min, and collecting a first supernatant; adjusting the pH value of the first fermentation solution to 8.5 by using a 5M NaOH solution, centrifuging for 10min at the rotating speed of 5000rpm and the temperature of 4 ℃, and adjusting the pH value of the collected supernatant to 3.2 to obtain a second supernatant; filtering the second supernatant through a 10KD ultrafiltration membrane, centrifuging at the rotating speed of 3000rpm and the temperature of 4 ℃, and collecting filtrate; concentrating the filtrate by 5 times at 60 ℃ by using a rotary evaporator to obtain a concentrated solution; adding 4 times volume of precooled absolute ethyl alcohol into the concentrated solution, stirring, centrifuging at the rotating speed of 5000rpm and the temperature of 4 ℃ for 10min, and collecting precipitate; re-dissolving the precipitate with pH7.0 phosphate buffer, centrifuging at 5000rpm and 4 deg.C for 10min, and collecting third supernatant; adjusting the pH value of the third supernatant to 3.2, adding 4 times of volume of precooled absolute ethyl alcohol, centrifuging for 10min at the rotating speed of 5000rpm and the temperature of 4 ℃, and collecting precipitates so as to obtain the polylysine glyceride. Therefore, the polylysine glyceride with high quantity and purity can be further obtained.
According to an embodiment of the invention, the fermentation treated medium comprises: 60g/L of glycerol, 10g/L of (NH)4)2SO40.8g/L KH2PO4·3H2O, 0.5g/L MgSO4·7H2O, KH of 1.36g/L2PO40.04g/L of ZnSO4·7H2O, 0.03g/L FeSO4·7H2O and 5g/L of yeast extract, wherein the pH value of the culture medium is 6.8. Therefore, the polylysine glyceride with high quantity and purity can be further obtained.
According to an embodiment of the invention, the fermentation process conditions are as follows: the fermentation temperature is 30 ℃, the rotation speed is 200rpm, the time is 72 hours, and the fermentation strain is Streptomyces albulus. Therefore, the polylysine glyceride with high quantity and purity can be further obtained.
In another aspect of the invention, the invention provides a polylysine glyceride. According to the embodiment of the invention, the polylysine glyceride has a structure shown in the following formula, wherein n is 21-34, and the polylysine glyceride is obtained by the method for preparing the polylysine glyceride.
In a further aspect of the invention, the invention proposes the use of polylysine glycerides as described above in the preparation of preservatives.
In yet another aspect of the invention, a method of preparing polylysine is provided. According to an embodiment of the invention, the method comprises: adopting glycerol as a substrate to perform fermentation treatment to obtain polylysine glyceride; and hydrolyzing the polylysine glyceride to obtain polylysine. Because the production strain is more tolerant to polylysine glyceride, polylysine with higher yield can be obtained by adopting a method of firstly obtaining a large amount of polylysine glyceride and then hydrolyzing the polylysine glyceride to obtain polylysine.
According to an embodiment of the invention, the hydrolysis treatment is performed in a microorganism. Thus, polylysine can be further obtained in high yield.
According to an embodiment of the invention, the hydrolysis treatment is carried out in vitro in a microorganism, the hydrolysis treatment comprising: and (3) dissolving the polylysine glyceride in a citric acid buffer solution with the pH value of 3.2, and adjusting the pH value of the mixed solution to 7.0 so as to obtain the polylysine. Thus, polylysine can be further obtained in high yield.
According to an embodiment of the present invention, the culture medium and fermentation treatment conditions of the fermentation treatment are as defined in the method for producing polylysine glyceride as described above. Thus, polylysine can be further obtained in high yield.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
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The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic flow diagram of a process for preparing polylysine glycerides, according to one embodiment of the present invention;
FIG. 2 shows a chromatogram of the composition of a fermentation broth according to an embodiment of the invention;
FIG. 3 shows an NMR chromatogram of polylysine glyceride according to an embodiment of the present invention;
FIG. 4 shows a chromatogram of a control fermentation broth composition, wherein: polylysine, according to an embodiment of the invention;
FIG. 5 shows a chromatogram of the composition of a fermentation broth of an experimental group according to an embodiment of the present invention, in which: polylysine, and: polylysine glyceride;
FIG. 6 shows a schematic diagram of an analysis of the effect of different carbon sources on polylysine glycerides, according to an embodiment of the present invention;
FIG. 7 shows a schematic analysis of the effect of different temperatures on the stability of polylysine glycerides, according to one embodiment of the present invention;
FIG. 8 shows a schematic analysis of the effect of different reaction times on the stability of polylysine glycerides, according to one embodiment of the present invention; and
FIG. 9 shows a schematic diagram of an analysis of the effect of different pH values on the stability of polylysine glyceride, according to one embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The present invention provides a method for preparing polylysine glyceride, use thereof, and a method for preparing polylysine, which will be described in detail below, respectively.
