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analysis and control

Hygiene monitoring in support of food safety: a review of methods and industry trends
Good Hygienic Practices are an essential to ensure food safety. They are required by law under national and international Food Hygiene Regulations and are frequently considered as pre-requisites to food safety systems based on Hazard Analysis such as HACCP .

Preventing food poisoning is a key focus of any food safety system. Food poisoning is usually caused by the proliferation of undesirable micro organisms for which some of the most common causes include inadequate segregation of raw materials and finished product, inadequate temperature control during processing and/or storage, cross-contamination and inadequate sanitation. Accordingly Good Hygienic Practices are a primary preventative measure and the monitoring of their effectiveness not only provides and early warning of potential problems but also evidence of due diligence. Optimising cleaning programs also reduces costs (both in materials and labour time), reduces environmental waste and has a product quality dividend by improving shelf life. An optimised system frequently provides savings of 2050% in chemical usage. Insufficient regard is given to the technology and practice of cleaning and sanitation, and a simple bucket chemistry approach usually leads to ineffective and wasteful process. The choice and application of detergents and sanitisers is a science in itself, where optimum conditions for chemical dosing and contact time and temperatures are critical. Detergents are designed to remove organic matter of the product residue from surfaces as a primary process prior to adding a sanitiser to disinfect the cleaned surface. The effective removal of product residue is of prime importance since it not only removes gross contamination

(organic matter and 90% of the micro organisms) but removes any product residue that could support the subsequent survival and growth of microbes. Accordingly the effective removal of product residue is more important than residual micro organisms. But how can the efficacy of cleaning processes be assessed? In this article we will review the available methods for hygiene assessment and monitoring.

Detergents are designed to remove organic matter of the product residue from surfaces as a primary process prior to adding a sanitiser to disinfect the cleaned surface

more convenient, user-friendly formats that save time and labour in the small or busy laboratory e.g. ready-poured plates, dehydrated plates, dipslides, allin-one swabs and dilution devices, identification test strips etc. However the results are generally available in 2472 hours, which is too slow to provide useful feedback information to the sanitation and manufacturing processes and ensure that high standards of food safety and quality are maintained.

The Ideal test

The primary objective of cleaning is to remove product debris, so the ideal test to measure the efficacy of cleaning and hygienic status is a test for product residue itself. This should give rapid results to facilitate immediate corrective action, and be simple enough to be performed on the production floor by the sanitation crew or supervisor without the need for a laboratory. The philosophy of considering soil rather than just microorganisms to assess cleanliness is not new (Armbuster, 1962)1, and Griffiths (1997)2 state that freedom from organic soil is thus a better indication of cleanliness. Similarly, the removal of active components is the fundamental part of cleaning validation in the pharmaceutical industry.

Traditional hygiene monitoring

Until the 1980s the only method available to measure the hygienic status of food contact surfaces was the conventional cultural method based on agar plate counts. These methods provide information about the number of microbes present on the surface and also have the advantage of being able to detect specific indicator organisms. However these methods tell us nothing about product residue left on the surface that can support the survival and growth of microbes. Microbiological tests need to be conducted in a laboratory by a skilled technician. These traditional tests have been packaged into

Alternative methods and technologies

There are several alternative methods for measuring the hygienic status of product contact

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surfaces that approach the ideals above. There are instrumental methods and simple visible colour tests.

ATP Bioluminescence
In the 1980s the detection of ATP by a bioluminescence assay was applied to the detection of contaminants in foods and hygiene monitoring. Pioneered initially by Lumac, this biochemical test uses an enzyme luciferase that emits light in the presence of ATP. The light is measured quantitatively in an instrument called a luminometer and results are available in 20 seconds. Since almost all organic matter contains ATP (the universal energy carrier), it is present in almost all foodstuffs in huge amounts. ATP is also present in viable microbes (albeit in tiny amounts). Therefore most of the ATP detected on product contact surfaces is derived from food residues. Microbes present on cleaned surfaces (typically <500 cfu/100cm2) are too low to be detected by their ATP content (see Stanley, 19893; Kyriakides, et al., 19914). Many reports over the past 20 years have shown a good correlation between surface cleanliness and plate counts (see Kyriakides and Patel, 19945; Illsley et al, 20006), such that it is now a widely accepted method of hygiene monitoring by industry, retailer and regulatory agencies. The first luminometers were large bench-top instruments designed for laboratory use, and the test reagents were provided as freeze dried powder in bottles or vials of 2550 tests. These first reagents had a short working life when rehydrated, usually 12 days at refrigerated temperatures. This meant that the test needed to done by a skilled analyst. Furthermore, reagents were wasteful and hence the test was not cheap. Smaller, portable luminometers were then made that could fit into a briefcase and were easily carried, however they

