Knife Decontamination by Cold Water Treatment Supplemented with Inspexx^TM 210—A Validation Study in an Abattoir

Abstract

To evaluate the decontamination effect of Inspexx 210, a cold-water disinfectant containing peracetic acid and peroctanoic acid (0.16%), knives contaminated with E. coli ATCC 25922 and S. aureus ATCC 29213 as well as naturally contaminated knives collected from different slaughtering steps of an abattoir were examined. Treatment with Inspexx 210 for 15 s reduced mean E. coli counts by 5.95 log CFU/cm² and mean S. aureus counts by 6.24 log CFU/cm². Contamination of the knives first with fat and thereafter with the bacteria led to lower mean reductions of 5.52 log CFU/cm² for E. coli and 3.58 log CFU/cm² for S. aureus. Contamination first with blood and thereafter with the bacteria had the greatest negative impact on the inactivation effect, with mean reductions of 3.70 log CFU/cm² for E. coli and 1.55 log CFU/cm² for S. aureus. Rinsing the blood with tap water before contamination with S. aureus led to the reduction of 5.50 log CFU/cm². Treatment with Inspexx 210 was also assessed for naturally contaminated knives from an abattoir. After treatment under routine conditions, colony counts were 0.09 log CFU/cm² (pigs) and 0.35 log CFU/cm² (sheep) in the wet area and below detection limit (pigs) and 0.91 (sheep) in the clean area. This study shows that Inspexx 210 can be an adequate alternative to knife decontamination with 82 °C hot water, but pre-rinsing is recommended especially in dirty process steps with a high contamination of blood and fat.

1. Introduction

Foodborne diseases have a major health impact in industrialized countries. In 2021, the European Food Safety Authority (EFSA) reported 4005 foodborne outbreaks, 32,543 cases of illness, 2495 hospitalizations and 31 deaths in the European Union [1]. Zoonotic pathogens are of special concern in food of animal origin and have to be controlled by a feed-to-food system.

Historically, in terms of meat production, the official veterinary meat inspection is the main intervention step in abattoirs to protect consumer health. It is based on ante-mortem examination and macroscopic post-mortem examination, thereby clinical manifestations or pathological–anatomical sign in the carcass and its organs are recognized. Premise for the functioning of this “safe food from healthy animals” approach is that health hazards are correlated with clinical or pathological–anatomical findings (e.g., bovine tuberculosis). However, healthy food animals were also recognized as carriers of a pathogen responsible for human illness. Because no clinical of pathological–anatomical signs are evident in the animals, such carriers are not detected int the traditional veterinary meat inspection. The significant microbial contamination of the carcass takes place during slaughter despite all advancements in slaughter technologies. Bacteria (e.g., pathogenic E. coli, S. aureus, Salmonella) on the carcass surfaces may originate from the animal itself, especially from the hides, the feet, or the gastrointestinal tract [2]. In addition, carcasses can be contaminated or cross-contaminated from various environmental sources such as equipment, knives or worker’s hands [3]. The extent of contamination and the relevance of individual sources are dependent on the cleanness of incoming livestock, the structural design of the slaughterhouse, the slaughter technology and the cleaning and disinfection regime. Strict adherence to good practices of slaughter hygiene is crucial to ensure meat safety at slaughter. Moreover, to limit cross-contamination by equipment and utensils like knives, in-process cleaning and disinfection should be performed after each use.

The European Union [4] and Swiss [5] regulation require that in abattoir utensils like knives be decontaminated with water at a minimum of 82 °C or with an alternative system with an equal effect. The use of hot water, however, has several limitations. On the one hand, hot water leads to a high energy consumption, a faster wearing of the knife blades, and results in the formation of water vapor. On the other hand, the heat-based denaturation of proteins on the surface of the knives can significantly influence the effect of bacteria inactivation. Moreover, due to the fact that 10% of accidents at slaughterhouses are burns, alternative systems could also improve safety at work [6,7].

Various systems have been tested to see if they could replace the traditional method. For example, temperatures of 60 °C with longer exposure, ultraviolet-C light, ultrasound, lactic acid, high voltage atmospheric cold plasma, or combinations of different methods were investigated [8,9,10,11,12].

A new possible alternative system is Inspexx 210 (Ecolab, Monheim am Rhein, Germany). It is a cold-water-based dip system containing two active compounds peracetic acid (PAA) and peroctanoic acid (POA) at a concentration of 0.16% (https://de-ch.ecolab.com/offerings/inprocess-disinfection, accessed on 15 April 2023). Compared to a decontamination with hot water, this system saves energy and water and is therefore more sustainable. So far, there are no published independent studies on the decontamination effect of the procedure. Moreover, based on a quality assurance system as, e.g., ISO:9001 or GFSI standards (IFS Food), food producing companies must validate processes when introducing them.

