A kind of bactericide and its preparation method and application

Abstract

The present invention belongs to the technical field of bactericides, and specifically relates to a bactericide and its preparation method and application. The present invention provides a bactericide, including D-amino acid and bactericidal components. By destroying the biofilm by D-amino acid, the bactericidal efficiency of FNA on bacteria is improved, thereby controlling the production of sulfide. In terms of bactericidal effect, more than 90% bactericidal effect is achieved, and the denitrifying bacteria are reduced to NO2 ‑ The utilization of FNA has improved the sustainability of sulfide control; in environmental protection, it has achieved a sterilization effect of more than 80% and a high efficiency in sulfide control with a low concentration of sodium nitrite solution. The dosage of sodium nitrite has been reduced, and the harm of N element to the environment has been reduced; in terms of sulfur control effect, it has produced a high efficiency sterilization effect and inhibited the sulfur production capacity of biofilm, reducing the sulfur production rate of biofilm, and can be used to control the production of sulfide in drainage pipes with thick biofilm, high sulfide production and long HRT.

Description

Technical Field

[0001] The present invention belongs to the technical field of fungicides, and in particular relates to a fungicide and a preparation method and application thereof. Background Art

[0002] Sulfide produced by urban drainage pipes is a common toxic and harmful pollutant. Anaerobic microorganisms in biofilms in sewers have been found to be the main factor causing sulfide production. Therefore, for the problems of sewer odor and corrosion, we believe that it is more effective to prevent the production of sulfide by inhibiting bacterial activity than to reduce it after it is discharged. Adding fungicides to sewers is an effective method. Free nitrite (FNA) is an effective fungicide, which is the protonated form of nitrite and can control sulfide emissions by inhibiting bacterial activity in sewers. However, the biofilm structure in the drainage pipe is complex, and the sulfate-reducing bacteria (SRB) that produce sulfide live in the deeper layers of the biofilm. When the FNA concentration is low, it cannot enter the deep layer of the biofilm to kill SRB. To enhance the bactericidal effect of FNA, it is necessary to increase the FNA concentration and extend the reaction time. In fact, when the FNA concentration is less than 0.1 mg-N / L, there will be a certain degree of bactericidal effect, but its effect is related to the FNA concentration. In order to achieve a deactivation efficiency of more than 90%, it is necessary to add 0.26 mg N / L of FNA (equivalent to 120 mg N-NO2, pH = 6) for 24 hours. However, higher concentrations of nitrite will increase the denitrification load of sewage treatment plants and damage the water ecology of urban water systems. In addition, FNA will be degraded by bacteria in the biofilm and long-term sulfur control cannot be achieved. Therefore, it is crucial to study how to effectively reduce the dosage of fungicides to reduce their harm to the environment.

[0003] Sulfide in sewers is mainly produced by sulfate-reducing bacteria (SRB) in the deeper layers of the biofilm. Biofilm is composed of bacteria and extracellular polymers (EPS), which is a protective mode of microbial growth. Microorganisms live in a closed matrix that is difficult for toxic substances to penetrate, so they can survive in harsh or adverse environments, and through the connection between microorganisms and EPS, the functional and structural integrity of the biofilm is achieved. Therefore, the biofilm provides a safe and stable living environment for SRB. The biofilm in the drainage pipe is highly heterogeneous, with a thickness of more than 1mm, and is composed of dozens or even hundreds of bacteria, making it more difficult to destroy than ordinary biofilms. Summary of the invention

[0004] The first aspect of the present invention aims to provide a composition.

[0005] The second aspect of the present invention is to provide a method for preparing the composition.

[0006] The third aspect of the present invention aims to provide an application of the composition.

[0007] The fourth aspect of the present invention aims to provide a product.

[0008] The fifth aspect of the present invention aims to provide an application.

[0009] The sixth aspect of the present invention aims to provide a sterilization method.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a composition, characterized in that:

[0012] The composition includes D-amino acid and bactericidal components.

[0013] Preferably, the D-amino acid includes at least one of D-tyrosine, D-leucine, D-methionine and D-tryptophan.

[0014] Preferably, the D-amino acids include D-tyrosine, D-leucine, D-methionine, and D-tryptophan.

[0015] Preferably, the final concentration of the D-amino acid is 40 μM to 25 mM.

[0016] Preferably, the final concentration of D-tyrosine is 30-50 μM.

[0017] Preferably, the final concentration of D-leucine is 6-10 mM.

[0018] Preferably, the final concentration of D-methionine is 6-10 mM.

[0019] Preferably, the final concentration of D-tryptophan is 6-10 mM.

