Food related allergies and intolerances
Year: 2020
Period 2
Lecture 0: Introduction
Food allergies are quite common: Up to 10% of the world population is affected.
More than 170 foods reported to cause allergic reactions, amongst them the major allergens (big 8) are:
milk, egg, peanut, tree nuts, wheat, soy, fish and crustacean.
Allergens in food à Processing/Labelling à Digestion à Immune response à Symptoms
Learning outcomes
• Describe the mechanism of action of major food allergens and other constituents causing adverse reactions and
distinguish between allergic and intolerance reactions;
• Assess the risks involved in using food raw materials containing potential allergens;
• Assess the dietary consequences of avoiding specific allergen containing food ingredients;
• Describe how such components can be avoided or eliminated and select basic processes to eliminate such components;
• Describe which assays can be used for identification of allergens, how they work and review their strengths and
weaknesses;
• Apply knowledge of regulations with regard to such components, food labelling and consumer information
Prior knowledge case
IMMUNOLOGICAL ASPECTS
Q1: What is the function of the immune system? To protect us against pathogens. The immune system protects us against
pathogens (e.g. virus, bacteria, fungi, or parasites).
Self, non-self-cells; MHC molecules and antigens
The key to a healthy immune system is its remarkable ability to distinguish between the body's own (self) cells and foreign
(non-self) cells. The body's immune defence normally coexist peacefully with cells that carry "self" marker molecules, but when
immune defenders encounter cells or organisms that carry "foreign" marker molecules they quickly launch an attack. These
"foreign” marker molecules are present on both foreign cells and the body's own cells that changed their DNA (modified cells).
The figure below shows a normal cell presenting a self-MHC molecule (Left).
Question: Among the four other cells presented below, choose the ones towards which the immune system will react.
Modified cell: modified
antigen (square)
Foreign cell: non-self-
antigen (colour)
Stressed cell: missing
MHC molecule
The immune system will recognize the self-MHC molecule and only react towards the modified cell, the foreign cell, or the
stressed cell.
Q2: The immune system is a network of cells, tissues and organs that work together to defend the body against diseases.
To do this, the immune system needs to be able to differentiate the normal components of the body, known as self-cells, from
the non-self-cells (e.g. foreign substances or infectious agents) and from the modified self-cells (e.g. tumour cells).
The self-identification molecules are called MHC molecules, while antigens are any substance that trigger an immune response.
Innate and adaptive immune system
The innate immune system is present in all organisms, rapidly activated (within minutes or hours) and constitutes the first
barrier of defence. It involves physical barriers, such as the epithelium to prevent the entrance of pathogens.
When pathogen still manages to enter the system, pathogen-associated molecular patterns (PAMPs; highly conserved
molecular structure in a large group of pathogens) can be recognized by pattern-recognition receptors (PRRs) of epithelial and
phagocytic cells, inducing the release of cytokines and the degradation of the pathogen via phagocytosis.
The remains of the pathogen can migrate to the surface of the cell to be presented to the adaptative immune system.
Cytokines are messenger molecules, that can have local effect (e.g. chemotaxis and attraction of phagocytic cells), increase
permeability and promote the activity of leukocytes or natural killer (NK) cells.
The adaptive immune system is a more tailored defence mechanism, as it is antigen-specific. It can take a few days before to be
protective. The remains of pathogen presented at the cell surface of antigen-presenting cells are recognized by helper T
lymphocytes (Th), leading to their activation. These Th cells activate both cytotoxic T cells (Tc) to become killer cells, and B
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,lymphocytes to produce antibodies. Some of the T and B lymphocytes become memory cells, which makes the host immune:
immunity is acquired.
Q3: Any substance that can trigger an immune response is called an antigen. The reactions associated with the activation of the
immune system can be separated in two parts: the
innate immunity and the adaptive immunity.
Innate immune system: immediate response, non-
specific, epithelial barriers, PAMPs and PRRs, phagocytes
(e.g. macrophages, neutrophils), natural killer cells,
dendritic cells.
The innate immunity is present in all organisms, rapidly
activated (within minutes or hours) and constitutes the
first barrier of defence of the organism. It involves
physical barriers (such as the epithelium), phagocytic
cells, dendritic cells, and NK cells.
Adaptive immune system: long-term response, specific,
T lymphocytes, B lymphocytes, memory, antibodies, dendritic cells.
The adaptive immunity is mediated mainly by lymphocytes B and T. It is characterized by its specificity (capacity to distinguish
specific antigens) and its memory (capacity to respond more intensively to repeated exposures to the same pathogen).
Adverse reactions: Aversions, non-toxic and toxic responses
Aversions are caused by a dislike of the food. Symptoms such as nausea or gastrointestinal discomfort can be developed and
are psychosomatic. Although there are most of the time relatively mild, some extreme forms exist, such as anorexia nervosa
and bulimia nervosa and should be taken seriously. (A young child always gets grumpy when his parents present to him green vegetables,
such as spinach, brussels sprouts, or green beans. He does not necessary show strong symptoms when he eats them, but always makes a face
before eating them and tries to convince his parents not to eat them.)
