Natural alcohol amino-acid ester antibacterial agent and preparation method thereof

Abstract

The invention relates to the technical field of bacteriostatic and antibacterial materials, in particular to a natural alcohol amino-acid ester antibacterial agent and a preparation method thereof.The preparation method comprises the steps that natural alcohol and five amino acids with protecting groups serve as raw materials, N-Boc-natural alcohol amino-acid ester is synthesized through an esterification reaction, then the Boc protecting groups are removed through concentrated hydrochloric acid, alkali washing is conducted through a NaOH solution, and the natural alcohol amino-acid ester antibacterial agent is obtained. Finally, ten natural alcohol amino-acid ester antibacterial agents are obtained. The natural alcohol amino-acid ester antibacterial agent prepared by the preparation method disclosed by the invention has good biocompatibility and hydrophobicity, and shows excellent broad-spectrum antibacterial activity on gram-negative bacteria and gram-positive bacteria. The antibacterial agent is simple to synthesize and low in cost. Amino acid is used as a raw material, biocompatibility is good, pollution to the environment is avoided, and the method can be widely applied to the fields of medical treatment, food, textile detection and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial and antimicrobial materials, and particularly to a natural alcohol amino acid ester antibacterial agent and a preparation method thereof. Background Art

[0002] At present, the extensive use and even abuse of traditional antibiotics have led to an increasingly serious problem of bacterial drug resistance, which has become a major challenge in the global public health field. According to statistics, the number of people who die from drug-resistant bacterial infections globally each year exceeds 700,000, and it is estimated that this figure may exceed 10 million by 2050. Traditional antibiotics such as sodium penicillin and levofloxacin have relatively single action targets, and bacteria can easily develop drug resistance through gene mutation or gene transfer. Moreover, about 60% of clinical infections are related to biofilms, which are composed of extracellular polysaccharides secreted by bacteria and form a physical barrier, seriously hindering the penetration of antibiotics and greatly reducing the therapeutic effect of traditional antibiotics on biofilm infections. Therefore, it is urgent to develop new antibacterial agents with anti-biofilm properties and low drug resistance.

[0003] Amino acids, as naturally occurring compounds, are widely used due to their low cost, easy availability, and environmental friendliness. Antimicrobial peptides (AMPs) are a class of polypeptide substances with antibacterial activity produced by organisms. They act by destroying the cell membranes of bacteria and have advantages such as broad-spectrum antibacterial and low drug resistance. However, antimicrobial peptides have some defects in practical applications, such as being easily degraded by proteases, having relatively high in vivo toxicity, and high production costs. The antibacterial activity of antimicrobial peptides is closely related to their cationicity, amphiphilicity, and hydrophobicity. Based on this, researchers have turned their attention to the design of antimicrobial peptide mimetics. It has been found that the activity of antimicrobial peptides is closely related to their positive charge, amphiphilicity, and hydrophobicity. Inspired by the design idea of antimicrobial peptide mimetics, attempts have been made to synthesize natural alcohol amino acid ester derivatives using amino acids as raw materials. Therefore, in view of the above current situation, it is urgent to develop a natural alcohol amino acid ester antibacterial agent and a preparation method thereof to overcome the deficiencies in current practical applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a natural alcohol amino acid ester antibacterial agent and a preparation method thereof to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A natural alcohol amino acid ester antibacterial agent, the structure of the antibacterial agent is shown in formula (I):

[0007]

[0008] Among them, R is selected from the amino acid side chains of L-phenylalanine, L-tryptophan, D-tryptophan, L-tyrosine or L-histidine, and R1 represents a natural alcohol, including one of L-menthol and borneol.

[0009] As a further solution of the present invention: the antibacterial agent is one of the following compounds: L-phenylalanine menthol ester, L-phenylalanine borneol ester, L-tyrosine menthol ester, L-tyrosine borneol ester, L-tryptophan menthol ester, L-tryptophan borneol ester, D-tryptophan menthol ester, D-tryptophan borneol ester, L-histidine menthol ester and L-histidine borneol ester.

[0010] A preparation method of the natural alcohol amino acid ester antibacterial agent according to the above, comprising the following steps:

[0011] (1) Esterify N-Boc-amino acid and natural alcohol in toluene solvent under the catalysis of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine;

[0012] (2) After the reaction is completed, spin-dry the solvent, extract with ethyl acetate and saturated NaCl solution, retain the organic phase, dry and filter, and add silica gel powder to the filtrate and spin-dry for standby;

[0013] (3) Purify the crude product by silica gel column chromatography, the eluent is a mixed solution of petroleum ether and ethyl acetate, collect the product spot solution and spin-dry;

[0014] (4) Add ethyl acetate and concentrated hydrochloric acid to remove the protecting group, and stir at room temperature for reaction;

[0015] (5) After the reaction solution in step (4) is washed with alkali and dried, the natural alcohol amino acid ester antibacterial agent is obtained.

[0016] As a further solution of the present invention: in step (1), the molar ratio of N-Boc-amino acid to natural alcohol is 1:1.2; the amount of N,N'-dicyclohexylcarbodiimide is 1.2 equivalents of the molar amount of N-Boc-amino acid; the amount of 4-dimethylaminopyridine is 0.1 equivalent of the molar amount of N-Boc-amino acid.

