Lupine,a source of new as well as hidden food allergens

The present review summarizes current knowledge about lupine allergy, potential sensitization routes, cross‐reactions between lupine and other legumes, and the respective IgE‐binding proteins. Since the 1990s, lupine flour is used as a substitute for or additive to other flours, mostly wheat flour, in several countries of the EU. In 1994, the first case of an immediate‐type allergy after ingestion of lupine flour‐containing pasta was reported. Since then, the number of published incidents following ingestion or inhalation of lupine flour is rising. So far, the Lupinus angustifolius β‐conglutin has been designated as the allergen Lup an 1 by the International Union of Immunological Societies Allergen Nomenclature Subcommittee. Initially, publications focussed on the fact that peanut‐allergic patients were at risk to develop anaphylaxis to lupine due to cross‐reactivity between peanut and lupine. At present, however, the ratio between cases of pre‐existing legume allergy (mostly peanut allergy) to de novo sensitization to lupine seed is nearly 1:1. Although in December 2006, lupine and products thereof were included in the EU foodstuff allergen list according to the Commission Directive 2006/142/EC amending Annex IIIA of Directive 2000/13/EC in order to prevent severe reactions caused by “hidden food allergens”, the majority of patients and medical personnel are still not aware of raw lupine seed as potentially dangerous food allergen.     

Comparison of different extraction solutions for the analysis of allergens in hen’s egg

An important requirement for the correct procedure of allergen analysis in hen’s egg is to obtain complete and unaltered protein extracts. Besides the aim of a quantitative extraction of the allergens from the matrix, it is equally important not to alter their allergenic potential during the extraction process. This paper describes and compares six extraction solutions for the analysis of whole-egg proteins and allergens. These requirements were examined via protein determination according to Bradford [Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Analytical Biochemistry, 72, 248–254] and Kjeldahl [Meyer, A. H. (2006). Lebensmittelrecht, Verlag C.H. Beck München, Stand: 1. February 2006, § 64, Lebensmittel- und Futtermittelgesetzbuch, Amtliche Sammlung von Untersuchungsmethoden, Nr. L 06.00-7] as well as the EAST-inhibition method. It could be demonstrated that the extraction with a urea solution (8 M) led to significant interferences during the protein determination, and substantially reduced the allergenic potential of egg proteins. With all other extraction solutions adequate protein contents could be extracted. The highest protein content was achieved by the extraction with phosphate buffered saline followed by a Tween 20 solution, physiological saline, water, and acetate buffer. The results show that none of these extracts – except for the urea solution (8 M) – was altered in its’ allergenic potential.     

Influence of food processing on the immunochemical stability of celery allergens

Celery roots were processed by microwave heating, cooking, drying, γ-irradiation, ultra high pressure treatment and high voltage impulse treatment. The immunochemical stabilities of the three known allergenic structures of celery were tested with sera from patients who were sensitised to celery. In addition, rabbit antisera were used to detect the allergens profilin and Api g 1 on celery immunoblots. The specificity and reactivity of IgE from the patients' sera were investigated by immunoblotting, by an enzyme allergosorbent test (EAST) and by dose-related IgE inhibition experiments. The results of all three methods agreed closely and indicated high antigenic and allergenic activity in native celery which was reduced by thermal processing. The heat-stability of the known celery allergens decreased in the following order: carbohydrate epitopes> profilin>Api g 1. In contrast, the allergenicity was only mildly reduced by non-thermal processing. The results obtained with human IgE were confirmed by an in vitro mediator-release assay that is based on rat basophil leukemia cells (RBL cells) which were passively sensitised with celery-specific murine IgE. With sera from mice that had been immunised with native celery, the native sample and non-thermal celery preparations elicited the strongest mediator release, whereas a weak response was obtained with samples from heat-processed celery. These results agreed closely with the data obtained in allergic patients whose IgE antibodies were directed against the major protein allergen Api g 1. Our results may be helpful in risk assessment and in selecting food preparations which can be consumed without symptoms by a subgroup of celery-allergic patients with a known sensitisation pattern. ©1997 SCI     

Characterisations of kabuli and desi chickpea starches cultivated in China

The characterisation of starches from kabuli and desi type chickpea seeds was investigated by monitoring amylose content, swelling power, solubility, synaeresis, water-binding capacity and turbidity properties. Total amylose and apparent amylsoe content were 31.80% and 29.93% for kabuli and 35.24% and 31.11% for desi, respectively. The shape of starch granules varied from round to oval or elliptic. The transition temperatures (T_o, T_p and T_c) were (62.237, 67.000 and 72.007 °C) and (59.396, 68.833 and 77.833 °C) for kabuli and desi starches, respectively. The ΔH value of kabuli type was higher than that of desi type. The crystal type of chickpea starches was a typical C_A-type pattern. Breakdown and setback viscosity of kabuli starch were lower than those of desi starch, indicating high heat and shear stability. Kabuli starch showed a higher value of M_w (5.382 × 10⁷ g/mol) than desi starch (3.536 × 10⁷ g/mol). Both kabuli and desi starches belonged to low glycaemic starches from measuring starch fractions and hydrolysis index.     