Method for preparing polylysine glyceride
In another aspect of the invention, the invention provides a process for the preparation of polylysine glycerides as described above. According to an embodiment of the invention, referring to fig. 1, the method comprises: s100 fermentation treatment and S200 extraction treatment. Therefore, the preparation method provided by the embodiment of the invention can obtain a large amount of polylysine glyceride and is simple and convenient to operate. The preparation method will be described in detail below.
According to an embodiment of the invention, referring to fig. 1, the method comprises:
s100 fermentation treatment
In this step, a fermentation treatment is carried out using glycerol as a substrate to obtain a fermentation product. Currently, in obtaining polylysine glyceride by a biological fermentation method, glycerol, glucose, and the like are generally used as a carbon source. The inventors have found that when glycerol is used as the sole carbon source, not only polylysine but also the polylysine glycerides according to the invention are present in the fermentation product. However, when glucose was used as a carbon source, no formation of polylysine glyceride was observed. Further, the present inventors carried out fermentation treatment using a medium containing glycerol as a sole carbon source, and isolated and extracted polylysine glyceride from the resulting fermentation product.
According to an embodiment of the invention, the medium of the fermentation process comprises: 60g/L of glycerol, 10g/L of (NH)4)2SO40.8g/L KH2PO4·3H2O, 0.5g/L MgSO4·7H2O, KH of 1.36g/L2PO40.04g/L of ZnSO4·7H2O, 0.03g/L FeSO4·7H2O and 5g/L yeast extract, and the pH value of the culture medium is 6.8. The inventor finds that the culture medium composition influences the yield of the polylysine glyceride, and further obtains the better culture medium composition through a large number of experiments, and the poly-lysine glyceride can be further improved under the conditionYield of lysine glyceride.
According to an embodiment of the invention, the fermentation process conditions are as follows: the fermentation temperature is 30 ℃, the rotation speed is 200rpm, the time is 72 hours, and the fermentation strain is Streptomyces albulus. The inventor finds that the fermentation treatment conditions and fermentation strains influence the yield of the polylysine glyceride, and further obtains the better fermentation treatment conditions and fermentation strains through a large number of experiments, and the yield of the polylysine glyceride can be further improved under the conditions.
According to an embodiment of the present invention, the extraction treatment is performed under acidic conditions. The inventors found that, by maintaining the pH of the fermentation product at 7.0, the polylysine glyceride was completely hydrolyzed into polylysine after 12 hours. Furthermore, the inventors found that hydrolysis of polylysine glyceride can be reduced as much as possible to obtain a large amount of polylysine glyceride by ensuring that the extraction treatment is performed under acidic conditions.
It is to be noted that the term "acidic" as used herein means having a pH of less than 7.0. According to the preferred embodiment of the invention, the extraction treatment is carried out under the condition of pH value of 3.2, so that the hydrolysis of the polylysine glyceride can be reduced to the maximum extent, and a large amount of polylysine glyceride can be obtained.
According to an embodiment of the present invention, the extraction process includes: carrying out solid-liquid separation on the fermentation product, and collecting a first supernatant; adjusting the pH value of the first supernatant to be alkaline, centrifuging, and adjusting the pH value of the collected supernatant to be acidic to obtain a second supernatant; filtering the second supernatant, and collecting filtrate; concentrating the filtrate to obtain a concentrated solution; precipitating the concentrated solution with ethanol, centrifuging, and collecting precipitate; re-dissolving the precipitate, centrifuging, and collecting a third supernatant; adjusting the pH value of the third supernatant to acidity to obtain a fourth supernatant; and performing ethanol precipitation on the fourth supernatant, centrifuging, and collecting the precipitate so as to obtain the polylysine glyceride. The inventor obtains the better extraction treatment condition through a large number of experiments, and a large number of polylysine glyceride can be obtained under the condition, and the purity is higher.
According to a specific embodiment of the present invention, the extraction process includes:
1) the fermentation product was centrifuged at 5000rpm for 20min and the first supernatant was collected.
2) Adjusting pH of the first fermentation solution to 8.5 with 5M NaOH, centrifuging at 5000rpm and 4 deg.C for 10min, and adjusting pH of the collected supernatant to 3.2 to obtain second supernatant. This was done within 15min to avoid product degradation, in order to remove the insoluble impurities from the fermentation broth.