[0017] As a further solution of the present invention: Add N-Boc-amino acid, natural alcohol, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to a dry reaction flask in sequence, evacuate and then introduce nitrogen, add toluene, stir and heat to 115 °C for reflux reaction for 24 hours.

[0018] As a further solution of the present invention: in steps (2) and (3), anhydrous magnesium sulfate is used for drying, and diatomite filtration is adopted, and 100-200 mesh silica gel powder is added to the filtered solution and spin-dried for standby;

[0019] The volume ratio of petroleum ether to ethyl acetate in the eluent is 3:1.

[0020] As a further scheme of the present invention: in step (4), the volume ratio of ethyl acetate to concentrated hydrochloric acid is 20:1, and the stirring reaction time at room temperature is 24 hours. After the reaction is completed, a product in the form of hydrochloride is obtained.

[0021] As a further scheme of the present invention: in step (5), the organic phase is washed with 5% NaOH solution and deionized water until neutral during alkali washing; anhydrous magnesium sulfate is used for drying.

[0022] An application of the natural alcohol amino acid ester antibacterial agent according to the above in the preparation of antibacterial drugs, wherein the antibacterial drugs are used to inhibit Gram-positive bacteria or Gram-negative bacteria.

[0023] As a further scheme of the present invention: the Gram-positive bacteria include Staphylococcus aureus, Bacillus subtilis or Enterococcus faecalis, and the Gram-negative bacteria include Escherichia coli or Pseudomonas aeruginosa.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. Using natural amino acids and natural alcohols as raw materials, a novel antibacterial agent is synthesized, which has the characteristics of simple synthesis and low cost;

[0026] 2. Amino acids are natural biomolecules. Compared with traditional synthetic organic antibacterial agents and metal antibacterial agents, they are biodegradable and environmentally friendly, and are rare green antibacterial agents. Description of the Drawings

[0027] Figure 1 It is a schematic diagram of the nuclear magnetic resonance carbon spectrum of L-histidine bornyl ester (L-His-Bor) in the embodiment of the present invention;

[0028] Among them, deuterated dimethyl sulfoxide: DMSO-d6, 25 °C, 126 MHz.

[0029] Figure 2 It is the antibacterial circle of the natural alcohol amino acid ester antibacterial agent against Escherichia coli in the embodiment of the present invention.

[0030] Figure 3 It is the antibacterial circle of the natural alcohol amino acid ester antibacterial agent against Staphylococcus aureus in the embodiment of the present invention.

[0031] Figure 4 It is a schematic diagram of the antibacterial circle diameter size of each compound against (a) E. coli and (b) S. aureus in the embodiment of the present invention.

[0032] Figure 5Schematic diagram of the preparation of CS / CMC-based films in an embodiment of the present invention.

[0033] Figure 6 The following are actual pictures of mangoes packaged with different films according to the embodiments of the present invention.

[0034] Figure 7 Schematic diagram of the effect of different films on the weight loss rate of mango in an embodiment of the present invention.

[0035] Figure 8 Schematic diagram of the effect of different films on the shelf life of mangoes in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0038] See also Figures 1-3 , a natural alcohol amino acid ester antibacterial agent provided by an embodiment of the present invention, the structure of the antibacterial agent is shown in formula (I):

[0039]

[0040] Wherein, R is selected from the amino acid side chain of L-phenylalanine, L-tryptophan, D-tryptophan, L-tyrosine or L-histidine, and R1 represents a natural alcohol, including one of L-menthol and borneol.

[0041] The antibacterial agent is one of the following compounds: L-phenylalanine menthol ester, L-phenylalanine bornyl ester, L-tyrosine menthol ester, L-tyrosine bornyl ester, L-tryptophan menthol ester, L-tryptophan bornyl ester, D-tryptophan menthol ester, D-tryptophan bornyl ester, L-histidine menthol ester and L-histidine bornyl ester.

[0042] A method for preparing the natural alcohol amino acid ester antibacterial agent according to the above-mentioned method comprises the following steps:

[0043] (1) Using N-Boc-amino acid and natural alcohol as raw materials, N,N'-dicyclohexylcarbodiimide (DCC) as a dehydration condensation reagent, and 4-dimethylaminopyridine (DMAP) as a catalyst, an esterification reaction is carried out in toluene solvent;

[0044] (2) After the reaction is completed, filter off the by-product DCU (dicyclohexylurea) of DCC, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, retain the organic phase, dehydrate with anhydrous magnesium sulfate and then filter. Add a little silica gel to the filtrate, rotary evaporate the ethyl acetate, and then place it in an oven at 60 °C for drying for 12 h;

[0045] (3) Purify the crude product by silica gel column chromatography. The eluent is a mixed solution of petroleum ether and ethyl acetate. Collect the product spot solution and rotary evaporate it to dryness;

[0046] (4) After dissolving the product in ethyl acetate, add concentrated hydrochloric acid to remove the protecting group, and stir and react at room temperature;

[0047] (5) After the solution obtained in step (4) is washed with alkali and dried, a natural alcohol amino acid ester antibacterial agent is obtained.

[0048] In step (1), the molar ratio of the N-Boc-amino acid to the natural alcohol in the feed is 1:1.2; the amount of N,N'-dicyclohexylcarbodiimide used is 1.2 equivalents of the molar amount of the N-Boc-amino acid; the amount of 4-dimethylaminopyridine used is 0.1 equivalent of the molar amount of the N-Boc-amino acid.