The effects of planting time on fatty acids and tocopherols in chickpea

In Turkey, as an important producer, chickpea is generally planted in March and April during spring. However, spring plantings have the disadvantage of unstable yield and quality from year to year due to irregularity in spring precipitation. In this research, fatty acids and tocopherol contents in different chickpea genotypes were investigated for consecutive 2 years with both autumn and spring plantings. Tocopherol analyses were carried out by using a high performance liquid chromatography. Gamma tocopherol was the major component of total tocopherol content and it ranged between 15.34 and 42.09 mg kg⁻¹. Alpha tocopherol content ranged between 4.55 and 10.69 mg kg⁻¹. Depending on planting time, the values of tocopherol components changed significantly. The mean values for alpha tocopherol were 6.77 mg kg⁻¹ for autumn and 7.55 mg kg⁻¹ for spring plantings; while the mean values for gamma tocopherol were 33.32 mg kg⁻¹ for autumn and 22.50 mg kg⁻¹ for spring plantings, respectively. Fatty acids, quantified using a gas chromatography, indicated that differences among the genotypes were significant. The values for autumn planting were generally higher than those of spring planting. The values of major fatty acids changed between 18.57 and 35.23% for oleic acid and 47.15 and 63.44% for linoleic acid. According to planting time the mean values for oleic acid changed between 21.23 and 30.52% and for linoleic acid between 51.77 and 60.79%.     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.

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Zusammenfassung. Allergene Lebensmittelbestandteile stellen für viele Nahrungsmittelallergiker in industrialisierten L?ndern ein reales Gesundheitsrisiko dar, wenn sie als nicht deklarierte Zutaten oder unerkannte Kontaminationen in Lebensmitteln enthalten sind. Diese so genannten versteckten Allergene k?nnen, teilweise in geringsten Mengen, schwere Reaktion bei sensibilisierten Personen auszul?sen, manchmal auch mit t?dlichem Ausgang. Zum Nachweis allergener Lebensmittelbestandteile wurden bereits verschiedene analytische Methoden, wie z. B. ELISA- und PCR-Verfahren, entwickelt. Allerdings sind diese Methoden nicht in der Lage, das allergene Potenzial solcher Bestandteile zu erfassen. Um jedoch die biologische Aktivit?t von Allergenen messen zu k?nnen wurde ein Testsystem entwickelt, das auf dem Mechanismus der Typ-I Allergie basiert. Dazu wurden basophile Leuk?miezellen der Ratte mit den Genen des humanen hochaffinen Rezeptors für IgE transfiziert. Die daraus resultierende Zelllinie exprimiert einen chim?ren Rezeptor (mit der IgE-bindenden humanen α-Kette) stabil auf ihrer Oberfl?che und ist somit in der Lage, auch allergenspezifisches IgE aus Allergikerseren zu binden, was mit der Ausgangszelllinie nicht m?glich ist. Die Vernetzung des rezeptorgebundenen IgEs durch das Allergen führt in der Folge zur Freisetzung entzündungsausl?sender Mediatoren, die im Zellkulturüberstand gemessen werden k?nnen. Diese transfizierte Zelllinie wurde zur Analyse einer Vielzahl von Allergenextrakten eingesetzt, darunter auch Extrakte von Lebensmitteln, die Haselnuss- oder Erdnussallergene enthielten. Der Vergleich des biologischen Testsystems mit den ELISA- und PCR-Verfahren für Hasel- und Erdnuss ergab eine ?hnliche Sensitivit?t und Spezifit?t. Die etablierte Zelllinie ist ein neues, wertvolles Hilfsmittel für den Nachweis von Allergenen in komplexen, zusammengesetzten Lebensmitteln und im Besonderen zur Erfassung des allergenen Potenzials solcher Nahrungsmittelbestandteile. Somit kann dieses neue Nachweisverfahren helfen, zus?tzliche wichtige Informationen für die Risikobewertung von Lebensmitteln zu gewinnen.

