Detailed Description
The invention relates to a bacteriostatic composition which can achieve the most effective inhibitory action on microorganisms represented by escherichia coli and staphylococcus aureus.
Organic acids used for bacteriostasis include Fumaric acid (Fumaric acid), Succinic acid (Succinic acid), L-Valine (L-Valine), Malic acid (Malic acid), Citric acid (Citric acid), Glycine (Glycine), and Hippuric acid (Hippuric acid). The proportion of the extract is consistent with the content of squama Manis, wherein in the total extract (CV), fumaric acid accounts for 0.1%, succinic acid accounts for 0.4%, L-valine accounts for 1.3%, malic acid accounts for 1.2%, citric acid accounts for 0.2%, glycine accounts for 0.1%, and hippuric acid accounts for 0.3%. Preferably, the above ratio is adjustable and effective.
The microorganisms referred to herein can comprise gram-negative bacteria, gram-positive bacteria, or comprise staphylococcus aureus as a representative of pathogenic staphylococci, coliform bacteria as a representative of conditional pathogenic bacteria or other pathogenic bacteria, such as mycobacterium tuberculosis.
The organic acids used in the experiments and constituting the components of the composition of the present invention are all artificially synthesized organic acids.
By the special content proportion of the composition, the better antibacterial effect is achieved compared with that of a single or few compositions with the same volume.
Example 1
Bacteriostatic tests were carried out based on the Minimum Inhibitory Concentration (MIC) method, and the above-described antibacterial metabolites were used for the detection of Minimum Inhibitory Concentrations (MIC) of Escherichia coli (e.coli; Escherichia coli) and Staphylococcus aureus (s.aureus; Staphylococcus aureus), Pseudomonas aeruginosa (p.aeruginosa; Pseudomonas aeruginosa) and Serratia marcescens (s.processes; Serratia marcocens), respectively.
(1) Experimental materials:
1) culture medium
A lysis Broth (LB; Lysogeny Broth) medium containing 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride was used.
2) Antibacterial drugs:
comprises the whole composition consisting of one or more of fumaric acid, succinic acid, L-valine, malic acid, citric acid, glycine and hippuric acid.
(2) The bacteriostatic test comprises the following steps:
1) preparation of bacterial suspension
And simultaneously recovering the preserved escherichia coli representing gram-negative bacteria and staphylococcus aureus representing gram-positive bacteria. A single colony having a diameter of about 1 mm was picked up on a plate of the test bacterium which had been purified by using an inoculating loop, inoculated into 2mL of LB broth, and cultured in a biochemical incubator at 37 ℃. The enriched logarithmic phase bacterial liquid is adjusted to 0.5 McLeod's turbidimetric standard (i.e. absorbance at 625 nm is 0.08-0.10) with LB broth, and contains about 1 × 108~2×108CFU·mL-1。
2) Preparation of antibacterial medicine
Preparing the combined antibacterial agent with concentration of 4C, 2C, C, 0.5C, 0.25C, 0.125C, 0.0625C, 0.03125C by two-fold dilution method, numbering 8 sterile test tubes in sequence, and adding 1ml sterile water into 2-8 test tubes. Preparing 2ml of 4C antibacterial drug in a first test tube, adding 1ml of solution in the first test tube into a second test tube, uniformly mixing, adding 1ml of solution in the second test tube into a third test tube, and sequentially diluting.
3) MIC assay
200. mu.L of LB broth was added to column 1 of a 96-well plate as a negative control, and 180. mu.L of the prepared bacterial suspension was added to columns 2-10 to a final concentration of 1X 105CFU·mL-1. And 20 mul of sterile water is added to the column 2 to serve as a positive control group, and 20 mul of medicaments are added to the columns 3-10 respectively, wherein the final concentration of the medicaments is 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.003125C. The respective inoculated drug sensitive plates were placed in an incubator at 37 ℃ for 24 hours, and MIC values were measured by observing turbidity of LB broth medium and detecting OD values, and the experiment was repeated 3 times.
The results are shown in Table 1, the minimum inhibitory concentration of each metabolite on Escherichia coli and Staphylococcus aureus is obtained, the concentrated antibacterial metabolites which independently exist are summarized, and malic acid is the best inhibitory effect on Escherichia coli and Staphylococcus aureus.
TABLE 1
In order to verify the optimal effect of malic acid and the composition thereof, the experiment further performed bacteriostatic tests on malic acid and other metabolites as compositions, and the results represented by MIC values are shown in Table 2.
