阅读说明：本技术 一种杀菌剂及其应用 (Bactericide and application thereof ) 是由 张德宁 奥兰多 徐洪利 高祥友 于 2020-12-08 设计创作，主要内容包括：本发明公开了一种杀菌剂及其应用,涉及工业领域,由羊毛硫细菌素和螯合剂混合而成。本申请实施例通过创新性的设计抗菌肽组合物,使其具有显著的抗菌效果和独特的抗菌机制,从而拓宽这些肽的抗菌活性谱,并开发它们在添加剂食品方面新的前景。螯合剂葡萄糖酸钠可使革兰氏阴性菌外膜失稳,并在菌膜内形成孔洞,使组合物具有抗革兰氏阴性菌的活性。该组合物能够诱导细菌壁的不稳定性,并在使用前不需要预处理或纯化就能提供广泛的抗菌活性。(The invention discloses a bactericide and application thereof, relates to the industrial field, and is prepared by mixing lantibiotic and a chelating agent. The embodiment of the application has the advantages that the antibacterial peptide composition is designed innovatively, so that the antibacterial peptide composition has obvious antibacterial effect and a unique antibacterial mechanism, the antibacterial activity spectrum of the peptides is widened, and a new prospect of the peptides in the aspect of additive food is developed. The chelating agent sodium gluconate can destabilize the outer membrane of gram-negative bacteria and form pores in the membrane, so that the composition has the activity of resisting gram-negative bacteria. The composition is capable of inducing instability of the bacterial wall and provides broad antimicrobial activity without the need for pretreatment or purification prior to use.)

1. A fungicide, characterized by: is prepared from lantibiotic and chelating agent through mixing.

2. The bactericide as claimed in claim 1, wherein the chelating agent is sodium gluconate.

3. The fungicide of claim 1, wherein said lantibiotic includes nisin, subtilin, epidermin, cinnamyl, duramycin, angiotensin converting enzyme inhibitory peptide.

4. A bactericide as claimed in claim 1, wherein the ratio of concentrations of said lantibiotic and chelating agent is from 1: 1-8: 1.

5. the application of the bactericide is characterized in that: can be used as food antiseptic.

Technical Field

The invention relates to the field of industry, in particular to a bactericide and application thereof.

Background

Lantibiotics are protein toxoids synthesized by the ribosomes of certain bacteria and have antibacterial activity against other classes of bacteria. Nisin is an antibacterial polypeptide, is a food additive with wide application, obvious effect and safety, and is a food preservative approved by European Union (E234 food additive) and FDA. In addition, more than 50 countries apply Nisin to food preservation and food preservation, with nearly 70 years of usage history. Due to the superior stability and efficient mechanisms of bacterial antimicrobial peptides, it is believed that a new generation of antibiotics of natural origin can be designed (Clardy et al (2006) nat. Biotechnol.24: 1541-.

The mechanism of inhibition by antibacterial polypeptides is strongly dependent on interaction with bacterial membranes (Breukink et al (1999) Science 286: 2361-2364). Lantibiotics kill bacteria by several mechanisms that bind to lipid II, affect bacterial cell wall synthesis and inhibit bacterial growth and/or cause the formation of cellular membrane pore structures, leading to leakage of the cell contents, directly leading to cell death (Kramer et al (2008) Microbiology 154: 1755-. Nisin forms a complex with lipid-II wall precursors, which affects the cell wall synthesis process and acts as a docking molecule, forming 225 nm-sized pores containing lipid-II molecules and can remain stable within a few seconds (Hsu et al (2004) nat. struct. mol. biol.11: 963-. The complex mechanism and dual mode of action by which antibacterial peptides exert their activity are the major contributors to their effects on antibiotic-resistant species (Kramer et al (2008) Microbiology 154: 1755-.

The main obstacle in clinical application of antibacterial peptides (represented by Nisin) is their short half-life in blood. In addition, their antimicrobial activity is limited by their narrow antimicrobial spectrum. However, nisin has potent antibacterial activity against a variety of gram-positive bacteria, including staphylococcus aureus, clostridium botulinum, listeria monocytogenes, bacillus cereus, and other gram-positive bacteria. However, nisin does not kill gram-negative bacteria because the plasma membrane of gram-negative bacteria is protected by an outer covering of lipopolysaccharide (Asaduzzaman and Sonomoto (2009) J.biosci.Bioeng.107: 475-87).

The spectrum of antibacterial activity of lantibiotic has been extensively studied. The bacteria are pretreated to destabilize their outer membranes by treatment with short-term, sublethal heat shock, osmotic shock or chelating agents (EDTA, citrate, lactoferrin, etc.). After destabilization of the outer membrane, the outer membrane of gram-negative bacteria is broken down and the bacteria become susceptible (Martin-Visscher et al (2011) FEMS Microbiol. Lett.317: 152-. Another approach is through removal of salt ions, organic milk-derived residues and pH adjustment, since salt ions were observed to decrease the permeability of the outer membrane, thus hindering activity against gram-negative bacterial species (Kuwano et al (2005) Int J antimicrobial Agents 26: 396-402).