Non-toxic responses include allergy and intolerance reactions. They are related to the individual rather than to the food. The
distinction between allergy and intolerance is based on the physiological response: the immune system is involved in allergy,
while it is not in intolerance. (Paul was sent to the emergency after eating a piece of cake at his friend’s birthday party. The cake indeed
contained traces of peanuts to which Paul is highly allergic.)
Toxic responses, also referred as food poisoning, is of particular concern for food producers. It is the result of the ingestion of a
toxin, such as residues of pesticides, heavy metals, or anti-microbial agents. Depending on the contaminant, the reaction will be
more or less immediate and more or less severe. (Silvia loves seafood. During a party at a restaurant, she ordered with some friends a
large platter of seafood, but when they came home later, they all suffered of strong stomach-ache and nausea. The symptoms disappeared
after 24-48h. After this party, Silvia continued to regularly eat seafood, but never suffered again of such a reaction.)
CHEMICAL ASPECTS
The building block of a protein is an amino acid. An amino acid contains an amino group (-NH2) and a carboxylic group (-COOH)
in its structure. The different amino acids are attached together via a peptide bond to form peptides (<100 amino acids) and
proteins (>100 amino acids).
The amino acid sequence of a protein is called the primary structure which is shown in Figure 3.
Due to non-covalent interactions between peptide bonds, the sequence will fold into repetitive spatial structures, such as α
helices, β sheets and random coils. This is called the secondary structure which is shown in Figure 1.
The conformation and orientation of the different α helices, β sheets and random coils determine the final shape of the
protein (3D structure). This is called the tertiary structure and is shown in Figure 2.
Some proteins are not formed by one, but by several polypeptide units. The way that these different units are associated
together is called the quaternary structure and is shown in Figure 4.
Q1: All food allergens are proteins. The structure of proteins is defined on four different levels: the primary, secondary, tertiary,
and quaternary structure.
The primary structure of the protein is related to the amino acid sequence and the number of amino acids. (2)
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,The secondary structure of the protein is uniform formation of hydrogen bridges resulting in an ordered repetition along the
peptide chain: helical structures or sheet structures. (4)
The tertiary structure of the protein is the final form of the folded peptide chain. It is stabilized by a number of forces. (3)
The quaternary structure of the protein refers to two or more (sometimes identical) polypeptide chains, kept together by way
of non-covalent bonds. (1)
Q2: The protein structure can change during processing. For each of the following processing steps, indicate if they would
modify the amino acid sequence and/or the 3D conformation of the protein.
Amino acid sequence: acid hydrolysis, enzymatic hydrolysis
3D conformation of the protein: acid hydrolysis, enzymatic hydrolysis, baking, blanching, boiling, cooking
Hydrolysis = chemical reaction that uses water to break down the compound. During hydrolysis, the peptide bonds between 2
amino acids will be cut, while high temperature will lead to the unfolding of the protein.
Hydrolysis will cut the peptide bonds between the amino acids. This will affect the amino acid sequence and depending on
which peptide bonds is cut, can in some cases also modify the conformation of the protein. During heating (e.g. cooking,
baking, blanching, boiling), proteins are denaturated, meaning that they unfold and lose their 3D configuration.
Q3: Various methods are available to characterize proteins in food samples. Which of the following statements are true?
• Liquid chromatography – mass spectrometry (LC-MS) can be used to identify and quantify different types of proteins
but does not provide information on their allergenicity. TRUE (Peptides/proteins can be separated by liquid
chromatography and identify based on their mass over charge ratio (m/z) and specific fragments with the mass
spectrometer. However, no information on the allergenicity of the protein can be deducted from LC-MS analysis.)
• SDS-PAGE provides information on the size of the proteins present, while ELISA can provide information on the
presence of allergens. TRUE (On a SDS-PAGE, proteins are separated on a gel based on their size. Using a molecular
weigh marker, it is therefore possible to get information on the size of the protein present in the sample. ELISA is an
immunoassay; specific antibodies can be used to detect the presence of allergens in a sample.)
• Both Dumas analysis and Bradford assay can be used to determine the total protein content. FALSE (Dumas measures
the total nitrogen content, including non-protein nitrogen. Bradford assay determines only soluble protein content.)
• Polymerase chain reaction (PCR) provides an accurate quantification of proteins. FALSE (PCR is a (semi)-quantitative
method for DNA.)
Q4: The most important and most common food allergies are called the big 8. Which of the following ingredients
together constitute "the big 8"? milk, egg, peanut, tree nuts, wheat, soy, fish and crustacean.
Lecture 1: General Aspects of the Immune System
Objectives of the lecture
• Basic structure of the immune system: innate vs adaptive; systemic vs mucosal
• Structure and function (tolerance vs immune activation) in gastro-intestinal tract
• Food allergy and food intolerance: inflammation and the allergic immune system? Inflammation is good if it is acute
because it puts the system (body) on alarm when there is something that shouldn’t be there (infection/tumour): it is a sign that the
immune system is working properly. The problem is if the inflammation is not resolved in time (10-14 days): chronic = bad. Allergy is a
type of a chronic inflammatory disease.