TABLE 2
Experimental results show that the MIC value of the two-component composition added with malic acid to escherichia coli and staphylococcus aureus is reduced, and the two-component composition has a higher inhibition effect on the escherichia coli and the staphylococcus aureus. Still further, random dispensing of the three compositions was performed and the bacteriostatic test was performed as shown in table 3.
TABLE 3
The combination of malic acid and fumaric acid has a weak rebound of bacteriostatic effect when combined with L-valine or citric acid, and similarly, malic acid and L-valine have the same result when combined with glycine or hippuric acid, and have a weak inhibitory effect on Escherichia coli and Staphylococcus aureus, and the MIC value rises to 0.4C. The composition formed by malic acid, hippuric acid and citric acid has optimal inhibition effect on escherichia coli, and the MIC value is 0.1C.
The experiment also carried out bacteriostatic tests on four compositions formed by distributing other metabolites and malic acid by selecting malic acid as a main component, and the results are shown in table 4.
TABLE 4
The results show that the combination of malic acid, fumaric acid, glycine and hippuric acid and the combination of malic acid, citric acid, glycine and hippuric acid are able to develop bacteriostatic effects with MIC as low as 0.1C.
In the experiment, five compositions consisting of malic acid as a main component and other metabolites are distributed to perform bacteriostasis tests, and the results are shown in table 5.
TABLE 5
The results show that malic acid, L-valine, citric acid, glycine, hippuric acid; malic acid, fumaric acid, succinic acid, L-valine, hippuric acid; the three compositions of malic acid, fumaric acid, L-valine, glycine and hippuric acid can have an inhibitory effect on escherichia coli with an MIC value of 0.1C. The composition comprises malic acid, succinic acid, L-valine, glycine and hippuric acid; malic acid, fumaric acid, L-valine, citric acid, hippuric acid; malic acid, fumaric acid, citric acid, glycine, hippuric acid; malic acid, succinic acid, L-valine, citric acid, hippuric acid; any one group of malic acid, succinic acid, citric acid, glycine and hippuric acid can have an inhibitory effect of 0.1C on Escherichia coli and Staphylococcus aureus. Compared with the prior art that a single organic acid or two organic acids are used as additives, the bacteriostatic effect of the bacteriostatic additive is not as good as that of a bacteriostatic additive formed by combining more than four organic acids.
In the experiment, the malic acid is selected as a main component, and other metabolites are distributed and mixed with the malic acid to form six compositions and seven metabolites for bacteriostasis test, and the results are shown in table 6.
TABLE 6
The results show that even if six or more metabolites are mixed, the bacteriostatic effect on staphylococcus aureus and enterobacter coli can reach the MIC value of 0.1C.
Further, the metabolite combinations comprising malic acid, hippuric acid, fumaric acid and glycine were tested for Escherichia coli, Staphylococcus metalens, Pseudomonas aeruginosa and Serratia marcescens, and the results are shown in Table 7:
TABLE 7
The results show that the organic acid metabolite composition consisting of malic acid, hippuric acid, fumaric acid and glycine has an MIC value of 0.1C for Escherichia coli and Staphylococcus aureus and an MIC value of 0.2C for Pseudomonas aeruginosa and Serratia marcescens. The organic acid metabolite composition comprising malic acid, hippuric acid, fumaric acid and glycine has growth inhibiting effect on bacteria represented by Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens, especially pathogenic bacteria.
Serratia marcescens, which is the smallest bacterium, belongs to gram-negative bacilli and is a conditional pathogen.
Pseudomonas aeruginosa is a gram-negative bacterium, and is an opportunistic infectious bacterium.
Escherichia coli, staphylococcus metal, pseudomonas aeruginosa and serratia marcescens are widely distributed in nature, can enter a human body through the positions of the mouth, the nose and the like of the human body, and cause the human body to generate pathogenic reactions such as inflammation and the like under certain conditions or environmental conditions. Therefore, the four strains are selected as test objects of the bacteriostasis test in the experiment.
Based on the experimental results, the composition consisting of malic acid, glycine and hippuric acid can form an effect of inhibiting the bacteria of staphylococcus aureus and escherichia coli to achieve an MIC value of 0.1C together with citric acid or fumaric acid.
The ratio of the composition for experiment can be 0.1% for fumaric acid, 0.4% for succinic acid, 1.3% for L-valine, 1.2% for malic acid, 0.2% for citric acid, 0.1% for glycine and 0.3% for hippuric acid, and the antibacterial composition can be prepared according to the above ratio.
Preferably, the composition for reducing the tendency of microorganisms present on the surface of the human body or in the environment in which the human body is located to multiply comprises: 0.125-0.482% of malic acid, 0.135-0.422% of L-valine, 0.021-0.081% of citric acid, 0.01-0.04% of succinic acid, 0.031-0.121% of hippuric acid, 0.001-0.041% of glycine and 0.01-0.041% of fumaric acid.