[0049] The raw material contents used in step (1) are N-Boc-amino acid (10 mmol, 1.00 equiv), natural alcohol (12 mmol, 1.20 equiv), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol, 1.20 equiv) and 4-dimethylaminopyridine (DMAP, 1 mmol, 0.10 equiv).

[0050] Add N-Boc-amino acid, natural alcohol, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to a dry reaction flask in sequence. After evacuating, introduce nitrogen, add toluene, stir and heat to 115 °C and reflux for 24 hours.

[0051] In steps (2) and (3), anhydrous magnesium sulfate is used for drying, and diatomaceous earth filtration is adopted. Add 100 - 200 mesh silica gel powder to the filtered solution and rotary evaporate it for standby;

[0052] The volume ratio of petroleum ether to ethyl acetate in the eluent is 3:1.

[0053] In step (3), the specific specification of the silica gel is 100 - 200 mesh. The specific operation process of the silica gel column chromatography method is as follows:

[0054] 1. Mix the silica gel with the eluent evenly and then pour it into the column. Tap the column to make the silica gel compact and flat. Finally, the height of the silica gel in the column is about 15 cm;

[0055] 2. Pour the dried sample silica gel mixture in step (2) into the column and pat it flat.

[0056] 3. When the liquid level of the eluent in the column is about 1 - 2 cm higher than the sample silica gel mixture, add a little quartz sand to press down the sample silica gel mixture.

[0057] 4. After adding the eluent, turn on the switch of the column and control the flow rate to be 1 - 2 drops per second.

[0058] In step (4), the volume ratio of ethyl acetate to concentrated hydrochloric acid is 20:1, the stirring reaction time at room temperature is 24 hours, and the product in the form of hydrochloride is obtained after the reaction.

[0059] In step (5), the basic washing uses 5% NaOH solution and deionized water to wash the organic phase to neutrality; the drying uses anhydrous magnesium sulfate.

[0060] An application of the natural alcohol amino acid ester antibacterial agent according to the above in the preparation of antibacterial drugs, wherein the antibacterial drugs are used to inhibit Gram - positive bacteria or Gram - negative bacteria.

[0061] The Gram - positive bacteria include Staphylococcus aureus, Bacillus subtilis or Enterococcus faecalis; the Gram - negative bacteria include Escherichia coli or Pseudomonas aeruginosa.

[0062] The present invention uses natural alcohols (L - menthol and borneol) and N - Boc - amino acids (including N - Boc - L - phenylalanine, N - Boc - L - tryptophan, N - Boc - D - tryptophan, N - Boc - L - tyrosine and N - Boc - L - histidine) as raw materials, DCC as a dehydrating agent and condensing agent, and DMAP as a catalyst through an esterification reaction to synthesize N - Boc - natural alcohol amino acid esters. Then, the Boc protecting group is removed with concentrated hydrochloric acid, and then washed with NaOH solution. Finally, 10 natural alcohol amino acid esters are obtained. The reaction can simply and quickly obtain the target product. The obtained natural alcohol amino acid ester compound antibacterial agent shows good antibacterial effects against Gram - positive bacteria including Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis, and Gram - negative bacteria including Escherichia coli and Pseudomonas aeruginosa, and has broad - spectrum antibacterial properties and anti - biofilm activity. The antibacterial agent is simple to synthesize, low in cost, and can be widely applied in fields such as medicine, food and textiles, and has broad application prospects.

[0063] Example 1:

[0064] Step 1: Add N-tert-butoxycarbonyl-L-phenylalanine (10 mmol), L-menthol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under N2 atmosphere, and stir and heat to 115 °C followed by refluxing for 24 h. After the reaction is completed, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomaceous earth, and add silica gel powder of 100 - 200 mesh to the filtered solution and rotary evaporate for standby.

[0065] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution and rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0066] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained after rotary evaporation to obtain L-phenylalanine menthol ester (L-Phe-Men).

[0067] L-Phe-Men: 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 174.47 (g, -CH-C=O-, 1C),

[0068] 137.85 (d, -C-CH2-, 1C), 129.27 (b, -CH-CH-CH-, 2C), 128.02 (c, -CH-C-CH-, 2C), 126.20 (a, -CH-CH-CH-, 1C), 73.23 (h, -O-CH-, 1C), 55.68 (f, -CH2-CH-NH2, 1C), 46.33 (n, -O-CH-CH-, 1C), 40.98 (i, -O-CH-CH2-, 1C), 33.66 (e, Ph-CH2, 1C), 30.74 (l, -CH2-CH2-, 1C), 25.51 (j, -CH-CH3, 1C), 22.85 (o, -CH-(CH3)2, 1C), 22.12 (m, -CH2-CH2-, 1C), 21.85 (p, -CH-(CH3)2, 2C), 20.56 (k, -CH-CH3, 1C).

[0069] Example 2:

[0070] Step 1: Add N-tert-butoxycarbonyl-L-phenylalanine (10 mmol), borneol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under a N2 atmosphere, stir and heat to 115 °C, and reflux for 24 h. After the reaction is completed, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomaceous earth, and add 100 - 200 mesh silica gel powder to the filtered solution and rotary evaporate for standby.

