RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 6 8 JUNE 2007, LOZENEC, BULGARIA

THE EFFECTIVENESS OF SANITATION KIT SANI-TEST AS A MONITOR OF THE HYGIENIC STATUS OF SURFACES
Daniela Botu, Ramona Mihilescu, Virgilia Popa and Beatrice tirbu Teofnescu
Abstract: For hygienic reasons, worktops, cookware, utensils and other equipment in contact with food must be free of invisible residue that food leaves behind, which could provide opportunities for microbial growth. The sanitation kit SANI-TEST indicates the hygiene level after cleaning by detecting residuals of protein which can be left behind after inadequate cleaning. In this study, we compared the test results with bacterial colony count. We studied the effect of various cleaners/disinfectants on test sensitivity. Field trials were carried out in a poultry slaughterhouse & processing company and a restaurant. Key words: proteins, surface, cleanness

INTRODUCTION Stringent hygiene procedures are key to the maintenance of end product quality. Failure to maintain hygiene standards can seriously impact on the final product quality, consistency and freshness [5, 2]. To be hygienic, working surfaces, ustensils and other equipments must be free of invisible food residues, which could provide opportunities for microbial growth. Since the presence of proteins invisible to the eye can indicate food residues, it is important to adequately assess the cleanness of the working surface with a suitable test. Protein remaining on the surface provides nutrients for bacterial growth and can lead to dangerous contamination. The advent of rapid, non instrument based hygiene tests, provide an opportunity to measure the cleaning effectiveness first hand on a day by day basis [1, 3]. The aim of this study was to validate the effectiveness of sanitation kit SANI-TEST as a monitor of the hygienic status of surfaces in a series of field trials and as a helpfull tool for quality management. SANI-TEST is a surface swab hygiene test that uses the colorimetric detection of proteins, to indicate cleanliness. The test was developed by Pasteur Institute and is based on an enhanced biuret reaction with use of bicinchoninic acid and copper sulphate reagents [4]. MATERIAL AND METHODS For assessment of the cleanness degree of surfaces, we used SANI-TEST, a kit developed by Pasteur Institute, Bucharest. The test is based on the Biuret reaction. The working surface needing to be analyzed is swabbed and put in contact with the reagents, under alkaline conditions. The intensity of the color determines the amount of proteins on the swab. A dark color (purple) indicates high protein concentration and therefore, a dirty surface. Conversely, a light color (green) indicates weak protein concentration and therefore, a clean surface. SANI-TEST provides a four-level colour chart for hygiene, allowing qualitative results assessing the cleanness of the surface. Three types of microorganisms, Escherichia coli O157, Enterococcus faecium and Salmonella anatum, were used in order to evaluate the detection limits of Sani-Test. Decimal dilutions in physiological saline were realized from bacterial cultures of 15-18h, in BHI (BioRad), unwashed and washed by centrifugation (17310 g), to eliminate the proteins media interferences, and plated on McConkey (HiMedia) and Columbia + 5% defrinated blood sheep agar (Oxoid), for E.coli and Salmonella anatum, and Enterococcus faecium respectively. 100l of each bacterial dilution were added to SANI-TEST reagent. After 10 minutes of incubation at room temperature (20 10C), the color developed by the test was read and interpreted according to the kit colour chart.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 6 8 JUNE 2007, LOZENEC, BULGARIA

Additional work was done by studying the effect of various cleaners/disinfectants on test sensitivity. As cleaners or disinfectants we used products that contained: sodium hypochlorite, chlorine, glutaraldehide, alkyl-dimethyl-benzyl-ammonium chloride, chlorhexidine, hydrogen peroxide, which were used on clean surfaces and surfaces contamined with BSA, 350g/100l. These products were used at normal concentrations, as well as at higher ones, following the working procedure recommended by kit producer. Field trials were carried out in a poultry slaughterhouse & processing company (31 samples), and a restaurant (50 samples), following the SANI-TEST instructions for use. The tests were done after routine cleaning/disinfection procedures of surfaces, before a new working day start. RESULTS AND DISCUSSION Based on the detection of protein residues by a colormetric reaction, SANI-TEST kit provides a semi-quantitative measure of surface hygiene. The SANI-TEST detects very small amount of proteins, 50g/100cm2. This kit indicates the hygiene level after cleaning by detecting residuals of proteins which can be left behind after inadequate cleaning. The reaction is time-dependent i.e. the colour develops with time and therefore it is important to record colour change after 10 min and disregard any colour change after this set time. Result interpretation was done according to the four-levels colour chart (Table 1).
Table 1 Protein concentration corresponding to colour level with SANI-TEST and colour change interpretation Level 1 2 3 4 Colour Green Grey Light purple Dark purple Protein amount (g) 0 50 50 100 100 500 500 - 1000 Colour change interpretation Clean surfaces Caution low levels of contamination present Significant levels of contamination present High levels of contamination present Pass Caution Fail