• What is dietary immunotherapy and how can we use this in food allergy? Using dietary components to correct the problem
you may have with diet/food
Functions of the immune system
Total area for protection: 2 m2 skin, 80 m2 lung, and 350 m2 gut tissue – it is constantly bombarded with infection: virus (HIV attacks T-
cells), parasite (malaria inside a red blood cell), bacteria (macrophage) = infections
Infection: non-self. Comes from the outside
Tumour: modified-self. Refers to your own body cells that have changed their DNA by exposure to radiation (UV), by metabolic activity, by
toxic compounds, dietary compounds. DNA may become damaged (normally it would be capable of restoring the damage, if not, it may
become a tumour cell). Immune system role: protect against tumours as well
Innate cells for immune defence
Neutrophils are the most abundant type of granulocytes and make up 40% to 70% of all white blood cells in humans. They
form an essential part of the innate immune system. They are able to get out of the blood stream quickly if there is a trouble in
a tissue; they are fast and flexible. In average they are in the blood stream for 6 hours only. Start an immune response.
Animals and even plants have innate immune system.
A picture of tumour in the blood stream, the white are NK-cells (natural killer cells). They are in the blood stream (not a lot of them) but they
are extremely effective. The number varies. By adopting a healthy lifestyle, the number of NK-cells rises, and you can make a good immune to
protect yourself against infections (viral, tumours). BRAVO (beweging, rookvrij, alcohol low, voiding, ontspanning). During the night the
immune system works best.
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, Haematopoiesis
All immune cells are formed in a process called haematopoiesis in the bone marrow (kostni mozeg).
From hematopoietic stem cell you get lymphoid progenitor, from which you get B-, T-, and NK-cells; and myeloid cell that gives rise to
granulocyte-monocyte (neutrophils, monocytes), etc.
There are many more macrophages in the brain than neurons. However,
we don’t know much of the function of macrophages in the brain.
A macrophage is a type of phagocyte, which is a cell responsible for
detecting, engulfing and destroying pathogens and apoptotic cells.
Macrophages are produced through the differentiation of monocytes,
which turn into macrophages when they leave the blood.
Innate immune recognition
Innate immune system is evolutionary very old. Here we see a picture of macrophage (blue)
and around it a virus, bacterium, and a parasite. These infectious organisms express certain
molecules on the cell membrane that are important for infectivity – to be able to infect the
macrophage. These molecules are called Pathogen Associated Molecular Patterns (PAMPs).
A special class of PAMPs in called DAMPs (Danger Associated …). These structures are very
well conserved in the evolution. So, the immune system has developed receptors over time
that are able to recognise these molecular patterns. These are called Pattern Recognition Receptors (PRRs). There are 6/7
different receptors (different classes), the one very well-known example is toll-like receptor (TLR). TLR not only bind infectious
organisms but also allergens. Humans have 10 different TLRs, fish have 40/50, plants have hundreds of them.
Protect against bacteria. G- bacteria: E.Coli, Salmonella. LPS (lipopolysaccharides on the membrane). TLR-4 receptor is able to recognise and
bind LPS. With one receptor you can bind all G- bacteria.
G+ (Lactobacillus, Staphylococcus) have Lipoteichoic acid (LTA) on the cell wall: TLR-2 receptor recognises LTA.
Basically, with 2 receptors you can bind any bacteria. You can kill any bacteria. Very efficient. Nobel prize for discovery.
Every 15-20min the number of bacteria doubles in your body. Exponential growth. The innate immune system gets a signal immediately and
kills the majority (90%) of the bacteria, the rest of bacteria start to multiply and if the job is done by the activation of adaptive immune system
(incredibly specific – can find all the bacteria in the body).
2 stages: rapid innate immune system first (very effective but not 100 %) and then adaptive immune system kicks in and kills the last bacteria
How immune cell is attacking the bacteria: the reason for that is chemotaxis. Chemotaxis is the movement of an organism in
response to a chemical stimulus. Somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements
according to certain chemicals in their environment.
Collaboration innate (rapid reaction) and adaptive immune system (antibodies already created from before). Humans have 9 classes of
antibodies. One of them being IgE (linked to the development of allergy).
5 signs of inflammation: heat, redness, swelling, pain, loss of function.
Systemic cytokines from liver into circulation
Cytokines are a large group of proteins, peptides or glycoproteins that are secreted by specific cells of immune system. Cytokines are a
category of signalling molecules that mediate and regulate immunity, inflammation, and haematopoiesis.
Pro-inflammatory cytokines (innate immune system), the majority of cytokines
in the body: Tumour Necrosis Factor (TNF), Interleukin-1α (IL-1α), Interleukin-
1β (IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8).
TNF is the first cytokine to be released if a tissue becomes inflamed. If you make a lot of
TNF, then it is dangerous – sepsis. Sepsis: living bacteria in the bloodstream. Generally G-
bacteria (E.Coli – ETEC, VTEC). Consequently, a lot of organs start to react: multiple organ
failure (kidney, liver, heart, brain stop working). Happens to babies who get infected with
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