3) Filtering the second supernatant with 10KD ultrafiltration membrane, centrifuging at 3000rpm and 4 deg.C, and collecting filtrate.
4) Concentrating the filtrate with a rotary evaporator at 60 deg.C for 5 times to obtain concentrated solution.
5) Adding 4 times volume of precooled anhydrous ethanol into the concentrated solution, stirring, centrifuging at the rotation speed of 5000rpm and the temperature of 4 ℃ for 10min, and collecting the precipitate. Thereby, to precipitate the polylysine glyceride.
6) The precipitate was re-dissolved with pH7.0 phosphate buffer, centrifuged at 5000rpm and 4 ℃ for 10min, and the third supernatant was collected. This was done as soon as possible within 15min to avoid product degradation.
7) Adjusting the pH value of the third supernatant to 3.2, adding 4 times volume of precooled absolute ethyl alcohol, centrifuging for 10min at the rotating speed of 5000rpm and the temperature of 4 ℃, and collecting precipitates so as to obtain polylysine glyceride. The degradation of polylysine glyceride is avoided by adjusting the pH value of the system to 3.2. Further, absolute ethyl alcohol is adopted to precipitate the polylysine glyceride, so that a large amount of polylysine glyceride with higher purity is obtained.
Through a large number of experiments, the inventor obtains the better extraction treatment conditions by combining the characteristics of the polylysine glyceride, so that the degradation of the polylysine glyceride is reduced as much as possible, and a large amount of polylysine glyceride with higher purity is obtained.
It should be noted that, although the pH of the system is adjusted to 8.5 (alkaline) in step 2), the time for centrifugation and collection of the supernatant is short, and the pH of the supernatant is immediately adjusted to 3.2 (acidic) in the subsequent step, so that the extraction process is performed under acidic conditions, and the yield of the polylysine glyceride is ensured. Similarly, in the step 6), the neutral buffer salt is adopted for redissolving and precipitating, centrifuging and collecting the supernatant in a short time, so that the extraction treatment is integrally ensured to be carried out under the acidic condition, and the yield of the polylysine glyceride is also ensured.
Polylysine glyceride
In one aspect of the invention, the invention provides a polylysine glyceride. According to the embodiment of the invention, the polylysine glyceride has the structure shown in the following formula, wherein n is 21-34, and the polylysine glyceride is obtained by the method for preparing the polylysine glyceride described above. In the existing polylysine glyceride structure, the carboxyl terminal of polylysine and the hydroxyl groups at the 1,3 positions of glycerol have esterification reaction. However, in the polylysine glyceride of the present invention, the carboxyl terminal of polylysine and the 2-position hydroxyl group of glycerol have esterification reaction, and the structures of the two are not the same. In addition, the inventor unexpectedly finds that the polylysine glyceride has a good preservative effect.
Use of
In a further aspect of the invention, the invention proposes the use of polylysine glycerides as described above in the preparation of preservatives. The inventor unexpectedly finds that the polylysine glyceride has a good preservative effect and can be used for preparing the preservative.
Process for preparing polylysine
In yet another aspect of the invention, a method of preparing polylysine is provided. According to an embodiment of the invention, the method comprises: adopting glycerol as a substrate to perform fermentation treatment to obtain polylysine glyceride; and hydrolyzing polylysine glyceride to obtain polylysine. The inventors found that, since the production strain is more tolerant to polylysine glyceride, a higher yield of polylysine can be obtained by using a method in which a large amount of polylysine glyceride is obtained and then hydrolyzed to obtain polylysine.
The hydrolysis treatment conditions are not strictly limited, and may be carried out in vivo or in vitro. According to a particular embodiment of the invention, the hydrolysis treatment is carried out in vitro on a microorganism, the hydrolysis treatment comprising: dissolving polylysine glyceride in a citric acid buffer solution with the pH value of 3.2, and adjusting the pH value of the mixed solution to 7.0 to obtain polylysine. The inventor finds that the polylysine glyceride can be degraded into the polylysine under the condition of pH value of 7.0, and the yield of the polylysine is higher. Thereby, polylysine is obtained in high yield.
According to the embodiment of the present invention, the culture medium for the fermentation treatment and the fermentation treatment conditions are as defined in the method for preparing polylysine glyceride as described above. Because the production strain is more tolerant to polylysine glyceride, polylysine with higher yield can be obtained by fermentation in a method of obtaining a large amount of polylysine glyceride and hydrolyzing the polylysine glyceride.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, polylysine glyceride was obtained as follows:
1. fermentation treatment
Streptomyces albulus NBRC 14147 is inoculated into a fermentation medium to carry out shake flask fermentation, and the fermentation medium is cultured at 30 ℃ and 200rpm for 72 hours to obtain a fermentation liquid.