The aim of the present study was to determine the bacterial reductions obtained by this new commercial system (i) on knives inoculated with E. coli or S. aureus (with and without additional fat and protein contamination) and (ii) on naturally contaminated knives under routine conditions in a slaughterhouse.

2. Materials and Methods

2.1. Challenge Test

The bacteria used in this study were E. coli (ATCC 25922) and S. aureus (ATCC 29213) as representatives for Gram-negative and Gram-positive bacteria, respectively. On knives commonly used in abattoirs (stainless steel, Swibo No. 5.8411.25 and 5.8411.26, Victorinox^®, Ibach-Schwyz, Switzerland), two areas of 5 cm² were inoculated with several colonies of the respective bacterium taken from sheep blood plates (incubated at 37 °C for 24 h) using sterile cotton swabs by smearing them on the defined surface. The tests were carried out in four different layouts: (i) a clean surface, (ii) a surface contaminated with fat (commercially available vegetable oil), (iii) a surface contaminated with protein (defibrinated sheep blood) or (iv) a surface contaminated with protein (defibrinated sheep blood) and then rinsed under a tap for 15 s before inoculation. After drying for five to ten minutes, the knives were taken to the abattoir to carry out the sampling.

The knives were exposed to Inspexx 210 (0.16% Inspexx 210 in water) for 15 s in a basin of 5 L with a constant influx of 0.5 L/min (water temperature between 7–10 °C). Using sterile cotton swabs, a sample from the inoculated area was taken before and after the intervention. For the sampling before the intervention, the sterile cotton swab was moistened with a 0.85% saline solution. After the intervention, the cotton swab was first moistened with, and then put into 1 mL of, a neutralization solution (1 g tryptone, 8.5 g NaCl, 30 g saponin, 1 g histidine, 3 g lecithin and 30 g tween80 solved in 1000 mL deionized water) to prevent the disinfectant from further effect. Fifty samples were taken for every test series.

2.2. Field Study

At a level of bleeding in the wet area and evisceration in the clean area, samples were taken from knives used in the production line during the slaughter of pigs and sheep. To collect the samples, a whole side of a knife (average approx. 68 cm²) was swabbed with sterile cotton swabs before and after intervention. For sampling after the intervention, the cotton swab was first moistened and then put into 1 mL of a neutralization solution (as described above). The time of exposure to Inspexx 210 (0.16%) was not standardized and differed depending on the employee and the position in the slaughter line. In general, knives were dived into the basin after every animal. For each area and animal species, 25 samples were gathered.

2.3. Microbiological Analysis

The cotton swabs were placed in stomacher bags where 5 mL (challenge test) or 10 mL (field study) of a 0.85% saline solution were added. After homogenizing for 30 s in a stomacher, aliquots of 1 mL were used to perform a decimal dilution series in the 0.85% saline solution. Volumes of 0.1 mL were then plated onto plate count agar (PC agar; Oxoid AG, Pratteln, Switzerland) and incubated for 24 h at 37 °C (challenge test) or for 72 h at 30 °C (field study). After the specified incubation time, the colonies grown on the plates were optically counted. The detection limit was 1 log CFU/cm² for the challenge tests and 0.84 CFU/cm² for the field tests. Results below the detection limit are reported as 0 log CFU/cm². Reduction rates before and after intervention were calculated based on the means.

Statistical analysis. Statistical analysis was performed using GraphPad Prism (Version 9.4.1, GraphPad Software, San Diego, CA, USA). All data were treated as log CFU/cm² and not normally distributed. The Mann–Whitney test was used to compare the results before and after intervention. To determine the statistical significance between the different test series after the intervention with the disinfectant, the Kruskal–Wallis test with post-hoc Dunn’s multiple comparisons test was performed. The results were considered statistically significant if p < 0.05.

