Method for removing mixed strain biofilm in food processing environment

Abstract

The invention provides a method for removing a mixed strain biofilm in a food processing environment, and particularly belongs to the technical field of microbial pollution treatment. A method for removing a mixed strain biofilm in a food processing environment comprises the following steps: immersing an ultrasonic probe into a liquid level of the food processing environment, carrying out ultrasonic treatment, and washing with sterile water; the frequency of the ultrasonic treatment is 20KHz, and the power of the ultrasonic treatment is 200W. The removing method can effectively remove the biofilm of the staphylococcus aureus and escherichia coli mixture species on the surface of the food processing environment material, and is short in treatment time, high in removing efficiency, safe, environmentally friendly and free of harmful substance residues.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pollution treatment, and in particular relates to a method for removing mixed bacterial biofilm in a food processing environment. Background Art

[0002] Biofilm is a complex micro-ecological system. It is a three-dimensional polysaccharide-protein complex composed of bacteria attached to the surface of an object and secreted extracellular polymers (exopolysaccharides, extracellular proteins and DNA). Biofilm is a protective barrier for bacteria in the natural environment, which can resist mechanical loss and the stimulation of bactericides. It can also increase the tolerance of bacteria to extreme environments and provide energy substances for bacteria inside the biofilm, which significantly improves the survival ability of the bacterial community. Therefore, compared with planktonic bacteria in the environment, bacterial communities that can produce biofilms naturally have more advantages. Biofilms are highly resistant and difficult to remove, and are the main cause of repeated cross-infection during processing and post-processing contamination. At present, most studies focus on single-species biofilms, but in fact, mixed-species biofilms are the main way of life for bacteria. The structure, development and function of biofilm communities are all affected by species interactions.

[0003] Biofilms in food processing environments have become an important source of bacterial cross-contamination. Staphylococcus aureus and Escherichia coli are the main foodborne harmful bacteria, and food poisoning incidents caused by these two bacteria contamination occur from time to time. Staphylococcus aureus is one of the most important pathogens for humans. It is a Gram-positive coccus that can cause skin and soft tissue infections, osteomyelitis, endocarditis, surgical site infections, pneumonia, sepsis and other diseases. Escherichia coli, a Gram-negative short rod, is a normal resident bacteria in the intestines of humans and animals. It is also the most common enterobacterial pathogen and an important cause of various infections such as urinary tract infections, bacterial pneumonia and sepsis.

[0004] At present, the commonly used method for removing biofilm in food processing is to use chlorine-containing disinfectants, such as sodium hypochlorite and quaternary ammonium salts, but biofilm is difficult to be effectively removed and is easy to damage food raw materials, produce disinfectant residues, and increase the risk of microbial resistance. At present, there is a lack of a more efficient method for removing mixed species biofilm. Summary of the invention

[0005] The object of the present invention is to provide a method for removing mixed bacterial biofilm in a food processing environment. The removal method of the present invention can effectively remove the mixed biofilm of Staphylococcus aureus and Escherichia coli on the surface of food processing environment materials, with short processing time, high removal efficiency, safety and environmental protection, and no harmful substances remaining.

[0006] The present invention provides a method for removing mixed bacterial biofilm in a food processing environment, comprising the following steps: immersing an ultrasonic probe into a liquid surface in the food processing environment, performing ultrasonic treatment, and washing with sterile water; the frequency of the ultrasonic treatment is 20KHz, and the power of the ultrasonic treatment is 200W.

[0007] Preferably, the ultrasonic treatment time is 1 to 5 minutes.

[0008] Preferably, the mixed bacterial species include Staphylococcus aureus and Escherichia coli.

[0009] Preferably, the food processing environment comprises a food processing pipeline and / or a food processing container surface.

[0010] Preferably, the material of the pipe includes stainless steel sheet or polypropylene.

[0011] Preferably, the ultrasonic probe enters the liquid surface by 2 to 4 cm.

