Elena Peñas

Savinase, the most suitable enzyme for releasing peptides from lentil (Lens culinaris var. Castellana) protein concentrates with multifunctional properties

The aim of this study was to produce multifunctional hydrolysates from lentil protein concentrates. Four different proteases (Alcalase, Savinase, Protamex, and Corolase 7089) and different hydrolysis times were evaluated for their degree and pattern of proteolysis and their angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activities. Alcalase and Savinase showed the highest proteolytic effectiveness (P ≤ 0.05), which resulted in higher yield of peptides. The hydrolysate produced by Savinase after 2 h of hydrolysis (S2) displayed the highest ACE-inhibitory (IC50 = 0.18 mg/mL) and antioxidant activity (1.22 μmol of Trolox equiv/mg of protein). Subsequent reverse-phase HPLC-tandem mass spectrometric analysis of 3 kDa permeates of S2 showed 32 peptides, mainly derived from convicilin, vicilin, and legumin containing bioactive amino acid sequences, which makes them potential contributors to ACE-inhibitory and antioxidant activities detected. The ACE-inhibitory and antioxidant activities of S2 were significantly improved after in vitro gastrointestinal digestion (P ≤ 0.05). Multifunctional hydrolysates could encourage value-added utilization of lentil proteins for the formulation of functional foods and nutraceuticals.

Biochemical and immunochemical characterization of different varieties of amaranth (Amaranthus L. ssp.) as a safe ingredient for gluten-free products.

Celiac disease is a food intolerance triggered by the ingestion of gluten-containing cereals; the only therapy is a strict gluten-free diet for life. In recent years, amaranth flour has received considerable attention as an interesting source for the formulation of gluten-free products due to its high nutritional value and low content of prolamins, the toxic proteins for celiacs. The aim of this study was to characterize 40 amaranth varieties using both SDS-PAGE/immunoblotting and ELISA to assess their possible tolerance by celiac subjects. All of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs. In most amaranth grains, the content of gluten-like proteins measured by ELISA was <20 ppm. The molecular characterization of amaranth proteins suggests that amaranth is safe for celiacs to consume. It is recommended that the most suitable amaranth varieties are those having the lowest content of proteins cross-reacting with anti-gliadin antibodies.

Biochemical and immunochemical evidences supporting the inclusion of quinoa (Chenopodium quinoa Willd.) as a gluten-free ingredient.

To date, the only acceptable therapeutic approach for celiac disease (CD) is a strict elimination from the diet of gluten-containing foods, but this diet does not always guarantee an adequate nutritional intake. Pseudocereals are receiving considerable attention as interesting alternatives for the formulation of gluten-free products, and quinoa grains arise as nutritive substitutes of conventional cereals. The aim of this study was the characterization of different quinoa samples corresponding to 11 quinoa varieties, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and immunoblotting techniques to assess their suitability for celiac subjects. Some of these varieties were grown in Italy to assess if the reproduction in a new habitat can guarantee the retention of the “safe” protein pattern. None of the quinoa varieties studied presented protein bands with electrophoretic mobility comparable with those of wheat gliadins, the toxic protein for celiac subjects. All the quinoa samples showed a low binding affinity for both specific anti-gliadin antibodies and IgAs from celiac subjects, confirming that quinoa can be considered as a safe ingredient for celiac patients. However, reliable varieties should be previously selected since the immuno cross-reactivity with anti-gliadin antibodies can vary significantly.

Characterization of the sensitization profile to lupin in peanut-allergic children and assessment of cross-reactivity risk.

Case reports of allergy to lupin, due to primary sensitization or cross‐reactions with other legumes, are increasing as a consequence of the augmented use of lupin flour in bakery, pasta formulations and other food items. The main allergens that have been associated with the sensitization to lupin are α‐ and β‐conglutins and, to a lesser extent, γ‐ and δ‐conglutin, but no conclusive data are available so far. The aim of this study was to characterize the sensitization pattern to lupin in a group of 12 Italian children allergic to peanut and identify the specific lupin proteins involved in the cross‐reactivity with peanut.

Immunochemical investigation of allergenic residues in experimental and commercially-available wines fined with egg white proteins.

Proteinaceous egg whites are widely used as a fining agent during the production of red wines. Residues of egg white in the final wine could present a risk for individuals allergic to eggs. This study investigated the presence of allergenic residues in both red and white wines fined with egg whites. Experimental and commercially available wines fined with egg whites, with or without subsequent bentonite fining, were studied. Unfined wines were used as negative controls. The physicochemical characteristics of each wine were determined to assess their possible role in enhancing or hindering the elimination of allergenic residues from wine. The amount of egg white protein residues was investigated both by a specifically developed/validated ELISA test and by immunoblotting. Both immunochemical tests used the same anti-total egg white protein antibody and were highly sensitive to the allergen. No egg white protein was detected in the wines studied in either immunochemical test, irrespective of the physicochemical characteristics of the wine, the type and dosage of the fining agent and the oenological process used. The risk of adverse reactions in egg-allergic individuals should therefore be considered negligible, but the exemption from labelling should be allowed only when the absence of residues is confirmed by analytical controls.

Children monosensitized to pine nuts have similar patterns of sensitization

Several cases of pine nut allergies and anaphylaxis have been reported in the literature, but only few pine nut allergens have been characterized. The aim of this research is to identify through immunoelectrophoretic techniques the major pine nut allergens in a group of children monosensitized to pine nuts.

Molecular characterization of allergens in raw and processed kiwifruit.