[0020] Preferably, the bactericidal component includes at least one of FNA, TTPC, ethylenediamine-N, THPS, chloride bactericide, cyanide bactericide, metal bactericide, antimicrobial peptide, phage, and enzyme.

[0021] Preferably, the bactericidal component comprises FNA.

[0022] Preferably, the FNA is derived from at least one of nitrite and nitrous acid.

[0023] Preferably, the FNA is derived from sodium nitrite.

[0024] Preferably, the final concentration of the FNA is 0.01-0.2 mg N / L.

[0025] Preferably, the molar ratio of D-tyrosine, D-leucine, D-methionine, and D-tryptophan is (3-5):(600-1000):(600:-1000):(600-1000).

[0026] The second aspect of the present invention provides a method for preparing the composition of the first aspect of the present invention, comprising mixing a D-amino acid with a bactericidal component to obtain.

[0027] The third aspect of the present invention provides use of the composition of the first aspect of the present invention in the preparation of a sterilization product.

[0028] The fourth aspect of the present invention provides a product comprising the composition of the first aspect of the present invention and auxiliary materials.

[0029] Preferably, the auxiliary material includes at least one of a desiccant, a pH adjuster, an antioxidant, and a fragrance.

[0030] The fifth aspect of the present invention provides the use of the composition of the first aspect of the present invention and the product of the fourth aspect in a1) to a4):

[0031] a1) Sterilization;

[0032] a2) reduce the content of extracellular polymers in biofilms;

[0033] a3) destroy bacterial biofilm;

[0034] a4) Suppress sulfur emissions.

[0035] Preferably, the bacteria include at least one of sulphur-reducing bacteria and denitrifying bacteria.

[0036] The sixth aspect of the present invention provides a sterilization method, characterized by using the composition of the first aspect of the present invention and / or the product of the fourth aspect of the present invention for treatment.

[0037] The final concentration of the FNA is 0.02-0.06 mg N / L.

[0038] The final concentration of the D-amino acid is 40 μM to 25 mM.

[0039] Preferably, the final concentration of D-tyrosine is 30-50 μM.

[0040] Preferably, the final concentration of D-leucine is 6-10 mM.

[0041] Preferably, the final concentration of D-methionine is 6-10 mM.

[0042] Preferably, the final concentration of D-tryptophan is 6-10 mM.

[0043] The beneficial effects of the present invention are:

[0044] The present invention provides a bactericide, including D-amino acids and bactericidal components. Through the destruction of biofilm by D-amino acids, the bactericidal efficiency of FNA on bacteria is improved, thereby controlling the production of sulfide. In terms of bactericidal effect, a bactericidal effect of more than 80% is achieved, the utilization of NO2- by denitrifying bacteria is reduced, and the sustainability of FNA in controlling sulfide is improved; in environmental protection, a bactericidal effect of more than 80% and an effect of efficiently controlling sulfide are achieved by using a low-concentration sodium nitrite solution. The amount of sodium nitrite used is reduced, and the harm of N element to the environment is reduced; in terms of sulfur control effect, an efficient bactericidal effect is produced, the sulfur production capacity of the biofilm is inhibited, and the sulfur production rate of the biofilm is reduced, and it can be used to control the production of sulfide in drainage pipes with thick biofilms, high sulfide production and long HRT. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic structural diagram of the biofilm preparation reactor of the present invention.

[0046] Figure 2 The effect of different concentrations of FNA on the concentration of total dissolved sulfide.

[0047] Figure 3 The effect of different concentrations of D-amino acid and FNA combination on the sulfurization-reduction ratio.

[0048] Figure 4 The effect of Examples 4-5 and Comparative Examples 1-3 on the total dissolved sulfide concentration within 48 hours after treating the biofilm.

[0049] Figure 5 NO2 within 48 hours after treating the biofilm in Examples 4-5 and Comparative Examples 1-3 - The result of concentration changes.

[0050] Figure 6 The effects of Examples 4-5 and Comparative Examples 1-3 on the sulfur production rate of SRB.

[0051] Figure 7 These are the fluorescence photography results after treating the biofilm in Examples 4-5 and Comparative Examples 1-3.

[0052] Figure 8 The statistical results of biofilm coverage and bacterial mortality after biofilm treatment in Examples 4-5 and Comparative Examples 1-3 are shown.