Disclosure of Invention

The invention aims to provide a bactericide and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a bactericide is prepared by mixing lantibiotic and chelating agent.

Further, the chelating agent is sodium gluconate.

Further, the lantibiotic includes nisin, subtilin, epidermin, cinnatin, duramycin, angiotensin converting enzyme inhibitory peptide.

Further, the concentration ratio of the lantibiotic to the chelating agent is 1: 1-8: 1.

has the advantages that: the beneficial effects of the embodiment of the application are that: the embodiment of the application has the advantages that the antibacterial peptide composition is designed innovatively, so that the antibacterial peptide composition has obvious antibacterial effect and a unique antibacterial mechanism, the antibacterial activity spectrum of the peptides is widened, and a new prospect of the peptides in the aspect of additive food is developed. The chelating agent sodium gluconate can destabilize the outer membrane of gram-negative bacteria and form pores in the membrane, so that the composition has the activity of resisting gram-negative bacteria. The composition is capable of inducing instability of the bacterial wall and provides broad antimicrobial activity without the need for pretreatment or purification prior to use.

Drawings

FIG. 1 shows the bactericidal effect of nisin and chelating agent sodium gluconate on Escherichia coli.

FIG. 2 shows the bactericidal effect of nisin and the chelating agent sodium gluconate on Klebsiella pneumoniae.

FIG. 3 shows the bactericidal effect of nisin and the chelating agent sodium gluconate on Salmonella typhimurium in the present invention.

FIG. 4 shows the bactericidal effect of nisin and the chelating agent sodium gluconate on Staphylococcus epidermidis in accordance with the present invention.

FIG. 5 shows the bactericidal effect of nisin and the chelating agent sodium gluconate on Staphylococcus aureus in accordance with the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.

Nisin and sodium gluconate serving as a chelating agent are used together, so that the broad bactericidal activity on gram-negative bacteria can be enhanced compared with that of the single use of the Nisin, and the broad bactericidal activity on gram-positive bacteria can also be enhanced.

The present invention provides a composition of Nisin or other lantibiotic bacteriocins in combination with the chelating agent sodium gluconate. The invention also provides for dissolving or suspending the composition in a suitable carrier to enhance the range of application of the antimicrobial agent. The invention has found that the 1:1 and 4:1 Nisin and sodium gluconate composition can well inhibit gram-negative bacteria such as escherichia coli, salmonella typhimurium, klebsiella pneumoniae, staphylococcus epidermidis, staphylococcus aureus and the like, and can be also applied to gram-positive bacteria in an expanded way to enhance the bacteriostatic effect of Nisin. The enhancement effect of the chelating agent sodium gluconate on Nisin activity is related to the concentration of sodium gluconate.

Since gram-positive and gram-negative bacteria are almost always present in food products at the same time, the killing action of Nisin compositions against gram-negative bacteria such as escherichia coli, salmonella typhi, klebsiella pneumoniae, and other gram-negative and gram-positive bacteria is very important. The bactericide is particularly useful for inhibiting the contamination of raw materials, processed foods and beverages with bacterial pathogens and other microorganisms. Potential uses associated with food products include the treatment of meat, particularly poultry, eggs, cheese and fish, and food packaging and equipment processing. Further uses include as food preservatives, such as in the processing of cheese, cream, milk, dairy products, and in the cleaning of poultry, fish, meat, vegetables, dairy products and food processing equipment.

The use of Nisin compositions is not limited to food-related uses, and Nisin compositions should be suitable for use in any situation where there is contamination by gram-negative and gram-positive bacteria. The compositions may be dissolved in a suitable carrier, such as an aqueous solvent or buffer, or suspended in a suitable liquid, colloid, or polymer matrix to produce a bactericidal agent. The composition or antiseptic can be used in ointments or pharmaceutical coatings, such as in the treatment of infections, wound dressings or surgical medical products, and can also be used for skin or oral irrigation, disinfection, or as a disinfectant for wipes and lotions. The bactericide can be used for cleaning medical instruments, sterilizing before operation, etc. Biocides have good utility where environmental disinfection is required, particularly where chemical biocides cannot be used due to the risk of corrosion or other harmful residues. The invention can also be applied as a bactericide in the presence of organic substances such as milk or serum.

Nisin belongs to lanthionine-containing peptide bacteriocins. Lantibiotics also include subtilin, epidermin, cinnamyl, duramycin, angiotensin converting enzyme inhibitory peptide and Pep 5 produced by different microorganisms. Therefore, we believe that, due to the molecular similarity, the combined use of other lantibiotics with sodium gluconate also inhibits both gram-negative and gram-positive bacteria.

