NOTE
Allergy to mackerel (Scomber scombrus):
effect of sterilisation treatment
Christine CHOPIN *, Nicolas LARDY, André DANIEL, Joël FLEURENCE
RÉSUMÉ Allergie au maquereau : influence de la stérilisation.
L’hypersensibilité aux produits de la mer est l’une des causes fréquentes d’al- lergie alimentaire. La production d’aliments « hypoallergiques » pourrait être l’une des solutions apportées à ce problème de santé publique. Notre étude montre que l’appertisation permettrait de réduire la reconnaissance des pro- téines par les IgE de séra de patients, et que cette reconnaissance diminue avec la durée du traitement thermique. Il serait intéressant de faire une carto- graphie des peptides responsables pour en connaître les propriétés et mieux comprendre les réactions biochimiques en cause dans le phénomène d’aller- gie.
Mots clés : poisson, traitement thermique, hypersensibilité.
SUMMARY
Allergy to seafood is one of the most common food allergies. Food technology could be employed to develop “hypoallergenic” foods. Heat treatment may reduce the potential allergenicity of mackerel. Serum collected from several patients showed that each serum had a specific response towards the various IgE binding peptides present in the product. The characterisation of allergens and the knowledge of their relative distribution could allow the setup of a dena- turation process to decrease the epitopic IgE recognition.
Keywords: fish, thermal process, hypersensibility.
Ifremer, rue de l’île d’Yeu, BP 21105, 44311 Nantes cedex 03, France.
* Correspondence Cchopin@ifremer.fr
1 - INTRODUCTION
Food allergy affects 1 to 5% of the population (EIGENMANN and SAMPSON, 1994) (YOUNGet al., 1994). Fish is one of the most common products involved in this reaction (AAS, 1966) (ANDREet al., 1994).
A new approach to prevent such allergies could be the development of
“hypoallergenic” foods. This could be achieved by structural modification of the allergenic peptides by thermal processing (TAYLOR, 1980) or by enzymatic hydrolysis (JOSTet al., 1987).
The present study aims to evaluate the effect of heat treatment (sterilisation) on the potential allergenicity of mackerel (Scomber scombrus) using a new chemi-luminescence detection method.
2 - MATERIALS AND METHODS
2.1 Steam sterilisation
The sterilisation process was achieved with the Stéripilote® from Gec Alsthom ACB society.
Products were prepared with fresh mackerel. Two hundred grams of muscle were canned in 1/4 size tins with 12 mL NaCl 30 g·L–1.
The temperature used was set at 120°C and the treatment lasted for 10, 20, 30 or 50 min. Three tins were prepared for each run.
2.2 Preparation of mackerel extracts
After draining, proteins were extracted from flesh with phosphate buffer 0.07 M pH 7.0 (2v:w), 3 min in an Ultra Turax®.
Samples were centrifuged 20 min, 20 000 g at 4°C. The protein concentra- tion was determined using the Bradford method (BRADFORD, 1976). Extracts were divided into fractions containing 10 µg of protein and preserved at – 20°C before use.
2.3 SDS - PAGE
Sodium- dodecyl sulfate polyacrylamide gel electrophoresis was performed with a 12% polyacrylamide gel in 20 mM Tris HCl, pH 8.3, 0.18 M glycine, and 0.1% SDS using a vertical Mini Protean® II apparatus (Biorad, Richmond, CA, USA). Two volumes of each fraction (10 µg of proteins) were mixed with one volume of Tris HCl 30 mM pH 6.8, EDTA 3 mM, SDS 3%, DTT 120 mM, glycerol 30%, bromophenol blue 0.05% and were heated at 100°C for 2 min before loa- ding. The separation was carried out using 35 mA for 1 h.
2.4 Silver staining of the gel
Gels were stained according to the procedure of BLUM et al., 1987, slightly modified. Proteins were fixed for 1 h in 40% methanol and 10% acetic acid. The gel was then washed three times in 30% ethanol and incubated in 0.02%
sodium thiosulfate for 1 min. It was then washed three times with ultrapure water, and stained (with 0.2% silver nitrate and 0.02% formaldehyde) for 20 min. The gel was washed and developed with 3% sodium carbonate, 0.05%
formaldehyde, 0.0005% sodium thiosulfate until the protein bands were suffi- ciently stained. Finally, staining was stopped with 0.5% glycine for 5 min and the gel washed with ultrapure water.
2.5 Relative allergenicity of each sample
2.5.1 Human sera
Patients who had an immediate adverse reaction to fish were investigated for the detection of specific IgE (CAP-RAST, Pharmacia, Uppsala, Sweden) against fish (f×3) in a medical laboratory specialized in allergy testing (CERBA Laboratory, Paris, France). One control blood was collected from a healthy sub- ject.
The blood was collected in dry tubes and centrifuged at 1 000 g for 5 min.
The sera were aliquoted and kept at – 80°C.
Twenty-one sera were tested against mackerel in our laboratory with the method described below. Four sera were positive: the ratio of the pixel intensity of the response obtained with the serum tested, to the pixel intensity obtained with the control (non-atopic subject) was greater than 3 (table 1).