[0053] Fig. 9 The composition changes of extracellular polymers in biofilms after the biofilms were treated with Examples 4-5 and Comparative Examples 1-3. DETAILED DESCRIPTION

[0054] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0055] Example 1 Sludge acclimatization and biofilm cultivation

[0056] Anaerobic sludge and domestic sewage from the sewer were inoculated in a closed reactor with a plexiglass carrier to simulate the sewer environment for acclimatization and biofilm cultivation. Figure 1 As shown, 32 pieces of 2×2×0.3 cm frosted organic glass sheets can be placed in it. The sludge comes from the secondary sedimentation tank of the sewage treatment plant containing sulfate-reducing bacteria and denitrifying bacteria, the total solid content (TS) of the sludge is 26 g / L, and the inoculation volume is 0.2 L.

[0057] Nutrient solution was added to the reactor. The total organic carbon (TOC) concentration of the nutrient solution was 150 mg C / L. The specific ingredients were: 122 mg / L glucose, 61 mg / L yeast extract, 163 mg / L sodium acetate, 115 mg / L ammonium chloride, 12 mg / L dipotassium hydrogen phosphate, 4.5 mg / L potassium dihydrogen phosphate, 177.5 mg / L sodium sulfate, and 25% (V / V) trace element solution (including FeCl3·6H20: 5 mg / L, H3BO3: 0.5 mg / L, CuSO4·5H20: 0.125 mg / L, KI: 0.2 mg / L, MnSO4·H20: 0.625 mg / L, ZnSO4·7H20: 0.375 mg / L, and CoCl2·6H20: 0.5 mg / L). The solution is pumped to the reactor. The hydraulic retention time (HRT) of the reactor is 6 hours. The pH of the reactor solution is adjusted to 5.9-6.1. When the reactor produces a relatively stable sulfide concentration (35 mgS / L) every day, the acclimation is considered complete. The total acclimation time is 8 months. At this time, the organic glass carrier is attached with a thick biofilm. The carrier containing the biofilm is transferred to the anaerobic bottle for further cultivation. The culture conditions of the anaerobic bottle are consistent with those in the reactor.

[0058] Example 2 FNA concentration screening

[0059] When the sulfur production reached 40 mg S / L after the anaerobic bottle with biofilm added in Example 1 was cultured, different concentrations of sodium nitrite were added to the anaerobic bottle to make the final concentration of FNA 0, 0.01, 0.02, 0.06, 0.1, 0.2 mg N / L. The concentration of total dissolved sulfide was determined using the methylene blue method at different time periods (0, 3, 17, 24, 42, 48 h).

[0060] The methylene blue detection method is as follows: add 20ml of sodium hydroxide to the colorimetric tube, add 50 microliters of the test solution, add deoxygenated deionized water to about 60ml, slowly add 10ml of N,N-dimethyl-p-phenylenediamine solution along the wall of the absorption tube, immediately cover the stopper and slowly invert once. Remove the stopper, slowly add 1ml of ammonium ferric sulfate solution along the wall of the absorption tube, immediately cover the stopper and shake well. After leaving it for 10 minutes, dilute to the mark with deoxygenated deionized water and shake well. Use a 10mm optical path cuvette, use deoxygenated deionized water as a reference, and measure the absorbance at a wavelength of 665nm.

[0061] Filter results such as Figure 2 shown.

[0062] according to Figure 2 As a result, in order to avoid environmental pollution problems caused by excessively high FNA concentrations, 0.02 and 0.06 mg N / mL of FNA were selected for subsequent experiments.

[0063] Example 3D - Amino Acid Concentration Screening

[0064] According to the results in Example 2, 0, 0.02, 0.06 mg N / mL of FNA were mixed with different concentrations of D-amino acid mixtures. The concentrations of the D-amino acid mixtures were grouped into 0, 6, 12, 24 mmoL / L, and were mixed with D-tyrosine, D-leucine, D-methionine and D-tryptophan. Wherein, the total 6 mmoL / L of D-amino acid mixtures included D-tyrosine 10 μM, D-leucine 2 mmoL, D-methionine 2 mmoL and D-tryptophan 2 mmoL; the total 12 mmoL / L of D-amino acid mixtures included D-tyrosine 20 μM, D-leucine 4 mmoL, D-methionine 4 mmoL and D-tryptophan 4 mmoL; the total 24 mmoL / L of D-amino acid mixtures included D-tyrosine 40 μM, D-leucine 8 mmoL, D-methionine 8 mmoL and D-tryptophan 8 mmoL.

[0065] When the sulfur production in the anaerobic bottle with biofilm added in Example 1 reaches 40 mg S / L, the 12 groups of D-amino acid and FNA mixed solutions with different concentrations are added to the anaerobic bottle to detect the sulfur reduction ratio (Sulfide reduction ratio %).

