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Food Allergen & Detection Methods
Vipa SurojanametakulInstitute of Food Research and Product
Development
Kasetsart University
Institute of Food Research and Product Development (IFRPD), Kasetsart University
October 22 th , 2015
Enjoyment
Taste
Nutrition
Health
Full
Economy
SavorSafety!
Shoji,2012
Food : What come the first importance ?
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Understanding Food
AllergyFood allergy is any abnormal reactions, occurs when the
immune system (IgE-mediated system) mistakenly
attacks a food protein (which is called “food allergen”) when
ingested by allergic individual.
Ingestion of the offending food may trigger the sudden release of
chemicals, including histamine, resulting in symptoms of an
allergic reaction, ranging from relatively mild to severe (as
anaphylaxis) or sometimes potential life–threatening.
The symptoms typically develop in less than 10 minutes up to 2
hours after consumption of the offending food
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Significant socioeconomic expense As top ten chronic disease in USA and is estimated
the medication cost for food allergy is ca, $ 25 Billion
Incredible health hazardSeverity and complexity of symptom is increasing and
more frequency cause of fatal consequence, i.e.
in USA 150 persons dried /year due to food allergy
Significant impacts to our society The prevalence is marked and moreover increasing in
both developed & developing countries,
especially in baby & children
How come Food allergy is a serious problem?
Currently, there is no known medication for curing food
allergy Institute of Food Research and Product Development (IFRPD), Kasetsart University
How can food allergy be
managed?
In order to help food allergic individuals, the
regulators of International and National
levels have introduced the regulation for
allergen labeling of prepackaged foods.
Presently, the most effective way of preventing
allergic reactions is the strict avoidance of the
offending allergenic food ( as self protection ).
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Food Ingredients for Food Allergen Labeling
a: Shrimp and crab are the only crustacean listed. Grain included wheat and buckwheat Others includes food for which labeling is recommended but not required:abalone, squid, salmon roe, oranges, cashew nut, kiwifruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, “matsutake” mushrooms, peaches, yams, apples and gelatinb: Shrimp and crab are the only crustacean listed. Grain included wheat and buckwheat. Mackerel is the only fish. Other includes pork, peaches and tomato.
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Thai government action related to Food allergen
Ministry of Public Health:Thai Food and Drug Administration is already announced
to enforce Food Labeling regulation including Food
Allergen labeling (following the Codex ) since June 6 th,2014.
Ministry of Agriculture and
Cooperative: National Bureau of
Agricultural Commodity and Food
Standards prepared the instruction, “Food
Allergens”, for Thai food exporter facilitating
to adjust themselves for North America, EU,
Japan and Australia markets etc.
Institute of Food Research and Product Development (IFRPD), Kasetsart University
What is Food Allergens ?
Food allergens
Protein or glycoproteins with the MW 10-70 Kdal(Not all proteins are allergens)
Naturally occurring (plants & animals) or newly introduced in GM foods
Have a specific surface features that antibodies
recognize
Comparatively stable to processing, cooking, proteolysis, and the digestive processes
Causing abnormal immunological reactions (immediate)
Institute of Food Research and Product Development (IFRPD), Kasetsart UniversitySource: Shoji, M and Obata,T.2010
Multi IgE binding sites (Contain more than 1
Epitope)
Processing impact conformational change
cause a change in solubility and allergenicity
properties
Structure
Protein with a globular compact structure
More than 160 foods have been associated with
allergic reaction
Eight types of allergenic food responsible for over
90% of all allergic reaction , named “ Big 8”
Cow’s milk
Egg
Fish Shellfish
Wheat Peanut Tree nut Soya
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Allergenic Foods
The amount of allergen required to elicit an allergic
reaction differs from person to person
No threshold level has been identified for most allergens
Major allergenic protein in foods
Food items Major allergenic protein
Milk casein / beta-lactoglobulin/ alpha-lactalbumin/Bovine
serum albumin, Immunoglobulins, Lactoferrin
Egg Egg white : ovalbumin /ovomucoid / lysozyme
/ovaltransferin
Egg yolk : α livetin
Fish parvalbumin
Shellfish tropomyosin (Cross-reactivity in various seafood : shrimp, crab, squid, abalone)
Wheat albumin, globulin, prolamin (gluten / gliadine)
Peanut Seed storage protein (globulin, albumin)/ conarachin/
arachin1-8
Soybean beta-conglycinin, gylcinin
Tree nut and Seeds Seed storage protein (albumins, globulin)
Fresh fruits and Vegetables Bet v1, latex
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Food
Allergen
Intentionally added Unintentionally added
Appropriate labelingAllergen testing
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Hidden allergen
Allergen presence in food
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Food allergen detection
Why Test for Allergens?