Preferably, the ratio of the components in the composition can be: 1 to 30 percent of malic acid, 0.1 to 20 percent of L-valine, 0.1 to 20 percent of citric acid, 0.1 to 20 percent of succinic acid, 0.1 to 20 percent of hippuric acid, 0.1 to 20 percent of glycine and 0.1 to 20 percent of fumaric acid.
Preferably, the ratio of the components in the composition can be: malic acid accounts for 10-40% of the total composition, L-valine accounts for 0.01-10% of the total composition, citric acid accounts for 0.01-10% of the total composition, succinic acid accounts for 0.01-20% of the total composition, hippuric acid accounts for 0.01-20% of the total composition, glycine accounts for 0.01-20% of the total composition, and fumaric acid accounts for 0.01-10% of the total composition.
Preferably, the malic acid can be in the range of about 0.1% to about 20%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20%, 20% to 60% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight, including all ranges and subranges therebetween, in the total composition.
Preferably, the L-valine can range from about 0.01% to about 1%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight, including all ranges and subranges therebetween, in the total composition.
Preferably, the citric acid can range from about 0.01% to about 2%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight, including all ranges and subranges therebetween, in the total composition.
Preferably, the succinic acid can range from about 0.1% to about 20%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight, including all ranges and subranges therebetween, in the total composition.
Preferably, the hippuric acid can range from about 0.1% to about 20%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20%, 20% to 60% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight of the total composition, including all ranges and subranges therebetween.
Preferably, the glycine can range from about 0.1% to about 2%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight, including all ranges and subranges therebetween, in the total composition.
Preferably, the fumaric acid can range from about 0.01% to about 20%, about 1% to about 10%, about 3% to about 8%, about 6% to about 12%, about 15% to about 18%, about 17% to about 20% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40% by weight, including all ranges and subranges therebetween, in the total composition.
Preferably, the combined range of malic acid, L-valine, citric acid, succinic acid, hippuric acid, glycine and fumaric acid can include, but is not limited to, the above-mentioned ratio and combined range.
The composition having an inhibitory effect against escherichia coli with an MIC value of 0.1C is represented by a composition of malic acid, hippuric acid and citric acid, while the composition having an inhibitory effect against escherichia coli and staphylococcus aureus with an MIC value of 0.1C is represented by malic acid, fumaric acid, glycine, hippuric acid; malic acid, citric acid, glycine, hippuric acid. Since all the components forming the composition are weakly acidic components and can be absorbed by the human body as food additives, the above-mentioned multi-group composition can be applied to various fields as components harmless to the human body. In the prior art, the methods and cognition using organic acids are: any organic acid has the bacteriostatic action, and the organic acid can be added into food or other disinfecting liquid contacting with human body in large dose within the legal allowance and has the bacteriostatic action. However, when used in large quantities as an additive, organic acids generate conversion products in the body, a small amount of the organic acids depends on metabolism, but a large amount of the organic acids cannot ensure that all the products can be metabolized out of the body in time, so that the large amount of the organic acids can cause harm to the body of the human body within a certain period of time. According to the invention, the optimal bacteriostatic composition is obtained by carrying out the bacteriostatic proportion on the organic acid which can effectively inhibit bacteria, and the addition amount of the edible organic acid can be reduced by increasing the variety of the edible organic acid, so that the food or other food is helped to form the influence on the health of a human body while the preservative effect is ensured.
Example 2
This embodiment is a further improvement of embodiment 1, and repeated contents are not described again.
Take the Liyan tablet for chronic pharyngitis treatment at present as an example. Aiming at chronic pharyngitis caused by bacterial infection, the sore throat relieving tablet can have a good effect on the disease. The ingredients of the sore throat relieving tablet comprise pangolin (prepared), ground beeltle, stiff silkworm, oyster (calcined) and figwort, and the pangolin (prepared) in the ingredients is a forbidden medicine because the pangolin (prepared) is taken from endangered organisms, so that the sore throat relieving tablet can not be used as a prescription medicine or a non-prescription medicine for treating patients. According to the invention, by researching the inhibition effect on flora in squama Manis, especially on Escherichia coli and Staphylococcus aureus, one or more groups of compositions with effective inhibition effect on Escherichia coli and Staphylococcus aureus are found. The main components of the medicine comprise: one or more of malic acid, hippuric acid, fumaric acid, glycine, succinic acid, L-valine and citric acid. When the pangolin scales of the liyan tablet are replaced, on one hand, the replacement components can adopt biological tissue extracts with similar components, and on the other hand, the replacement components can also be metabolite replacement components which are synthesized by people and effectively inhibit bacteria. Preferably, when an artificially synthesized compound is used as the substitute component, it can be a composition designed in this experiment to have one of the most excellent inhibitory effects on gram-negative bacteria represented by escherichia coli and gram-positive bacteria represented by staphylococcus aureus: malic acid, fumaric acid, succinic acid, L-valine, citric acid and glycine. The addition of the organic acid can bring fruity taste to the tablets, which is popular with children, thereby increasing the enthusiasm of the infants for taking the medicine.