Table 1
Adsorption to crude mackerel protein extract of serum IgE from patients who had allergic reaction to fish expressed as the ratio of the pixel intensity
of the response with the serum tested to the pixel intensity obtained with the control (nonatopic subject)
Serum 7.63 97 B 67
Score 6.3 5.8 6.5 5.25
2.5.2 IgE reactivity of the extracts
An amount of 10 µg of each extract or the crude mackerel sample was applied to a nitrocellulose membrane, 0.2 µm Trans-Blot® Transfert Medium (Bio-Rad, Richmond, CA, USA) using the microfiltration system Bio-Dot Appara- tus (Biorad).
Un-occupied protein binding sites were blocked by incubation with 0.3%
(v:v) Tween 20, 20 mM Tris-HCl pH 7.4, and 0.15 M NaCl for 1 h (DORYet al., 1998). After three washings with 0.05% (v:v) Tween 20, 20 mM Tris-HCl pH 7.4, and 0.15 M NaCl, the nitrocellulose membrane was incubated for 3 h with 1 mL of serum diluted 1:10 in 0.1% (v:v) Tween 20, 20 mM Tris-HCl pH 7.4 and 0.15 M NaCl. The membrane was washed three times with the 0.05% Tween
buffer and incubated for 3 h, at room temperature, with 2 mL anti-IgE from Epoq diluted 1/10 in the 0.1% Tween buffer. The membrane was washed three times and incubated overnight with anti-mouse IgG (whole molecule) horse radish peroxydase conjugate (Amersham International) diluted 1/500 in the 0.1% Tween buffer.
The membrane was rinsed (0.05% Tween buffer) at room temperature. After draining the excess wash buffer, the membrane was incubated 1 min in the detection reagent ECL® (Amersham International). The membrane was washed briefly and put on a sheet of Saran Wrap®. The wrapped blots were placed in a x-ray film cassette (HypercassetteTM, Amersham International) in contact with a sheet of autoradiography film (HyperfilmTM, Amersham International) for 2 h and 30 min.
The film was then removed and developed immediately.
The relative allergenicity for each extract and each serum was estimated as the ratio of the fraction allergenicity to the crude extract allergenicity.
3 - RESULTS AND DISCUSSION
The whole series of processed preparations was subjected to SDS-PAGE and blotted onto nitrocellulose membrane. Electrophoresis showed changes in protein patterns. Peptides with high molecular weight were more highly solubili- zed in the sterilised samples than in the crude extract, partly due to the action of NaCl on the myofibrillar protein solubilisation. Additionaly, denaturation seems to have been the more effective at 120°C, 50 min (figure 1).
Figure 1
SDS-PAGE of the sterilized extracts M: molecular weight marker (Sigma).
1: untreated sample; 2: 10 min 120°C; 3: 20 min 120°C; 4: 30 min 120°C; 5: 50 min 120°C.
105 kDa
M 1 2 3 4 5 M
82 kDa 49 kDa
33.3 kDa 28,6 kDa 19,4 kDa
Table 2
Hypersensibility results by chemiluminescence method in relation with the thermal treatment
Serum 67
Crude 10 20 30 50
Serum 67
Crude 10 20 30 50
Serum 67
Crude 10 20 30 50
Serum 67
Crude 10 20 30 50
100 90 80 70 60 50 40 30 20 10 0
raw 10 min 20 min 30 min 50 min
7.63 97 B 67
Figure 2
Relative intensity of immune response using the 4 different sera (3 repetitions)
A control sample was run with all samples consisting of a serum of a nona- topic individual and was always negative. Heating decreases the antibody bin- ding of all four sera (table 2).
The relative affinity for IgE of the samples decreased with the severity of the thermal treatment. The lowest immune response was obtained with the samples treated for 50 min (figure 2).
The crude extract obtained with a low ionic strengh buffer contained mostly sarcoplasmic proteins. Parvalbumin, the major thermostable allergen described in the literature (MATSUDAand NAKAMURA, 1993), was present in high quantities in the crude extract. In the samples however, its relative concentration was reduced because of the partial solubilisation of myofibrillar components in the presence of NaCl. This could explain the lower IgE affinity of the heated samples.
No diminution was observed with the serum 7.63 after 20 min at 120°C. Fifty min of heating deleted IgE binding only in serum 67. This result shows that the response is serum dependant. Thus more than one component is involved in this reaction and it is clear that several epitopes exist in the product.
4 - CONCLUSION
Our study shows that IgE-reactive components are still present after a sterili- sation process in mackerel.
It would be interesting to complete our study by characterising of the IgE reactive peptides in order to determine the epitopes and the biochemical reac- tions which are involved in the allergy phenomenon. Nevertheless, this work shows that technological processing allows to minimize the fish allergenic potential. Our group has previously detected, in cod, allergen bands which have not previously been described and which have a distribution that is dependent on fish freshness (DORYet al., 1998). Heat denaturation of some peptides inhi- bits the immune response. Enzymatic hydrolysis of milk proteins reduces sensi- tivity of children to milk (HALKENand HOST, 1997). Cod surimi, obtained by a prolonged washing of fish flesh, contains only one 63 kDa reactive peptide (MATAet al., 1994).
Clearly further effort is needed to produce “hypoallergenic” foods to limit the increasing incidence of food allergy. Several international organizations, have expressed concern regarding this problem, and have begun to develop actions in this field (F.A.O., 1993; US Food and Drug Administration, 1995; European Commission, 1997).
Received 11 January 1999, revised 8 October 1999, accepted 23 February 2000.
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