QUALITY MANAGEMENT: CONTROL OF SURFACE CLEANNESS BY SANI-TEST

DANIELA BOTU, RAMONA MIHILESCU, BEATRICE TIRBU, VIRGILIA POPA S.N. Institutul Pasteur S.A., pasteur.biomol@pasteur.ro
Keywords: proteins, surface, cleanness

Summary
To be hygienic, 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. The kit does not directly indicate microbial activity.

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 (Tebbutt, 1999, Numa, 1997). 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. 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 (Atwell, 1997, Sasawaga, 1997). 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 helpful 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 (Smith, 1985).
1. MATERIAL AND METHOD

For assessment of the cleanness degree of surfaces, we used SANITEST, 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
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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 color chart for hygiene, allowing qualitative results assessing the cleanness of the surface. Field trials were carried out in a poultry slaughterhouse & processing company (31 samples) and a restaurant (50 samples), following the SANITEST instructions for use. The tests were done after routine cleaning/disinfection procedures of surfaces, before a new working day start. Additional work was done by evaluating the effect of temperature on rate and sensitivity SANI-TEST chemical reaction, by incubating the samples of various protein concentrations at 370C (bovine serum albumine: 0, 10, 25, 50, 100, 250, 500, 750 i 1000g protein/100l) and recording the time needed for color exchange. We studied the effect of various cleaners/disinfectants on test sensitivity. As cleaners or disinfectants we used products that contained: sodium hypochlorite, chlorine, glutaraldehide, alkyl-dimethyl-benzylammonium 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.
2. RESULTS AND DISSCUSIONS

The SANI-TEST kit uses the Biuret reaction to detect the presence of proteins on working surfaces. 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 kit is not intended as a microbiological monitoring tool, as it does not directly indicate microbial activity. The 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 & processing company and a restaurant. The reaction is time-dependent i.e. the color develops with time and therefore it is important to record color change after 10 min and disregard any color change after this set time. However the devices can be incubated for
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longer times at ambient temperature (15 25 C) if sensitivity greater than 50 g /test is required. Result interpretation was done according to the four-levels color chart (Table 1).
Table 1 Protein concentration corresponding to color level with SANI-TEST and color change interpretation Level 1 2 3 4 Color Green Grey Light purple Dark purple Protein amount (g) 0 50 50 100 100 500 500 - 1000 Color change interpretation Clean surfaces Caution low levels of contamination present Significant levels of contamination present High levels of contamination present Pass Caution

Fail

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. Poultry slaughterhouse & processing company For the purpose of this trial, 31 areas were analyzed. The results were scored as levels of contamination (Table 2).
Table 2 Results of SANI-TEST, poultry slaughterhouse & processing company Level 3&4 Level 1 green Level 2 grey Tested areas purple/dark purple PASS CAUTION FAIL Conveyor belt X Stripping conveyor X Bandsaw blade X Weigh bin X Table 1 X Blade 1 X Transfer belt X Hygiene trolley X Mesh sieves X Ledge X

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Large bin Table 2 Blade 2 Chiller walls Freezer walls Underneath wash basin Golves / production On/off switch Control panels Underneath weigh bin Steps to weigher Door handle/blast chiller Cutting table 1 Cutting table 2 Packing belt Packing table Holding chiller entrance Tunnel belt Knife Bagging area Start button conveyor TOTAL

X X X X X X X X X X X X X X X X X X X X 22 5 X 4

Analyzing the results, 13 % of those areas tested (from total of 31 samples) were shown to have significant level of contamination producing a purple or dark purple color results; 16% showed low levels of contamination grey or caution result and 71% were clean a green color result. Restaurant There were analyzed 50 areas from a restaurant and the results are shown in table below (Table 3). The trial was performed before of a new work day.
Table 3 Results of SANI-TEST, restaurant Tested areas kitchen table 1 kitchen table 2 glasses (5 pieces) Level 3&4 Level 1 green Level 2 grey purple/dark purple PASS CAUTION FAIL X X 3X 2X

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cups (3 pieces) plates (10 pieces) forks (6 pieces) spoons (6 pieces) knives (6 pieces) pots (3 pieces) fryingpan (2 pieces) refrigerator (inside) refrigerator door handle underneath wash basin kitchen ledge bar table kitchen floor kitchen wall tiles TOTAL

3X 8X 4X 6X 6X 2X 1X X

2X 1X

1X 1X

1X X X X

X X X 37 9 4

The results obtained by applying SANI-TEST on restaurant samples show that 8 % of those areas tested (from total of 50 samples) were shown to have significant level of contamination producing a purple or dark purple color results; 18% showed low levels of contamination grey or caution result; 74% were clean a green color result. 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. Effect of temperature on rate and sensitivity of SANI-TEST chemical reaction Additional work was carried out on the test kinetics and this showed that the rate of reaction was dependent on the incubation temperature of the assay as well as the amount of protein present.
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In figure bellow it is shown the time taken to reach the different color levels for a range of protein (BSA) concentration.
40 35 Time to colour level (min) 30 25 20 15 10 5 0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 level 2 level 3 level 4

g BSA/100 l

Fig. 1. SANI-TEST the time taken to reach color levels for different protein (BSA) concentrations