[0071] Step 2: Purify the crude product by silica gel column chromatography. The eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution, rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0072] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained by rotary evaporation to obtain L-phenylalanine bornyl ester (L-Phe-Bor).

[0073] L-Phe-Bor: 13 13C NMR (126 MHz, DMSO-d6): δ (ppm) = 175.24 (g, -CH-C=O-, 1C),

[0074] 138.03 (d, -CH-C-CH-, 1C), 129.30 (b, -CH-CH-CH-, 2C), 128.28 (c, -CH-C-CH-, 2C), 126.40 (a, -CH-CH-CH-, 1C), 79.13 (h, -O-CH-, 1C), 56.15 (f, -CH-NH2, 1C), 48.47 - 47.56 (o, k, -C-C-, 2C), 44.38 (j, -CH2-CH-CH2-, 1C), 36.27 (e, i, -Ph-CH2-, -O-CH-CH2-, 2C), 26.82 (m, n, -CH2-CH2-, 2C), 19.61 (l, -C-(CH3)2, 2C), 12.27 (p, -C-CH3, 1C).

[0075] Example 3:

[0076] Step 1: Add N-Boc-L-tyrosine (10 mmol), L-menthol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under a N2 atmosphere, stir and heat to 115 °C, and reflux for 24 h. After the reaction is completed, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomaceous earth, and add 100 - 200 mesh silica gel powder to the filtered solution and rotary evaporate for standby.

[0077] Step 2: Purify the crude product by silica gel column chromatography. The eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the solution of the product spot, rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0078] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained by rotary evaporation to obtain L-tyrosine menthol ester (L-Tyr-Men).

[0079] L-Tyr-Men: 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 169.61 (g, -CH-C=O, 1C),

[0080] 155.72 (a, -C-OH, 1C), 129.94 (c, -CH-C-CH-, 2C), 128.49 (d, -CH-C-CH-, 1C), 114.65 (b, -CH-C-CH-, 2C), 72.99 (h, -O-CH-, 1C), 55.70 (f, -CH-NH2, 1C), 49.39 (n, -CH-CH-, 1C), 44.87 (i, -O-CH-CH2-, 1C), 34.20 (e, Ph-CH2-, 1C), 30.87~30.09 (j, l, -CH-CH2-, 2C), 25.35 (o, -CH-(CH3)2, 1C), 24.62 (m, -CH2-CH2-, 1C), 20.80~20.36 (k, p, -CH-CH3, -CH-(CH3)2, 3C).

[0081] Example 4:

[0082] Step 1: Add N-tert-butoxycarbonyl-L-tyrosine (10 mmol), borneol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under the condition of N2 purging, and stir and heat to 115 °C and reflux for 24 h. After the reaction is completed, spin-dry the solvent with a rotary evaporator, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomite, and add the filtered solution to silica gel powder of 100 - 200 mesh and spin-dry for standby.

[0083] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution and spin-dry it, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0084] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and spin-dry the solvent. Finally, dry the viscous liquid obtained by spin-drying to obtain L-tyrosine bornyl ester (L-Tyr-Bor).

[0085] L-Tyr-Bor: 13 C NMR (126 MHz, CDCl3-d6): δ (ppm) = 172.14 (g, -CH-C=O, 1C),

[0086] 149.43 (a, -C-OH, 1C), 131.33 (c, -CH-C-CH, 2C), 128.91 (d, -CH-C-CH, 1C), 116.74 (b, -CH-C-CH-, 2C), 80.21 (h, -O-CH-, 1C), 65.71 (f, -CH-NH2, 1C), 49.16 (k, -C-CH3, 1C), 47.93 (n, -C-(CH3)2, 1C), 45.05 (j, -CH2-CH-CH2-, 1C), 39.40 (i, -O-CH-CH2-, 1C), 37.25 (e, Ph-CH2-, 1C), 28.27 (l, -CH2-CH2-, 1C), 26.90 (m, -CH2-CH2-, 1C), 19.47 (p, -C-(CH3)2, 2C), 13.36 (o, -C-CH3, 1C).

[0087] Example 5:

[0088] Step 1: Add N-Boc-L-tryptophan (10 mmol), L-menthol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under N2 atmosphere, and stir and heat to 115 °C followed by refluxing for 24 h. After the reaction is completed, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomaceous earth, and add 100 - 200 mesh silica gel powder to the filtered solution and rotary evaporate for standby.

[0089] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution and rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0090] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained by rotary evaporation to obtain L-tryptophan menthol ester (L-Trp-Men).

