A quick and simple way to detect organic residue and a wide range of allergens, protein swabs are a popular choice across the food and beverage production industry. In fact, Food Safety Magazine carried out a survey towards the end of 2018 which revealed that 17% of sites rely on protein-specific tests for their allergen management.

Despite their popularity, many sites have expressed frustration at the limitations of protein swabs. One of the key complaints is that protein-specific tests are qualitative – generating non-specific and non-numerical results. There is also the question of the reduced efficiency that occurs when they are tasked with detecting a substance that has low levels of residual protein contamination.

With this in mind, we have put together an honest comparison of protein swabs and a newer methodology: the A3 system. This article explores the benefits and limitations of both approaches, helping you to decide on the best solution for your site and applications.

If you currently use protein swabs at your site, but have limited to no experience with the A3 system, the following video will guide you through the essentials:

For more information on the A3 system, you can also follow the link below to watch a webinar on Hygiene Monitoring in High Care Environments. Hosted by Klipspringer Director Alex Carlyon, our in-house A3 specialist Radek Tameczka, and scientific advisor Dr. Stephan Speidel, this webinar explores the limitations of conventional hygiene monitoring methods and provides real-world examples of A3 verification at High Care production sites.

The detection of raw fish or meat

If your site handles the production of raw fish or meat, this is one area of the industry where it is especially important to understand the different detection sensitivities of protein swabs and the A3 system. Failing to do so could result in cross-contamination and could pose a serious risk to the safety of your product.

Below are the results of comparative tests carried out by Wataru Saito and Mikio Bakke for the Kikkoman Biochemifa Company. These tests relate to the sanitation monitoring of stainless steel when exposed to raw fish and meat.

The orange markers reflect the four points at which testing was carried out. The first test took place before the surface was washed, the second after it was rinsed with cold water for fifteen seconds, the third after a fifteen second rinse with water at 50°C, and the fourth after the surface was cleaned with detergent and a sponge.

As you can see, the benchmark value of 200 RLU was put in place for the A3 system and it was only reached after the final test when the surface had been thoroughly washed with detergent.

In contrast, the protein swab detected salmon and chicken residue before the first wash and detected a trace of chicken after the second – turning purple to reflect these findings. Aside from this, the protein swab incorrectly passed the stainless steel surface, failing to detect the raw fish and meat. 

These findings suggest that if the denaturation of proteins occurs through processes such as thermal processing or exposure to chemical agents, the results of protein-specific tests will be compromised. The accuracy of the A3 system; however, is unimpacted by such changes.

The detection of nuts

Further research from the Kikkoman Biochemifa Company includes a comparison of the following methodologies for the detection of peanut residue:

  • Protein Swabs
  • The A3 System
  • Conventional ATP Tests
  • Lateral Flow Detection Methods (LFD)

During this study, 10g portions of peanuts were mixed with 90ml of sterile distilled water and homogenised to generate 10-fold dilutions, before 10-fold serial dilutions were carried out.

100 RLU was established as an action level, with the calculated concentration equivalent displayed in the figure below. The concentration of total protein was also evaluated by the ELISA and Bradford assays to produce the theoretical dilution factors that are plotted with the blue and red Xs.

As you can see, the protein swab test could only detect samples up to a 10²-fold dilution, making it only slightly more effective than the conventional ATP tests.

In contrast, the A3 test proved to be much more effective. In fact, the system was sensitive enough to detect the 10ppm (parts per million) levels of peanut protein calculated by the ELISA and Bradford assays – proving to be the most sensitive of the three methodologies.

The detection of milk

If your site handles milk or dairy products, it would also be worth evaluating the findings of a Case Study carried out at the Saitama Kikkoman Company Processing Plant.

In this study, the effectiveness of an A3 system was compared to that of conventional ATP tests and protein swabs.

Products using raw milk were selected and nine sampling points were tested:

  • A silicon spatula
  • Wire mesh at raw material inlet
  • The insertion slot for raw materials
  • Nearby surface to the insertion slot
  • Inner wall of mixing tank
  • Impeller of mixing tank
  • Hopper
  • Nozzle
  • Stainless steel saucer of filling machine

These sampling points were tested at three stages: before cleaning, after a rinse with 75°C water, then after a wash with 90°C water and an alkaline cleaner, followed by a rinse with 75°C water.

The ATP test could only detect samples up to a 10²-fold dilution, generating a reading of just 13 RLU. Similarly, the protein swab could only detect residue up to a 10²-fold dilution. In contrast, the A3 system was still providing a reading for the 10⁵-fold dilution, generating a RLU value of 86.

An ELISA test for milk protein was run alongside these tests. It revealed the extent to which milk protein decreased throughout the cleaning process: 10-40 μg before the first wash and 0.1 μgo or less after the final wash. 

