Study on the bactericidal effect of different organic acids on Escherichia coli and its application in slaughterhouse

Author(s): Ding Xiao-yan, Bai Yu-hui, Zhao Qi, Wang He-Jia and Xu Shi-Xin* The purpose of this experiment is to study the bactericidal effects of organic acids in a short time on Escherichia coli in order to screen the best organic acid disinfectants for slaughterhouse. Lactic acid, malic acid and citric acid that conform to the standards of food additives were selected, and the sterilization effect of different concentrations of organic aci ... Abstract View Full Article View DOI: 10.17352/ijvsr.000078

Microbial contamination is inevitable during the slaughtering and processing of broilers, and crosscontamination among carcass, treated water and equipment can also increase the level of microbial contamination of the carcass. Escherichia coli is a common food-borne pathogen in chicken, and it is also an internationally recognized important indicator of food hygiene. In the broiler slaughtering and processing, drug-resistant strains in broiler carcasses can also be isolated, resulting in cross-contamination in environment and carcass. Drug resistant strains can be passed from generation to generation through resistant genes or resistant plasmids, and even transferred to different strains, which makes the resistance of bacteria extremely complex, and the development of antibiotic resistant bacteria in animals poses a potential risk to humans. Zhao [1] also found that although pre-cooling can reduce bacterial contamination in broiler chickens, the bacterial contamination rate will increase during the subsequent segmentation and conveying process, and the cross-contamination of the segmentation and conveying process is determined to be the key control point, followed by pre-cooling pool water. The same is true of Wang [2], Zeng [3], and Rasschaert [4] on bacterial contamination of broiler slaughtering and processing.
The disinfectant used in the pre-cooling cleaning process in China is sodium hypochlorite [5]. Sodium hypochlorite has the advantages of high effi ciency and broad-spectrum, but it is unstable when encountering organic matter, greatly reducing the content of active chlorine, reducing the sterilization ability. However, there is also a certain controversy that the toxicity of the reaction product of sodium hypochlorite and organic matter. Thus the European Union has prohibited the use of sodium hypochlorite in direct contact with food [6], and Islamic countries that export livestock and poultry products to the EU also has prohibited the use of sodium hypochlorite [7]. Therefore, livestock and poultry products which are export to the European Union and Islamic countries, must be sterilized with chlorine-free chemical disinfectants. Organic acids have Citation: Xiao good bactericidal effect, non-toxic and harmless to the human body, and are not easy to produce drug resistance. They are also approved by the US Food and Drug Administration (FDA) as "Generally Recognized as Safe" (GRAS) foods Additives [8], the US Department of Agriculture regulates 1.5%~2.5% lactic acid, citric acid and acetic acid to treat carcasses. The United States has used peroxyacetic acid in carcass rinsing and precooling tanks [9].
In this paper, three organic acids that meet the national standards of food additives were selected to study the bactericidal effect. At the same time, lactic acid was used in combination with SDS, which is an ingredient allowed for food disinfectants. Response surface analysis was used to optimize the concentration of lactic acid and SDS and processing time. Two kinds of disinfectants were tested for stability, comparing the bactericidal effect under the conditions of 54 0 C acceleration test and interference of organic matter. Finally, the best disinfectant was selected for on-site application in the slaughterhouse.

Materials
Strains used: Laboratory tests were carried out on twelve strains of Escherichia coli ( Figure 1, Table 1). In the experiment of screening disinfectants, single strain(HB6-1) were used.
The coincidence rate of drug resistance phenotype and gene in 12 strains of E. coli was 88%.The drug-resistance of -lactams predicted by whole genome sequencing analysis was completely consistent with the drug-resistance phenotype(no drug-resistance gene was found in the susceptible strains, while drug-resistance genes were found in all the resistant strains). The coincidence rate of sulfonamides resistance of phenotype and resistance gene was 95%, fl orfenicol was 95%, aminoglycosides was 90%, polypeptide antibiotic was 90%.
The coincidence degree of both quinolones and tetracyclines was less than 90%, the coincidence rate was 75% and 70%, respectively.
Strains were cultured at 37 0 C on nutrient agar(Oxoid) for 18-24 h. Colony material was passaged in normal saline to a concentration of 8 log CFU mL-1, which was verifi ed by mcreynolds turbidimeter. Screening of compound organic acids: According to the screening results of single acid, it was found that lactic acid had the best bactericidal effect. Therefore, the combination of lactic acid (0.1-2%) and SDS (10-500 ppm) was chosen. The method was same as above.   Organic matter interference test: In order to simulate the organic matter existed in the slaughterhouse, 10%, 15%, and 20% fetal bovine serum were added to the disinfectants to determine the killing effect.

Application in slaughterhouse
In order to verify the bactericidal effect of the compound disinfectant in slaughterhouse, the experiment was carried out in two slaughterhouse workshops in Hebei and Anhui. According to previous investigations on the key points of contamination of food-borne pathogens in the slaughter process of broilers, splitting and packaging were identifi ed as the key point of contamination. And two workers were selected for carcass segmentation. The operation of the experimental group was as follows: 30 whole chicken carcasses after precooling were randomly selected. For each division, a whole chicken carcass, the gloves and knives used by the workers were disinfected. After soaking for 1 minute, the surfaces of gloves and cutting tools were directly scraped with a sterile cotton swab, and 30 samples were taken from the surface of gloves and knives. Workers use the gloves and knives disinfected with compound disinfectant to cut the whole chicken carcass, and 30 samples were taken from the surface of the chicken. 30 whole chicken carcasses on the pre-cooling line were taken randomly as the control group. The control group was not dealt with disinfectant. Other operation was same as above. After sampling, the swabs were placed immediately in the transport medium and transported to the laboratory in an ice box for testing.