Compared with the existing Nisin product on the market, the Nisin and sodium gluconate composition has very obvious effect on gram-negative bacteria as a bactericide. In addition, the Nisin and sodium gluconate composition has the antibacterial effect, and meets the long-term urgent requirements of food preservation science. Food preservation science has been lacking in an acceptable, natural, non-toxic agent that is effective against a variety of bacteria. In order to prove that the composition containing Nisin and sodium gluconate has very obvious inhibition effect on both gram-negative bacteria and gram-positive bacteria, a plurality of experiments are carried out on the composition. These experiments are exemplary only, and the patent is not limited to these examples.

Example 1:

the bactericidal effect of nisin and the chelating agent sodium gluconate on gram-negative bacteria escherichia coli is shown in fig. 1.

The test was carried out under the conditions of 37 ℃ and pH 6.0 with a control, and the initial viable count of Escherichia coli was 10⁵CFU/ml. The effect of the bactericide was determined using the survival rate of bacteria after 24 hours as an index. In the control group without sodium gluconate, the killing effect of nisin on escherichia coli was not significant. However, the treatment group to which sodium gluconate was added exhibited strong activity against E.coli. The activity increased with increasing nisin concentration. The combination of nisin and sodium gluconate as a fungicide shows improved effectiveness in killing escherichia coli. Therefore, a bactericide containing Nisin and the chelating agent sodium gluconate is considered to be a food preservative effective against various gram-negative bacteria.

Example 2:

bactericidal effect of nisin and chelating agent sodium gluconate on gram-negative bacteria Klebsiella pneumoniae (Klebsiella pneumoniae)

At 37 deg.C and pH of 6.0, the initial concentration is 10⁵CFU/ml, evaluated on the composition containing Nisin and sodium gluconate and on the gram-negative bacteria klebsiella pneumoniae kill with Nisin alone (figure 2). In the absence of sodium gluconate, Nisin has no obvious bactericidal effect on klebsiella pneumoniae. However, with the addition of sodium gluconate, the bactericide shows a significant activity against klebsiella pneumoniae. With increasing nisin concentration, the bactericidal activity increases.

Example 3:

bactericidal effect of nisin and chelating agent sodium gluconate on gram-negative bacterium salmonella typhimurium (S. typhimurium)

The killing activity of Nisin and sodium gluconate against the gram-negative bacterium salmonella typhimurium is shown in fig. 3. The Nisin control group without sodium gluconate had no significant activity on the typhimurium. However, when Nisin is used in combination with sodium gluconate, the bactericide exhibits significant activity against typhimurium. The enhancement of nisin by sodium gluconate is concentration dependent.

Example 4:

bactericidal effect of nisin and chelating agent sodium gluconate on gram-positive bacteria Staphylococcus epidermidis

FIG. 4 shows the effect of a fungicide containing streptococcin and sodium gluconate on Staphylococcus epidermidis. The bactericide of the sodium gluconate combined with the streptococcin can obviously improve the bactericidal activity of the streptococcin on staphylococcus epidermidis. The combined use of Nisin and sodium gluconate is indeed effective.

Example 5:

bactericidal effect of nisin and chelating agent sodium gluconate on gram-positive bacteria Staphylococcus aureus (Staphylococcus aureus)

The data in fig. 5 show that Nisin and sodium gluconate have a stronger bactericidal effect on Staphylococcus aureus (Staphylococcus aureus) than Nisin alone. The sterilization of the chelating agent sodium gluconate matched with Nisin needs to be in a certain concentration range.

Example 6:

a bactericide is prepared by mixing lantibiotic and chelating agent. The chelating agent is sodium gluconate. The lantibiotic includes nisin, subtilin, epidermin, cinnamyl element, duramycin and angiotensin converting enzyme inhibitory peptide. The concentration ratio of the lantibiotic to the chelating agent is 1: 1.

Example 7:

a bactericide is prepared by mixing lantibiotic and chelating agent. The chelating agent is sodium gluconate. The lantibiotic includes nisin, subtilin, epidermin, cinnamyl element, duramycin and angiotensin converting enzyme inhibitory peptide. The concentration ratio of the lantibiotic to the chelating agent is 8: 1.

Example 8:

a bactericide is prepared by mixing lantibiotic and chelating agent. The chelating agent is sodium gluconate. The lantibiotic includes nisin, subtilin, epidermin, cinnamyl element, duramycin and angiotensin converting enzyme inhibitory peptide. The concentration ratio of the lantibiotic to the chelating agent is 4: 1.

Example 9:

an application of the bactericide based on example 8 to food preservatives.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.