[0066] The sulfide reduction ratio was measured by: after 48 hours of treatment, the sulfide was determined by the methylene blue method in Example 2, and the sulfide generation rate within 48 hours was calculated. The control group was used as a control, and the sulfide generation rate of the other groups was divided by the sulfide generation rate of the control group to obtain the sulfide generation ratio of the ordinate in the figure.

[0067] The results are as follows Figure 3 As shown, the addition of D-amino acids increased the sulfur reduction ratio, among which 12 and 24 mmol / L D-amino acid total concentrations had better effects, and a total D-amino acid concentration of 24 mmol / L was selected for subsequent experiments.

[0068] Embodiment 4 A kind of bactericide

[0069] A bactericide, comprising 7.2 mg D-tyrosine, 1.2 g D-methionine, 1.04 g D-leucine, 1.6 g D-tryptophan, and 56.32 mg sodium nitrite. The dosage is for treating 1 L of liquid to be sterilized, and the final concentration after dissolution is 40 μmol / L D-tyrosine, 8 mmol / L D-leucine, 8 mmol / L D-methionine, 8 mmol / L D-tryptophan, and 1.428 μmol / L sodium nitrite.

[0070] Example 5 A bactericide

[0071] A bactericide, comprising 7.2 mg D-tyrosine, 1.2 g D-methionine, 1.04 g D-leucine, 1.6 g D-tryptophan, and 168.95 mg sodium nitrite. The dosage is for treating 1 L of liquid to be sterilized, and the final concentration after dissolution is 40 μmol / L D-tyrosine, 8 mmol / L D-leucine, 8 mmol / L D-methionine, 8 mmol / L D-tryptophan, and 4.285 μmol / L sodium nitrite.

[0072] Comparative Example 1D - Amino Acid Mixture

[0073] A D-amino acid mixture: containing 7.2 mg D-tyrosine, 1.2 g D-methionine, 1.04 g D-leucine, and 1.6 g D-tryptophan. The amount is used to treat 1 L of liquid to be sterilized. The final concentration after dissolution is 40 μmol / L D-tyrosine, 8 mmol / L D-leucine, 8 mmol / L D-methionine, and 8 mmol / L D-tryptophan.

[0074] Comparative Example 2 A bactericide

[0075] A bactericide comprises 56.32 mg of sodium nitrite, which is used to treat 1 L of liquid to be sterilized. The final concentration after dissolution is 1.428 μmol / L sodium nitrite, namely 0.02 mg N / L FNA.

[0076] Comparative Example 3: A bactericide

[0077] A bactericide comprises 168.95 mg of sodium nitrite, which is used to treat 1 L of liquid to be sterilized. The final concentration after dissolution is 4.285 μmol / L sodium nitrite, namely 0.06 mg N / L FNA.

[0078] Effect Example 1 Long-term Control of Sulfide Concentration by Mixed Bactericides

[0079] When the sulfur production reaches 40 mg S / L after the anaerobic bottle with biofilm added in Example 1 is cultured, the reagents of Examples 4-5 and Comparative Examples 1-3 are added to the anaerobic bottle for treatment. The amount used per 1L of liquid is as shown in Examples 4-5 and Comparative Examples 1-3, and the control group is a simple nutrient solution.

[0080] The concentrations of sulfide and nitrite ions (NO2 - ) concentration. The sulfide detection method is the methylene blue detection method, and the specific process is the same as that of Example 2; NO2 - The detection method is ion chromatography analysis.

[0081] The ion chromatography analysis method is as follows: filter the test solution with a 0.22 μm filter head, fill a 2 ml injection bottle with the test solution, and analyze the nitrite concentration using an ion chromatograph (IC-16, Shimadzu, Japan) equipped with a SI-35 4D column. Calculate the nitrite concentration based on the nitrite standard curve.

[0082] The results of the sulfide concentration experiment are as follows Figure 4 As shown, after the D-amino acid solution and FNA were mixed and added, the sulfide concentration continued to decrease, and after 48 hours, the sulfide concentration was close to 0 mgS / L. It is worth noting that although Comparative Examples 2 and 3 without the addition of D-amino acid can achieve good sulfur control effects within 24 hours, the sulfide concentration increases after 24 hours, indicating that FNA alone cannot achieve a long-term sulfur control effect, and SRB bacteria may resume growth.

[0083] NO2 - The concentration test results are as follows Figure 5 As shown, compared with Comparative Examples 2 and 3, the NO2 - The concentration decrease rate is slower, and the presence of D-amino acids slows down the oxidation of nitrite by SRB, making the effective bactericidal component FNA last longer.

