阅读说明：本技术 一种高效清除细菌生物被膜的方法 (Method for efficiently removing bacterial biofilm ) 是由 赵勇 童金蓉 黄振华 赵晟 张昭寰 韩乔 于 2021-01-15 设计创作，主要内容包括：本发明公开了一种高效清除细菌生物被膜的方法,属于微生物领域。该方法采用二槽隔膜电解水装置制得酸性电解水,用其迅速处理生物被膜不低于5min,再用含0.5g/100mL NaS-2O-3的PBS缓冲液中和不低于5min,其中生物被膜为预形成、包含有被膜菌且经无菌磷酸缓冲溶液PBS洗涤去除未附着菌的生物被膜。本发明利用酸性电解水具有低pH值、高氧化还原电位和一定的有效氯等特性以达到破坏生物被膜和清除被膜菌的双重功效,高效且安全。(The invention discloses a method for efficiently removing bacterial biofilm, belonging to the field of microorganisms. The method adopts a two-tank diaphragm water electrolysis device to prepare acidic electrolyzed water, the acidic electrolyzed water is used for rapidly treating the biofilm for not less than 5min, and then 0.5g/100mL NaS is contained 2 O 3 And (4) neutralizing with PBS buffer solution for not less than 5min, wherein the biofilm is pre-formed and contains enveloped bacteria, and washing with sterile phosphate buffer solution PBS to remove the biofilm of non-attached bacteria. The invention utilizes the characteristics of low pH value, high oxidation-reduction potential, certain available chlorine and the like of the acidic electrolyzed water to achieve the dual effects of destroying the biofilm and removing the tunica mucosa bacteria, and is efficient and safe.)

1. A method for efficiently removing bacterial biofilm is characterized in that acidic electrolyzed water is firstly used for treating the biofilm for not less than 5min, and then 0.5g/100mL NaS is contained₂O₃The neutralization time of the PBS buffer solution is not less than 5 min; the biofilm is a preformed biofilm which contains enveloped bacteria and is washed by sterile phosphate buffer solution PBS to remove non-attached bacteria.

2. The method for removing bacterial biofilms with high efficiency according to claim 1, wherein the acidic electrolyzed water is prepared by a two-tank diaphragm electrolyzed water device.

3. The method for removing bacterial biofilm according to claim 2, wherein the acidic electrolyzed water is used for rapidly treating the biofilm for 5 min.

4. The method for efficiently removing a bacterial biofilm according to claim 1, wherein said bacteria are selected from the group consisting of escherichia coli, vibrio parahaemolyticus, and listeria monocytogenes.

5. A method for removing bacterial biofilm according to claim 1, wherein said PBS buffer is neutralized for 5 min.

Technical Field

The invention belongs to the field of microorganisms, and relates to a method for efficiently removing a bacterial biofilm.

Background

Food-borne pathogenic bacteria are the leading cause of food-borne diseases. In the food processing environment, common food-borne pathogenic bacteria such as escherichia coli, listeria monocytogenes, vibrio parahaemolyticus and the like which cause more food pollution are adhered to food, processing contact surfaces and non-food processing contact surfaces (such as walls, sewers, dead corners and the like) under certain conditions to form biofilms. Biofilm refers to a membrane which is formed by bacteria attached to the surface of a contact object to secrete polymers such as polysaccharide and protein and wrap the bacteria in the membrane, and most of the bacteria exist in a form of biofilm attached to the surface of an animate and inanimate object in the natural environment, and the life style can help the bacteria to resist various adverse factors in the environment such as peracid or alkalescence. However, the biofilm bacteria are hundreds or even thousands of times resistant to disinfectants than floating bacteria, and due to their difficult removal characteristics, even after severe washing and disinfection procedures in food processing, biofilms can linger on food contact surfaces and compromise food safety. It is estimated that about 65% of human bacterial infections are caused by biofilms, which form in the food industry at the culprit of causing food-borne diseases.