The results of the evaluation concerning the limits detection of the kit, made by the bacterial colony count method on three bacterial strains, Escherichia coli O157, Enterococcus faecium and Salmonella anatum, are shown in Table 2.
Table 2 Comparison between SANI-TEST results and bacterial colony count McFarland index > 7.5 1.1 0.2 0.1 0.1 0.1 0.0 6.1 0.7 0.1 0.0 0.1 0.0 0.1 Cfu / ml N/A N/A N/A N/A 141 21 1 N/A N/A N/A N/A 66.5 SANI-TEST results Colour / Protein level amount (g) Grey / 2 50 - 100 Green / 1 0 - 50 Green / 1 0 - 50 Green / 1 0 - 50 Green / 1 0 - 50 Green / 1 0 - 50 Green / 1 0 - 50 Grey / 2 50 - 100 Green / 1 0 - 50 Green / 1 0 - 50 Green / 1 0 - 50 Green / 1 0 - 50

Sample / dilution E. coli, undiluted, washed culture E. coli, 10-1 E. coli, 10-2 E. coli, 10-3 E. coli, 10-4 E. coli, 10-5 E. coli, 10-6 S. anatum, undiluted, wahed culture S. anatum, 10-1 S. anatum, 10-2 S. anatum, 10-3 S. anatum, 10-4

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 6 8 JUNE 2007, LOZENEC, BULGARIA
S. anatum, 10-5 S. anatum, 10-6 E. faecium, undiluted culture (+ growth medium) E. faecium, undiluted, wahed culture E. faecium, 10-1 E. faecium, 10-2 E. faecium, 10-3 E. faecium, 10-4 E. faecium, 10-5 E. faecium, 10-6 0.0 0.0 6.3 4.8 0.6 0.1 0.0 0.1 0.0 0.0 0.0 12.5 2 N/A N/A N/A N/A 389 77 7 0 Green / 1 Green / 1 Dark purple / 4 Green / 1 Green / 1 Green / 1 Green / 1 Green / 1 Green / 1 Green / 1 0 - 50 0 - 50 500 - 1000 0 - 50 0 - 50 0 - 50 0 - 50 0 - 50 0 - 50 0 - 50

The results recorded in Table 2 demonstrated that SANI-TEST is able to detect bacterial cultures of Escherichia coli O 157 of 1 x 106 cfu/ml, Salmonella anatum of 2 x 106 cfu/ml and Enterococcus faecium of 7 x 105 cfu/ml, but the kit is designed as a rapid test for the cleanness evaluation in the field and not to replace the classical microbiological tests (more reliable but time consuming). As we mentioned before, we performed a study about the effect of cleaners/disinfectants on the SANI-TEST results. The cleaners / disinfectants were used at the normal working concentration (1x) and at the other concentration (10x) relative to the normal working concentration (Table 3).
Table 3. Effect of cleaners/disinfectants on SANI-TEST results Effect Concentration Positive (protein 350 Negative g BSA) (no protein) 0.9% (1x) No effect No effect Sodium hypochlorite / disinfectant (level 3 purple) (level 1 green) Chlorine / anionic surfactant 1.2% (1x) No effect No effect (cleaner) (level 3 purple) (level 1 green) 0.25% (1x) No effect No effect glutaraldehide / disinfectant (level 3 purple) (level 1 green) 2.5% (10x) Enhancement of colour False positive (level 2 change (from level 3 to grey in 10 min.) glutaraldehide / disinfectant 4 purple to dark purple) Alkyl-dimethyl-benzyl-ammonium 25 (1x) No effect No effect chloride / sanitiser (level 3 purple) (level 1 green) 0.67% (1x) No effect No effect chlorhexidine / disinfectant (level 3 purple) (level 1 green) 0.01% (not Enhancement of colour False positive (level 3/4 known) change (from level 3 to purple/dark purple in 10 disinfectant 4 purple to dark min.) purple) disinfectant 0.001% (not No effect False positive (level 1/2 known) green/grey in 10 min.) (level 3 purple) Type / active ingredient

It can be observed that there is no interference with most common sanitisers at normal working concentrations. However high concentrations of alkaline cleaners may cause the enhancement of colour and false positive results in some instances. Peroxide based disinfectants can cause a false positive colour change reaction with SANI-TEST. Another aim of this study was to validate the effectiveness of SANI-TEST as a monitor of the hygienic status of surfaces in a series of field trials: poultry slaughterhouse

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 6 8 JUNE 2007, LOZENEC, BULGARIA