3. Results

3.1. Challenge Test

On inoculated knives with a clean surface, the initial amount of bacteria was 6.14 log CFU/cm² for E. coli and 7.07 log CFU/cm² for S. aureus. Treatment with Inspexx 210 for 15 s led to a significant reduction of 5.95 log CFU/cm² for E. coli and 6.24 log CFU/cm² for S. aureus (p < 0.0001). Inoculated knives with a fat contaminated surface showed an initial count of 6.00 log CFU/cm² for E. coli and 6.67 log CFU/cm² for S. aureus. The treatment resulted in a significant reduction of 5.52 log CFU/cm² for E. coli and 3.58 log CFU/cm² for S. aureus (p < 0.0001). After the initial amount of 5.21 log CFU/cm² for E. coli and 7.07 log CFU/cm² for S. aureus on inoculated knives contaminated with protein, the intervention with Inspexx 210 significantly reduced the bacteria by 3.70 log CFU/cm² for E. coli and 1.55 log CFU/cm² for S. aureus (p < 0.0001). Inoculating the knives with S. aureus after rinsing the blood led to the significant reduction of 5.50 log CFU/cm² from the initial amount of 7.40 log CFU/cm² (p < 0.0001) (Figure 1,

Table 1. Reduction in E. coli and S. aureus inoculated on stainless steel knives after the intervention with Inspexx 210 for 15 s. Tests were run with knives being (i) clean, (ii) contaminated with fat (vegetable oil), (iii) contaminated with protein (defibrinated sheep blood) or (iv) contaminated with protein and then being rinsed before inoculation ($\overline{\mathrm{X}}$ = mean log CFU/cm², ai = ante-interventionem, pi = post-interventionem, SD = standard deviation).

		$\overline{\mathbf{X}}$ (ai)	SD (ai)	$\overline{\mathbf{X}}$ (pi)	SD (pi)	Reduction
E. coli
	Clean	6.14	0.55	0.19	0.50	5.95
	Fat	6.00	0.67	0.47	0.96	5.52
	Protein	5.21	0.42	1.52	1.31	3.70
S. aureus
	Clean	7.70	0.33	1.46	1.87	6.24
	Fat	6.67	0.53	3.08	1.05	3.58
	Protein	7.07	0.52	5.51	0.88	1.55
	Protein rinsed	7.40	0.25	1.89	1.95	5.50

).

3.2. Field Study

Naturally contaminated knives in the wet area at the level of bleeding had an initial count of 1.41 log CFU/cm² in the pig, and 2.10 log CFU/cm² in the sheep production line, respectively. After the non-standardized exposure to Inspexx 120 in the basin, there was a significant reduction of 1.32 log CFU/cm² in the pig, and 1.74 log CFU/cm² in the sheep line (p < 0.0001), respectively. In the clean area at the level of evisceration, the initial amount of contamination was 0.83 log CFU/cm² on knives used to eviscerate pigs and 2.40 log CFU/cm² on those used to eviscerate sheep. The disinfection led to a significant reduction of 0.83 log CFU/cm² below the detection limit in the pig and 1.49 log CFU/cm² in the sheep production line (p < 0.0001) (Figure 2,

Table 2. Reduction in total viable counts on naturally contaminated stainless steel knives used in the production line of the wet and clean area during the slaughter of pigs and sheep. The time of intervention with Inspexx 210 differed depending on the employee and the position in the slaughter line ($\overline{\mathrm{X}}$ = mean log CFU/cm², ai = ante-interventionem, pi = post-interventionem, SD = standard deviation).

		$\overline{\mathbf{X}}$ (ai)	SD (ai)	$\overline{\mathbf{X}}$ (pi)	SD (pi)	Reduction
Wet area
	Pigs	1.41	0.96	0.09	0.24	1.32
	Sheep	2.10	0.62	0.35	0.55	1.74
Clean area
	Pigs	0.83	0.75	0.00	0.00	0.83
	Sheep	2.40	0.52	0.91	0.63	1.49

).

4. Discussion

To ensure food safety at slaughter, additional measures to the conventional meat inspection are crucial to counter the threats posed by latent zoonoses. In the daily practice, strict compliance with good practices of slaughter hygiene constitutes a significant means to avoid carcass contamination. In this context, effective in-process cleaning and disinfection of knives after each use is essential. The European Union [4] and Swiss [5] regulation require that in abattoirs utensils like knives be decontaminated with water at a minimum of 82 °C or with an alternative system with an equal effect. Here, we evaluated the Inspexx 210 system as a cold-water-based dip system containing two active compounds peracetic acid (PAA) and peroctanoic acid (POA) at a concentration of 0.16%. The aim was to determine the bacterial reductions obtained by this new commercial system (i) on knives inoculated with E. coli or S. aureus (with and without additional fat and protein contamination) and (ii) on naturally contaminated knives under routine conditions in a slaughterhouse.

