Preparation method of high polymerization degree epsilon-polylysine

Abstract

The application relates to the field of biology and new medicine technology, and discloses a preparation method of high-polymerization-degree epsilon-polylysine. After the fermentation liquid after culture is inactivated, the inactivated fermentation liquid is filtered to obtain clear epsilon-polylysine filtrate, starch is added to obtain epsilon-polylysine-starch suspension, filtration separation is carried out to obtain a mixture of epsilon-polylysine and starch, the mixture is dissolved through an aqueous hydrochloric acid solution, epsilon-polylysine solution is obtained after filtration, concentration is carried out through a rolling membrane to obtain epsilon-polylysine concentrated solution, activated carbon is added to carry out decolorization, the decolorized liquid is sprayed to dry to obtain epsilon-polylysine finished product. The epsilon-polylysine has the property of positive charge, and the epsilon-polylysine in the solution is separated according to the principle that the epsilon-polylysine and starch form a precipitate, problems existing in the production of the three main extraction methods (salting-out method, organic solvent extraction method and ion exchange method) are avoided, and the method can be used for industrialized production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology and new medicine technology, and particularly relates to a preparation method of high-polymerization-degree epsilon-polylysine. BACKGROUND

[0002] Polylysine is a polypeptide with bacteriostatic effect produced by fermentation of Streptococcus albus, and is a natural, safe and healthy antibacterial food preservative. It has obvious inhibitory effect on gram-positive bacteria, gram-negative bacteria and yeast, and can be applied to fruits and vegetables, beans, rice and products, wheat flour and products, coarse grain products, meat products, condiments, beverages, cosmetics and daily chemical products as a preservative. The polylysine with a molecular weight of 3600-4300 has the best bacteriostatic activity, and the polylysine loses the bacteriostatic activity when the molecular weight is lower than 1300.

[0003] The epsilon-polylysine is an extracellular polypeptide, and the main extraction methods include salting-out method, organic solvent extraction method and ion exchange method. The salting-out method produces a large amount of high-concentration wastewater, which causes certain pollution to the environment. The organic solvent extraction method uses a large amount of organic solvents, and the volatilization of the organic solvents in the operation process causes certain harm to the human body and the environment, and the operation needs to be carried out in an explosion-proof workshop, which increases the investment. The ion exchange method produces a large amount of wastewater in the resin regeneration process.

[0004] There is a lack of a preparation method of epsilon-polylysine suitable for industrial production, and a high-polymerization-degree and pure polylysine product can be obtained. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a preparation method of high-polymerization-degree epsilon-polylysine, which solves the problems in the background art.

[0006] To solve the above technical problems, according to one aspect of the present application, more specifically, a preparation method of high-polymerization-degree epsilon-polylysine, comprising the following steps:

[0007] S1, pre-treatment of the fermentation liquor: after fermentation tank discharge, the bacteria are inactivated, and the inactivated fermentation liquor is filtered to obtain a clear epsilon-polylysine filtrate;

[0008] S2, a certain amount of starch is added to the clear epsilon-polylysine filtrate to obtain an epsilon-polylysine-starch suspension;

[0009] S3, the epsilon-polylysine-starch suspension is filtered and separated to obtain a mixture of epsilon-polylysine and starch;

[0010] S4, the mixture is dissolved with a certain concentration of hydrochloric acid aqueous solution, and an epsilon-polylysine solution is obtained after filtration;

[0011] S5, the epsilon-polylysine solution is concentrated using a roll membrane to obtain an epsilon-polylysine concentrate;

[0012] S6, activated carbon is added to the epsilon-polylysine concentrate to decolorize to obtain a decolorized solution;

[0013] S7, the decolorized solution is spray dried to obtain epsilon-polylysine finished product.

[0014] Further, the sterilization method of the bacteria in step S1 is any one of acid addition, base addition or heating, preferably heating to 80-90 DEG C, and maintaining for 1-2 hours;

[0015] The filtration method is any one or combination of ceramic membrane filtration, plate and frame filtration, centrifuge separation, preferably using ceramic membrane filtration.

[0016] Further, the amount of starch added in step S2 is 50%-80% of the mass of epsilon-polylysine.

[0017] Further, the separation method of the suspension in step S3 is any one of ceramic membrane, tubular centrifuge and disc centrifuge, preferably using ceramic membrane separation.

[0018] Further, the dissolution method in step S4 is: first adding salt-free water, and then adjusting the pH to 4.4-4.8 with hydrochloric acid.

[0019] Further, the amount of salt-free water is 20-50 times the amount of epsilon-polylysine.

[0020] Further, the molecular weight of the roll membrane used for concentration in step S5 is 3000D-20000D.

[0021] Further, the activated carbon selected in step S6 is powdered activated carbon with a particle size of 200-300 mesh, and the decolorization temperature is about 50 DEG C, and the duration is 40min-1h.

[0022] Further, the spray drying machine is used for spray drying operation in step S7.

