CN107805651B

CN107805651B - Synergist for promoting synthesis of Brevibacillus laterosporus antibacterial peptide and application thereof

Info

Publication number: CN107805651B
Application number: CN201711020778.9A
Authority: CN
Inventors: 王志新; 贾英民; 宁亚维; 李兴峰; 杨柯
Original assignee: Hebei University of Science and Technology
Current assignee: Hebei University of Science and Technology
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2021-06-15
Anticipated expiration: 2037-10-27
Also published as: CN107805651A

Abstract

The invention provides a synergist for promoting synthesis of antibacterial peptide of Brevibacillus laterosporus and application thereof, wherein active ingredients of the synergist comprise glutamic acid, threonine and succinic acid, and the molar ratio is (10-50): (20-170): (10-60). When the synergist is used for fermenting the brevibacillus laterosporus, the yield of the antibacterial peptide is improved by 25-90 percent, and the OD of the thallus is₆₀₀The value is improved by 40 to 80 percent. The synergist has the advantages of easily obtained raw materials, simple use method, remarkable antibacterial peptide synthesis promoting effect, high antibacterial peptide yield, promotion of thallus growth, production efficiency improvement, fermentation period shortening, and good economic benefit.

Description

Synergist for promoting synthesis of Brevibacillus laterosporus antibacterial peptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a synergist for promoting synthesis of a Brevibacillus laterosporus antibacterial peptide and application thereof.

Background

Antibacterial peptides (antibiotic peptides) are short for active polypeptides with the effect of inhibiting or killing bacteria and fungi, and because the phenomenon of abuse of antibiotics is increasingly increased at present, super bacteria continuously appear, and the search for a novel antibiotic substitute is an urgent problem to be solved. The antibacterial peptide has the characteristics of stable physicochemical properties, wide antibacterial spectrum, high-efficiency antibacterial effect, unique antibacterial mechanism, safety and no residue, becomes an ideal substitute of antibiotics, and has attracted extensive attention and research of people in recent years.

Brevibacillus laterosporus (Brevibacillus laterosporus) is a gram-positive bacterium widely present in soil, seawater, etc., and is a facultative anaerobic bacterium belonging to the genus Brevibacillus. A large characteristic of Lateosporus is that active small peptides with antibacterial action can be synthesized. At present, researchers at home and abroad separate and obtain a plurality of strains of Brevibacillus laterosporus for producing antibacterial peptide, but the fermentation production of the antibacterial peptide mostly stays in the optimization of nutrient components such as fermentation carbon and nitrogen sources and fermentation conditions, the synthesis path and the metabolic regulation of the antibacterial peptide are blank, and the research on adding amino acid or organic acid in the fermentation process is still to be developed. Therefore, the industrial production of antibacterial peptides faces many problems such as low fermentation potency, high cost, etc. In recent years, how to obtain high yield of antibacterial peptide through regulation of fermentation metabolism becomes a key point of research.

For microorganisms, amino acids are the basic units that make up proteins and can also be used as energy substances, as well as precursors of many important nitrogen-containing compounds and also as basic building blocks for secondary metabolites. When the microorganism takes in amino acid from the external environment, the amino acid can be decomposed to form intermediate products in important metabolic pathways, or synthesize proteins or polypeptides required by the bacteria. Organic acids are ubiquitous in metabolic pathways and can serve as important intermediate metabolites entering metabolic pathways to provide energy and also serve as precursors of many secondary metabolites.

Therefore, it is necessary to research amino acids and organic acids in the fermentation synthesis pathway of antibacterial peptides, so as to promote the synthesis amount and improve the synthesis efficiency. The invention forms the antibacterial peptide fermentation process with simple process and high production efficiency by adding the synergist containing amino acid and organic acid in the antibacterial peptide fermentation process.

Disclosure of Invention

The invention aims to provide an antibacterial peptide synthesis synergist which has high production efficiency and simple addition process aiming at the problems of low yield, poor efficiency and the like of the conventional antibacterial peptide fermentation. The synergist is simply added and mixed in the fermentation process, so that the capability of the brevibacillus laterosporus for fermenting and synthesizing the antibacterial peptide can be greatly improved, the growth of the brevibacillus laterosporus is promoted, and a foundation is laid for the mass production and large-scale preparation of the antibacterial peptide.

The technical scheme of the invention is as follows:

a synergist for promoting the synthesis of antibacterial peptide of Brevibacillus laterosporus contains glutamic acid, threonine and succinic acid as active components.

Specifically, the synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus has the active components of glutamic acid, threonine and succinic acid with the molar ratio of (10-50): (20-170): (10-60).

Furthermore, the synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus has the active components of glutamic acid, threonine and succinic acid with the molar ratio of (10-30): (40-80): (15-45).