The extracellular polysaccharide plays an important role in forming a biofilm and resisting external pressure, can prevent antibiotics, disinfectants and the like from contacting with bacteria wrapped in the biofilm so as to play a role in shielding, weakens the killing effect of the bacteria on the biofilm, not only increases the resistance of the bacterial biofilm to the external pressure, but also can protect the integrity of the biofilm structure. Therefore, the control of the pollution of various pathogenic bacteria in food, particularly the prevention of biofilm cross-contamination of food by destroying extracellular polysaccharide is important content for controlling the occurrence of food-borne diseases. Common methods for controlling the biofilm comprise high-pressure spray treatment, nitrous acid, hydrogen peroxide, hypochlorous acid and the like, the methods have certain effect of destroying the biofilm, and chlorine dioxide and citric acid/methylene blue/p-hydroxybenzoic acid can effectively kill the included tunica bacteria, but the methods do not simultaneously have the dual effects of destroying the extracellular polysaccharide of the biofilm and removing the tunica bacteria. The acidic electrolyzed water is taken as an environment-friendly bactericide, has the characteristics of low pH value, high oxidation-reduction potential, certain available chlorine and the like, and mainly changes the cell membrane potential of microorganisms by interfering the balance of a membrane, so that the cell permeability is enhanced, the bacteria swell and the cell metabolic enzyme are damaged, and the intracellular substances overflow and dissolve, thereby achieving the effect of killing the microorganisms. The acidic electrolyzed water is widely applied to sterilization and disinfection in the industries of medical treatment, food processing, environmental sanitation and the like, and has the characteristics of strong sterilization capacity, wide spectrum, rapidness and the like, but the removal effect of the acidic electrolyzed water on the biofilm of the food-borne pathogenic bacteria is rarely reported.

Disclosure of Invention

The invention aims to provide a method for efficiently removing a bacterial biofilm, which has the double functions of destroying bacteria, particularly the extracellular polysaccharide of the biofilm of food-borne pathogenic bacteria and removing the biofilm bacteria.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a method for efficiently removing bacterial biofilm comprises treating biofilm with acidic electrolyzed water for not less than 5min, and adding 0.5g/100mL NaS₂O₃The neutralization time of the PBS buffer solution is not less than 5 min; the biofilm is a preformed biofilm which contains enveloped bacteria and is washed by sterile phosphate buffer solution PBS to remove non-attached bacteria.

Preferably, the acidic electrolyzed water is prepared by a two-tank diaphragm water electrolysis device.

Preferably, the bacteria are selected from escherichia coli, vibrio parahaemolyticus or listeria monocytogenes.

More preferably, the bacterium is selected from escherichia coli ATCC 25404, vibrio parahaemolyticus S36, or listeria monocytogenes WaX 12.

Preferably, the acidic electrolyzed water rapidly treats the biofilm for 5 min.

Preferably, the PBS buffer is neutralized for 5 min.

Compared with the prior art, the invention has the beneficial effects that: the acidic electrolyzed water has the characteristics of low pH value, high oxidation-reduction potential, certain available chlorine and the like, is an environment-friendly, cheap and easily-obtained biochemical preparation, can remove bacterial biofilm, and has the double effects of destroying the biofilm and removing tunica mucosa bacteria in a short time.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention to its proper form. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:

FIG. 1 is a scanning electron microscope observation of the change of biofilm of Escherichia coli (A), Vibrio parahaemolyticus (B) and Listeria monocytogenes (C) after untreated (a), treated with sterile water (B) and treated with acidic electrolyzed water (C).

FIG. 2 shows the effect of acidic electrolyzed water on exopolysaccharides of Escherichia coli, Vibrio parahaemolyticus and Listeria monocytogenes.

Detailed Description

The technical solution of the present invention is further specifically described below with reference to specific examples and drawings. It should be understood that the following specific examples are illustrative only and are not limiting upon the present invention.