& processing company and a restaurant. During the trials, both high and low risk areas were tested. Areas were cleaned as per established cleaning procedure. After cleaning, swab samples of surfaces were taken using SANI-TEST. For the poultry slaughterhouse & processing company, 31 samples were analyzed, consisting in areas which could be critical for hygiene (i.e. cutting or packing tables, blades, knives, weigh bins, conveyor belts, freezers). The results were scored as levels of contamination. As one can see in figure 1, 13 % of those areas tested (4 from a total of 31 samples) were shown to have significant level of contamination producing a purple or dark purple colour results (i.e. table, start button conveyor, control panel); 16% (5/31) of analyzed areas showed low levels of contamination grey or caution result and 71% (22/31) were clean a green colour result. A similar trial of a restaurant hygiene was carried out and there were analyzed 50 areas specific to this location, for example, kitchen tables and floor, refrigerators ans dishes, the trial being performed before of a new work day. The results obtained by applying SANI-TEST on restaurant samples show that 8% (4/50) of those areas tested (forks, pots and refrigerators door handle) were shown to have significant level of contamination producing a purple or dark purple colour results; 18% meaning 9 areas from a total of 50 (some dishes, a kitchen table and kitchen floor) showed low levels of contamination grey or caution result; 74% (37/50) were clean a green colour result (figure 1).
Percentage

Percentage

80 70 60 50 40 30 20 10 0
SANI-TEST clean SANI-TEST caution SANI-TEST fail

80 70 60 50 40 30 20 10 0
SANI-TEST clean SANI-TEST caution SANI-TEST fail

Fig.1. SANI-TEST results. Poultry slaughterhouse & processing company (A) and restaurant (B)

A rapid hygiene test is a useful way to identify areas that need improved cleaning performance in a traditionally neglected area. The results above have shown there are significant levels of contamination in many direct product contact points even after normal cleaning. The results obtained in this study show that it is reasonable to achieve a level 1 hygiene standard on surfaces cleaned by standard methods in working food premises. Failure to achieve level 1 indicates that further cleaning is necessary. This survey has demonstrated the value of rapid methods which although not fully reliable for highlighting the bacterial risk, allow on-the-spot remedial action to be suggested and explained to management. The results from trials demonstrate the effectiveness of SANI-TEST as a method of hygiene monitoring. Based on the detection of protein residues by a colorimetric reaction, the test provide a semi-quantitative measure of surface hygiene. Levels of contamination on surface are indicated by the colour produced in the test solution. The reaction takes up to 10 minutes, but highly contaminated surfaces will produce a fail result in less than this

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 6 8 JUNE 2007, LOZENEC, BULGARIA

time frame. Designed to monitor cleaning effectiveness, SANI-TEST will quickly show if an area has been cleaned properly or requires further attention before use. This will reduce the risk of contamination and represents a benefit for food safety and human / animal health. As key benefits, SANI-TEST quickly indicates whether cleaning has been effective, provides a tool to help the improvement of hygienic performance and fits the need of HACCP regime. CONCLUSIONS AND FUTURE WORK SANI-TEST offers: a way to control and improve hygiene standards in animal and poultry industries, hospitals and human health, food and beverage manufacturing; a simple to use and interpret test requiring no instrument or specialist skills; a test that can be used by technical services or the licensee themselves; can be very usefull for quality management and HACCP requirements. REFERENCES [1]. Atwell R. and S. Powel. 1997. The Konica Hygiene Monitoring System. Manchester Metropolitan University Summary Report. [2]. Numa, M., M. Miyazaki, K. Umehara and H. Kurata. 1997. Method of monitoring Washing Efficacy for Sanitation Control sanitation degree index as a process control method. Japonese Journal of Food Microbiology 13 (4) . [3]. Sasawaga, N. 1997. Hygiene Control by means of Washing efficiency management using Swab'N'Check. Japan Food Science, 36. [4]. Smith, P.K, R.L. Krohn,G.T. Hermanson, A.K. Mallia, F.H Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke and K. Olson, 1985. Measurement of Protein using Bicinchoninic acid. Anal. Biochem. 150, pp. 76-85. [5]. Tebbutt G.M. 1999. Comparison of traditional and rapid methods for assessing the risk of bacterial contamination from cutting boards. International Journal of Environmental Health Research, 9, pp. 67-74. ABOUT THE AUTHORS Daniela Botu, National Society Pasteur Institute, 333 Calea Giulesti Str., Bucharest 6, 060269, Romania, E-mail: daniela.botus@pasteur.ro Ramona Mihilescu, National Society Pasteur Institute, 333 Calea Giulesti Str., Bucharest 6, 060269, Romania, E-mail: ramona.mihailescu@pasteur.ro Virgilia Popa, National Society Pasteur Institute, 333 Calea Giulesti Str., Bucharest 6, 060269, Romania, E-mail: virgilia.popa@pasteur.ro Beatrice tirbu Teofnescu, National Society Pasteur Institute, 333 Calea Giulesti Str., Bucharest 6, 060269, Romania, E-mail: beatrice.stirbu@gmail.com