generally required two hands to operate and so the instruments were used on a bench or desktop but away from the laboratory close to the production area. There are several commercial systems for measuring ATP bioluminescence and hygiene applications and the current trend is to add other features to the instrument to extend its application either to other ATP based assays, or other tests such as pH and temperature. The next

of the drawbacks. The snapshot device is stable for 12 months at refrigerated temperatures and is very robust, being tolerant to temperature abuse at 21C for 46 weeks without significant loss of activity or performance. The ATP test is a biological assay that uses an enzyme to generate the test result. Biological tests are usually more specific and more sensitive than chemical tests that are themselves usually more precise and reproducible. The ATP test is very sensitive and will detect very small amounts of product residue, typically <1ppm depending on the foodstuff. Cleaned surfaces can be expected to show 1050fmols of ATP depending on the surface type and processing material. Most ATP systems can detect approx 1fmol ATP which exceeds the requirements of even the most demanding customers, and so sensitivity is of little significance when comparing systems. Griffiths et al (1997) compared several commercial systems and stated that swabbing (for sample collection) is the biggest source of variation with the ATP test and also that manufacturers sensitivity claims should be interpreted with caution. The total

Pioneered initially by Lumac (now Hygiena), this biochemical test uses an enzyme luciferase that emits light in the presence of ATP
development in instrumentation was the truly portable palm-sized instrument where the challenge is to reduce the capital cost of the instrument without compromising performance of the test. In addition, advances in miniturisation and computerisation enable small instruments to have a large capacity for data storage and analysis. Instruments can display simple statistics as well as download to a computer. Simple but sophisticated software provides the ability to program sample identification data back to the instrument and download data for further data manipulation, record keeping, trend analysis and due diligence. Reagents for ATP bioluminescence and their packaging have also been improved to provide singleshot, all-in-one tests systems that offer convenience and ease of use. However the majority of these use the same freeze-dried reagent technology and reproducibility can be compromised in single-shot devices. A novel liquidstable luciferase has been developed that has none

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protein or simple sugars. Protein tests generally detect protein and amino acids only and are applicable to foodstuffs that are high in protein such as meats, whereas sugar tests detect a much broader range for foodstuff. There are several different commercial protein test kits that operate on different detection technologies and some require a total test time of 1 minute while others require 10 minutes to demonstrate the absence of contamination and verify cleaning has been conducted properly. Several protein tests change colour from green to purple in 110 minutes depending on the contamination level. SpotCheck and SpotCheckPlus detect simple sugars and changes from colourless to green where the speed and intensity of the colour change over a 60 second period are indicative of the level of contamination.

simplest, fastest, most sensitive technique for rapid hygiene monitoring. It correlates well with contaminations levels and is widely accepted. Rapid Hygiene tests provide additional information in a timely manner to supplement food safety programs by facilitating immediate corrective action, providing evidence of due diligence, optimising manufacturing processes and reducing costs whilst providing a product quality dividend.

ATP bioluminescence in the dairy industry

Most businesses today are trying to achieve more with less and the diary industry is no exception. Cost saving measures such as just-in-time and reduced inventory are being used and rapid tests for quality and safety can offer significant benefits. The implementation of management systems for food safety and quality provide evidence of due diligence and traceability that enable quality and shelf life improvements whilst reducing the risk of downgraded product, product recalls and bad press that all have major cost implications. These requirements and associated audits increase the demands on staff operating management systems. Skilled technical labour can be difficult to find and pressures to reduce labour costs can result in a lack of skills in some technical functions particularly in the laboratory or hygiene departments that are in the frontline of monitoring and protecting quality and safety of products. These pressures demand that test methods are simple, convenient and low cost. ATP bioluminescence is being used for several test applications within the dairy industry (Kyriakides 19945), and recent developments have made it more cost effective. There are two main applications: 1. The direct replacement for the

sensitivity of a detection system is a function of the swab and swabbing solutions ability to remove the bioburden from the surface, the ability of the extractant to extract cellular ATP and the ability of the reagents and instrument to use the ATP to generate light.