  • Square: before washing
  • Triangle: after first wash
  • Circle: after final wash

The above chart illustrates a clear correlation between the results produced by the A3 system and the ELISA test for milk protein. This correlation only existed for the less sensitive protein swabs and ATP tests up to a 10²-fold dilution. In contrast, the A3 system supported the findings of the ELISA testing throughout the entire cleaning process – acting as the perfect example of how the A3 methodology could work in tandem with your site’s allergen control system.

The detection of allergens

Someone who suffers with a food allergy will experience an allergic reaction when their immune system recognises proteins in certain foods. This is why protein-specific tests are often linked to allergen management.

The A3 system detects all three forms of the adenosine molecule: ATP, ADP, and AMP. It does not; however, detect allergenic proteins, which means allergens cannot be identified or assayed directly. In fact, it’s important to stress that the A3 system cannot replace allergen testing and should never be relied upon for this function.

With this in mind, it would be easy to assume that protein swabs should play a much more central role in your site’s allergen control program than the A3 system.

However, this is challenged when you factor in the heightened sensitivity of the A3 system and its unique ability to determine the level of organic residue on a surface. After all, if a surface is free from organic residue, it follows that there will be no allergen residue left to cross contaminate your product.

Consequently, the A3 system can work in conjunction with your allergen testing, with the potential to reduce the frequency of your tests and the ability to provide a much more accurate picture than the less sensitive and often incorrect protein swabs.

To support this point, Saito and Bakke also carried out a thorough comparison of the rapid hygiene tests used for the detection of allergenic foods. This process involved taking 40 products that are regulated as allergens across the USA, Canada, EU or Asia, then homogenising and serially diluting them with water.

Using the data collected during this testing, we can determine which methodology had the superior detection limits for which product. There are three categories to consider: products where the A3 system displayed a heightened sensitivity, products where the sensitivity of the A3 system and protein swabs was comparable, and products where the protein swabs displayed a heightened sensitivity.

A3

A3 & Protein Swabs

Protein Swabs

Canned tuna, mackerel (raw), salmon (raw), shrimp, crab, yogurt, cream cheese, chicken (raw), pork (raw), beef (raw), abalone, squid, salmon roe, peanuts, soybean, bread, beer, almond, hazelnut, cashew, mustard, sesami, matsutake mushroom, coconut milk, celery, tomato, orange, kiwi fruit, banana, peach, apple, yam, walnut, and buckwheat

Milk, ice cream, whole egg and wheat flour (boiled)

Gelatin

These tests revealed that the A3 system offered heightened sensitivity for 35 products, there weren’t any areas where conventional ATP test proved superior, there were four products where the A3 system and the protein swabs were equally effective, and there was one area where the protein swabs acted as the more effective methodology. The fact that protein swabs were more sensitive to gelatin is unsurprising, as gelatin is an extracted animal protein.


On the subject of allergen control, it is also worth noting that we have launched a new range of Rapid Allergen Test Kits for: Egg (Ovalbumin), Casein, Gluten, Soya, Peanut, Crustacean, and Buckwheat.

These Lateral Flow Tests offer the following benefits:

  • Minimum accuracy rate of 0.5ppm
  • Simple, two-step process (eliminates Extraction Buffer Solution)
  • Assay time of 10 minutes
  • Stored at 2-30°C (no need for fridge)
  • Lifespan of 12 months

Key Points to Consider

Building on the case studies and testing referenced throughout this article, below are the key points that you should consider when deciding if protein swabs or the A3 system are the best fit for your site.

Protein Swabs

  • Detects protein and a wide range of allergens
  • Instrument free and convenient to use
  • More effective for the detection of the extracted animal protein: gelatin
  • Results in 10 minutes
  • Qualitative results
  • Reduced efficiency when dealing with low levels of protein contamination
  • Less effective than the A3 system for verifying the presence/amount of remaining food debris

The A3 System

  • Detects organic residue and supports your allergen control program
  • Can be used to assess the effectiveness of cleaning on site
  • Results in 10 seconds
  • Processes like heating and cleaning result in the denaturing of protein and ATP, but results will still be in measurable parameters for AMP and ADP
  • Quantitative results
  • More effective than protein swabs for verifying the presence/amount of remaining food debris
  • Unable to directly detect allergenic proteins

That brings us to the end of our honest comparison of protein swabs and the A3 system. We hope that it has helped to illustrate the fact that although the A3 system is unable to detect specific allergenic proteins, it is more effective than protein swabs when verifying the amount food debris left on a surface – able to function even after thermal processing and exposure to chemical agents. The system can also be used to measure sanitation efficiency at your site and support your allergen control program.

If you would like further guidance in this area or would be interested in learning more about the A3 system mentioned in this article, you can contact us on 01473461800 or sales@klipspringer.com. Alternatively, you can fill out the contact form below and one of our friendly team members will be in touch. 

If you would like to learn more about the A3 system, the Klipspringer team would be happy to help. Share your details below to arrange a free consultation.