Furthermore, the synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus has the active components of glutamic acid, threonine and succinic acid with the molar ratio of 10 (60-80) to (40-45).

Preferably, the synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus is an aqueous solution, wherein the aqueous solution contains 0.1-0.5mol/L glutamic acid, 0.2-1.7mol/L threonine and 0.1-0.6mol/L succinic acid.

Further preferably, the synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus is an aqueous solution, wherein the aqueous solution contains 0.1-0.3mol/L glutamic acid, 0.4-0.8mol/L threonine and 0.15-0.45mol/L succinic acid.

Still more preferably, the synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus is an aqueous solution, wherein the aqueous solution contains 0.1mol/L glutamic acid, 0.6-0.8mol/L threonine and 0.40-0.45mol/L succinic acid.

In a preferred embodiment of the present invention, the synergist for promoting the synthesis of the antibacterial peptide of brevibacillus laterosporus is an aqueous solution, which contains: 0.1mol/L of glutamic acid, 0.8mol/L of threonine and 0.45mol/L of succinic acid.

In another preferred embodiment of the present invention, the synergist for promoting the synthesis of the antibacterial peptide of brevibacillus laterosporus is an aqueous solution, which comprises: 0.1mol/L of glutamic acid, 0.6mol/L of threonine and 0.40mol/L of succinic acid.

The synergist for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus can be prepared according to a conventional method in the field. When the synergist for promoting the synthesis of the Brevibacillus laterosporus antibacterial peptide is aqueous solution, slowly heating the solution until glutamic acid is completely dissolved in the preparation process.

Further, the glutamic acid is L-glutamic acid or L-sodium glutamate, and the threonine is L-threonine.

Further, the glutamic acid, threonine and succinic acid are of analytical or industrial grade.

The invention also comprises a culture medium containing the synergist and used for promoting the synthesis of the antibacterial peptide of the brevibacillus laterosporus. In addition to the above mentioned synergists, other components of the medium are available according to the general knowledge in the art.

The invention also comprises the application of the synergist for promoting the synthesis of the antibacterial peptide of the Brevibacillus laterosporus or the culture medium in the process of producing the antibacterial peptide by fermenting the Brevibacillus laterosporus.

The synergist can be directly added into a culture medium for producing the antibacterial peptide by fermenting Brevibacillus laterosporus (Brevibacillus laterosporus).

The synergist can be used for shake flask fermentation culture of the Brevibacillus laterosporus antibacterial peptide and fermentation culture of fermentors with different grades.

Further, the final concentrations of glutamic acid, threonine and succinic acid in the medium were respectively: glutamic acid 10-30mmol/L, threonine 20-80mmol/L and succinic acid 10-50 mmol/L; preferably glutamic acid 10-30mmol/L, threonine 40-80mmol/L and succinic acid 15-45 mmol/L.

In a preferred embodiment of the invention, the final concentrations of threonine and succinic acid in the culture medium are: 10mmol/L, threonine 80mmol/L and succinic acid 45 mmol/L.

In another preferred embodiment of the present invention, the final concentrations of threonine and succinic acid in the medium are: 10mmol/L threonine 60mmol/L and succinic acid 40 mmol/L.

Further, the brevibacillus laterosporus includes, but is not limited to, b.laterosporu AS 1.2827, b.laterosporu AS 1.2739, b.laterosporu AS 1.864.

The microorganisms mentioned in the present invention are known strains and are commercially available.

The invention provides a synergist based on the improvement of the fermentation synthesis amount of the Brevibacillus laterosporus antibacterial peptide, and the synergist is directly added into a culture medium of the synergist to improve the growth of thalli, promote the generation of the antibacterial peptide, improve the production efficiency and reduce the production cost. Glutamic acid in the synergist has a remarkable promoting effect on the growth of the brevibacillus laterosporus, and when glutamic acid exists in fermentation liquor, the bacterial strain can perform transamination effect by utilizing the glutamic acid, enters tricarboxylic acid cycle decomposition energy supply and participates in protein synthesis. Threonine and succinic acid in the synergist have a remarkable effect on promoting the synthesis of the antibacterial peptide, and during the synthesis of the antibacterial peptide, the threonine and succinic acid are beneficial to enhancing the secondary metabolic pathway of bacteria, especially the synthetic capacity of the antibacterial peptide.

When the synergist is used for fermentation culture of the Brevibacillus laterosporus antibacterial peptide, the use mode is simple, the synergist is directly added into a fermentation culture medium, and the optimal content of each component of the synergist in the culture medium is 10-30mmol/L of glutamic acid, 20-80mmol/L of threonine and 10-50mmol/L of succinic acid. The addition amount can promote the synergistic effect among the components of the synergist, so as to achieve the purposes of promoting the growth of thalli and the synthesis of the antibacterial peptide, and the yield of the antibacterial peptide is obviously improved.

