Allergens

ZEULAB launchs PROTEON DUO MILK EXPRESS, an easy, rapid, and reliable test for dual detection of β-lactoglobulin and casein in foodstuff, rinse water and surfaces. Specially designed for the food industry and related businesses. Main characteristics of the test: Double rapid test: milk and its by-products detection in 10 min. Versatile: valid for highly processed food. Simple:no previous training or additional equipment are required Economic: 2 detections with only one rapid dip-stick test. Food allergies represent a major health problem in developed countries. Undeclared allergens as contaminants in foodstuffs suppose a high risk for allergic people. According to different studies, the incidence of cow’s milk protein allergy is 0.3-7%. Milk is the first food introduced into children’s diet after breastfeeding. Therefore, cow’s milk protein is the first allergen which develops an allergic reaction1. Milk allergy, together with egg and fish, is the main cause of allergy in infants and children younger than 3 years-old. Although 85% of patients become milk tolerant in adulthood, 40% of children allergic to milk also develop allergies to other food and 28% to environmental agents. The only way to avoid an allergic reaction is to stop ingesting the allergen. Therefore, providing a correct ingredient list to the consumer is of a paramount importance. According to the European Union (Annex II, Regulation (EU) 1169/2011 2,3) and other countries regulation, such as USA4 and Japan5, milk or any milk by-products must be listed in the label. Many non-dairy products can contain milk or its by-products as ingredients, as well as being present as hidden allergens in a lot of products. Therefore, they must be monitored to ensure a correct labelling. Caseinates, for example, are used to stabilize emulsions, as well as to increase shelf life, improve nutritional content, and enhance flavors and aromas in products such as baked goods, meat products, cream soups or coffee creamers6. Regarding whey proteins, their functional properties include gelation, emulsification, foaming, flavour or aroma in products such varied as meats, beverages, breads, cakes, or soups7. PROTEON DUO MILK EXPRESS is a double test for detection of milk by combining the presence of β-lactoglobulin (β-LG), as indicator of whey proteins, and casein showing presence of caseinates. It is an immunochromatographic test in strip format, based on specific antibodies obtained against β-LG and casein which are bound to red particles, will react with these proteins when they are present in the sample, producing one or two red test lines. In addition, a blue line (control line) indicates that the test has worked properly. Food or working surfaces containing milk residues above the limit of detection will show, two red lines. In case of samples containing either of whey or caseinates, a control blue line and a β-LG or casein red line will appear, respectively. PROTEON DUO MILK EXPRESS detects the main milk allergen indicators. Therefore, it is especially useful when the source of milk in unknown, such as control of suppliers or in industries where different production processes using whole milk, whey or caseinates are carried out. Single tests targeting only one of these groups of proteins are suitable for detection of milk, but they can miss out the presence of some by-products. High temperatures or high pressures as well as cleaning procedures with alkaline detergent can alter the protein structure. Therefore, the double detection of β-LG and casein is also recommended in those circumstances as the probability of detecting milk increases. PROTEON DUO MILK EXPRESS has been validated, according to the guideline AOAC 2016 8,9, 10 with a wide range of matrices, including pastries, cold meat, pâté, ice-cream, infant formulas, beverages or alcoholic drinks. 1Bobe et al. (2007) Composition of Milk Protein and Milk Fatty Acids Is Stable for Cows Differing in Genetic Merit for Milk Production. J Dairy Sci. 90: 3955-3960. 2Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers (http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32011R1169&from=ES) 3Guidance on food allergen management for food manufactures (2013), Food and Drink Europe, Brussels, Belgium (http://www.fooddrinkeurope.eu/uploads/press-releases_documents/temp_file_FINAL_Allergen_A4_web1.pdf) 4Department of health and human services, Food and Drug Administration (https://www.gpo.gov/fdsys/pkg/FR-2016-05-27/pdf/2016-11865.pdf) 5Consumer Affairs Agency, Government of Japan (http://www.caa.go.jp/en/) 6https://www.ulprospector.com/en/eu/Food/Detail/16980/419669/Calcium-Caseinate 7http://www.milkingredients.ca/index-eng.php?id=195 8Appendix M: Validation Procedures for Quantitative. Food Allergen ELISA Methods: Community Guidance and Best Practices. AOAC 2012 (http://www.eoma.aoac.org/app_m.pdf) 9Appendix F: Guidelines for Standard Method Performance Requirements. Official Methods of Analysis (2016), AOAC INTERNATIONAL, Rockville, MD, USA (http://www.eoma.aoac.org/app_f.pdf) 10Standard Method Performance Requirements (SMPRs®) for Detection and Quantitation of Selected Food Allergens. AOAC SMPR 2016.002. AOAC INTERNATIONAL, Rockville, MD, USA (https://www.aoac.org/aoac_prod_imis/AOAC_Docs/SMPRs/SMPR%202016_002.pdf).

