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

√

√

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√ 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