The treatment of clean knives contaminated with E. coli ATCC 25922 and S. aureus ATCC 29213 with Inspexx 210 for 15 s led to a significant reduction of 5.95 log CFU/cm² and 6.24 log CFU/cm², thus showing a strong effect both on Gram-negative and Gram-positive bacteria. In a previous study, Goulter et al. [12] compared, also for E. coli ATCC 25922, different exposure times at different hot water temperatures. In that study, a reduction of 4.65 log CFU/cm² of E. coli after an exposure time of 20 s was measured (15 s exposure time was not performed). Further, Taormina and Dorsa [13] found a reduction of 3.02 log CFU/cm² for E. coli O157:H7 on knives immersed in 82 °C water for 15 s. The comparison of these earlier data with the new evaluated cold-water treatment in the present study shows that Inspexx 210 can have just as good an effect as the traditional disinfection system. Unfortunately, there are no studies on 82 °C water disinfection of knives inoculated with S. aureus.

Fat and protein contamination on knives can lead to insufficient disinfection [14]. Investigation by Snijders et al. [14] indicated that when fats or proteins are absent, even a high bacterial contamination on the blades of the knives is eliminated by immersion in water at ≤82 °C. On the other hand, if a high amount of fat and protein contamination is present, even a much longer exposition time in water at this temperature does not provide satisfactory results.

This was especially shown for the combination “protein contamination and S. aureus” in the current study. Therefore, additional tests were carried out with pre-rinsing the knives after blood contamination in order to produce only a fine biofilm. These tests revealed similar results for S. aureus to those obtained from testing clean inoculated knives. These data could be an indication that pre-rinsing blood-contaminated knives is necessary before the intervention with Inspexx 210.

The present study demonstrates that Inspexx 210 has a comparable if not better effect on bacterial reduction than the traditional method under challenge conditions. On naturally contaminated knives used during the slaughter of pigs and sheep in the wet area at the step of bleeding, 0.09 log CFU/cm² (pigs) and 0.35 log CFU/cm² (sheep) were counted after the disinfection with Inspexx 210. These numbers are lower than colony counts described by Eustace et al. [7], where at the same slaughter process step, 0.73 log CFU/cm² (sheep) and 2.34 log CFU/cm² (beef) were found after cleaning knives with water heated to 82 °C. At evisceration, using Inspexx 210, counted CFU on the knives of the abattoir workers were below the detection limit (pigs) and 0.91 log CFU/cm² (sheep). Higher values were reported by Eustace et al., where the decontamination of knives with 82 °C hot water resulted in 1.81 log CFU/cm² (sheep) and 1.35 log CFU/cm² (beef). Surprisingly, in the present study, the initial CFU count during the slaughter of sheep was higher in the clean than in the wet area. This could be due to a contamination of the tested knives with organic material rather than the work step having a higher bacterial load in general, especially if the counts are compared to those of the pig slaughter line.

Studies have shown that PAA can not only have an insufficient effect, but may also fix biofilms, thus leading to the selection of biofilm building bacteria such as E. coli, S. aureus and L. monocytogenes [15,16]. By contrast, POA, which is more hydrophobic than PAA, may have a higher ability to penetrate cell membranes. Moreover, there is evidence that POA penetrates biofilms better than PAA [17]. Hence, Inspexx 210 should be effective also against biofilms, although further studies are needed in this context.

This study has some limitations, (1) We focused on one E. coli and one S. aureus strain as representatives for Gram-negative and Gram-positive bacteria, respectively. However, further studies could be performed to evaluate the effect of Inspexx 210 on more strains of these species and also on other bacteria species (e.g., L. monocytogenes, S. enterica, Clostridium spp.) (2). It should be noted that native animal fat and blood could influence the decontamination effect differently than the plant oil and defibrinated sheep blood used for the challenge tests in this study. (3) The effect of pre-rinsing the knives under different conditions should be further evaluated. This could also be achieved using a spraying system that combines pre-rinsing and disinfection in one step, which is a system already offered by Ecolab. Such a system would save the employees time and could in particular be implemented at positions in the slaughter line where high contamination is expected. (4) Only the microbial effectiveness of this new treatment was evaluated, while other effects on the equipment (e.g., knife blades) or the cost analysis were not examined.

5. Conclusions

Effective in-process cleaning and disinfection of knives after each use is essential to inhibit cross-contamination and ensure food safety at slaughter. Inspexx 210 can be used as an adequate alternative to knife decontamination with 82 °C hot water. Nevertheless, this study has shown an insufficient effect on S. aureus-inoculated knives contaminated with blood. Therefore, it is recommended to pre-rinse knives whenever possible, but especially in process steps involving a high contamination of blood and fat.