Analytical methods are needed for a correct management and control of potential allergen cross-contamination, as well as the reinforcement of the quality of the manufacturing and cleaning processes. According to European regulation No 1169/2011, it is mandatory to report specific allergens present in a product. Direct methods are recommended because they allow the detection of specific allergenic proteins, avoiding false positives, because they are able to distinguish accurately the presence of a particular allergen. The main guides which advise on the risk management of food allergens recommend the use of rapid immunochromatographic tests LFIC for routine verification controls. Rapid tests stand out for their easy of use and rapidity, as well as their specificity. They can be used in site by workers and do not require a reader equipment, since the interpretation is visual. A method incorrectly used in the industry is the one based on ATP detection. ATP is a molecule used by living organisms (animals, plants and microorganisms) to provide energy to chemical reactions. Detection is performed using bioluminescence assays, and so a special reader device is required. This method detects biological contamination, being useful for showing the cleanliness level. However, ATP detection is not equivalent to allergenic protein’s detection on surfaces. The reason of use in such cases is usually based on the assumption that if ATP has been removed from the surface, allergenic proteins would have been removed too, but there is no evidence about it. Besides, some components of the detergents or even the water could interfere in the detection of ATP. Furthermore, microorganisms that live in the water are a source of ATP, so that basal levels have to be established in each facility. Another technique which is used to assess the presence of allergens is the quantification of total proteins. It is an indirect method based on a color change. It is a simple technique with some limitations. The main inconvenient is that it does not detect the specific allergenic protein but total proteins. This, together with its high detection limit (3 µg of total protein versus 40 ng/cm2 in the case of gluten rapid test, for example), makes false negatives possible, so allergens can be present when the signal is negative. Also, a special equipment is required for read the results. Below, a table where the main advantages and disadvantages of these techniques are shown: Zeulab manufactures rapid tests LFIC: PROTEON EXPRESS. These kits not only offer allergen detection on surfaces, but also in final product and rising waters. They are simple tools, perfect for suppliers control (raw materials, semi-processed products, processed products, etc), as well as for manufacturing and cleaning processes verification, and cross-contamination control. Their used is extended to industrial manufacturing, retail and kitchens in the catering sector.

Food allergies are the exaggerated response of our immune system against the consumption, contact or inhalation of a food, its derivatives or an additive contained in the same. There is an increasingly important public health problem since its prevalence is increasing. Management of allergens in the food industry is not a new system, but it is already an integral part of the food security control. An effective allergen management system should take into account all operations, from the supply of raw materials to production, handling or packaging. The legislation only requires to inform of the presence of 14 allergens in foods, either because they are included in the list of ingredients or because a cross contamination occurs during its production or distribution. However, the management of allergens must be integrated into the Good Manufacturing Practices (GMP) and into the Hazard Analysis and Critical Control Points (HACCP) aiming to avoid contaminations and minimize the unintentional appearance of allergens in products. Certainly, this process involves being aware of the raw material composition used in the processes, as well as organizing them, carrying out the production in an orderly manner and implementing a very exhaustive cleaning. Our primary goal as a company involved in allergen security is to inform customers about its products, through a series of posts about the major risks the operators in the chain are facing during the different stages in development, production and distribution,. Also, our objective is to advise them about the preventive tools that they have at their disposal to ensure the correct labelling of processed products, in order to protect sensible customers to these kinds of foods.

