Review of the regulatory management of food allergens

6 Exemption of ingredients derived from allergenic foods

There is increasing recognition that some food ingredients derived from allergenic sources present negligible risk to the majority of allergic consumers. However, the effect of food processing operations on different allergens in various food matrices is not always predictable. The following discussion highlights some of the issues in this area.

6.1 Impact of food processing on protein allergenicity

Food allergens are generally proteins of molecular weight more than 9 kDa. The sites on the protein which bind IgE antibodies, known as epitopes, may be conformational or linear. As the name suggests conformational epitopes are dependent on the 3-dimensional folding of a protein for IgE-binding. Consequently, conformational epitopes are more likely to be associated with larger proteins (~>25 kDa) because, unlike short polypeptides they can undergo extensive folding. Such epitopes are also readily inactivated by denaturation of the protein. Linear epitopes are determined by the specific sequence of amino acids in a protein and, therefore, remain active even when the protein is unfolded. Linear epitopes may have a particular clinical significance such as correlation with persistent food allergy (Beyer et al., 2003; Chatchatee et al., 2001; Järvinen et al., 2002).

Food is processed using a variety of techniques including mechanical processing, separation, distillation, thermal processing, biochemical treatment, high pressure treatment, electric field treatment and irradiation (Thomas et al., 2007). In general, allergenic proteins are resistant to processes commonly used in food manufacturing with most allergens retaining their allergenicity after treatment by heat and/or proteolysis. The structural characteristics of a protein influence its stability under various processing conditions and potentially its allergenicity. In addition to the intrinsic properties of the protein, the overall composition of the food, and the past processing history may affect the allergenic potential of processed food. Therefore, in complex food matrices, the overall effect of processing on the allergenicity of food proteins cannot always be predicted (Wal, 2003; Mills et al., 2007).
Scientific investigations of the impact of food processing on allergenicity are further challenged by the fact that proteins can lose solubility as a result of food processing or storage. Consequently, information available on the impact of food processing is largely limited to the soluble proteins that can be extracted for serological or clinical studies.

6.1.1 Thermal processing

Thermal processing is widely used in food manufacturing and most commercial food operations include one or more thermal treatment steps. Thermal food processing methods include boiling, steaming, baking, roasting, drying and pasteurisation. These processes use hot surfaces, steam injection, hot air and microwave heating.

The effect of thermal processing may increase or decrease the allergenicity of proteins depending on a number of factors including the temperature, the duration of heat treatment and the type of thermal processing used, e.g., in the presence or absence of water. However, there are no clear rules regarding the consequences of thermal processing on the allergenicity of food proteins in various food matrices (Wal, 2003; Mondoulet et al., 2005; Mills et al, 2009).
Thermal treatment may cause proteins to undergo significant modifications that affect their physical and chemical characteristics. The loss of tertiary structure is typically followed by unfolding causing aloss of secondary structure, cleavage of disulphide bonds, formation of intra-/intermolecular interactions, rearrangement of disulphide bonds and aggregation. Changes in protein structure result in the loss of conformational epitopes and potentially the loss of allergenicity (Davis and Williams, 1998; Hefle, 1999; Davis et al., 2001; Wal, 2003; Mills et al., 2007; Sathe and Sharma, 2009).
One of the main thermally induced chemical modifications of protein is the Maillard reaction. The reaction occurs when amino acids are heated in the presence of reducing sugars resulting in the spontaneous, non-enzymatic, glycation of proteins. Glycation can affect the structural characteristics and physicochemical properties of a protein. The Maillard reaction is believed to aggregate allergenic proteins thus enhancing their allergenicity by increasing the IgE-binding capacity. Novel epitopes can also be introduced, for example, through changes in a protein’s resistance to digestion as a consequence of the Maillard reaction. The IgE-binding capacity of roasted peanuts was approximately 90-fold higher than that of raw peanuts of the same cultivars (Maleki et al., 2000a; Hansen et al., 2003; Mills et al., 2007; Mills et al., 2009).

6.1.2 Enzymatic treatment

Biochemical food processing often involves the use of enzymes including proteases, oxidases or transglutaminases (Paschke, 2009).The allergenicity of some food proteins can be reduced by enzymatic treatment. For example, proteolysis of milk followed by further processing such as ultrafiltration, is used to produce hypoallergenic infant formulas. Hypoallergenic wheat flour can be produced by using bromelain enzyme to cleave the wheat glutenin IgE-binding epitope (Wichers, 2007; Mills et al., 2009). However, enzyme-mediated proteolysis did not destroy the IgE-reactivity of the major peanut allergen Ara h 1 (Maleki et al., 2000b). Therefore, knowledge of the protein structure and the sequence of the IgE epitope, can provide powerful tools to use targeted processes to reduce protein allergenicity.