[0023] The preparation method of high polymerization degree epsilon-polylysine has the following advantages:

[0024] The present application uses the principle that epsilon-polylysine has a positive charge and forms a precipitate with starch to separate epsilon-polylysine in solution, avoiding the problems existing in the three main extraction methods (salting-out method, organic solvent extraction method and ion exchange method), and can be used for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0025] The application will be described in further detail below with reference to the drawings and specific implementation methods.

[0026] Figure 1 The structural diagram of the application is shown. DETAILED DESCRIPTION

[0027] The application will be described in further detail below with reference to the drawings and specific implementation methods. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0028] As Figure 1 shown, according to one aspect of the application, a preparation method of high-polymerization-degree ε-polylysine is provided, including the following steps:

[0029] S1, pretreatment of the fermentation broth: after the fermentation tank is discharged, the bacteria are inactivated (any one of adding acid, adding alkali or heating, preferably heating to 80-90℃, and maintaining for 1-2h), the inactivated fermentation broth is filtered through a ceramic membrane to obtain a clear ε-polylysine filtrate, the pH is adjusted to 7.0 by using a sodium hydroxide aqueous solution, and the content of polylysine in the filtrate is 25g / L;

[0030] S2, starch in an amount of 50%-80% of the mass of the ε-polylysine is added to the clear ε-polylysine filtrate to obtain an ε-polylysine-starch suspension.

[0031] S3, the ε-polylysine-starch suspension is separated by ceramic membrane filtration to obtain a mixture of ε-polylysine and starch.

[0032] S4, 20-50 times the amount of ε-polylysine is added to the mixture, and then the pH is adjusted to 4.4-4.8 by using hydrochloric acid for dissolution, and ε-polylysine solution is obtained after filtration.

[0033] S5, the ε-polylysine solution is concentrated by using a roll-type membrane with a molecular weight of 3000D-20000D to obtain an ε-polylysine concentrate.

[0034] S6, powdered activated carbon with a particle size of 200-300 mesh is added to the ε-polylysine concentrate, the temperature is controlled at about 50℃, and the decolorization operation is continued for 40min-1h to obtain a decolorized solution.

[0035] S7, the decolorized solution is spray-dried by using a spray dryer to obtain ε-polylysine finished product.

[0036] Example One

[0037] The fermentation broth was put into a tank, heated to 80°C, and kept for 2 hours to complete sterilization. The sterilized fermentation broth was filtered using a ceramic membrane to obtain a filtrate, and the pH was adjusted to 7.0 using a sodium hydroxide aqueous solution. The content of polylysine in the filtrate was 25 g / L;

[0038] To 1 m3 of the filtrate, 12 kg of starch was added, and stirred for 2 hours to form a suspension. The suspension was filtered using a ceramic membrane. When the circulation volume in the ceramic membrane reached 10% of the original volume, 100 L of salt-free water was added for top washing to obtain a polylysine-starch mixture. 450 L of salt-free water was added, and the pH was adjusted to 4.4 using hydrochloric acid under stirring to obtain a solution of 480 L. The solution was filtered using a ceramic membrane after being stirred for 3 hours. When the circulation volume in the ceramic membrane reached 10% of the original volume, 100 L of salt-free water was added for top washing to obtain a polylysine hydrochloride solution with a content of 25 g / L. The solution was concentrated to 150 g / L using a 3000D rolling membrane, and 200 L of salt-free water was continuously added for top washing. The concentration was stopped to obtain a concentrated solution of 135 L. 0.5 kg of activated carbon was added to the 135 L concentrated solution, stirred for 1 hour, and then filtered to obtain a decolorized solution. The decolorized solution was spray-dried to obtain 22 kg of polylysine.

[0039] Example Two

[0040] The fermentation broth was put into a tank, heated to 80°C, and kept for 2 hours to complete sterilization. The sterilized fermentation broth was filtered using a ceramic membrane to obtain a filtrate, and the pH was adjusted to 7.0 using a sodium hydroxide aqueous solution. The content of polylysine in the filtrate was 25 g / L;

[0041] To 1 m3 of the filtrate, 20 kg of starch was added, and stirred for 2 hours to form a suspension. The suspension was filtered using a ceramic membrane. When the circulation volume in the ceramic membrane reached 10% of the original volume, 100 L of salt-free water was added for top washing to obtain a polylysine-starch mixture. 450 L of salt-free water was added, and the pH was adjusted to 4.8 using hydrochloric acid under stirring to obtain a solution of 480 L. The solution was filtered using a ceramic membrane after being stirred for 3 hours. When the circulation volume in the ceramic membrane reached 10% of the original volume, 100 L of salt-free water was added for top washing to obtain a polylysine hydrochloride solution with a content of 26 g / L. The solution was concentrated to 150 g / L using a 20000D rolling membrane, and 200 L of salt-free water was continuously added for top washing. The concentration was stopped to obtain a concentrated solution of 135 L. 0.5 kg of activated carbon was added to the 135 L concentrated solution, stirred for 1 hour, and then filtered to obtain a decolorized solution. The decolorized solution was spray-dried to obtain 23 kg of polylysine.