Example 3
This embodiment is a further improvement of embodiment 1 or embodiment 2, and repeated contents are not described again.
The composition can be used in various foods as additive composition for inhibiting Escherichia coli and Staphylococcus aureus in foods. For example, the natural flavor is increased by the proportion of malic acid and citric acid in some natural fruit juice, and the preservative effect is realized by inhibiting the growth of escherichia coli and staphylococcus aureus by matching the fruit juice in vacuum or sealed package. The main components of the additive comprise: one or more of malic acid, hippuric acid, fumaric acid, glycine, succinic acid, L-valine and citric acid. The presence or combination of malic acid and citric acid, respectively, results in an additive having a fruity flavor.
Example 4
This embodiment is a further improvement on embodiment 1, embodiment 2, or embodiment 3, and repeated contents are not repeated.
The composition can also be used for killing bacteria at home or under ordinary living conditions. For example, in the household life, bacteriostatic bacteria maintenance of the environment or bacteriostatic washing of clothes, especially staphylococcus aureus harmful to the human body, is required. The disinfection of alcohol or 84 disinfectant is also harmful to the human body and is not suitable for families with pregnant women or children. The invention can form the liquid disinfectant suitable for families with children or pregnant women based on the composition of the weakly acidic components and other harmless disinfectant components.
The composition of organic acids can be applied as an additive in detergents or bactericides. The composition as additive is used for clothes or environmental bacteriostasis and deodorization. The composition is provided with a first composition comprising a PH modifier and a second composition for increasing the disinfecting effect and time. The first composition comprises any one of two combinations: malic acid, glycine, hippuric acid, citric acid; malic acid, glycine, hippuric acid and fumaric acid. The second composition comprises a hydrocolloid.
Hydrocolloids, which contain polysaccharides and proteins, form a gel network. When the gel network wraps escherichia coli or staphylococcus aureus carried by a target applied by detergent or bactericide, the first composition existing in the hydrocolloid enters the thallus from the thallus based on the pressure of the gel network on the thallus, the first composition and the second composition act together to form organic acid with synergistic effect, and H is released inside the thallus+Thus, the alkaline environment inside the cells is destroyed, and the activity of the cells is inhibited. The first composition is introduced into the hydrocolloid, and acts on the inner part of the thallus of escherichia coli or staphylococcus aureus along with the coating of the hydrocolloid on escherichia coli or staphylococcus aureus carried by a target for applying the detergent or the bactericide.
The molecular diameter of the organic acid used to act on the intracellular environment of the bacterium can be smaller than the diameter of the cell membrane channel of escherichia coli and staphylococcus aureus so that the organic acid molecule can enter the interior of the bacterium cell from the cell membrane channel and destroy the alkaline environment of the interior of the bacterium cell to inhibit the activity of the bacterium cell.
The joint inhibition effect of the escherichia coli and the staphylococcus aureus is realized through the synergistic effect of a first composition consisting of malic acid, glycine and hippuric acid and a second composition consisting of citric acid or fumaric acid.
Example 5
This embodiment is a further improvement on embodiment 1, embodiment 2, embodiment 3, or embodiment 4, and repeated contents are not repeated.
The present invention contemplates a composition for spreading onto a target surface and killing and inhibiting escherichia coli and staphylococcus aureus on the target surface by spraying, atomizing or otherwise forming a small particle gas or liquid.
The composition consists of combined liquid or gas particles. The liquid or gas particles comprise a particle core consisting of organic acid with high bacteriostasis effect and an outer coating film which is covered on the particle core and consists of a zirconium oxide-tin oxide complex with a nano-grade inorganic micropore form neutralized by a silane coupling agent and is used for protecting and increasing the adhesion capability.