6.1.3 Physical/ chemical separation of proteins

Processing operations that physically or chemically separate and remove proteins from food, such as distillation, filtration and solvent extraction can reduce the allergenicity of some food ingredients. Specific examples of ingredients derived using physical separation processes are discussed below.

Distillates
Distillation is one of the oldest methods of separating and purifying substances. Distillation is used to separate liquids from nonvolatile substances, or to separate two or more liquids that have different boiling points.
Distillation relies on the difference in the boiling points of the components in the aqueous solution to be separated. The mixture is heated to the boiling point so that components with lower boiling temperature will preferentially vaporise first. The vapour is then cooled to liquefy and the resulting liquid is collected. Initially, low boiling components are collected but as the distillation proceeds, these components are depleted from the starting mixture and higher boiling components begin to distil over. In commercial distillation, the operation is usually well controlled to prevent higher boiling components in the starting material from being carried over to the distilled product.
In the food industry, distillation is commonly used for alcoholic beverages and to purify alcohol for use as a solvent in the formulation of flavours and other food ingredients. Alcohol is produced by fermentation of sugars from various sources, including allergenic foods such as cereal grains and milk whey. Fermentation alone does not eliminate the allergenic proteins present in the mixture, and fermentation products usually contain proteins and protein fragments. The alcohol content is maintained at 12-15% because the fermenting yeast is destroyed at high alcohol concentrations.
Alcohol distillation is used mainly to achieve higher alcohol content but it also removes proteins and other substances present in the fermented product. There is general scientific agreement that non-volatile substances such as sugars (e.g. lactose from whey) and proteins do not distil and therefore, would not be present in the distilled product. The European Food Safety Authority (EFSA) considered a number of analytical studies using total protein and protein-specific detection methods. EFSA concluded that these studies provided supporting evidence that proteins from cereal grains and whey, as well as lactose, were not detectable in distilled products (EFSA, 2007a; EFSA 2007b). Based on these studies, determined that, in a properly controlled process, distillates made from whey and cereals are unlikely to trigger a severe allergic reaction in susceptible individuals (EFSA, 2007a; EFSA, 2007b). Under European Commission legislation, these products are exempt from allergen declaration.
Recently, Cressey et al. (2010) reported on the analysis of distilled ethanol from whey provided by a New Zealand manufacturer. Thirty five samples were analysed for residual protein using Enzyme linked immune-sorbent assay (ELISA) specific for the milk whey protein β-lactoglobulin ((β-LG) with a limit of detection (LOD) 2.5 mg/L. No samples contained detectable β-LG. Absence of whey proteins was further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis.
Distillation products may be processed further to produce foods and ingredients. Down-stream products, such as vinegar derived from distilled alcohol, would not be expected to contain whey proteins. Cressey et al. (2010) analysed seven commercial samples of vinegar produced in New Zealand by secondary fermentation of distilled whey ethanol, for residual whey proteins. Based on ELISA method, no samples contained detectable β-LG at a detection limit of 2.5 ppm (mg/L) and no residues of whey protein were detected by LC-MS.

Glucose derived from cereal grain starch

Glucose syrups are extremely versatile sweeteners, and are widely used in confectionery products, soft drinks, sports drinks, jams, sauces and ice creams. Wheat starch is commonly used for the commercial manufacture of glucose syrup in Australia.

Wheat starch is also known to contain various proteins, including gluten, the protein involved in coeliac disease and in allergic reactions to wheat. The amount of protein associated with the starch fraction can vary considerably depending on the method of preparation.
Starch granules contain intrinsic proteins embedded in the starch matrix – mainly enzymes involved in starch synthesis (Rahman et al., 1995). In addition, a large number of proteins are associated with the surface of the starch granule (Kasarda et al., 2008). The majority of these proteins were identified as gluten (glutenins and gliadins) and non-gluten (albumins and globulins) proteins. Because starch synthesis occurs in a separate cellular compartment to gluten and other storage proteins, the presence of these proteins in starch is most likely due to the breakdown of organelles during grain maturation. Also identified on the surface of starch granules were proteins which protect the grain from biotic and abiotic stresses (Kasarda et al., 2008).
Glucose is produced by enzymatic hydrolysis of starch. The enzyme alpha-amylase is used for the liquefaction (or thinning) of starch into dextrins, and another enzyme, for example amylogucosidase, is used for the final saccharification resulting in a syrup of high glucose content. The hydrolysis degrades the starch granules releasing the proteins and lipids. Further steps include centrifugation and/ or filtration, physical screening and ion exchange.