The data above demonstrates that a direct relationship exists between sensitivity and rate of the chemical reaction. Sensitivity of the test procedure was examined at 370C over time (Table 5).
Table 5. SANI-TEST the time taken to reach color levels for different protein (BSA) concentrations, incubation at 370C BSA (g/100 l) 1000 750 500 250 100 50 25 10 0 Time (min) 0 1-2 1 1 1 1 1 1 1 1 2 3-4 3 1-2 1-2 1 1 1 1 1 4 4 3-4 2-3 2 1-2 1 1 1 1 6 4 4 3-4 2-3 1-2 1-2 1 1 1 8 4 4 4 3 2 1-2 1 1 1 10 4 4 3 2 1-2 1 1 1 12 4 3 2 1-2 1 1 1 14 3 2 2 1 1 1 16 3 2-3 2 1 1 1 18 3-4 2-3 2 1-2 1 1 20 3-4 2-3 2 1-2 1 1 25 4 3 2-3 1-2 1 1

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30 35 40 45 50 60 70

4 4

3 3 3 3-4 4 4 4

2-3 2-3 2-3 3 3 3 3

1-2 2 2 2 2-3 2-3 2-3

1 1 1-2 1-2 2 2 2

1 1 1 1 1 1 1-2

Key: 1-green; 1-2-green-grey; 2-grey; 2-3-grey-purple; 3- light purple; 3-4-mid purple; 4-dark purple

It can be observed that lengthening the test incubation time substantially increased the assay sensitivity by allowing the chemical reaction to continue over time. The rate of this color change reaction was also observed to increase when carried out at 370C, compared to ambient temperature (22-250C). The 10 g/100l sample could be detected (clearly different to control) after 50 minutes at 37C. The 25 g/100l sample gave a clear positive (level 2) after 35 minutes and was different to the control (0g), although the color change (level 1/2) at 20 minutes was not distinct enough at this stage to give a clear positive. The control swabs (no addition of protein) started to change to a grey color after 70 minutes. 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 6).
Table 6 Effect of cleaners/disinfectants on SANI-TEST results Effect Type / active Concentration Positive (protein Negative ingredient 350 g BSA) (no protein) Sodium hypochlorite 0.9% (1x) No effect No effect / disinfectant (level 3 purple) (level 1 green) Chlorine / anionic 1.2% (1x) No effect No effect surfactant (cleaner) (level 3 purple) (level 1 green) glutaraldehide / 0.25% (1x) No effect No effect disinfectant (level 3 purple) (level 1 green)

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glutaraldehide / disinfectant

2.5% (10x)

Alkyl-dimethylbenzyl-ammonium chloride / sanitiser chlorhexidine / disinfectant disinfectant

25 (1x) 0.67% (1x)

Enhancement of color change (from level 3 to 4 purple to dark purple) No effect (level 3 purple)

False positive (level 2 grey in 10 min.) No effect (level 1 green) No effect (level 1 green) False positive (level 3/4 purple/dark purple in 10 min.) False positive (level 1/2 green/grey in 10 min.)

disinfectant

No effect (level 3 purple) 0.01% (not known) Enhancement of color change (from level 3 to 4 purple to dark purple) 0.001% (not known) No effect (level 3 purple)

It can be observed that there is no interference with most common sanitizers at normal working concentrations. However high concentrations of alkaline cleaners may cause the enhancement of color and false positive results in some instances. Peroxide based disinfectants can cause a false positive color change reaction with SANI-TEST. 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 color 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 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.

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3. CONCLUSIONS

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 useful for quality management and HACCP requirements.
REFERENCES

1. Atwell R, S. Powel - The Konica Hygiene Monitoring System. Manchester Metropolitan University Summary Report, 1997. 2. Numa, M., M. Miyazaki, K. Umehara, H. Kurata - Method of monitoring Washing Efficacy for Sanitation Control sanitation degree index as a process control method. Japanese Journal of Food Microbiology 13 (4), 1997. 3. Sasawaga, N. - Hygiene Control by means of Washing efficiency management using Swab'N'Check. Japan Food Science, 36, 1997. 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, K. Olson - Measurement of Protein using Bicinchoninic acid. Anal. Biochem. 150, 76-85, 1985. 5. Tebbutt G.M. - Comparison of traditional and rapid methods for assessing the risk of bacterial contamination from cutting boards. International Journal of Environmental Health Research, 9, 67-74, 1999.

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