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Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates

A chickpea protein hydrolysate produced with pepsin and pancreatin was used for the affinity purification of chickpea chelating peptides. Three chelating peptide fractions were obtained after affinity chromatography with immobilised copper. These peptide fractions showed a higher chelating activity and histidine contents than the original protein hydrolysate. Chelating activity was positively correlated with the histidine content of the purified fractions. Different subfractions were also obtained after gel filtration chromatography from the affinity purified peptide fractions. Some of these subfractions showed a higher chelating activity and histidine contents than the original fractions. These results suggest that a combination of high His contents, around 20–30%, and small peptide size provide the best chelating activities. Thus sequential purification with affinity and gel filtration chromatography is a useful procedure for the purification of chickpea peptides with high chelating activity. These results show that a range of chelating peptides are generated during digestion of the chickpea proteins that, after metal chelation, may prevent the generation of reactive oxygen species (ROS) and favour metal absorption.     

Seed protein characterisation and nitrogen fixation rates in the chickpea mutant hyprosola and its parent

The amino acid composition of the seed proteins of Hyprosola, a high protein, high yielding mutant of chickpea (Cicer arietinum L), differed little from that of its parental cultivar, Faridpur-1. Oser's essential amino acid index was 76-5 in the mutant and 76-7 in the parent. Hyprosola seed has higher crude protein, extractable protein, globulin and albumin contents than Faridpur-1. The mutant had an albumin/globulin ratio of 0-23 compared with the parental value of 0-21. The mutant oligomeric proteins differed quantitatively and qualitatively from the parent on cellulose acetate electrophoretograms. The polypeptide composition of globulins and albumins fractionated by SDS-PAGE differed markedly in the intensity of many bands but not in the mobility of any polypeptides. Nitrogen fixation (acetylene reduction) rates were similar in the two cultivars, and in both hydrogen evolution utilised 45 % of the electron flow through nitrogenase.     

Isolation and characterisation of the isoflavones from sprouted chickpea seeds

Seven isoflavones were isolated from sprouted chickpea seeds by chromatography on silica gel column, polyamide column, sephadex LH-20 column and preparing thin-layer chromatography (TLC), respectively. The structures were characterised with one- and two-dimensional NMR in combination with mass and IR spectrometry. The obtained isoflavones were biochanin A (5,7-dihydroxyflavone-4′-methoxyflavone, (1); calycosin (7,3′-dihydroxy-4′-methoxyisoflavone, (2); formononetin (7-hydroxy-4′-methoxyisoflavone, (3); genistein (5,7,4′-trihydroxyisoflavone, (4); trifolirhizin (maackiain-3-O-β-d-glucopyranoside, (5); ononin (7-O-β-d-glucosyl-7-hydroxy-4′-methoxyisoflavone, (6); sissotrin (7-O-β-d-glucosyl-5,7-dihydroxy-4′-methoxyisoflavone, (7).     

Combined effects of high-pressure and enzymatic treatments on the hydrolysis of chickpea protein isolates and antioxidant activity of the hydrolysates

A chickpea protein isolate (CPI) was pretreated before hydrolysis under a pressure that varied between 100 and 600MPa. The hydrolysis rate increased significantly with pressure above 300MPa. At 40min, the DH of the control was 15.3%, while the DH of the CPI treated at 300MPa was 18.5%, which reached 23.74% post treatment at 400MPa. The pretreatment of CPI above 300MPa enhanced the superoxide anion capturing rate of enzymatic hydrolysis. Pretreatment at 400MPa significantly reduced the hydrolysis time with the release of antioxidant peptides. While hydrolysis by Alcalase during treatment at high pressure (100-300MPa) significantly increased the degree of hydrolysis (DH), its maximum value peaked after hydrolysis at 200MPa for 30min. In addition, hydrolysates obtained at high pressure (100-300MPa) had a higher superoxide anion capturing rate. High-pressure treatment at 200MPa for 20min resulted in products with high antioxidative activity. The molecular-weight (MW) determination of the enzymatic hydrolysates indicated that hydrolysis at high pressure could significantly increase the amount of low-molecular-weight peptides.     

Genotypic diversity in physico-chemical,pasting and gel textural properties of chickpea (Cicer arietinum L.)