The antibacterial peptide synthesis synergist for brevibacillus laterosporus developed by the invention can greatly improve the synthesis amount of antibacterial peptide in the culture process of brevibacillus laterosporus, and simultaneously, the metabolism of thalli is more vigorous, the biomass is increased, thereby shortening the production period and improving the production efficiency.

The synergist disclosed by the invention is easy to obtain raw materials of all components, extremely simple in use method, good in controllability, strong in reliability, simple in large-scale application and wide in market prospect, and can be simply added.

After the synergist is used, the yield of the antibacterial peptide reaches 2250-₆₀₀The value is improved by 40-80%, and the economic benefit is good.

The antibacterial peptide produced by the invention has stable property and wide antibacterial spectrum, is used for preparing various biological preservatives, and is widely applied to the fields of food, medicine, feed, agriculture and the like.

Detailed Description

The present invention will be described in detail with reference to the following embodiments. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the starting materials used are commercially available unless otherwise specified.

EXAMPLE 1 antimicrobial peptide production at different potentiator concentrations

Adding different contents of synergists into a fermentation medium (a carbon-nitrogen source is glucose and peptone respectively) of Brevibacillus laterosporus AS1.864, carrying out shake-flask culture at 30 ℃ and 250r/min for 24h, and measuring the bacteriostatic activity of the antibacterial peptide, wherein the final concentration of each component of the synergists and the yield of the antibacterial peptide are shown in the following table 1. Through SPSS software multiple comparison analysis, the CK control group and 9 experiment groups added with the synergist have significant difference (P is less than 0.05), and the synergist is added to obviously promote the yield of the antibacterial peptide.

TABLE 1 yield of antimicrobial peptides at different content of synergist components in the culture medium

Note: CK means no synergist added.

EXAMPLE 2 Effect of synergistic application of synergists on antimicrobial peptide production

In a fermentation medium of Brevibacillus laterosporus AS1.864 composed of glucose and peptone, 10mmol/L glutamic acid, 80mmol/L threonine and 45mmol/L succinic acid are respectively added, and a control group is formed by singly adding 10mmol/L glutamic acid, 80mmol/L threonine and 45mmol/L succinic acid, and a blank group is formed by adding no synergist. Respectively inoculating the culture medium with Brevibacillus laterosporus seeds, and performing shake culture at 30 ℃ and 250r/min for 24 h. The antibacterial peptide yield of the experiment group added with the synergist is higher than that of the control group added with the glutamic acid, the threonine and the succinic acid alone, and the antibacterial peptide yield of the control group added with the glutamic acid, the threonine and the succinic acid alone is higher than that of the blank group not added with the synergist. The results are shown in Table 2. Through multiple analysis and comparison, the blank group has obvious difference (P <0.05) compared with the synergist group, and the blank group has obvious difference (P <0.05) compared with the threonine group and the succinic acid group, so that the antibacterial peptide synthesis is promoted by adding the synergist or adding threonine or succinic acid independently; the synergistic agent group has obvious difference (P <0.05) compared with a control group added with glutamic acid, threonine and succinic acid separately, thereby showing that the three components of the synergistic agent have synergistic promotion effect on the synthesis of the antibacterial peptide.

TABLE 2 Effect of synergist synergy on antibacterial peptide production

EXAMPLE 3 use of potentiators for antimicrobial peptide production in corn steep liquor fermentation Medium

Adding synergist into Brevibacillus laterosporus AS1.864 fermentation culture medium composed of glucose and corn steep liquor AS carbon-nitrogen source, wherein the final concentrations of glutamic acid, threonine and succinic acid in the culture medium are 10mmol/L, 80mmol/L and 45mmol/L respectively, shake-flask culturing at 30 deg.C and 250r/min for 24h, determining titer of antibacterial peptide AS 2340AU/mL, and determining thallus OD₆₀₀The value is 3.0, compared with the group without the synergist, the yield of the antibacterial peptide is improved by 90.0 percent, and the OD of thalli is₆₀₀The value increased by about 50.0%.

Example 4 synergist for antimicrobial peptide production on 5L fermenter Scale

The synergist was used for fermenter-scale production of antimicrobial peptides and was added to a fermentation medium made of glucose, peptone, and glutamic acid, threonine, and succinic acid at final concentrations of 10mmol/L, 60mmol/L, and 40mmol/L, respectively. 3L of culture medium liquid loading amount in a 5L fermentation tank, autoclaving, cooling, inoculating Brevibacillus laterosporus AS1.864 according to 5% (v/v) inoculation amount, performing fermentation culture under the conditions of aeration rate of 1.6vvm, 30 ℃ and 300r/min, wherein the yield of the antibacterial peptide reaches the maximum at 20h, is 2630.0AU/mL, is increased by 66.5% compared with a control group without the synergist, and the OD of thalli is increased by 66.5% compared with that of a control group without the synergist₆₀₀The value is improved by nearly 50.0%.