Colour hygiene test

Several different technologies can be harnessed to provide a simple colour test that is visible to the naked eye, do not require an instrument and give results in 110 minutes. Accordingly these tests are appropriate for small and large food processors but also for catering outlets and food service applications. These tests are generally less sensitive than ATP bioluminescence but provide a simple, semi-quantitative hygiene test. These biochemical tests can detect ATP, or NADPH (another energy-carrying molecule present in organic matter and living organisms), or protein, or simple sugars. Some produce a single colour change while others produce a multiple colour change depending on the level of contamination. There are singleshot, all-in-one test formats and reagent kits in bottle format. The two most common commercially available colour hygiene tests are those detecting

The two most common commercially available colour hygiene tests are those detecting protein or simple sugars

There is an acceptance that rapid hygiene monitoring methods that detect food residues on product contact surfaces provide a direct and relevant measurement of cleaning efficiency and hygiene. Rapid Hygiene test methods are not intended to replace the traditional cultural methods. The developments in technology and convenience packaging provide a variety of technologies and products that are user-friendly, affordable and applicable to almost all food processors and caterers. The ATP hygiene test is the

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bacterial test for long life sterile products 2. A rapid test for cleaning efficiency and hygiene monitoring. The sterility application test requires that the long life product is incubated at elevated temperature for ~24 hours to allow any surviving organism to grow to detectable levels (>500,000 per ml) prior to testing that gives results in less than 1 minute. Laboratory instrumentation can be either small manual instruments or fully automated systems with reduced labour requirements. Accordingly, long life products can be released to the market earlier giving major savings (10,000s) in warehousing costs and reduced capital tied up in inventory. Other test applications for ATP bioluminescence include raw material screening and forcing tests to predict the shelf stability of diary products, but these are not well established because the ATP content of the product is much higher than that of microorganisms and it is difficult to separate and differentiate the two sources of ATP. The most widely used test application of ATP bioluminescence is that of rapid hygiene monitoring because it delivers a direct, objective test with simple, easy-to-use equipment at low cost. The technology and application has been established for over 30 years and is widely recognised as a test of cleaning efficiency (Griffiths 19972). It can be applied to equipment and processes for milk collection, storage and haulage and all subsequent production equipment where cleaned manually or by clean-in-place systems. The benefits include:

Figure 1: Effective monitoring drives continuous improvement of hygiene standards

optimisation of cleaning chemicals giving potential savings of 2550%.

Result in 15 seconds permitting immediate corrective actions such as re-cleaning. Rapid results also enable fast traceback and troubleshooting to quickly identify and prevent problems from escalating out of control. Reduced risk of cross contamination that facilitates the production of high quality products with best

simultaneously removed by the cleaning processes. Microbes present on clean product contact surfaces (typically <500cfu/100cm2) are too low to be detected by their ATP content (Stanley, 19893; Kyriakides et al 19914). A study of the cleaning of 465 milk haulage tankers was conducted and six internal sampling areas were compared using both the traditional microbiological methods and the rapid ATP test. The following conclusions were drawn;

The microbiological method produced a false view of the hygienic status of surfaces The ATP test method gave a good indication of cleanliness Implementation of the routine ATP test improved tanker cleaning by 77% as shown by a reduction in failure rates from 30 to 7% (see Figure 1).

shelf life.