Example 1

The following steps are taken as examples to remove biofilms of escherichia coli ATCC 25404, vibrio parahaemolyticus S36 and listeria monocytogenes WaX12 by acidic electrolyzed water respectively:

(1) biofilm formation: escherichia coli ATCC 25404, Vibrio parahaemolyticus S36 and Listeria monocytogenes WaX12 were streaked on LB, TCBS and PALCAM medium, respectively, and left to stand at 37 ℃ for overnight culture. Picking single colony to 5mL liquid culture medium, shake culturing at 37 deg.C and 180r/min for 7-8h to OD_600nmAbout 0.6. The volume ratio of the bacterial suspension to the culture medium is 1: 99 (inoculate 10. mu.L of bacterial suspension in990 μ L of medium) was added to a 24-well cell culture plate at 1 mL/well and cultured for 48h to form a biofilm.

(2) Preparation of acidic electrolyzed water and treatment of biofilm: the acid electrolyzed water is prepared by adopting a two-tank diaphragm water electrolysis device and adjusting parameters such as voltage, current, water electrolysis time, electrolyte mass concentration and the like. Because the available chlorine in the obtained acidic electrolyzed water is easy to lose, the biofilm can be quickly treated after preparation. Washing the formed biofilm 3 times with sterile Phosphate Buffered Saline (PBS) to remove non-attached cells; treating with sterile water and acidic electrolyzed water for 5min, and adding NaS 0.5g/100mL₂O₃Was neutralized for 5min in PBS buffer.

Performance testing

(1) Scanning electron microscope for observing biofilm

The treated biofilm was fixed with 4% glutaraldehyde solution for 2 h. After fixation, gradient dehydration is carried out by using 30%, 50%, 70%, 90% and 100% absolute ethyl alcohol respectively (30%, 50%, 70% and 90% absolute ethyl alcohol are dehydrated for one time in sequence, 10min is carried out each time, 100% absolute ethyl alcohol is dehydrated for 2 times), and the mixture is dried overnight at room temperature and observed under a scanning electron microscope.

(2) Determination of biofilm live bacterial count

Wiping the culture hole with sterile cotton ball for several times under aseptic condition, transferring the cotton ball into test tube containing 10mL of 0.85% NaCl solution, placing the test tube into ultrasonic instrument, and performing 50Hz ultrasonic treatment for 15min to disperse the bacterial colony on the cotton ball in the solution. After gradient dilution with 0.85% NaCl solution, the cells were cultured at 37 ℃ for 24h on the corresponding plates and counted, and the viable cell clearance (%) (number of untreated viable cells-number of treated viable cells)/number of untreated viable cells × 100 was expressed as Log CFU/mL.

(3) MTT method for measuring activity of cells in biological envelope

Adding 1mL of fresh liquid culture medium and 100 μ L of pre-prepared MTT (tetramethylazozolium) dye solution with final concentration of 5mg/mL into the treated biofilm, incubating at 37 deg.C in dark for 1h, adding 1mL of dimethyl sulfoxide into a fume hood to dissolve and precipitate for 30min, and detecting OD with a microplate reader_570nmThe decrease rate of cell activity (OD)_570nm(untreated) -OD_570nm(after treatment))/OD_570nm(untreated).

(4) Determination of viable count in raffinate

The treated residue was collected and plate counted by gradient dilution with 0.85% NaCl solution, the results are expressed as Log CFU/mL.

(5) Determination of extracellular polysaccharides

After the biofilm culture is finished, the OD of the bacterial liquid is measured by an enzyme-linked immunosorbent assay_595nmLight absorption value of (a). The medium in the wells was then carefully discarded and washed 3 times with sterile PBS buffer to remove floating bacteria that had not yet formed a biofilm. Add 1mL0.01M potassium chloride solution heavy suspension for 5min, then each sample hole ultrasound 5s, gap 5s, cycle 5 times. After the ultrasonic treatment is finished, transferring the bacterial liquid into a 1.5mL sterile centrifugal tube, and centrifuging for 20min at the rotation speed of 4000g/min at the temperature of 4 ℃. The supernatant was then filtered through a 0.22 μm diameter filter to remove impurities. Determination of exopolysaccharides: draw 100. mu.L of the filtrate into a 1.5mL sterile centrifuge tube, add 200. mu.L of 98% concentrated sulfuric acid, and stand at room temperature for 30 min. Subsequently, 25. mu.L of a 6% phenol solution was added and incubated for 5min in a metal bath at 90 ℃. Transferring 200 μ L of sample into 96-well microporous plate, and detecting OD with enzyme-labeling instrument_490nmThe value of light absorption of (D) and the calculated ratio OD_570nm/OD_595nmThe relative content of the polysaccharide in the sample is obtained.