Consumer safety
Ensure accuracy of
food labeling
Cleaning effectiveness
Consumer complaints
(Lauren S. Jackson ,2010)
Uses of Allergen Tests
Raw material/ingredients
Effectiveness of allergen
control program (cleaning/
environment)
Finish products for labeling
compliance
Investigate/confirm
consumer complaints
Regulatory agency effort
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Allergen Detection
Immunochemical methods (protein based)
ELISA
Lateral flow devices (dipsticks)
DNA-based methodsPCR
Mass spectrometry
Generic/non-specific (cleaning)
Protein
ATP
Visual inspection
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Enzyme-Linked Immunoassay (ELISA)
Antibody-based detection of allergenic protein or
other protein in food
Available in well (micro plate) and lateral flow
formats( dip stick)
Sandwich format most common
The most commonly used as screening test
ELISA analysis take between 1 –2 h to complete
Quantitative & qualitative
Kits available for most of the major allergens
Used for ingredients, finished products, cleaning
fluids, swabs, environmental samples
(Lauren S. Jackson, JIFSAN Advisory Council Spring Symposium , 2010 )
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Sandwich ELISA
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Lateral Flow Devices (LFD) - Dipstick Tests
• Rapid qualitative test of allergen (can be semi-
quantitative with reader)
• Suitable for industrial purpose
• Available for many allergenic foods
• Typically used for environmental sampling,
cleaning verification, screening of foods
• True measurement of presence of allergenic food
• Satisfactory sensitive (DL~ 5 ppm)
Enzyme-Linked Immunosorbent Assay (ELISA)
Advantages• High selective to allergen residue
• High sensitive (in low ppm range)
• Powerful - qualitative /quantitative tool
• Crude sample use
• Many samples possible for one analysis (not for LFD)
Disadvantages• Long developmental time
• Cross-reactivity possible
• Matrix effect (polyphenol, pH, oil etc.)
• Trained staff requirement ( but not for LFD)
• Lack of reference material, need do in house validation
Institute of Food Research and Product Development (IFRPD), Kasetsart University
ELISA Points of Consideration
Antibody Specificity – total protein vs. allergen
Polyclonal vs. Monoclonal Antibodies
Calibrators
Effects of Processing on Detection
Efficiency of Extraction method
Sensitivity Limits
(Joe Baumert, 2011)
Institute of Food Research and Product Development (IFRPD), Kasetsart University
PCR Method
Confirmation test for allergen detection by ELISA
Specific –to the source (DNA) but not to the allergenic proteins
Sensitive (high)
Qualitative /Semi-quantitative
Depends on specific primers
Available for many allergenic food sources
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Polymerase chain reaction (PCR)Advantages
• Detected origin of allergic food (species specific)
• DNA stable than protein
• Suitable for confirmation
• Short developmental time
Disadvantages
• Not detected allergenic protein
• False positive result
• Not practical for in-plant use
• Required trained staff and isolated lab
• DNA is low in some high protein food (milk, egg white, protein isolated)
• Some foods cannot be differentiated (beef/milk; egg/chicken)
• Food matrix & processing can negatively impact on DNA extraction and detection
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Institute of Food Research and Product Development (IFRPD), Kasetsart University
General Protein Tests
Detects protein regardless of source but not specific for
allergenic protein
Can be used for routine sanitation verification but should
first validate the cleaning methods with specific ELISAs
Chemical reaction (Biuret method)
ATP/Bioluminescence Tests
Does not prove presence of protein and None are specific for allergens
Method selection
• The specificity of the test method : protein, allergenic protein,
DNA, ATP
• The sensitivity of the test method : detection limit of target
• Recommended to validate removal of allergenic residue using
specific ELISA
• Testing for total protein or ATP can be helpful in some cases.
They can provide a good quick check on sanitation effectiveness
during routine cleaning
Institute of Food Research and Product Development (IFRPD), Kasetsart University
(Joe Baumert, 2011)
• Allergen-specific ELISAs will allow very specific and
sensitive detection for qualitative and quantitative of allergenic residue
Conclusion
Food allergy is regarded as a serious health hazard, globally
To protect the health hazard of food allergy, food allergen
labeling regulation has been enforced in many countries,
including Thailand
Undeclared food allergen is become an emerging issue
related to food safety hazard
√
√
√
√ Food allergen detection for accurate and reliable result is
needed to meet the requirement of food allergen labeling regulation
ELISA is a very powerful tool for allergenic residue testing
FQA (ศูนย์บริการประกันคุณภาพอาหาร) Food Allergen test : ELISA technique
Wheat Soy beanGluten MilkPeanut Egg
Web site: http//www. Ifrpd-foodallergy.com ข้อมูลองค์ความรู้เกี่ยวกับภูมิแพ้อาหาร สารก่อภูมิแพ้ กฎระเบียบ ว่าด้วยการแสดงฉลากสารก่อภูมิแพ้ เทคนิคการตรวจวิเคราะห์... ฯลฯ
Food Allergen Detection Service (IFRPD, Kasetsart U.)
Institute of Food Research and Product Development (IFRPD), Kasetsart University
Thank you for your
attention
Institute of Food Research and Product Development (IFRPD), Kasetsart University
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