[0091] L-Trp-Men: 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 174.87 (k, -CH-C=O-, 1C),

[0092] 136.07 (f, -C-C-, 1C), 127.54 (h, -CH-C-NH-, 1C), 123.58 (e, -C-C-, 1C), 120.66 (b, -CH-CH-, 1C), 118.24 (a, c, -CH-CH-, 2C), 111.08 (d, -CH-C-, 1C), 110.31 (g, -CH-C-NH-, 1C), 73.05 (l, -O-CH-, 1C), 55.08 (j, -CH-NH2, 1C), 49.43 (n, -CH-CH, 1C), 46.24 (m, -O-CH-CH2-, 1C), 34.06 (r, i, -CH2-CH2-,-CH2-CH-, 2C), 30.41 (s, -CH-CH3, 1C), 25.08 (o, -CH-(CH3)2, 1C), 22.21 (q, -CH2-CH2-, 1C), 20.20 (p, t, -CH-(CH3)2, -CH-CH3, 3C)

[0093] Example 6:

[0094] Step 1: Add N-Boc-L-tryptophan (10 mmol), borneol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) into a 100 mL dry reaction flask in sequence. Then evacuate the reaction flask, and subsequently add toluene under the condition of N2 bubbling, and stir and heat to 115 °C followed by refluxing for 24 h. After the reaction is completed, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomite, and add the filtered solution to silica gel powder of 100 - 200 mesh and rotary evaporate for standby.

[0095] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution and rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0096] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained by rotary evaporation to obtain L-tryptophan bornyl ester (L-Trp-Bor).

[0097] L-Trp-Bor: 13 13C NMR (126 MHz, CDCl3-d): δ (ppm) = 175.79 (k, -CH-C=O-, 1C), 136.66

[0098] (f, -C-C-, 1C), 127.54 (h, -C-NH-, 1C), 123.26, 122.21 (b, e, -CH-CH-C-C-, 2C), 119.29 - 118.56 (a, d, -CH-CH-, 2C), 111.40 (c, -CH-CH-, 1C), 99.36 (g, -CH-C-NH-, 1C), 80.94 (l, -O-CH-, 1C), 55.54 (j, -CH-NH2, 1C), 49.16 (s, -C-CH3, 1C), 47.61 (o, -C-(CH3)2, 1C), 45.19 (n, -CH2-CH-CH2-, 1C), 36.53 (m, -O-CH-CH2-, 1C), 31.19 (i, -C-CH2-CH-, 1C), 28.27 (r, -CH2-CH2-, 1C), 27.36 (q, -CH2-CH2-, 1C), 19.29 (p, -C-(CH3)2, 2C), 13.68 (t, -C-CH3, 1C).

[0099] Example 7:

[0100] Step 1: Add N-Boc-D-tryptophan (10 mmol), L-menthol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) into a 100 mL dry reaction flask in sequence. Then evacuate the reaction flask, and subsequently add toluene under N2 atmosphere, stir and heat to 115 °C, then reflux for 24 h. After the reaction, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomaceous earth, and add 100 - 200 mesh silica gel powder to the filtered solution and rotary evaporate for standby.

[0101] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution, rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0102] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained by rotary evaporation to obtain D-tryptophan menthol ester (D-Trp-Men).

[0103] D-Trp-Men: 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 174.69 (k, -CH-C=O-, 1C),

[0104] 136.34 (e, -C-C-, 1C), 127.36 (h, -CH-C-NH-, 1C), 123.26 (f, -C-C-, 1C), 121.11 (a, -CH-CH-, 1C), 118.10 (b, c, -CH-CH-, 2C), 111.54 (d, -CH-CH-, 1C), 110.17 (g, -CH-C-NH-, 1C), 72.87 (l, -O-CH-CH-, 1C), 55.68 (j, -CH-NH2, 1C), 46.10 (r, -CH-CH-, 1C), 40.90 (m, -O-CH-CH2-, 1C), 31.19 (p, i, n, -CH2-CH-NH2, -CH2-CH2-CH-, 3C), 25.26 (s, -CH-(CH3)2, 1C), 21.89 (q, t, -CH2-CH2-, -CH-(CH3)2, 3C), 20.66 (o, -CH-CH3, 1C).

[0105] Example 8:

[0106] Step 1: Add N-Boc-D-tryptophan (10 mmol), borneol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under a N2 atmosphere, and stir and heat to 115 °C followed by refluxing for 24 h. After the reaction is completed, rotary evaporate the solvent, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomaceous earth, and add 100 - 200 mesh silica gel powder to the filtered solution and rotary evaporate for standby.

[0107] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution, rotary evaporate to dryness, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0108] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and rotary evaporate the solvent. Finally, dry the viscous liquid obtained by rotary evaporation to obtain D-tryptophan bornyl ester (D-Trp-Bor).

[0109] D-Trp-Bor: 13 C NMR (126 MHz, CDCl3-d): δ (ppm) = 175.65 (k, -CH-C=O-, 1C),

[0110] 136.34 (f, -C-C-, 1C), 127.27 (h, -C-NH-, 1C), 123.07, 122.27 (b, e, -CH-CH-CH-C-, 2C), 119.61~118.86 (a, d, -CH-CH-, 2C), 111.38 (c, -CH-CH-, 1C), 99.84 (g, -CH-C-NH-, 1C), 80.77 (l, -O-CH-CH2-, 1C), 55.34 (j, -CH-NH2, 1C), 48.94 (q, -C-CH3, 1C), 47.72 (s, -C-(CH3)2, 1C), 45.06 (n, -CH2-CH-CH2-, 1C), 36.79 (m, -CH2-CH-CH2-, 1C), 31.50 (i, -CH2-CH-NH2, 1C), 28.19 (p, -C-CH2-, 1C), 27.25 (o, -CH2-CH2-, 1C), 20.10~18.84 (t, -C-(CH3)2, 2C), 13.56 (r, -C-CH3, 1C).

