The HACCP-driven hygiene procedures in place in modern food production facilities are an essential part of ensuring safety and quality in the food supply chain. Clean food handling and processing equipment is a prerequisite for good hygiene, but the effectiveness of cleaning must also be monitored and recorded. The techniques available for hygiene monitoring have advanced considerably over the last 20 years with the development of ATP-bioluminescence as the method of choice. The latest test equipment is capable of producing rapid results on site that can be used in real time to validate cleaning and detect the build up of soiling before it becomes a source of contamination.
Preventing contamination, especially microbial contamination, of the food supply is one of the underlying principles of ensuring food safety and quality. Food products contaminated with bacterial pathogens are potential vehicles for human infection and large microbial populations can cause rapid spoilage and dramatically reduce product shelf life. One of the most basic preventative measures available to food manufacturers is effective cleaning of all food contact surfaces, utensils and equipment. Soiled surfaces can act as a reservoir for microbial contamination and allow the build up of complex biofilms, which protect microorganisms from cleaning and sanitising agents and can be extremely difficult to remove once established. It is just as important to keep non-food contact areas, like floors and drains, clean, since any permanent source of contamination can act as a beachhead for re-colonisation of food processing equipment.
A clean facility is the best starting point for hygienic food production and there is no substitute for proper cleaning and sanitising procedures. As one might expect, the requirement for clean premises and equipment is written into EU food law. EC food hygiene Regulation 852/2004 stresses the importance of HACCP-based food safety systems and best food hygiene practice in all food handling operations and places the responsibility for controlling food hazards squarely with the manufacturer. It also contains requirements for factory designs and materials that facilitate cleaning and the following specific requirement for food contact surfaces:
“All articles, fittings and equipment with which food comes into contact are to be effectively cleaned and, where necessary, disinfected. Cleaning and disinfection are to take place at a frequency sufficient to avoid any risk of contamination.”
The obligation for food businesses to clean processing equipment effectively is plain, but there is also a need to demonstrate that cleaning procedures are effective and to assess and document cleaning operations on a day-to-day basis. While a simple visual check may be useful, it is far too limited as a means of confirming that hygienic processing conditions have been achieved, or identifying contaminated locations. Heavy visible soiling often correlates closely with high levels of microbial contamination, but a surface that looks clean can actually be harbouring sufficient organic material to support the growth of substantial numbers of bacteria, or even an established biofilm. What is needed is to combine visual inspection with a sensitive, scientifically objective method of hygiene monitoring.
Until the late 1980s, microbiological testing was virtually the only practical hygiene monitoring method available. Using a swab, sponge or contact plate to sample the microbial population on a surface can provide valuable information about the level of contamination. The sampling device is used to remove microorganisms from a known area (typically 100 cm2) of a surface such as a worktop or conveyor, or from equipment known to be a potential source of contamination, such as a valve or pump. The material collected is then suspended in a suitable diluent and transferred to a microbiological culture medium, usually in agar plates. After a period of incubation the degree of contamination can be measured by counting any visible colonies. The main advantage of microbiological monitoring is that it provides a direct indication of microbial contamination, and it is also possible to test for the presence of specific organisms, such as foodborne pathogens.
Unfortunately, there are also a number of limitations, most notably the time taken to obtain results. The culture step normally requires at least 24 hours of incubation and must be carried out in a properly equipped laboratory, which is often off-site. This means that results are only ever available retrospectively and are of limited value for HACCP monitoring purposes, or for rapid cleaning assessment. Even if swabs are taken immediately after cleaning and disinfection, at least a full day of production is likely to have been completed before there is any information available on contamination levels. Results can also be badly affected by residual antimicrobial activity remaining on surfaces after sanitising, so that a special diluent containing inactivating compounds is required to prevent inhibition in culture.