Generation of data that provides evidence of due diligence for auditors and trend analysis for continuous improvement

SystemSURE Plus
Advances in solidstate technology have enabled new instruments to be developed that deliver performance, convenience and robustness at lower cost. One such instrument is SystemSURE Plus (Hygiena International) which also has the following unique features;

Direct detection of product residue that should have been removed by cleaning Improved cleaning efficiency and

The rapid ATP hygiene test measures ATP from all sources which is mostly derived from food residue. This application is not a replacement bacteria test, however, there is a good relationship between the two methods, primarily because both product residues and microbes are

Low background that enables reliable detections at low ATP

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levels therefore reducing false positives

Table 1: Comparative performance of two ATP test systems in a leading UK dairy

Parameter Total samples Total Passes; - Biotrace UniLite - SystemSURE Plus Total Fails; - Biotrace UniLite - SystemSURE Plus Passes by both systems Fails by both systems Fail by Biotrace / Pass by Hygiena Pass by Biotrace / Fail by Hygiena True Positive False Positive False Negative True Negative Sensitivity Specificity Accuracy

All sample locations 278 240 240 38 38 237 33 2 (0.7%) 3 ( 1.1%) 237 3 2 36 99.2% 92.3% 98.2%

The RLU output scale is quantitative and 1 RLU = 1fmol ATP because high RLU numbers do not mean higher sensitivity Self-calibration that significantly reduces service and maintenance costs Secure data download to PC by either of two software options including advanced, simplified trend analysis (Sure Trend) with pre-defined and customised report generation at the click of a button Other features include sampling plans, re-test tag and trace function, on-screen statistical review of results without the need to download to PC.

Figure 2: Trend analysis of rapid hygiene test results in a dairy production line

Sampling devices are available for solid surfaces (Ultrasnap) and for liquid samples (Aquasnap) such as CIP rinses and other water samples. All sampling devices have a simple snap and squeeze activation step with 12-months shelf life and tolerance to ambient temperature abuse. Sample devices are slim and lightweight thus combining ease-of-use with reduced environmental impact. SystemSURE is used by many UK and International dairy companies whose evaluations have demonstrated equivalent performance to other systems. A leading UK dairy processor compared SystemSURE Plus with the Biotrace UniLite system in a working dairy situation and found >98% agreement in terms of sensitivity, specificity and accuracy (see Table 1).

In addition, SystemSURE Plus reveals trend analysis data sooner and more clearly than Biotrace. Figure 2 shows a similar trend for both systems, however, all the Biotrace results are passes (RLU <150) and no failures. The systemSURE data (at equivalent RLU Pass/Fail settings) shows three fails. This is a function of the low background and low signal to noise ratio of SystemSURE. It is very important to know the inherent variability of the system

at low ATP levels in order to properly assess the overall tolerance and performance of any system. In summary, improvements in technology enable business to do more with less by making processes more efficient, eliminating waste and reducing risk. Rapid test methods based on ATP bioluminescence are well proven and have a broad range of applications. There are no significant differences in performance between available systems. Modern solid-state ATP systems deliver simplicity, convenience, performance and reliability at best value. Martin Easter BSc, PhD General Manager Hygiena International Ltd www.hygiena.net

References 1. Armbuster, E. H. (1962) Evaluation of surface contamination. J. of Environmental Health. 25: 26 - 29. 2. Griffith, C. J., Davidson, C. A., Peters, A. C. and Fielding, L. M. (1997) Towards a strategic cleaning assessment programme: hygiene monitoring and ATP luminometry, an options appraisal Food Science and Technology Today (1997) 11 (1) pg 15 -24. 3. Stanley , P.E. (1989) A review of Bioluminescent ATP techniques in rapid microbiology. J. Biolumin. Chemilumin. 4: 376 -380 4. Kyriakides, A. L., Costello, S. M., Easter, M. C. and Johnson, I. (1991) Rapid hygiene monitoring using ATP bioluminescence, in Bioluminescence and Chemiluminescence: Current Status (eds. Stanley, P. E. and Kricka, L. J). John Wiley and Sons, Chichester, pp. 519 - 522. 5. Kyriakides, A. L. and Patel, P. D. (1994) Luminescent techniques for microbiological analysis of foods. In Rapid Analysis Techniques in Food Microbiology . (eds. P. Patel). Blackie Academic and Professional, Glasgow, pp.196 - 231. 6. Illsley, R. A., Jackson, E. D., McRae, K. B. and Feirtag, J. M. (2000) Dairy, Food and Environmental Sanitation., 20; (7), 522 - 526.

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