CN101486794B - Preparation method of high antibacterial activity ε-poly-L-lysine component - Google Patents

Abstract

The present invention relates to a preparation method of ε-poly-L-lysine (ε-PL) component with high antibacterial activity, which adopts microfiltration, ultrafiltration and nanofiltration combined multistage separation technology to obtain ε with different molecular weight distributions -PL, through antibacterial experiments to determine the molecular weight range of the best antibacterial active components, and then collect and dry to obtain high antibacterial active component ε-PL products. Applying the method of the present invention can obtain high-purity ε-PL in different molecular weight ranges, because the membrane separation technology is used, it will not have any impact on the activity of ε-PL, and it is beneficial to the production of ε-PL with various molecular weights. Research.

Description

Preparation method of high antibacterial activity ε-poly-L-lysine component

technical field

The invention belongs to the technical field of biochemical industry, in particular to a preparation method of ε-poly-L-lysine component with high antibacterial activity.

Background technique

ε-poly-L-lysine (ε-poly-L-lysine, referred to as ε-PL) is an amino acid homopolymer biosynthesized by Streptomyces albicans and other strains. The ε-amino group of the acid is connected with the amide bond formed by the α-carboxyl group of another monomer L-lysine. ε-PL has good water solubility, slightly soluble in ethanol, high thermal stability, broad antibacterial spectrum, and can resist the activity of Gram-positive bacteria, Gram-negative bacteria, fungi, heat-resistant bacillus and viruses. Break down into L-lysine. L-Lysine is one of the eight essential amino acids for the human body, so it is a safe and efficient natural biological preservative. In addition, ε-PL can also be used as a gene transport carrier, drug coating material, electronic material, environmental protection material, etc., and has a very wide application prospect. In recent years, with the improvement of strain performance and fermentation control means, the fermentation production efficiency of ε-PL has been continuously improved.

At present, the main patents related to the production of ε-PL published in my country are:

1. On July 12, 2000, the State Intellectual Property Office announced the invention patent titled "Strain and Production Method for Large-scale Production of ε-poly-L-lysine" applied by Japan's Toshishi Iwata and others in China. The patent number is 97182253.0. This patent provides a relatively conventional method for improving the production capacity of strain ε-PL and producing ε-PL on a large scale.

2. On November 16, 2005, the State Intellectual Property Office announced the invention patent of "Method for Preparing ε-PL and Its Salts Using Lispora militaris PL6-3" applied by Nanjing University of Technology, the application number is 200510037774.2. This patent proposes a method for producing ε-PL by fermenting PL-3 obtained from B. militaris through screening.

3. On November 28, 2007, the State Intellectual Property Office announced the invention patent of "method for producing ε-poly-L-lysine by reflow process" applied by the applicant himself, and the application number is 200610013800.2. This patent proposes a method for producing ε-PL by a reflux process, that is, during the fermentation process of ε-PL, the breakthrough liquid in the later stage of the extraction process is used as a fed-in solution for fed-batch fermentation to produce ε-PL. The later breakthrough liquid is used as feed liquid, which improves the yield of ε-PL, reduces the discharge of fermentation waste liquid and hydrochloric acid waste liquid for elution, and reduces environmental pollution. On April 9, 2007, the applicant continued to apply for "a mutagenous strain Streptomyces albicans TUST2 that produces a large amount of ε-PL and uses the mutagenized strain TUST2 to produce ε-PL and The method of its salt", the invention patent, application number 200710057098.4. This patent utilizes Streptomyces albicans TUST2 (CGMCC No.1986), a production strain of ε-PL that was screened, mutated and identified from the soil in Hainan Province, my country, to ferment and produce ε-PL with a yield of 10-30 g/L.

Studies have shown that the molecular weight distribution of ε-PL produced by microbial fermentation is wide, and ε-PL with different degrees of polymerization has different physical and chemical properties and uses; in addition, the molecular weight of ε-PL is different, and its antibacterial effect is very different .

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, to provide a kind of preparation method of high bacteriostatic activity epsilon-poly-L-lysine component, the epsilon-PL that this method produces according to existing technology according to different molecular weight (different Polymerization degree) was graded, and the ε-PL fraction with high antibacterial activity was collected.