Nevertheless, microbiological sampling and testing remains a valuable technique, particularly for monitoring vulnerable equipment and fittings for pathogen contamination. Foodborne pathogens like Listeria monocytogenes are able to persist and multiply under cool wet conditions and are a major problem in facilities handling chilled foods. Microbiological testing remains the best way to monitor Listeria contamination and is a legal requirement in US meat processing plants.
The rapid alternative
The serious limitations inherent in microbiological testing as a hygiene monitoring method led to growing interest in more rapid methods during the 1980s as HACCP developed and the importance of rapid testing that could be carried out on the factory floor was recognised. Many techniques were investigated, most of which relied on detecting microbial cells more quickly than culture methods. One of these was the ATP-bioluminescence method, first developed commercially as a laboratory-based rapid microbial detection and enumeration method by Dutch company Lumac.
Adenosine-5′-triphosphate (ATP) is a compound found in all metabolically active living cells as a convenient means of storing energy at a molecular level. The energy stored in ATP molecules can be detected and quantified by means of a bioluminescence reaction, such as the one responsible for the light produced by the firefly. This reaction is catalysed by the enzyme luciferase and utilises energy stored in ATP to break down a substrate (luciferin) and release visible light in the following two-step reaction.
D-luciferin + ATP → Luciferyl adenylate complex + PPi
Luciferyl adenylate complex + O2 → Oxyluciferin + AMP + CO2 + Light
As the oxyluciferin molecule produced returns from an excited to a stable state, energy is released as a photon, so that there is a direct relationship between the ATP present and the light produced. By measuring the amount of light generated in the reaction using a luminometer, the level of ATP present can be estimated within a matter of seconds. Typically, the result is expressed in relative light units (RLU) and is directly proportional to the quantity of ATP.
ATP-bioluminescence was originally employed to detect and quantify the ATP present in microbial cells and estimate the population present. But all metabolically active cells contain ATP, and food residues contain much more than the microbial cells. To measure only microbial ATP, it is necessary to remove the non-microbial ATP first. This can be done by adding reagents containing enzymes and surfactants to extract and eliminate non-microbial ATP and then lysing any microbial cells present to release microbial ATP. Unfortunately, these additional steps mean that the assay takes longer to complete and loses sensitivity, and the additional reagents add cost. When large amounts of non-microbial ATP are present – almost always the case in unprocessed food samples – the method is less than ideal as a means of detecting microbial contamination. ATP-bioluminescence is still widely used to enumerate microbial contamination in pharmaceutical laboratories and for sterility testing of UHT processed food products, but is rarely used for other applications in food microbiology laboratories.
The breakthrough for hygiene monitoring came with the realisation that, by estimating the total amount of ATP present, it would be possible to quantify the amount of organic matter present on a surface, and therefore assess its cleanliness. So instead of being a technique for assessing levels of microbial contamination, ATP-bioluminescence became a rapid method to monitor factory hygiene, neatly turning the method’s drawbacks into assets. It is known that removal of organic food residues from processing equipment also removes about 90% of the microorganisms present and helps prevent the survival and subsequent growth of those that remain. This means that the amount of organic material present has a direct relationship with the potential for microbial contamination.
Early hygiene monitoring applications of ATP-bioluminescence used large, laboratory-based luminometers and relatively expensive freeze-dried test reagents, which had a very short life once rehydrated. Swabs had to be transported from the factory floor to the laboratory for testing. Smaller, portable luminometers and more convenient reagents were then developed that could be used outside the laboratory by less highly skilled staff. This made it possible to use the technology on-site, so that hygiene staff could use the results to assess the effectiveness of each clean and either pass equipment for production, or order further cleaning.
Today’s luminometers are extremely portable, hand-held devices containing the computing power needed to handle large amounts of sample data that can be integrated with electronic HACCP databases and other systems. Reagents have also undergone development and can now be supplied in ‘single-shot’ dispensers with an integral swab, which can be fitted directly into the luminometer to take a reading immediately after sampling. These systems are extremely robust and easy to operate, so that very little training is required and hygiene staff can use them to validate cleaning operations and commence production without delay.