[0012] The present invention provides a method for removing mixed bacterial biofilm in a food processing environment. The present invention provides a non-thermal sterilization method based on ultrasonic treatment, which can effectively remove the biofilm of mixed species of Staphylococcus aureus and Escherichia coli on the surface of food processing environment materials, with short processing time, high removal efficiency, safety and environmental protection, no harmful substances remain, and no adverse effects on the food processing environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0014] Figure 1 It is a schematic diagram of the structure of the ultrasonic device used in the present invention;

[0015] Figure 2 This is a diagram showing the effect of the embodiment of the present invention on a stainless steel plate;

[0016] Figure 3 This is a diagram showing the effect of an embodiment of the present invention on a polypropylene plate. DETAILED DESCRIPTION

[0017] The present invention provides a method for removing mixed bacterial biofilm in a food processing environment, comprising the following steps: immersing an ultrasonic probe in the liquid surface of the food processing environment, performing ultrasonic treatment, and rinsing with sterile water; the frequency of the ultrasonic treatment is 20KHz, and the power of the ultrasonic treatment is 200W. In the present invention, the ultrasonic treatment time is 1 to 5 minutes, specifically 1 minute, 2 minutes, 3 minutes, 4 minutes or 5 minutes. The probe-type ultrasonic equipment has high energy density and can efficiently remove mixed bacterial biofilm. In the present invention, the mixed bacterial species include Staphylococcus aureus and Escherichia coli. In the present invention, the food processing environment includes a food processing pipeline and / or a food processing container surface. During the flow of liquid in the pipeline, bacteria adhere to the surface of the pipe wall and continue to adhere and multiply to form a biofilm, which has the characteristics of high resistance and difficulty in removal, resulting in repeated cross infection during processing, and the removal method of the present invention uses ultrasound to remove the biofilm attached to the pipeline. In the present invention, the material of the pipeline includes stainless steel sheet or polypropylene. In a specific embodiment, a stainless steel sheet and a polyethylene sheet are placed in water to simulate the bacterial growth environment in the pipeline. In the present invention, the ultrasonic probe enters the liquid surface 2 to 4 cm, specifically 2 cm, 3 cm or 4 cm. Compared with existing common technologies such as disinfectants, the method of the present invention can remove most of the biofilms on the surface of the food processing environment, and the processing time is short and the removal efficiency is high; on the other hand, it is safe and environmentally friendly, no harmful substances remain, and will not cause adverse effects on the food processing environment.

[0018] In order to further illustrate the present invention, a method for removing mixed bacterial biofilm in a food processing environment provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Stainless steel sheets and polypropylene (PP) sheets, two materials to which biofilms are often attached during food processing, were used, with specifications of 20×20×4mm; Staphylococcus aureus and Escherichia coli were selected from ATCC 25923 and O157:H7 of Beina Biotechnology; the ultrasonic cell crusher used was purchased from Ningbo Xinzhi Biotechnology Co., Ltd., model SCIENTZ-IID.

[0020] Biofilm culture:

[0021] The resuspended bacterial suspensions of Staphylococcus aureus and Escherichia coli were adjusted to 1×10 7CFU / mL, 4 mL of Staphylococcus aureus liquid was added to a 6-well polystyrene plate with a stainless steel sheet / polypropylene sheet as a single Staphylococcus aureus group, and 4 mL of Escherichia coli liquid was added to a 6-well plate as a single Escherichia coli group. 2 mL of Staphylococcus aureus and 2 mL of Escherichia coli liquid were added to a 6-well plate as a mixed bacterial group, with 3 parallels in each group, and tryptone soy broth medium was used as a blank control. The 6-well plate was placed in a 37°C incubator for 72 hours.

[0022] Ultrasonic removal of biofilm:

[0023] After the biofilm matures, take out the stainless steel sheet / polypropylene sheet and place it in a beaker filled with 50 mL of sterile water. Immerse the ultrasonic probe 2 cm below the liquid surface and perform ultrasonic treatment at a frequency of 20 KHz and a power of 200 W (e.g. Figure 1 shown).