[0111] Example 9:

[0112] Step 1: N-Boc-L-histidine (10 mmol), L-menthol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) were successively added to a 100 mL dry reaction flask. Then the reaction flask was evacuated, and then toluene was added under N2 atmosphere, and the mixture was stirred and heated to 115 °C and refluxed for 24 h. After the reaction, the solvent was evaporated by a rotary evaporator, and the mixture was extracted with ethyl acetate and saturated NaCl solution, and the organic phase was retained. The organic phase was dried with anhydrous magnesium sulfate, filtered through diatomaceous earth, and the filtered solution was added with silica gel powder of 100-200 mesh and evaporated to dryness for standby.

[0113] Step 2: The crude product was purified by silica gel column chromatography, and the eluent was a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). The product spot solution was collected and evaporated to dryness, and EtAc and concentrated hydrochloric acid were added for deprotection reaction, and the reaction was stirred at room temperature for 24 h.

[0114] Step 3: The organic phase was washed with 5% NaOH solution and deionized water until neutral, and then the organic phase was dried with anhydrous magnesium sulfate and the solvent was evaporated. Finally, the viscous liquid obtained after evaporation was dried to obtain L-histidine menthol ester (L-His-Men).

[0115] L-His-Men: 13 C NMR (126 MHz, CDCl3-d): δ (ppm) = 170.55 (f, -CH-C=O-, 1C),

[0116] 74.03 (g, -O-CH-, 1C), 49.75 (e, -CH-NH2, 1C), 46.55 (m, -CH-CH-, 1C), 40.66 (h, -CH2-CH-, 1C), 33.76 (k, -CH-CH2-, 1C), 31.03 (d,i, -CH2-CH-NH2, -CH-CH3, 2C), 25.96 (n, -CH-(CH3)2, 1C), 23.23 (l, -CH2-CH2-, 1C), 22.27~20.50 (j,o, -CH-CH3, -CH-(CH3)2, 3C).

[0117] Example 10:

[0118] Step 1: Add N-Boc-L-histidine (10 mmol), borneol (12 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol), and 4-dimethylaminopyridine (DMAP, 1 mmol) successively into a 100 mL dry reaction flask. Then evacuate the reaction flask, and subsequently add toluene under N2 atmosphere, stir and heat to 115 °C, and reflux for 24 h. After the reaction, spin-dry the solvent with a rotary evaporator, extract with ethyl acetate and saturated NaCl solution, and retain the organic phase. Dry the organic phase with anhydrous magnesium sulfate, filter through diatomite, and add 100 - 200 mesh silica gel powder to the filtered solution and spin-dry for standby.

[0119] Step 2: Purify the crude product by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether (PE) and ethyl acetate (EtAc). Collect the product spot solution, spin-dry, add EtAc and concentrated hydrochloric acid for deprotection reaction, and stir at room temperature for 24 h.

[0120] Step 3: Wash the organic phase with 5% NaOH solution and deionized water until neutral, then dry the organic phase with anhydrous magnesium sulfate and spin-dry the solvent. Finally, dry the viscous liquid obtained by spin-drying to obtain L-histidine bornyl ester (L-His-Bor).

[0121] L-His-Bor: 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 170.59 (f, -CH-C=O-, 1C),

[0122] 78.52 (g, -O-CH-, 1C), 48.38 (e, -CH-NH2, 1C), 47.33 (l, n, -C-CH3, -C-(CH3)2, 2C), 43.96 (i, -CH-CH2-, 1C), 35.89 (h, -CH-CH2-, 1C), 27.82 (d, -CH2-CH-NH2, 1C), 26.45 (k, j, -CH2-CH2-, 2C), 18.70, 13.36 (o, m, -C-(CH3)2, -C-CH3, 3C).

[0123] Application Example 1: Test of inhibition zone diameter (diffusion method with drug sensitivity test paper):

[0124] (1) Preparation of bacterial suspension:

[0125] Pour the solid medium after high-temperature sterilization into a disposable petri dish (90 mm in diameter). After the solid medium has cooled and solidified, use an inoculation loop to pick up a small amount of the purchased strain and streak it on the agar surface. Then, place the streaked petri dish upside down in an incubator at 37°C. After 12 h, use an inoculation loop to pick a single bacterial colony from the petri dish and transfer it to a conical flask containing 100 mL of liquid medium. Place it in a shaker and shake it (37°C, 120 rpm). After culturing for 4 - 6 h, take it out and store it in a refrigerator at 4°C for later use.

[0126] (2) Testing the diameter of the inhibition zone by the drug sensitivity test paper diffusion method:

[0127] Absorb 100 μL of the bacterial solution and drop it into a disposable petri dish. Then, pour 10 mL of the solid medium into the petri dish and gently shake the petri dish to evenly disperse the bacterial solution in the medium. When the solid medium has fully solidified, place a drug sensitivity test paper (6 mm in diameter) in the middle of the petri dish and gently press it with forceps to make it completely adhere to the agar medium. Use a pipette to take 20 μL of the natural alcohol amino acid ester solution with a concentration of 5000 μg / mL (the solvent is DMSO) and drop it in the middle of the drug sensitivity test paper. After static diffusion, place it upside down in a biochemical incubator at 37°C and culture for 12 h. After the culture is completed, the diameter of the transparent circular area observed on the agar surface is the diameter of the inhibition zone. Use a vernier caliper to measure it and take a photo to record the results of the inhibition zone.