Wherein, the fermentation medium is as follows:
60g/L of glycerol; (NH)4)2SO4 10g/L；KH2PO4·3H2O 0.8g/L；MgSO4·7H2O 0.5g/L；KH2PO41.36；ZnSO4·7H2O 0.04g/L；FeSO47H2O 0.03 g/L; yeast extract 5g/L, pH 6.8.
2. Extraction process
The scheme is as follows: solid-liquid separation of fermentation liquor, pH adjustment to alkalinity, pH adjustment to acidity, ultrafiltration to remove macromolecular heteroprotein, ethanol precipitation, redissolving under a neutral condition to remove insoluble protein, pH adjustment to acidity, and ethanol precipitation to obtain the product.
The method comprises the following specific steps:
1) performing solid-liquid separation on the fermentation liquor at 5000rpm for 20 min;
2) adjusting pH to 8.5 with 5M NaOH; centrifuging at 5000rpm and 4 deg.C for 10min, removing precipitate, collecting supernatant, and adjusting pH to 3.2;
3) filtering the supernatant with 10KD ultrafiltration membrane at 3000rpm at 4 deg.C, and collecting filtrate;
4) concentrating the filtrate by 5 times at 60 deg.C with rotary evaporator to obtain concentrated solution;
5) adding 4 times volume of precooled absolute ethyl alcohol into the concentrated solution, stirring, centrifuging at the rotating speed of 5000rpm and the temperature of 4 ℃ for 10min, and collecting the precipitate;
6) re-dissolving the precipitate with pH7.0 phosphate buffer solution, centrifuging at 5000rpm and 4 deg.C for 10min, and removing insoluble foreign protein;
7) quickly adjusting the pH of the supernatant to 3.2, adding 4 times volume of precooled absolute ethyl alcohol, centrifuging at the rotating speed of 5000rpm and the temperature of 4 ℃ for 10min, and centrifuging and collecting precipitates;
8) freeze-drying to obtain light yellow powder.
The whole extraction process is basically finished under the acidic condition.
The extracted product is analyzed by LC-MS, and the result (figure 2) shows that the extracted product is almost the same as the product in the fermentation liquor, is not degraded, and has single product and belongs to glyceride.
Example 2 structural identification of polylysine glycerides
Dissolving appropriate amount of glyceride in heavy water (D)2O), 13C-Nuclear Magnetic Resonance (NMR) identification was performed. The result is shown in fig. 3, and the structure of the glyceride is determined by the esterification reaction between the carboxyl terminal of polylysine and the hydroxyl group at the 2-position of glycerol through map analysis, so as to obtain the final product, 2-epsilon-poly (L-lysyl) -glycerol.
Example 3 Activity assay of polylysine glycerides
Escherichia coli 8099 (3.7X 10)9cfu/ml), E.coli O157: H7 (1.05X 10)9cfu/ml) and ATCC6538 (1.9X 10)8cfu/mL) were diluted in 10-fold gradient to 100-fold to obtain bacterial suspensions, and 4.5mL of 1g/L polylysine glyceride was added to 0.5mL of the diluted bacterial suspension and mixed well (volume 9: 1). Sucking 1ml of the above mixed solution, placing in a flat plate, pouring with LB medium cooled to 45 deg.C, and rotating the flat plate to make it uniform. After solidification, the culture was inverted for 48h and counted. Calculating the bacteriostasis rate and the sterilization rate: x ═ A-B)/A × 100% (wherein: X-inhibition rate,%; A-average colony number of control sample; B-average colony number of test sample)
The results are shown in the following table, and it can be seen that polylysine glyceride has significant bacteriostatic activity.
Example 4
In this example, the effect of different carbon sources on polylysine glycerides was investigated.
Experimental groups: the fermentation treatment according to example 1 was carried out;
control group: the fermentation process of example 1 was followed, with the difference that the fermentation medium was as follows: 50g/L of glucose; (NH)4)2SO4 10g/L；K2HPO4·3H2O 0.8g/L；MgSO4·7H2O 0.5g/L；KH2PO4 1.36g/L；ZnSO4·7H2O 0.04g/L；FeSO4·7H2O0.03 g/L; 5g/L of yeast extract.