[0024] Biofilm removal rate determination:

[0025] Remove the stainless steel sheet and polypropylene sheet, dry them and fix them with methanol for 10 minutes, add 2mL 0.1% crystal violet dye solution after drying for 10 minutes, and dye for 15 minutes. Pour off the dye solution, wash with sterile water, transfer to a sterile 6-well plate after natural air drying, add 4mL 33% glacial acetic acid and let it stand for 10 minutes, and measure the OD 570 The group without ultrasonic treatment was set as the control group. Biofilm removal rate:

[0026]

[0027] Among them, US is the ultrasonic treatment group and C is the control group.

[0028] Example 1

[0029] The stainless steel sheet and the polypropylene sheet attached with the mixed bacterial biofilm of Staphylococcus aureus and Escherichia coli were placed in a beaker of 50 mL sterile water and subjected to ultrasonic treatment at a frequency of 20 KHz and a power of 200 W for 1 min.

[0030] Example 2

[0031] The ultrasonic treatment time in Example 1 was changed to 2 min; the rest was the same as in Example 1.

[0032] Example 3

[0033] The ultrasonic treatment time in Example 1 was changed to 5 min; the rest was the same as Example 1.

[0034] Comparative Example 1

[0035] The stainless steel sheet and polypropylene sheet with only Staphylococcus aureus biofilm attached were placed in a beaker containing 50 mL of sterile water and ultrasonically treated for 1 min.

[0036] Comparative Example 2

[0037] The stainless steel sheet and polypropylene sheet with only E. coli biofilm attached were placed in a beaker containing 50 mL of sterile water and ultrasonically treated for 1 min.

[0038] Comparative Example 3

[0039] The stainless steel sheet and the polypropylene sheet attached with the mixed bacterial biofilm of Staphylococcus aureus and Escherichia coli were placed in a beaker containing 50 mL of 0.5% sodium hypochlorite and treated for 1 min.

[0040] Biofilm removal effect

[0041] The stainless steel sheets and polypropylene sheets obtained after ultrasonic treatment in the above-mentioned Examples 1 to 3 and Comparative Examples 1 to 3 were stained with crystal violet to determine the biofilm biomass, and a blank control (not ultrasonically treated) was set up to conduct the same experiment to compare the removal effects of different treatment methods on the biofilm. The specific data are shown in Tables 1 and 2, among which the treatment results of Examples 1 to 3 at different times are shown in Tables 1 and 2. Figure 2 and Figure 3 shown.

[0042] Table 1 Removal rate of biofilm on stainless steel plate under different treatments (%)

[0043] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Clearance 73.23±4.62 85.77±3.95 97.21±0.94 84.85±2.43 63.25±12.45 31.57±16.94

[0044] Table 2 Removal rate of biofilm on polypropylene plate under different treatments (%)

[0045] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Clearance 63.13±18.41 91.73±5.37 93.27±2.22 86.02±4.77 61.97±8.94 48.78±11.73

[0046] According to the data in Table 1 and Table 2 and Figure 2 and Figure 3 It can be seen that the mixed biofilm of Staphylococcus aureus and Escherichia coli is more difficult to remove than the single-species biofilm of Staphylococcus aureus, and ultrasound has a significant effect on the removal of mixed-species biofilms. Ultrasonic treatment with a power of 200W for 5 minutes can remove 97.21% and 93.27% of the biofilm on the material surface.

[0047] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for removing mixed bacterial biofilm in a food processing environment, characterized in that: The method comprises the following steps: immersing an ultrasonic probe into a liquid surface of a food processing environment, performing ultrasonic treatment, and washing with sterile water; the frequency of the ultrasonic treatment is 20KHz, and the power of the ultrasonic treatment is 200W.

2. The cleaning method according to claim 1, characterized in that: The ultrasonic treatment time is 1 to 5 minutes.

3. The cleaning method according to claim 1, characterized in that: The mixed bacterial species include Staphylococcus aureus and Escherichia coli.

4. The cleaning method according to claim 1, characterized in that: The food processing environment includes food processing pipelines and / or food processing container surfaces.

5. The cleaning method according to claim 1, characterized in that: The material of the pipeline includes stainless steel sheet or polypropylene.

6. The cleaning method according to claim 1, characterized in that: The ultrasonic probe is inserted 2 to 4 cm into the liquid surface.

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