Twenty chickpea cultivars were evaluated for genetic diversity in seeds (physical, hydration and cooking), flours (composition, pasting and gel textural) and starch (swelling, thermal, amylose content and amylopectin structure) properties. Frequency distribution and principal component analysis revealed significant differences among the cultivars studied. Pasting temperature, peak viscosity, breakdown, final viscosity and setback of flours ranged from 75.0 to 87.1 °C, 564 to 853 cP, 32 to 123 cP, 573 to 969 cP, and 84 to 185 cP, respectively. Amylose content of starch ranged from 28.26% to 52.82%. Amylopectin unit chains of DP 6–12, 13–18, 19–24 and 25–30 ranged from 36.2% to 43.25%, 36.44% to 38.68%, 14.86% to 18.22% and 4.95% to 6.9%, respectively. To establish the relationships between different properties Pearson correlation coefficients (r) were computed. Cluster analysis for grain and flour characteristics was also done to see the association between chickpea cultivars.     

Thermal processing effects on the functional properties and microstructure of lentil, chickpea, and pea flours

Pulses are rich in nutrients. The existence of anti-nutritional components and the length of time required for preparation have, however, limited their frequency of use compared to recommended intake levels. Anti-nutritional components in pulses can be largely removed by heat treatment. Additionally pre-treatment of pulses with heat and processing of seeds into flour could further enhance their use by decreasing processing and preparation times. In this study, trypsin inhibitor activity, functional properties, and microstructural characteristics of flours prepared from different varieties of lentil, chickpea, and pea as affected by roasting and boiling were evaluated. Both thermal treatments resulted in significant reduction (p < 0.05) in trypsin inhibitor activity ranging from − 95.6% to − 37.8%. Scanning electron microscopy (SEM) results showed that the roasted pulse flours had similar microstructure (i.e., starch granule and protein matrix structure) to the raw samples. For the pre-boiled flours, amorphous flakes were observed by SEM with no presence of intact starch granules. This is likely due to gelatinization of starch during cooking. Interestingly, flours treated by boiling exhibited significantly higher (p < 0.05) fat binding capacity, water holding capacity, and gelling capacity, while protein solubility was significantly reduced compared to the raw and roasted pulse flours. Overall, thermal treatments either had no impact or impacted to different extents the emulsifying and foaming properties of the flours. Our results suggest that thermally-treated pulse flours may have very good potential to be used as value-added food ingredients for food applications due to their improved nutritional value and, in some instances, superior functionality.     

Influence of preparation methods on physicochemical and gelation properties of chickpea protein isolates

Two protein isolates were prepared from defatted chickpea seed flour by applying alkaline extraction followed by isoelectric precipitation or ultrafiltration (TpI and TUF, respectively), while another one (TF) was obtained by a combination of protein extraction at a mildly acidic environment and ultrafiltration processes. The isolates differed in composition, with the TpI and TF containing mainly the chickpea globulins and the albumins, respectively, whereas the TUF isolate consisted of both types of proteins with the globulin fraction dominating over the albumins. The differences in protein composition between the isolates as well as the impact of extraction conditions were reflected in their protein solubility, surface hydrophobicity, sulfhydryl group content, thermal properties and the onset of gelation during heating. On the other hand, the protein isolate gelling behavior depended mainly on the method applied for their preparation rather than the protein composition, with the isolates obtained by ultrafiltration exhibiting lower gelling concentrations and gel networks of higher elasticity at protein contents below 12% (w/v).     

Emulsifying properties of chickpea protein isolates: Influence of pH and NaCl

Structure and emulsifying properties of chickpea protein isolates (CPI) as a function of protein concentration, oil volume, pH and ionic strength were studied. The optimum protein concentration 2 g l⁻¹ used to determine the emulsifying properties was obtained. Emulsifying activity index (EAI) increased from 244 to 376 m² g⁻¹ with pH from 3.0 to 11.0 except the protein isoelectric point (pI 5.0), where the EAI was 20 m² g⁻¹ and emulsion droplet size was the largest. At lower ionic strengths (0.0–0.1 M NaCl, pH 7.0), EAI decreased from 253 to 72.4 m² g⁻¹; however, it increased from 72.4 to 231.4 m² g⁻¹ at higher ionic strengths (0.1–1.0 M NaCl). A positive relation between EAI and surface hydrophobicity (S₀) of CPI at various ionic strengths was obtained, while EAI was independent of S₀ under different pH values. α-Helix was the major configuration of CPI at the pI or lower ionic strength.     

Advances on the molecular characterization,clinical relevance,and detection methods of Gadiform parvalbumin allergens

Gadiform order includes several fish families, from which Gadidae and Merlucciidae are part of, comprising the most commercially important and highly appreciated fish species, such as cod, pollock, haddock, and hake. Parvalbumins, classified as calcium-binding proteins, are considered the main components involved in the majority of fish allergies. Nine and thirteen parvalbumins were identified in different fish species from Gadidae and Merlucciidae families, respectively. This review intends to describe their molecular characterization and the clinical relevance, as well as the prevalence of fish allergy. In addition, the main protein- and DNA-based methods to detect fish allergens are fully reviewed owing to their importance in the safeguard of sensitized/allergic individuals.     

Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH)

Chickpea protein hydrolysate (CPH) was fractionated by gel filtration on Sephadex G-25. The antioxidant and free radical-scavenging activities of four CPH fractions (Fra.I, Fra.II, Fra.III, and Fra.IV) were measured using reducing power, inhibition of linoleic acid autoxidation, and 1,1-diphenyl-2- pycrylhydrazyl (DPPH)/superoxide/hydroxyl radical-scavenging assay. The antioxidant activity of Fra.IV (81.13%) was closer to that of α-tocopherol (83.66%) but lower than that of BHT (99.71%) in the linoleic acid oxidation system. Amino acid analyses showed that Fra.IV with the strongest antioxidant activity also had the highest total hydrophobic amino acids content (38.94% THAA) and hydrophobicity (125.62 kcal/mol amino acid residue) compared with the other three fractions. The molecular weight distribution of Fra.IV was found to vary from 200 to 3000 Da.     

Isolation and characterization of natural Ara h 6: evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat

Peanut allergy is a significant health problem because of its prevalence and the potential severity of the allergic reaction. The characterization of peanut allergens is crucial to the understanding of the mechanism of peanut allergy. Recently, we described cloning of the peanut allergen Ara h 6. The aim of this study was isolation and further characterization of nAra h 6. We purified nAra h 6 from crude peanut extract using gel filtration and anion exchange chromatography. The preparation was further characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with subsequent immunoblotting. Stability of nAra h 6 was studied by an in vitro digestibility assay as well as by resistance against thermal processing. Sequencing of nAra h 6 identified the N-terminal amino acid sequence as MRRERGRQGDSSS. Further results clearly demonstrated stability of nAra h 6 against pepsin digestion and heating. Immunoglobulin G (IgE) binding analysis and its biological activity shown by RBL 25/30-test of natural Ara h 6 supported the importance of this peanut allergen. Investigation of nAra h 6 revealed evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat.     

Influence of germination techniques on phytic acid and polyphenols content of chickpea (Cicer arietinum L.) sprouts

Influence of germination time and type of illumination on phytic acid and polyphenols of chickpea was investigated. With blue light illumination, a significant decrease (p < 0.01) in phytic acid content was observed, while all other illuminations had no effect on this parameter. Germination time up to 48 h significantly reduced (p < 0.01) the phytic acid content from 1.01% to 0.6% while beyond that time it increased significantly (p < 0.01) reaching the maximum value of 0.9% after 120 h which is still significantly lower than control (1.01%). A similar trend was observed for methanol extractable polyphenols as for phytic acid. It decreased significantly (p < 0.01) with 24 h and 48 h germination time while after that it increased significantly (p < 0.01) and the maximum value was noted with 120 h germination. Red light significantly diminished methanol extractable phenols as compared to dark, fluorescent, yellow and irradiation illuminations. Effect of germination time and type of illumination was highly significant (p < 0.01) for water and acidic methanol extractable phenols. Acidic methanol extractable phenols significantly increased (p < 0.01) from 0.055% to 0.14% within the first 24 h germination while beyond that it decreased significantly reaching the minimum value as for control. Germination under dark and irradiated chickpea seed enhanced the methanol extractable phenols content followed by fluorescent and yellow illuminations. Lowest values for methanol extractable phenols were noted for red followed by green and blue illuminations. Water extractable polyphenols decreased significantly with the increase in germination time. Significantly higher water extractable polyphenols content were noted under blue light germinating samples followed by irradiated samples and lower values for germination under yellow light.     

Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein

The chickpea vicilin-like globulin was isolated and chromatographed on Sepharose CL-6B and Sephacryl S-300. The native globulin with a molecular weight of 140 kDa was resolved in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seven polypeptide bands in the range of 12.4-67 kDa. The solubility profile of the protein in water and NaCl solutions was typical of a legume globulin. The purified vicilin-like globulin, native and heated, was hydrolyzed by pepsin, trypsin and chymotrypsin. The hydrolysis patterns indicated that the native vicilin-like protein was only partially degraded by the enzymes in comparison with casein. Heating increased its susceptibility to hydrolysis relative to the native form, for all the enzymes. However, the results obtained by the pH-drop method revealed that the in vitro digestibility of the vicilin-like protein was not altered by heating, while 11S-like and total globulins suffered a small increase, indicating that the structural characteristics of storage globulins may be important factors limiting the protein digestion.