[0128] The results are as Figure 4 shown. It can be intuitively seen from the figure that for Escherichia coli, L-Trp-Men has the largest inhibition zone diameter, with a diameter of 17.92 ± 0.10 mm. For Staphylococcus aureus, L-Trp-Bor and D-Trp-Bor have similar antibacterial effects, and the inhibition zone diameters are 21.20 ± 0.28 mm and 22.40 ± 0.01 mm respectively. The natural alcohol amino acid esters with larger inhibition zone diameters not only have excellent antibacterial activity but also have good permeability, which is very beneficial for the absorption of antibacterial drugs in the human body. Note: Figure 4 Error bars were set according to experimental errors.

[0129] Application Example 2: Minimum inhibitory concentration test (TTC method):

[0130] First, prepare a liquid medium containing 0.02% TTC color developer. Add 100 μL of the liquid medium containing TTC to each well of a 96-well plate (8 rows and 12 columns). Then, add 100 μL of a natural alcohol amino acid ester solution with a concentration of 5000 μg / mL (the solvent is DMSO) to the first column. After mixing evenly, perform two-fold dilutions in sequence. Finally, add 100 μL of the diluted bacterial solution to each well. The concentrations of the antibacterial agents in the 12 wells of each row are 1250 μg / mL, 625 μg / mL, 312.5 μg / mL,..., 2.44 μg / mL, 1.22 μg / mL, and 0.61 μg / mL from left to right. Finally, place the well plate in a biochemical incubator at 37 °C and incubate for 24 h, and observe the color change in the well plate. Among them, the group without antibacterial agent and the group without bacteria are used as the positive control group and the negative control group, respectively. The concentration of the antibacterial agent corresponding to the well that does not show red at the rightmost side of the well plate is the MIC value, and all experiments are carried out with 3 parallel tests. The experimental results are shown in Table 1.

[0131] Observing the MIC values in the table, it can be found that the prepared natural alcohol amino acid ester antibacterial agent has good broad-spectrum antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, and may be a substitute for traditional antibiotics in the future. It is worth noting that the compounds L-Trp-Men and L-Trp-Bor derived from L-tryptophan and two natural alcohols show the strongest broad-spectrum antibacterial efficacy, and their MIC values against the five experimental strains are all less than 10 μg / mL. The MIC values of L-Trp-Men against S. aureus, B. subtilis, E. faecalis, E. coli, and P. aeruginosa are 1.22 μg / mL, 0.61 μg / mL, 4.88 μg / mL, 4.88 μg / mL, and 2.44 μg / mL, respectively. The MIC values of L-Trp-Bor are 2.44 μg / mL, 0.61 μg / mL, 9.75 μg / mL, 4.88 μg / mL, and 9.75 μg / mL, respectively. In comparison, the antibacterial efficacy of L-Trp-Men is slightly higher than that of L-Trp-Bor.

[0132] Note: This experiment was carried out in a 96-well plate, and three parallel experiments were conducted for each antibacterial agent at the same time. Since the MIC values were all consistent, there was no standard error. The experimental results are as Figure 4 shown in Table 1.

[0133] Table 1 MIC values of natural alcohol amino acid ester compounds against Gram-positive bacteria and Gram-negative bacteria

[0134]

[0135] Application Example 3: Fruit preservation:

[0136] (1) The two antibacterial agents with the best antibacterial effects, L-Trp-Men and L-Trp-Bor, were added as antibacterial active ingredients to the film-forming matrix of corn starch (CS) and sodium carboxymethyl cellulose (CMC). At the same time, the natural antioxidant curcumin (Cur) was added to prepare a fully biobased and biodegradable CS / CMC-based antibacterial and antioxidant film. The preparation process of the CS / CMC-based film is as Figure 5 shown: First, 2.5 g of CS and 2.5 g of CMC were weighed and mixed with 100 mL of deionized water, and stirred at 80 °C for 2 h. Subsequently, the two obtained solutions were mixed together, and 2 g of glycerol was added. The mixture was continuously stirred at 80 °C for 2 h. After the mixture cooled, the bioactive ingredients pre-dissolved in ethanol were added at room temperature and stirred for 1 h. The bioactive ingredients included L-Trp-Men, L-Trp-Bor, and curcumin (Cur), and the mass fraction of each was 2% (calculated based on the total mass of CS and CMC). After 30 minutes of ultrasonic degassing treatment, 20 mL of the obtained film-forming solution was poured into a petri dish with a diameter of 90 mm and dried at 45 °C for 24 h. According to the added active ingredients, a total of 3 different films were prepared, including: (1) CS / CMC (without active ingredients); (2) CS / CMC / Men2 / Cur2 (containing 2% L-Trp-Men and Cur); (3) CS / CMC / Bor2 / Cur2 (containing 2% L-Trp-Bor and Cur).

[0137] (2) In this experiment, mangoes were used as the research object to evaluate the application effect of CS / CMC-based films in fruit preservation. In the experiment, CS / CMC-based films and common polyethylene (PE) films on the market were used to package mangoes, and the unpackaged mangoes were used as the control group. The effects of different films on the preservation effect of mangoes were evaluated by observing the changes in fruit appearance. The experimental results are as Figure 6 shown.