Technical scheme of the present invention is:

A kind of preparation method of high bacteriostatic activity epsilon-poly-L-lysine component, the steps of its preparation method are:

(1). After centrifuging the ε-PL fermentation broth to remove bacteria and protein, obtain the ε-PL filtrate by decolorization;

(2). Add the ε-PL filtrate to the pre-assembled microfiltration, ultrafiltration and nanofiltration membrane combination separation device with various molecular weight cut-offs, separate and obtain ε-PL component filtrates with various molecular weight distributions ;

(3). Verify that the molecular weight of the ε-PL filtrate ranges from 2KDa to 5KDa, and obtain ε-PL products with high antibacterial activity through concentration, low-temperature freeze-drying or spray drying.

Moreover, the material of the filter membrane is polysulfone, polyamide, bacterial cellulose, regenerated cellulose or other filter membranes with a suitable pH value of 2 to 14, the pore size of the microfiltration membrane is 0.2 μm, ultrafiltration and nanofiltration The molecular weight cut-offs of the membranes are 25KDa, 10kDa, 5kDa, 2kDa, and 1kDa, respectively. Microfiltration, ultrafiltration and nanofiltration membrane combination separation devices are used for separation and purification.

Moreover, the working pressure of the filtration and classification treatment can be 0.05-1.0 MPa, the pH of the fermentation broth filtration is neutral, and the temperature is 20-40°C.

Advantage and positive effect of the present invention are:

In the present invention, ε-poly-L-lysine is graded according to the molecular weight of the membrane combination technology, and the molecular weight part with high bacteriostatic activity is determined through antibacterial experiments, so as to prepare ε with a suitable molecular weight distribution with high bacteriostatic activity. - Poly-L-lysine products. Applying the method of the present invention can obtain high-purity ε-PL filtrates in different molecular weight ranges, because the membrane separation technology is used, it will not have any impact on the activity of ε-PL, and it is beneficial to the ε-PL of various molecular weights Research.

Detailed ways

The present invention is further described below in conjunction with embodiment; Following embodiment is illustrative, not limiting, can not limit protection scope of the present invention with following embodiment.

It should be noted that the fermentation broth of the present invention is prepared according to the method in the patent "Method for producing ε-poly-L-lysine by reflux process", and the strain used is "a mutagenized strain that produces a large amount of ε-PL Streptomyces albicans TUST2 and the method for producing ε-PL and its salts by fermentation using the mutagenized strain TUST2" are strains in the patent.

The material of the filter membrane used in the present invention can be polysulfone, polyamide, bacterial cellulose, regenerated cellulose or other filter membranes with a suitable pH value of 2 to 14, the pore size of the microfiltration membrane is 0.2 μm, ultrafiltration and nanofiltration membranes The molecular weight cut-offs are 25KDa, 10kDa, 5kDa, 2kDa, 1kDa, respectively, and are separated and purified by a combination of microfiltration, ultrafiltration and nanofiltration membrane separation devices.

The working pressure of the filtration and classification treatment of the present invention can be 0.05-1.0 MPa, the pH of fermentation broth filtration is neutral, and the temperature is 25 DEG C.

The invention collects the ε-PL solution with the highest antibacterial activity and the molecular weight range of 2KDa to 5KDa, and sprays or freezes it to make the ε-PL product, which is used as a food preservative.

Provide the embodiment of the present invention below:

Example 1:

Content 20g/L ε-PL fermentation broth was centrifuged to separate bacteria, and the solution obtained after decolorization could be separated by membrane combination separation device.

Take 2000ml ε-PL filtrate and pass through 0.2μm microfiltration membrane, 25KDa, 10KDa, 5KDa, 2KDa, 1KDa ultrafiltration membrane and nanofiltration membrane step by step. Among them, the operating pressure of microfiltration is 0.05MPa, the operating pressure of ultrafiltration membrane with molecular weight cut off of 25KDa, 10KDa and 5KDa is 0.15MPa, the operating pressure of ultrafiltration membrane with molecular weight cut off of 2KDa is 0.2MPa, and the operating pressure of ultrafiltration membrane with molecular weight cut off of 1KDa is 0.2MPa. The operating pressure is 0.7MPa.