The results are shown in FIGS. 4 to 6. After fermentation, the products produced in the control group were detected by mass spectrometry as polylysine having a degree of polymerization of n 25-31 (fig. 4), while the products in the experimental group were polylysine glyceride having a degree of polymerization of n 25-30 and polylysine having a degree of polymerization of n 25-31 (fig. 5). In addition, the yield of polylysine in the experimental group was higher than that in the control group (FIG. 6). Therefore, the glycerol is used as the only carbon source for fermentation treatment, and the polylysine glyceride can be obtained at the same time.
Example 5
1. Study of the Effect of different temperatures on the stability of polylysine glyceride
When 1mL of each of the fermentation liquids obtained in step 1 of example 1 was left at pH7.0 at 25, 30, 35, 40, 45 and 50 ℃ for 12 hours, LC-MS results (FIG. 7) showed that the polylysine glyceride was more completely hydrolyzed into polylysine at a temperature higher than 40 ℃ and thus no polylysine glyceride could be obtained.
2. Study of the influence of different reaction times on the stability of polylysine glyceride
1mL of each of the fermentation liquids obtained in step 1 of example 1 was reacted at pH7.0 and 40 ℃ for 1, 5, 7 and 12 hours, respectively, and the degree of hydrolysis was compared with the time of hydrolysis. The LC-MS result (figure 8) shows that when the reaction time reaches 5h, the polylysine glyceride is hydrolyzed to polylysine more completely, so that the polylysine glyceride cannot be obtained.
3. Study on the influence of different pH values on the stability of polylysine glyceride
The pH value of the fermentation liquid obtained in the step 1 of the example 1 is stabilized to be about 3.2, so that the pH value of 3.2 is initially selected to be compared with the pH value of 7.0, the fermentation liquid is reacted for 12 hours at the temperature of 40 ℃, and the influence of different pH values on the hydrolysis of the polylysine glyceride is compared. The LC-MS result (figure 9) shows that the polylysine glyceride is not hydrolyzed after 12 hours of hydrolysis under the condition of pH3.2, and the polylysine glyceride is more completely hydrolyzed into the polylysine under the condition of pH7.0, so that the polylysine glyceride cannot be obtained.
Thus, a large amount of polylysine glyceride can be obtained as long as it is ensured that the pH of the system is maintained under acidic conditions (preferably 3.2) during the extraction. And under the condition of ensuring the acidic extraction, the extraction temperature and time have no obvious influence on the yield of the polylysine glyceride.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (2)
1. A polylysine glyceride, having a structure represented by the formula:
wherein n is 25 to 30,
the polylysine glyceride is obtained by the following steps:
(1) fermentation treatment:
inoculating Streptomyces albulus NBRC 14147 into a fermentation medium for shake flask fermentation, and culturing at 30 ℃ and 200rpm for 72 hours to obtain a fermentation liquid;
wherein, the fermentation medium is as follows:
60g/L of glycerol; (NH)4)2SO4 10g/L；KH2PO4·3H2O 0.8g/L；MgSO4·7H2O 0.5g/L；KH2PO41.36；ZnSO4·7H2O 0.04g/L；FeSO47H2O 0.03 g/L; 5g/L yeast extract, pH 6.8;
(2) extraction treatment:
1) performing solid-liquid separation on the fermentation liquor at 5000rpm for 20 min;
2) adjusting pH to 8.5 with 5M NaOH; centrifuging at 5000rpm and 4 deg.C for 10min, removing precipitate, collecting supernatant, and adjusting pH to 3.2;
3) filtering the supernatant with 10KD ultrafiltration membrane at 3000rpm at 4 deg.C, and collecting filtrate;
4) concentrating the filtrate by 5 times at 60 deg.C with rotary evaporator to obtain concentrated solution;
5) adding 4 times volume of precooled absolute ethyl alcohol into the concentrated solution, stirring, centrifuging at the rotating speed of 5000rpm and the temperature of 4 ℃ for 10min, and collecting the precipitate;
6) re-dissolving the precipitate with pH7.0 phosphate buffer solution, centrifuging at 5000rpm and 4 deg.C for 10min, and removing insoluble foreign protein;
7) quickly adjusting the pH of the supernatant to 3.2, adding 4 times volume of precooled absolute ethyl alcohol, centrifuging at the rotating speed of 5000rpm and the temperature of 4 ℃ for 10min, and centrifuging and collecting precipitates;
8) and (5) freeze-drying to obtain the polylysine glyceride.
2. Use of a polylysine glyceride according to claim 1 in the manufacture of a preservative.
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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 |
| CN102363797A (en) * | 2011-10-28 | 2012-02-29 | 天津科技大学 | Method for producing epsilon-poly-L-lysine |
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