Example 5 synergist for antimicrobial peptide production in 30L fermenter Scale

Potentiators were used for pilot scale studies in a 30L fermentor with final concentrations of glutamic acid, threonine and succinic acid in brevibacillus laterosporus AS1.864 fermentation medium (glucose, peptone configured medium) of 10mmol/L, 60mmol/L and 40mmol/L, respectively. The liquid loading of the medium in the 30L fermenter was 20L, and the culture conditions were: the strain inoculation amount is 5 percent, the ventilation amount is 1.2vvm, the temperature is 30 ℃, the fermentation time is 300r/min, the yield of the antibacterial peptide reaches 2700.0AU/mL after 18 hours of fermentation, compared with a control group without the synergist, the yield of the antibacterial peptide is improved by 68.5 percent, and the OD of thalli is₆₀₀The value is improved by 40.3%.

EXAMPLE 6 bacteriostatic Profile of antimicrobial peptides

The antimicrobial spectra of the antimicrobial peptides prepared in examples 1-5 were studied, and the antimicrobial properties of the antimicrobial peptides against various indicator bacteria were determined by the Oxford cup method. The results are given in Table 3 below.

TABLE 3 antibacterial spectra of antibacterial peptides

Note: , + represents the diameter of the inhibition zone of >2.0cm, and + represents the diameter of the inhibition zone of 1.0-2.0 cm.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A synergist for promoting the synthesis of antibacterial peptide of Brevibacillus laterosporusBrevibacillus laterosporu) The application in the process of producing the antibacterial peptide by fermentation is characterized in that the synergist is glutamic acid, threonine and succinic acid; the mol ratio of glutamic acid, threonine and succinic acid in the synergist is (10-50): 20-170: (10-60); the Brevibacillus laterosporus isB. laterosporu AS 1.864。

2. The use according to claim 1, wherein the molar ratio of glutamic acid, threonine and succinic acid in the potentiator is (10-30): (40-80): (15-45).

3. The use of claim 1, wherein the molar ratio of glutamic acid, threonine and succinic acid in the potentiating agent is 10 (60-80) to (40-45).

4. The use according to claim 1, wherein the potentiator is an aqueous solution containing 0.1-0.5mol/L of glutamic acid, 0.2-1.7mol/L of threonine, and 0.1-0.6mol/L of succinic acid.

5. The use according to claim 1, wherein the potentiator is an aqueous solution containing 0.1-0.3mol/L of glutamic acid, 0.4-0.8mol/L of threonine, and 0.15-0.45mol/L of succinic acid.

6. The use according to claim 1, wherein the potentiator is an aqueous solution containing 0.1mol/L of glutamic acid, 0.6-0.8mol/L of threonine, and 0.40-0.45mol/L of succinic acid.

7. Use according to claim 1, wherein the potentiating agent is an aqueous solution comprising: 0.1mol/L of glutamic acid, 0.8mol/L of threonine and 0.45mol/L of succinic acid;

or, the synergist is an aqueous solution, which contains: 0.1mol/L of glutamic acid, 0.6mol/L of threonine and 0.40mol/L of succinic acid.

8. The use according to claim 1, wherein the glutamic acid is L-glutamic acid or sodium L-glutamate and the threonine is L-threonine.

9. A culture medium containing a synergist and used for promoting the synthesis of an antibacterial peptide of brevibacillus laterosporus in brevibacillus laterosporus (B) ((B))Brevibacillus laterosporu) The application in the process of producing the antibacterial peptide by fermentation;

characterized in that the synergist is glutamic acid, threonine and succinic acid; the final concentrations of glutamate, threonine and succinate in the medium were: glutamic acid 10-30mmol/L, threonine 20-80mmol/L and succinic acid 10-50 mmol/L; the Brevibacillus laterosporus isB. laterosporu AS 1.864。

10. The use according to claim 9, wherein the final concentrations of glutamate, threonine and succinate in the culture medium are: glutamic acid 10-30mmol/L, threonine 40-80mmol/L and succinic acid 15-45 mmol/L.

11. The use according to claim 9, wherein threonine and succinic acid are present in the culture medium at respective final concentrations of: 10mmol/L, threonine 80mmol/L and succinic acid 45 mmol/L;

alternatively, the final concentrations of threonine and succinic acid in the medium are: 10mmol/L threonine 60mmol/L and succinic acid 40 mmol/L.