[0042] Example Three

[0043] After the fermentation broth is put into a tank, it is heated to 80℃ first, and kept for 2 hours to complete sterilization. The fermentation broth after sterilization is filtered using a ceramic membrane to obtain a filtrate, and the pH is adjusted to 7.0 using a sodium hydroxide aqueous solution. The content of polylysine in the filtrate is 25 g / L;

[0044] 15 kg of starch is added to 1 m3 of the filtrate, and stirred for 2 hours to form a suspension. The suspension is filtered using a ceramic membrane. When the circulation in the ceramic membrane reaches 10% of the original volume, 100 L of salt-free water is added for top washing to obtain a polylysine-starch mixture. 450 L of salt-free water is added thereto, and the pH is adjusted to 4.6 using hydrochloric acid under stirring to obtain a solution of 480 L. After being fully stirred for 3 hours, the solution is filtered using a ceramic membrane. When the circulation in the ceramic membrane reaches 10% of the original volume, 100 L of salt-free water is added for top washing to obtain a polylysine hydrochloride solution with a content of 25 g / L. The solution is concentrated to 150 g / L using a 10000D roll-type membrane, and 200 L of salt-free water is continuously added for top washing, and the concentration is stopped to obtain a concentrated solution of 135 L. 0.5 kg of activated carbon is added to the 135 L concentrated solution, stirred for 1 hour, and then filtered to obtain a decolorized solution. The decolorized solution is spray-dried to obtain 22 kg of polylysine.

[0045] The working principle of the device is as follows: the ε-polylysine has a positive charge, and the ε-polylysine in the solution is separated by forming a precipitate with starch, which avoids the problems existing in the salt fractionation method, the organic solvent extraction method and the ion exchange method (the salt fractionation method produces a large amount of high-concentration wastewater, which causes certain pollution to the environment; the organic solvent extraction method uses a large amount of organic solvent, and the volatilization of the organic solvent during operation will cause certain harm to the human body and the environment, and the operation needs to be carried out in an explosion-proof workshop, which increases the investment; the ion exchange method produces a large amount of wastewater during resin regeneration) and can be used for industrial production.

[0046] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled persons in the technical field within the essential scope of the present application also belong to the protection scope of the present application.

Claims

1. A method for producing high-polymerization ε-polylysine, characterized by, The following steps are: S1, pretreatment of the fermentation broth: after fermentation, the bacteria are inactivated, and the inactivated fermentation broth is filtered to obtain a clear ε-polylysine filtrate; S2, starch is added to the clear ε-polylysine filtrate to obtain an ε-polylysine-starch suspension, and the amount of starch added is 50%-80% of the mass of ε-polylysine; S3, the ε-polylysine-starch suspension is filtered and separated to obtain a mixture of ε-polylysine and starch; S4, the mixture is dissolved with a certain concentration of hydrochloric acid aqueous solution, and ε-polylysine solution is obtained after filtration; S5, the ε-polylysine solution is concentrated using a rolling membrane to obtain an ε-polylysine concentrate; S6, activated carbon is added to the ε-polylysine concentrate to decolorize to obtain a decolorized solution; S7, the decolorized solution is spray dried to obtain ε-polylysine finished product.

2. The method for preparing high-polymerization-degree ε-polylysine according to claim 1, characterized in that: The sterilization method of the bacteria in step S1 is any one of acid addition, base addition or heating; The filtration method is any one or a combination of ceramic membrane filtration, plate and frame filtration, and centrifuge separation.

3. The method for preparing high-polymerization-degree ε-polylysine according to claim 2, characterized in that: When the bacteria in step S1 are heated, the temperature is raised to 80-90℃ and maintained for 1-2h.

4. The method for preparing high-polymerization-degree ε-polylysine according to claim 1, characterized in that: In step S3, the separation method of the suspension is any one of ceramic membrane, tubular centrifuge and disc centrifuge.

5. The method for preparing high-polymerization-degree ε-polylysine according to claim 1, characterized in that: In step S4, the dissolution method is to first add salt-free water, and then adjust the pH to 4.4-4.8 with hydrochloric acid.

6. The method for preparing high-polymerization-degree ε-polylysine according to claim 5, characterized in that: The amount of salt-free water is 20-50 times the amount of ε-polylysine.

7. The method for preparing high-polymerization-degree ε-polylysine according to claim 1, characterized in that: The molecular weight of the rolling membrane used for concentration in step S5 is 3000D-20000D.

8. The method for preparing high-polymerization-degree ε-polylysine according to claim 1, characterized in that: In step S6, the activated carbon selected is powdered activated carbon with a particle size of 200-300 mesh, and the decolorization temperature is about 50℃, lasting for 40min-1h.

9. The method for preparing high-polymerization-degree ε-polylysine according to claim 1, characterized in that: In step S7, a spray dryer is used for spray drying.

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