By dispersing the zirconium oxide-tin oxide complex in the form of nano-scale inorganic micropores neutralized by the silane coupling agent wrapping the organic acid in the manner of aerosol, aerosol or small-particle liquid in clothes and living environment, when the zirconium oxide-tin oxide complex in the form of nano-scale inorganic micropores neutralized by the silane coupling agent wrapping the organic acid touches escherichia coli and staphylococcus aureus on the target surface, the outer membrane releases organic acid under pressure and allows the organic acid to enter the bacteria body through the cell membrane on the bacteria surface, thereby inhibiting the bacterial activity. The nano-scale inorganic micropores formed by the zirconia-tin oxide composite can allow organic acid molecules to enter and exit and limit the obstruction of the decomposition and attachment of the particles by large-volume particles. The zirconia-tin oxide composite can effectively maintain the hardness of the outer coating film and the toughness of preventing the collapse of nano-micropores when the outer coating film of particles existing in the form of aerosol, liquid or gas is formed.
Preferably, the ratio of the silane coupling agent to the nano-inorganic microporous zirconia-tin oxide composite is 1:40 to 1: 120. The ratio of the nano-inorganic microporous zirconium oxide-tin oxide composite to the organic acid is 1:1 to 1: 10.
Microparticles composed of organism-inorganic bodies coupled with a silane coupling agent can move toward a target surface in a state of a diameter of at least less than 10 μm. The above microparticles have excellent dispersion stability in an aqueous phase for dilution or homogeneous liquid phase, a high adhesion rate of the organic acid carried on the target surface, and a high volatility of the organic acid core reduced by the coating of the outer membrane. The organic-inorganic hybrid membrane is characterized in that an intermediate phase which is low in cost and can stably couple an internal organic layer and an external inorganic layer is utilized, the inorganic layer which is coupled with the organic layer and does not generate other chemical reactions is added under the condition of not damaging the dissociation of hydrogen ions of the organic layer, and the problems of poor adhesion, no effect and volatility of the organic layer on a target surface are solved through the property of the inorganic layer. Preferably, the silane coupling agent coupling can be an aqueous silane coupling agent coupling.
The preparation method of the composition comprises the following steps:
(1) weighing: weighing the silane coupling agent coupling and zirconia-tin oxide composite (which can be present in the form of a sol) in proportion by weight, and weighing the organic acid composition in terms of volume;
(2) mixing: putting the zirconium oxide and tin oxide complex into organic acid in batches, uniformly mixing, slowly pouring a silane coupling agent along with stirring action for coupling, and stirring until three phases are uniform to obtain the concentrated composition.
The concentrated composition can be diluted with water before use, and sprayed to the target surface to be sterilized such as environment or clothes by using equipment such as an aerosol sprayer and a spray can.
The organic acid in the particle core can be combined with the organic acid which is related in the embodiment and can achieve the bacteriostasis effect on escherichia coli and staphylococcus aureus and the MIC of which is 0.1C. The organic acid composition wrapped by the outer involucra has good stability in water and has high-efficiency inhibition effect on escherichia coli and staphylococcus aureus.
Example 6
This embodiment is a further improvement on embodiment 1, embodiment 2, embodiment 3, embodiment 4, or embodiment 5, and repeated contents are not repeated.
Aiming at the organic acid composition which can be permanently attached to the surface and acts on bacteria, the invention can design a bacteriostatic ointment or spray with one or more of malic acid, hippuric acid, fumaric acid, glycine, succinic acid, L-valine and citric acid, and the bacteriostatic ointment or spray can act on the skin surface or mucous membrane of the face, the arms, important organs and the like. The organic acid composition has obvious antibacterial effect on the multiplication of various pathogenic skin bacteria such as gram-negative bacteria and gram-positive bacteria represented by escherichia coli and staphylococcus aureus.
Preferably, the antibacterial ointment comprises traditional Chinese medicine ointment, traditional Chinese medicine powder, mussel mucin and an organic acid composition containing one or more of malic acid, hippuric acid, fumaric acid, glycine, succinic acid, L-valine and citric acid.
Preferably, the bacteriostatic spray comprises pentanediol, 1, 3-propanediol and an organic acid composition containing one or more of malic acid, hippuric acid, fumaric acid, glycine, succinic acid, L-valine and citric acid.
It should be noted that the above-mentioned embodiments are exemplary, and that those skilled in the art, having benefit of the present disclosure, may devise various arrangements that are within the scope of the present disclosure and that fall within the scope of the invention. It is to be understood by persons skilled in the art that the present description is illustrative and not restrictive of the claims. The scope of the invention is defined by the claims and their equivalents. The present description contains several inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", each indicating that the respective paragraph discloses a separate concept, the applicant reserves the right to submit divisional applications according to each inventive concept. Throughout this document, the features referred to as "preferably" are only an optional feature and should not be understood as necessarily requiring that such applicant reserves the right to disclaim or delete the associated preferred feature at any time.