Food allergens are components of foodstuff and additives that when digested, in contact or inhale cause an immune reaction known as food allergy. Although some allergies occur more frequently than others, and there are about 160 foods that can cause allergic reactions, the most common ones are those to the cow milk, eggs, legumes (including soy), wheat/gluten, fish, shellfish, fruits, and nuts, such as peanuts and walnuts. The declaration of allergens is mandatory, even if the possibility of the allergen to be present is small. At present, European regulation No 1169/2011 expands the UE Regulation with specific requirements regarding the declaration of allergens in food. This regulation insists on most food allergy incidents can be traced back to non-prepacked food. The information on potential allergens is considered very important and affects both commercial and collective catering service, retailers selling unpackaged food, and official labeling. In order to ensure a high level of protection for consumers and to facilitate their choice, products put on the market must be safe and adequately labeled: any substance or ingredient causing intolerances or allergies used in the manufacture or preparation of a food and still present in the finished product, even if in an altered form, must be declared. There are several methods for assessing the presence of allergens in food matrices, surfaces and rinsing waters, which are grouped into two: – Direct methods detect allergenic proteins (gluten, soy, milk β-lactoglobulin, etc.) in a specific and sensitive way. Some of these methods are: Rapid immunochromatographic strips test (presence/absence of allergen). It is an immunochemical technique which uses specific antibodies. They are really easy-to-use test and can be easily run on-site as not qualified personnel, neither laboratory equipment is needed. ELISA kits (quantitative analysis of specific allergens). It is also an immunochemical technique. They require laboratory equipment and qualified personnel Mass spectrometry, which require highly qualified personnel and highly expensive laboratory equipment. –  Indirect methods do not detect allergenic proteins:   Genetic techniques. The genes of allergenic proteins, ie, their DNA thanks to PCR assays. They are        only useful when protein detection assays are not available. Total proteins detection (allergenic proteins plus those which are not). There are different        techniques, e.g. colorimetric techniques like Biuret, Bradford and BCA methods. Neither these        methods are specific.   Detection of ATP, a molecule used by all living organisms to provide energy in chemical reactions, is considered an indirect method. However, this technique is not allergen specific and it is only useful for indicating the cleanliness level, so that it should not be considered as such. Every method has advantages and disadvantages according to their rapidity, reliability, ease of use, etc. There is not a perfect method but some of them can suit better than others the users’ needs. Customers must be informed on specific allergens. On this basis, direct detection methods would be the appropriate techniques to correctly and specifically declare the allergens which may be present in a product or a meal. In addition, direct detection methods are more sensitive and avoid false positives, as they distinguish the specific presence of allergens. Food and Drink Europe launched in 2013 its Guidance on Food Allergen Management for Food Manufacturers. This guide recommends the use of on-site rapid strips LFIC for routine check-ups and ELISA kits for result confirmation on a regular basis. In case of ambiguous results, it is advisable to carry out confirmatory tests by PCR. However, because of the low sensitivity of the PCR technique for certain allergens, it should only be used when no other detection technology is available.

Have you ever wondered if the cleaning that you do is really effective? The answer is especially important when we talk about cross-contamination through food allergens. People who are allergic to foods may suffer a reaction in the presence of very small amounts of the allergen (in the order of parts per million or ppm). This reaction can be mild like a hive or very serious as is the case of the dreaded anaphylactic shock. In recent years, due to the growing importance of this problem, the food industry is making an effort to improve the information on the content of food allergens in products offered to the consumer. However, there are still many products where the reference “may contain…” appears on the label, theoretically the result of the impossibility of controlling cross-contamination with ingredients of other products that are manufactured in the same facility. This labelling generates confusion and reduce the range of foods available for allergic consumers (DunnGalvin et al., 2015;) Marchisotto et al., 2016; Muraro et al., 2014). As well as the increase in the number of alert notifications RASFF shows them the threat posed to their health food allergens present in products that do not appear on the labelling. As we discussed in our blog, in order to reduces the use of the words «may contain» and reduce the presence of non- intentional allergens, it is important that all members of the food industry consider allergens as a danger and are integrated within a HACCP system. An effective tool to confirm that the practices of handling and cleaning are being done correctly is the control of allergens on work surfaces and analytical tools. But don’t forget that to make useful application of analytical techniques the user has to have information in order to understand what the applicability and scope of the test being performed is. For this reason ZEULAB in collaboration with the University of Zaragoza and Madrid’s Hortaleza District Health Department, has conducted a study to demonstrate the analytical conditions for the realization of this type of analysis. The work that has been published recently in the journal Food Control shows the development of a method that combines the recovery of material allergen of surfaces through the use of swabs and further analysis both qualitatively, with a rapid strip, and quantitatively through a test ELISA (Galan-Malo et al., 2017). The objective of this work has been to develop a method that is both as simple as possible without losing technical performance and has initially focused on the analysis of residues of milk and egg. Three important aspects of the method have been evaluated in the study: swab material, composition of the extraction solution, and temperature and time of removal before the trial. Swabs made of five different materials were analyzed and of these selected the higher yield was obtained. The results indicated that cotton was the material that worst captures the remains of milk and egg. So, that famous phrase saying «cotton does not deceive» does not apply in this context. Therefore, it is very important that the material supplied or recommended by the manufacturer is used for analysis of allergens on surfaces and that swabs from another source, are not used since they could compromise the results of the analysis. In this work, the analysis of allergens in two types of surfaces were evaluated namely melamine and stainless steel. These two types of surface are located in the facilities of the food industry or in the kitchens of canteens and of which there was not much information available about the effectiveness of the methods of analysis. The results showed that the ELISA test is able to detect levels as low as 0.04 µg of egg and 0.2 µg of milk. The rapid tests put their detection limit at 0.07 µg of egg and 0.6 µg of milk depending on the analyzed surface. From a practical point of view, the rapid tests are very useful and have proven their validity for the routine check after cleaning of equipment and facilities. In a few minutes they let you verify if the cleaning procedure has been correctly carried out and whether the measures of containment against cross-contamination are adequate and effective. On the other hand, the ELISA tests due to its greater sensitivity and especially to provide quantitative results, are a very useful tool for the validation of cleaning procedures. This type of testing should be conducted periodically or after the introduction of relevant changes in the cleaning procedure. A proper use of these tools will improve allergy risk management plans and reduce the indiscriminate use of preventive labeling.