Isolation and purifi cation of Escherichia coli:
The sample was purifi ed and passaged 2 times by MacConkey. A single red colony was picked and inoculated on nutrient agar. The purifi ed strains were identifi ed by PCR. The method to extract Verifi cation of optimal conditions: In order to judge the feasibility of the plan, 12 strains of Escherichia coli with different drug resistance phenotype isolated from each process in a slaughterhouse in Hebei were randomly selected. The bacterial suspension of 12 strains of Escherichia co li was diluted 10 4 times and then plated as a positive control. The experimental method was same as quantitative sterilization test.  (6) Table 3: PhoA primer sequence [11].

Experimental design and results
According to the preliminary screening results, the  Table   4.

Model establishment and signifi cance test
Response surface analysis was used to regression fi t the bactericidal effect of the compound preparation, and the standard regression equation of bactericidal rate was obtained.
The F test was used to conduct variance analysis on the test data to evaluate the statistical signifi cance of the model. Variance analysis was conducted on this model, and the results were shown in Table 5. The signifi cance test of model coeffi cients was shown in Table 6. Table 5 shows that the P value of overall model is 0.05, indicating a signifi cant difference at different levels of the model equation. Table 6 shows that the fi rst ter m C(P<0.05) and the interaction item AC(P<0.05) were statistically signifi cant; the fi rst term A (0.05<P<0.1) was statistically signifi cant.

Response surface analysis and parameter optimization
It can be seen from Figure 4 that the concentration of lactic acid and the concentration of SDS are the factors that have the greatest infl uence on the bactericide rate, with the most obvious interaction. With the increase of the concentration of lactic acid and SDS, the bactericide rate increased gradually. Based on the results and regression equations, the Design Expert11 software was used to obtain the optimal test combination for each factor (0.5% lactic acid concentration, 1min treatment time, 50 ppm SDS concentration), and obtained a predicted response value of 100% inhibition. 12   Citation: Xiao-

Results of application in slaughterhouse
The compound of 0.5% lactic acid and 50 ppm SDS was used is the raw materials allowed to be used for food disinfectants, which can be directly used to disinfect food, catering utensils, tools, equipment or food packaging materials and containers

Degrees of freedom
The mean square     Physical and chemical methods are usually used to reduce the microbial contamination rate and carry amount of chicken in broiler slaughterhouse. The physical disinfection methods include mainly ultraviolet irradiation and high-temperature scalding. Usually, when a day's work is completed, the workshop is sterilized by ultraviolet irradiation. 58-62 0 C high-temperature water was used to scald the hair in the scalding pool, and some heat-labile microorganisms die due to high temperature. Chemical methods mainly cover chemical disinfectants, and disinfectants are usually added to the pre-cooling pool to reduce bacteria. The change of bacterial contamination rate caused by cross-contamination is an important parameter in the risk assessment model of broiler slaughterhouse [17]. Bacteria remaining in the pre-cooled washing water can migrate to the surface of the uncontaminated chicken, causing cross-contamination. In the process of dividing and conveying, the carcass repeatedly passes through pollution sources such as processing equipment, cutting boards, and workers' hands. If the disinfection is not thorough enough, the number of bacteria on the surface of the carcass will increase, which will lead to cross-contamination of the product before packaging. The single and compound formulas of organic acids selected in this test can achieve high-effi ciency bactericidal effects in a short period of time, and are less affected by organic matter interference. Therefore, the application of sodium hypochlorite in slaughterhouses can be considered in the future. Therefore, it can be considered to replace sodium hypochlorite in slaughterhouse application in the future.

Conclusion
Using quantitative bactericidal test, the best effect of 3% lactic acid on multi-drug resistant Escherichia coli was screened. Response surface methodology was used to optimize the three conditions of lactic acid, SDS and time. After mixing with the normal strain (which had no resistance to the 14 detected antimicrobial agents) for 1min, zero bacteria remained. The strains with different drug resistance were treated for 1min without any residual bacteria. This indicates that the treatment mode of this formulation can completely and effi ciently kill both normal and drug-resistant strains, providing a solution   to the concern that drug-resistant genes of drug-resistant strains enter the human body through the food chain.
Through 54 0 C accelerated test and organic interference test, it was found that the two formulations had good stability, indicating that the bactericidal or bacteriostasis validity of the two disinfectants was kept at room temperature for at least 1 year. The compound disinfectant is little affected by the interference of organic matter, 10% of organic matter will not affect its effect at all, but the high concentration of organic matter will affect the bactericidal power of lactic acid, and the low concentration of organic matter has little infl uence on it.
The combination of 0.5% lactic acid and 50 ppm SDS applied in the slaughterhouse could reduce the E. coli on the knives and gloves, and the bactericidal rate reached 77%, which was not the same as the experimental results of organic matter interference, which may be related to the E. coli carrying rate in the chicken carcass. In a nutshell, the effect of organic acid disinfectants is similar to that of traditional disinfection methods, but organic acids are safer and less affected by organic matter. So they will have a very wide application prospect in slaughterhouses in the future.