[0084] Furthermore, this example explores the effect of mixed fungicides on the sulfur production rate of SRB. After the biofilm in the anaerobic bottle was treated with the control group (Control, pure nutrient solution) and the experimental group (Comparative Examples 1-3, Examples 4-5) for 48 hours, all were replaced with pure nutrient solution and re-cultured for another 48 hours, and the sulfur production rate of the final biofilm was detected. The experimental results are shown in Figure 6 As shown, the sulfur production rate of the biofilm treated with comparative examples 1-3 increased slightly compared with the control group after re-cultivation for 48 hours. The reason may be that the simple FNA treatment for 48 hours reduced the bacterial density. When the FNA was removed and re-cultivated, the bacterial density decreased, the survival pressure decreased, and the activity increased, and the sulfur production rate increased; and the simple D-amino acid may have a regulatory effect on the growth of bacteria, slightly increasing the sulfur production rate. After the biofilm treated with examples 4-5 was re-cultivated for 48 hours, its sulfur production rate was significantly reduced to 50% compared with the control group, indicating that the bactericide of examples 4-5 has a longer-lasting sulfur control effect.

[0085] Effect Example 2: Effect of mixed bactericide on biofilm removal and sterilization

[0086] When the sulfur production in the anaerobic bottle with biofilm added in Example 1 reaches 40 mg S / L after cultivation, the reagents of Examples 4-5 and Comparative Examples 1-3 are added to the anaerobic bottle for treatment, and the control group is a simple nutrient solution.

[0087] Biofilms were stained using the LIVE / DEADTM BacLightTM Bacterial Viability Kit (ThermoFisher) 48 h after treatment. Cells were double stained with propidium iodide (PI; final concentration: 30 μM) and SYTO 9 (final concentration: 5 μM) and incubated in the dark for 30 min at room temperature. Images were acquired on an Olympus FV3000 confocal laser scanning microscope. Red (PI) fluorescence above 616 nm represents dead bacteria. Green (SYTO 9) fluorescence above 521 nm represents live bacteria. Bacterial coverage (%) and attached cell survival (%) were calculated using ImageJ processing and analysis software. The staining results are shown in the figure. Figure 7 As shown, it can be seen that the biofilms treated in Comparative Examples 1-3 and the control group were dense and had no significant changes, while after treatment in Examples 4-5, the biofilms were significantly eliminated. The statistical results of bacterial coverage are shown in Figure 8 As shown in a, the survival rate of attached cells is Figure 8 As shown in b, after the D-amino acid solution and FNA were mixed and added, the proportion of dead bacteria increased by 62% and the bacterial coverage rate decreased by 75%.

[0088] At the same time, the composition of the extracellular polymer (EPS) of the biofilm was analyzed using fluorescent isothiocyanate (FITC) (green), Nile red (red) and calcofluor white (blue) staining. The staining process was carried out in the dark at room temperature, and the excess dye solution was washed with 0.01M PBS buffer after each staining. The stained slides were observed using a laser confocal scanning microscope (Olympus FV3000). Green (FITC) fluorescence was above 516 nanometers, red (Nile red) fluorescence was above 635 nanometers, and blue (calcofluor white) fluorescence was above 432 nanometers. Ten locations were randomly selected on the slide and photographed. ImageJ image analysis software was used to analyze the quantity and proportion of lipids, proteins and polysaccharides. The statistical results are as follows. Fig. 9 As shown, the presence of D-amino acids reduced EPS by about 85%.

Claims

1. A composition, characterized in that: The composition includes D-amino acids and FNA; The D-amino acids include D-tyrosine, D-leucine, D-methionine and D-tryptophan; The FNA is derived from at least one of nitrite and nitrous acid; The molar ratio of D-tyrosine, D-leucine, D-methionine and D-tryptophan is (3-5): (600-1000): (600-1000): (600-1000); The molar ratio of the FNA to the D-amino acid is (1-5):(40-25000).

2. A method for preparing the composition according to claim 1, comprising mixing D-amino acid with FNA to obtain.

3. Use of the composition according to claim 1 in the preparation of a sterilization product.

4. A product comprising the composition of claim 1 and excipients; The auxiliary material includes at least one of a desiccant, a pH regulator, an antioxidant, and a fragrance.

5. Use of the composition according to claim 1 and / or the product according to claim 4 in suppressing sulfur emissions.

6. A sterilization method, characterized in that: Treatment with the composition according to claim 1 and / or the product according to claim 4; The final concentration of FNA is 0.02-0.06 mg N / L; The final concentration of D-tyrosine is 30-50 μM; The final concentration of D-leucine is 6-10 mM; The final concentration of D-methionine is 6-10 mM; The final concentration of D-tryptophan is 6-10 mM.

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