The countdown to the coming into force of Regulation (EU) 1169/2011 starts. From 13th December of this year, all commercialized food has to highlight if any allergens of mandatory mention are present in their composition. Even unpackaged food and menus offering in catering or restaurants are included in this regulation. Professionals along the chain from farm to folk must be aware of what ingredients are used in the different steps of manufacturing and handling to identify allergens correctly. In this way, final consumers can be informed correctly about allergens content, making this law useful. Hidden allergens will be one of the most dangerous enemies that workers must face to follow the law. But, where are allergens hidden? Unknown allergens have mainly two origins: 1) They can be part of the composition of a processed food as additive or preservative. For instance, egg can be added to clarification processing in wines. Also, soy lecithin is used like emulsifier to mix ingredients. 2) Cross-contacts (also known as cross contamination). When the ingredients are manipulated in powder form such flour, aerosols can appear. These particles remain in suspension in the air. Then, this powder flecks begins to deposit on the working surfaces, even on surrounding areas after cleaning.  Other hideous source of cross contacts is hard-to-reach areas in equipments and utensils which are difficult to clean properly. In these areas can remain rests of allergens that can be transferred to the next manufactured product. How to avoid hidden allergens to appear in the food product sold to the consumer? Professionals have to work with a “Hazard analysis and critical control points” system, also known as HACCP. This systematic preventive approach helps to develop a risk management plan in a logical and efficient way. However, allergen analysis presence is needed to verify that the guidelines followed really work. Where can we analyze allergens? Ingredients used in manufactured food should be analyzed not only to confirm suppliers information, but also transport and storage are correct. Besides, working, equipment and utensils surfaces needs allergen analysis to verify manufacturing processes and cleaning systems avoid cross contact phenomenon. How to analyzed allergens presence? Among all existing methodologies, ELISA tests and immunochromatographic strips are the ones that show better sensitivity vs cost. The Proteon line by ZEULAB offers several ELISA kits and rapid strips that allow analyzing allergens in a wide range of food. The first one provides quantitative results but small laboratory equipment is required to carry out the test. The strips tests results are qualitative but the testing time is just a few minutes with no need of trained personal or infrastructures. Besides, ZEULAB also supplies a specially design kit to analyze gluten, egg or milk in working surfaces. This kit integrates all components needed to control in an easy way without any additional material. This kind of tests permits to control the cleaning of facilities at the end of the workday or even during inspection and audit activities. Therefore it becomes a valuable tool for production and quality supervisors as well as for food industry and catering companies.

Food allergy involves adverse reaction of immunity system to determined foods (mostly proteins). Food allergies represent an important health problem affecting mainly children (6-8%). Symptoms can vary form mild (hives) to severe reactions such as anaphylaxis. The amount of food allergen that can trigger an allergic reaction is different in each patient and trace of allergens (little microgram) can be enough to induce a reaction. Hence, rigorous information about foods that contain allergens is very important to allow allergic people manage their diet. Egg allergy is one of the most common in children, generally aged less than 5 years old. Egg is an ingredient widely used food industry to manufacture products such as tortillas, bread and bakery. Egg is not only added in food as a basic ingredient, but also as additive or supplementary condiment. Moreover, white egg is used as clarification agent in wine industry. Altogether, egg can be a hidden allergen and it can be present in a non declared way in food. According to European legislation (Annex II of Regulation (EU) 1169/2011) egg and thereof require mandatory mention in food labels. In order to claim correct information label in food, it is important to perform assays to verify egg presence/absence in food, especially when egg is used as additive. Moreover, analytical controls are needed to verify measures to avoid cross contamination in facilities where food manufacturing is realized with and without egg. ZEULAB releases a new ELISA Sandwich test to detect egg, “PROTEON Ovalbumin”, in an effort to offer analytical tools to the food sector in this issue. Ovalbumin is the major protein in white egg (54%) and together ovomucoid, lysozyme and ovotransferrin are the main allergens in egg. «PROTEON Ovalbumin» allows quantifying egg quantity in whole egg powder ppm in an accurate way. This test is available for a wide range of matrix in sectors such as bakery and wine industry. Besides, ZEULAB has improved “PROTEON Ovomucoid”. Ovomucoid is a heat stable glycoprotein that represents 11% of white egg proteins. So this ELISA Sandwich is designed to detect egg in food matrix treated at high temperatures where proteins could be denaturalized. Both tests are available in 48 and 96 well plate format and results are traceable with the NIST 8445 reference material.