Cereals such as wheat and barley contain gums which increase the viscosity and reduce the filterability of aqueous extracts of the cereals including glucose syrups.

In 2007, EFSA evaluated data on glucose syrups derived from barley and wheat. EFSA noted that most of the protein is removed during starch manufacturing and that different purification steps, in particular the active carbon treatment, removes proteins and other nitrogen-containing compounds (EFSA, 2007c; 2007d). Residual gluten and peptides were detected by mass spectrometry and liquid chromatography analysis (0.3-1.4 mg/kg) in the final glucose syrup. Gluten levels were below 25.3 mg/kg using a gluten-specific ELISA with (3.1 mg/kg LOD). EFSA considered the analytical and dietary exposure data in the context of the clinical evidence for coeliac disease and wheat allergy, and concluded that it is not very likely that this product will trigger a severe allergic reaction in susceptible individuals. Under European Commission legislation, glucose syrups derived from wheat and barley are exempt from allergen declaration.
Six samples from different runs of wheat glucose syrup manufactured in Australia were analysed using a gluten-specific ELISA method and the Bradford protein assay (Cressey et al., 2010). Gluten was below the LOD, i.e. <3 mg/kg, in three out of six samples; and was 8, 15 and 22 mg/kg in the remaining three samples. Total protein levels detected were 8-16 mg/kg.
Refined oils
Many of the edible oils and fats are derived from the major allergenic foods i.e., soybeans, peanuts, tree nuts and sesame seeds. Crude oils are minimally processed and usually contain various levels of protein from the source food. Martín-Hernández et al. (2008) reported that the protein profile of the cold-pressed soy oil is very similar to that of soy flour. Teuber et al (1997) analysed the protein content in a number of commercially available oils and concluded that oils that underwent least processing at lower temperature had higher protein concentrations.
Crude oil can be further processed to produce refined oil or N/RBD oil. Refining involves a series of steps including degumming, neutralising, bleaching and deodorising. Such highly refined oils contain no detectable, or extremely low, protein levels (Taylor and Hefle, 2001; Martín-Hernández et al., 2008).
Although a number of DBPCFC studies showed that highly refined oils do not provoke allergic reactions in susceptible consumers (Taylor et al., 1981; Bush et al., 1985; Hourihane et al., 1997; Crevel et al, 2000), the debate on the safety of these oils for allergic consumers, particularly peanut oil, remains unsettled. Some of the concerns raised relate to the small number and/or insufficient clinical characterisation of allergic individuals tested, the limited number of oils tested compared to the range of products and blends available commercially, and the lack of standardised and validated methodology that can be used routinely for maintaining process specifications (EFSA, 2004b; Hildago and Zamora, 2006; Wilchers, 2007). Recent publications describe improved methodology for the detection of protein in oil (Ramazotti et al., 2008; Jablonski et al., 2010).
Nevertheless, based on more thorough investigations, scientific consensus now exists that refined soybean oil, produced by hot solvent-extraction, bleaching and deodorising, is not likely to cause severe allergic reactions in soy-allergic individuals (Taylor et al., 2004a; EFSA, 2007e).

6.1.4 Conclusions

• 
  Food processing can alter the allergenicity of food proteins. The impact of a given process may differ from one allergenic food to another. Proteins may undergo significant physical and chemical modifications as a result of food processing. The intrinsic characteristics of the proteins and the food matrix, as well as the processing method and processing environment affect the outcome. In addition to processes discussed in this report, other processes may be considered on a case-by-case basis, as appropriate.
  

• 
  Processes that physically or chemically separate food constituents can result in undetectable, or only residual, levels of protein in the processed products. However, reliable and easy to use protein detection methodologies are generally required to ensure process specifications are consistently achieved. Consideration of clinically relevant data is also required to determine the safety of food products derived from allergenic sources.
  

• 
  A well-controlled distillation operation is probably unique in its ability to eliminate non volatile substances, including proteins, from liquid mixtures.
  

6.1.5 Recommendation

• 
  FSANZ to consider, on a case-by-case basis, the scientific and clinical data available on the impact of food processing on the allergenicity of food ingredients derived from allergenic sources. In consultation with the food industry, FSANZ to develop options to reflect the evidence base through guidance and/or regulatory amendments.
  

7.