The rejection rate of the microfiltration membrane to ε-PL filtrate is 0, and the volume of the permeate is about 2000ml; after passing through the 25KDa ultrafiltration membrane, the volume ratio of the permeate to the concentrate is about 10:1; after passing through the 10KDa ultrafiltration membrane , the volume ratio of the permeate to the retentate is about 7:1; after passing through the 5KDa ultrafiltration membrane, the volume ratio of the permeate to the retentate is about 7:1; after passing through the 2KDa ultrafiltration membrane, the ratio of the permeate to the retentate The volume ratio of the liquid is about 2:1; after finally passing through the 1KDa nanofiltration membrane, the volume ratio of the permeate to the concentrate is about 1:1.

Then antibacterial experiments were carried out for each of the different components.

Through the results of antibacterial experiment verification (see attached table 1), it is concluded that the molecular weight segment with the best antibacterial effect is 2KDa ~ 5KDa. This part of the solution is collected, concentrated and freeze-dried to make a high antibacterial activity ε-PL product .

Example 2:

The ε-PL product powder is prepared into 2L of 20g/L solution (pH is neutral, temperature is 25°C), and then separated by membrane filtration through microfiltration, ultrafiltration and nanofiltration combined membrane separation device in sequence. Among them, the operating pressure of microfiltration is 0.1MPa, the operating pressure of ultrafiltration membrane with molecular weight cut off of 25KDa, 10KDa and 5KDa is 0.2MPa, the operating pressure of ultrafiltration membrane with molecular weight cut off of 2KDa is 0.25MPa, and the operating pressure of ultrafiltration membrane with molecular weight cut off of 1KDa is 0.25MPa. The operating pressure is 1MPa.

The above 2000ml ε-PL solution completely permeated the microfiltration membrane; after that, it passed through the 25KDa ultrafiltration membrane, and the volume ratio of the permeate to the retentate was about 10:1; then passed through the 10KDa ultrafiltration membrane, the permeate to the retentate The volume ratio of the permeate to the retentate is about 8:1; then after passing through the 5KDa ultrafiltration membrane, the volume ratio of the permeate to the retentate is about 7:1; then through the 2KDa ultrafiltration membrane, the volume ratio of the permeate to the retentate is about 2:1; after finally passing through the 1KDa nanofiltration membrane, the volume ratio of the permeate to the retentate is about 1:1.

Then antibacterial experiments were carried out for each of the different components.

According to the results of antibacterial experiments, it can be concluded that the molecular weight segment with the best antibacterial effect is 2KDa ~ 5KDa. This part of the solution is collected, concentrated and then freeze-dried to produce a high antibacterial activity ε-PL product.

Table 1

Claims (2)

1. the preparation method of a high bacteriostatic activity epsilon-poly-L-lysine component, it is characterized in that: preparation method's step is:

(1). epsilon-poly-L-lysine ε-PL fermented liquid behind centrifugal removal thalline and albumen, is obtained ε-PL filtrate by decolouring;

(2). epsilon-poly-L-lysine ε-PL filtrate is joined in micro-filtration, ultrafiltration and the nanofiltration membrane combination tripping device of the various molecular weight cut-offs that assemble in advance, separate and obtain respectively the ε-PL component filtrate of various molecular weight distribution;

(3). checking epsilon-poly-L-lysine ε-PL filtrate molecular weight ranges is in 2KDa～5KDa, and by the ε-PL product that concentrates, frozen drying or spraying drying obtain high bacteriostatic activity;

The material of described filter membrane is polysulfones, polymeric amide, bacteria cellulose, regenerated cellulose or other appropriate pH values at 2～14 filter membrane, the aperture of its microfiltration membrane is 0.2 μ m, the molecular weight cut-off of ultrafiltration and nanofiltration membrane is respectively 25KDa, 10kDa, 5kDa, 2kDa, 1kDa, adopts micro-filtration, ultrafiltration and nanofiltration membrane combination tripping device to carry out separation and purification.

2. the preparation method of high bacteriostatic activity epsilon-poly-L-lysine component according to claim 1 is characterized in that: the operating pressure of described filtration stage treatment is at 0.05～1.0Mpa, and the pH of filtering fermentation liquor is neutral, and temperature is 20～40 ℃.