The data results are all expressed in x ± s, and the data obtained were analyzed using SPSS17.0 software.

Analysis of results

(1) Influence of acidic electrolyzed water on morphology of biological coating

The change of the bacterial biofilm before and after treatment is observed by using a scanning electron microscope, and the result shows that the acidic electrolyzed water has a certain effect of removing the biofilm of escherichia coli, vibrio parahaemolyticus and listeria monocytogenes. FIG. 1(A-a, B-a, C-a) shows the biofilm morphological structures of Escherichia coli, Vibrio parahaemolyticus and Listeria monocytogenes, all of which are coated with extracellular polymer in high order, and the enveloped bacteria are aggregated and connected in a network shape. However, after the acidic electrolyzed water is treated (as shown in figures 1A-C, B-C and C-C), biofilm bacteria are obviously reduced, and the morphological structure of the biofilm is destroyed, which shows that the acidic electrolyzed water has obvious effect on removing the biofilm. However, after the treatment with sterile water (as shown in FIGS. 1A-B, B-B, C-B), the morphological structure of the biofilm has not changed significantly, and the tunica bacteria are still wrapped in the extracellular polymeric substance.

(2) Effect of acidic electrolyzed Water on enveloped bacteria

After the acidic electrolyzed water is treated, the viable count and the cell activity in the biofilm of the three food-borne pathogenic bacteria are both obviously reduced (the table 1 shows the removal rate of the viable count and the cell activity reduction rate in the biofilm after the acidic electrolyzed water and the sterile water are treated and the viable count in the residual liquid after the treatment), but the treatment of the sterile water has no obvious influence. After the escherichia coli biofilm is treated by acidic electrolyzed water, the viable count is reduced by 66.65%, and the cell activity is reduced by 81.15%; after the vibrio parahaemolyticus is treated by the acidic electrolyzed water, the viable count is reduced by 51.69 percent, and the cell activity is reduced by 77.41 percent; after the listeria monocytogenes is treated by acidic electrolyzed water, the viable count is reduced by 81.52 percent, and the cell activity is respectively reduced by 89.28 percent.

In addition, after the food-borne pathogenic bacteria biofilm is treated by acidic electrolyzed water, the number of viable bacteria in residual liquid is obviously lower than a detection value; after the sterile water treatment, a large amount of viable bacteria still exist in the residual liquid. Compared with the sterile water treatment, the residual liquid after the acid electrolyzed water treatment can not cause secondary pollution to the environment, and can be used as a green and safe biofilm removing method.

TABLE 1

(3) Effect of acidic electrolyzed Water on exopolysaccharides

After the acidic electrolyzed water treatment, the extracellular polysaccharide of the three food-borne pathogenic bacteria is obviously reduced, and the content (OD) of the extracellular polysaccharide of the escherichia coli is reduced_490nm/OD_595nm) From 2.1 to 1.4, the reduction is 33.33 percentThe vibrio parahaemolyticus is reduced by 41.37% from 2.9 to 1.7, and the listeria monocytogenes is reduced by 53.07% from 2.6 to 1.22, while the exopolysaccharide content of the three food-borne pathogenic bacteria is not significantly reduced after the treatment with sterile water (as shown in fig. 2). Thus, the acidic electrolyzed water can not only remove tunica adventitia bacteria, but also reduce the content of extracellular polysaccharide.

After the acidic electrolyzed water is treated, the structure of the bacterial biofilm is damaged, the viable count and the cell activity in the biofilm state are obviously reduced, the viable count in the treated residual liquid is lower than a detection value, and the content of extracellular polysaccharide is also obviously reduced, so that the effect of efficiently removing the bacterial biofilm is achieved, and the acidic electrolyzed water can be used for removing the biofilm and the tunica bacteria of the food-borne pathogenic bacteria.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.