A number of handheld luminometers designed specifically for hygiene monitoring are now commercially available. One of the best known is the SystemSURE Plus™ system supplied by US-based Hygiena. This is the latest development in a line of SystemSURE devices and is extremely small and light, weighing less than 300g. It can store sample location codes in programmable test plans and can record up to 2000 test results in a database. Reagents for surface tests are supplied in a pen-like sample collection device called Ultrasnap™, which does not use freeze-dried enzymes, but a liquid-stable reagent. The integral, pre-moistened swab is used to take the sample and replaced in the device. The reagents are then released and the Ultrasnap is placed in the luminometer to read the amount of light produced in RLU. The system is designed to be quick and simple to use. Hygiena also produces Snapshot™, a universal ATP test for use with other luminometer designs, such as the Lightning MVP® from BioControl Systems and Charm Sciences’ NovaLUM instrument.
A similar hygiene monitoring system is marketed by 3M™, in the shape of the Clean-Trace™ luminometer and surface ATP test, which also uses a liquid-stable enzyme preparation. These instruments are now so easy to use that they have become the method of choice in the food processing industry for hygiene monitoring. Costs have come down as the technology has developed, with hand-held luminometers now being priced at €750 – 3,000 and the price-per-test typically €1-2. It has been estimated that at least 80% of European and North American food processing facilities currently use ATP bioluminescence for hygiene monitoring.
Practical application of ATP-bioluminescence
Although the technology for hygiene monitoring by ATP-bioluminescence is now readily available, there are a number of points to consider in how it should be used.
1. Sample locations – The technique is best suited for testing food contact surfaces like conveyors, cutting boards and utensils and hard to clean areas, such as corners, joints and seals. But it may also be useful to monitor non-contact locations that can act as reservoirs of cross contamination. Examples might include sinks and machine parts close to food contact areas. A survey of the production line and historical data on cleaning can help to identify suitable sample locations where there is the greatest risk of contamination and soiling. Typically a 10 x 10 cm area would be swabbed, or as much of the surface as possible in hard to clean places.
2. Calibration – It is important to establish ‘threshold’ levels of ATP (expressed as RLU) for each sample location. This can be done by taking a number of readings at the same location over several days when cleaning is observed and known to have been carried out to an appropriate standard. The mean reading then becomes the threshold or ‘pass’ level. A ‘fail’ level is typically set at two or three times the threshold value and means that a re-clean is needed, with any readings between pass and fail being treated as ‘caution’ level.
3. When to take samples – Ideally ATP testing should be done after cleaning, but before the sanitising or disinfection step. This is partly because some sanitizers can cause interference in the test by reacting with the reagents, but also helps to prevent the waste of expensive sanitizers on surfaces and equipment that will require re-cleaning. It is also important to test as soon as possible after cleaning, as ATP is broken down quickly outside living cells. If the samples are taken after an interval of more than two hours, the results may be invalid and give a false impression of the cleanliness of the sample location.
A properly set up ATP bioluminescence hygiene monitoring system can provide data in real time to help hygiene staff assess the effectiveness of cleaning and determine whether it is safe to begin production. The data can also be used over longer periods for trend analysis to see whether there is build up of soiling and contamination at certain points, so that it can be dealt with at an early stage. Detailed monitoring also helps to optimise cleaning procedures and ensure that the plant is not ‘over-cleaned’, which could help cut wastage of cleaning chemicals and reduce costs. The principal disadvantage is that the readings obtained do not correlate directly with bacterial contamination. A low RLU value may still mean high levels of bacteria. It is advisable to carry out some microbiological testing in addition to ATP, especially where Listeria is a potential problem. Nevertheless, ATP-bioluminescence can reasonably claim to have revolutionised hygiene monitoring in the food processing industry, driving up standards and reducing contamination levels in food. Its contribution to food safety continues to be considerable.