[0138] As can be seen from the figure, the two antibacterial and antioxidant films CS / CMC / Men2 / Cur2 and CS / CMC / Bor2 / Cur2 prepared by us can significantly improve the preservation time of mangoes compared with commercial polyethylene (PE) food wrap.

[0139] (3) The effects of different films on the weight loss rate of mangoes were measured. Compared with the unpackaged control group and polyethylene (PE) films, all CS / CMC-based films could significantly inhibit the water loss of mangoes and showed good water retention performance. Among them, the water retention effects of CS / CMC / Men2 / Cur2 and CS / CMC / Bor2 / Cur2 films were the most prominent, effectively slowing down the water evaporation of mangoes during storage. The experimental results are as Figure 7 shown.

[0140] (4) Determine the effects of different films on the shelf life of mangoes. The results show that all CS / CMC-based films can significantly extend the storage period of mangoes and delay their spoilage rate. In particular, the CS / CMC / Bor2 / Cur2 film has the most significant preservation effect, which can provide a longer shelf life for mangoes and effectively maintain the freshness of the fruits. The experimental results are as Figure 8 shown.

[0141] It should be noted that in the present invention, it should be understood that although this specification is described according to the embodiments, not each embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A natural alcohol amino acid ester antibacterial agent, characterized in that, The structure of the antibacterial agent is shown in formula (I): Wherein, R is selected from the amino acid side chains of L-phenylalanine, L-tryptophan, D-tryptophan, L-tyrosine or L-histidine, and R1 represents a natural alcohol, including one of L-menthol and borneol.

2. The natural alcohol amino acid ester antibacterial agent according to claim 1, wherein The antibacterial agent is one of the following compounds: L-phenylalanine menthol ester, L-phenylalanine borneol ester, L-tyrosine menthol ester, L-tyrosine borneol ester, L-tryptophan menthol ester, L-tryptophan borneol ester, D-tryptophan menthol ester, D-tryptophan borneol ester, L-histidine menthol ester and L-histidine borneol ester.

3. A preparation method of the natural alcohol amino acid ester antibacterial agent according to claim 1 or 2, characterized in that, It includes the following steps: (1) Esterification reaction is carried out between N-Boc-amino acid and natural alcohol in toluene solvent under the catalysis of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine; (2) After the reaction is completed, the solvent is evaporated to dryness, extracted with ethyl acetate and saturated NaCl solution, the organic phase is retained, dried and filtered, and silica gel powder is added to the filtrate and evaporated to dryness for standby; (3) The crude product is purified by silica gel column chromatography, and the eluent is a mixed solution of petroleum ether and ethyl acetate, and the product spot solution is collected and evaporated to dryness; (4) Ethyl acetate and concentrated hydrochloric acid are added to deprotect the group, and the reaction is stirred at room temperature; (5) After the reaction solution in step (4) is washed with alkali and dried, a natural alcohol amino acid ester antibacterial agent is obtained.

4. The preparation method of the natural alcohol amino acid ester antibacterial agent according to claim 3, characterized in that, In step (1), the molar ratio of the N-Boc-amino acid to the natural alcohol is 1:1.2; the dosage of N,N'-dicyclohexylcarbodiimide is 1.2 equivalents of the molar amount of N-Boc-amino acid; the dosage of 4-dimethylaminopyridine is 0.1 equivalent of the molar amount of N-Boc-amino acid.

5. The preparation method of the natural alcohol amino acid ester antibacterial agent according to claim 4, characterized in that, The N-Boc-amino acid, natural alcohol, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are successively added to a dry reaction flask, evacuated and then nitrogen is introduced, toluene is added, and the mixture is stirred and heated to reflux at 115 °C for 24 hours.

6. The preparation method of the natural alcohol amino acid ester antibacterial agent according to claim 3, characterized in that In steps (2) and (3), anhydrous magnesium sulfate is used for drying, and diatomite filtration is adopted. Silica gel powder of 100-200 mesh is added to the filtered solution and evaporated to dryness for standby; The volume ratio of petroleum ether to ethyl acetate in the eluent is 3:

1.

7. The preparation method of the natural alcohol amino acid ester antibacterial agent according to claim 3, characterized in that, In step (4), the volume ratio of ethyl acetate to concentrated hydrochloric acid is 20:1, and the reaction is stirred at room temperature for 24 hours. After the reaction is completed, the product in the form of hydrochloride is obtained.

8. The preparation method of the natural alcohol amino acid ester antibacterial agent according to claim 3, characterized in that, In step (5), the organic phase is washed with 5% NaOH solution and deionized water until neutral during alkali washing; anhydrous magnesium sulfate is used for drying.

9. Use of the natural alcohol amino acid ester antibacterial agent according to claim 1 or 2 in the preparation of antibacterial drugs, characterized in that, The antibacterial drug is used to inhibit Gram-positive bacteria or Gram-negative bacteria.

10. Use of the natural alcohol amino acid ester antibacterial agent according to claim 9, characterized in that, The Gram-positive bacteria include Staphylococcus aureus, Bacillus subtilis or Enterococcus faecalis, and the Gram-negative bacteria include Escherichia coli or Pseudomonas aeruginosa.

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