The prevalence of allergy to kiwifruit is increasing in Europe since the last two decades. Different proteins have been identified as kiwifruit allergens; even though with geographic differences, Act d 1, a cysteine protease protein of 30 kDa, and Act d 2, a thaumatin‐like protein of 24 kDa, are normally considered the most important. The aim of this study was (i) to identify at molecular level the sensitization pattern in a group of well‐characterized patients allergic to kiwifruit and (ii) to assess the role of technological treatments on kiwifruit allergenic potential.

Clinical monosensitivity to salmon and rainbow trout: a case report

To the Editor, Seafood plays an important role in human nutrition; the increasing consumption of certain fishes due to their healthy effects has led to a raising in the number of cases related to allergic reactions (1). The major fish allergen is parvalbumin (2), an acidic, calcium-binding 12 kDa protein resistant to heat and gastrointestinal digestion. Fish allergic patients are often sensitized to more than one species due to the cross-reactivity of their betaparvalbumins. However, until now, only few cases of monosensitivity to fish have been described (3–7). We report the case of a 14-year-old boy with a history of food allergy (milk) from the first year of life. At age 4, he presented his first allergic reaction to fish, after ingesting a few appetizers containing smoked salmon. In a few minutes, he suffered from rush cutaneous and lip edema, which regressed after oral administration of corticosteroids. Skin prick tests, performed with commercial extracts (Lofarma, Milano, Italy), and prick by prick tests showed negative results to mollusks, crustaceans, and fishes, including salmon. Two years later, few minutes after the ingestion of broiled salmon, he showed a new allergic reaction including lip and nose edema, which regressed after cortisone treatment. At the age 11, a small quantity of broiled rainbow trout (Salmonidae) determined a sudden and severe intense throat itching; this reaction regressed spontaneously in a few minutes. After this new event, the boy stopped consuming salmon, rainbow trout, and other freshwater fishes. He had safely consumed codfish, halibut, anchovies, tuna fish, and mackerel, apart from a case of reaction to fish sticks (containing only cod fish) where a cross-contamination with salmon in the production chain was hypothesized. As monosensitization to fish is quite unusual, the patient was tested at molecular level to confirm the specific pattern of reactivity by immunoblotting. IgE multiplex test for the most usual allergens showed a positive response only for Gadus callarias (cod fish) muscle IU = 0.03. Further investigation by ISAC test showed the presence of serum-specific IgEs versus the allergens Onc m from rainbow trout (Oncorhynchus mykiss) ISU = 2.10 and Sal s from salmon (Salmon salar) ISU = 2.22. ISAC tests that were reported negative were obtained with fishes from the families of Gadidae (cod fish, Gad m), Soleidae (sole, Sol so), and Scombridae (tuna and similar fishes, Thu a). Finally, a negative result was found for Anisakis simplex (Ani s). Proteins from codfish (Gadus morhua), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), sole (Solea vulgaris), and tuna fish (Thunnus albacares) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions. Fish species were from a local supermarket and were analyzed as such (raw) and cooked (boiled in water at 100°C for 20 min) to simulate the usual domestic preparation. All the samples were freeze-dried, ground, and the resulting flour was suspended in sample buffer (containing 0.063 M TRIS–HCl pH 6.8, 1.88% glycerol, 0.5% SDS, 2.5% b-mercaptoethanol) at a final concentration of 7.5 mg/ml (w/v), corresponding to 4 mg/ml of proteins approximately. A pre-stained molecular weight standard solution (broad range; Bio-Rad, Richmond, CA, USA) was run in parallel to the samples. SDS-PAGE and immunoblotting were performed according to Ballabio et al. (8). Briefly, after SDS-PAGE, proteins were transferred to a PVDF membrane by Western blotting, blocked, and washed with gelatin solutions to prevent non-specific adsorption of the immunological reagents. The membrane was then immersed in 10 ml of 0.25% gelatin solution containing 0.3 ml of the patient’s serum. Antigen–IgE complexes were detected using 10 ll of goat antihuman IgE antibodies labeled with alkaline phosphatase in the presence of the specific substrates. To verify the specificity of immunoreactivity, immunoblotting was performed under identical conditions using the serum of two children highly allergic to foods different from fish (results not shown) with faint unspecific immunoreactivity. Figure 1 shows the electrophoretic profiles of fishes included in the study, tested as such (left panel) and after boiling (right panel). All the samples yielded a complex protein pattern, with some differences from the qualitative and quantitative point of view. Raw codfish, rainbow trout, and salmon showed some protein bands having low molecular weight (MW); in particular, two bands (L and M) were less abundant or absent in sole and tuna fish. Parvalbumin was only visible in codfish and rainbow trout (band L, MW 12–14 kDa). Cooked samples presented several differences in their electrophoretic pattern when compared to raw fish; in particular, cooked samples showed a larger number of protein bands in the MW region from 6 kDa to 30 kDa. A band having a MW of about 12–14 kDa, corresponding to parvalbumins, was clearly evident in all the samples analyzed, although the intensity of this band in the sole was very low (band L). Immunoblotting analysis (Fig. 2) showed that serum allergen-specific IgEs were mainly reactive for two protein bands having a molecular weight of approximately 35 kDa (G) and 75 kDa (C) in raw salmon. These two protein bands were also immunoreactive in the other fish species tested, with the exception of codfish, in which the IgE recognized the 35 kDa (G) protein and a second band having a MW>100 kDa (B). Several bands were recognized by serum IgEs in rainbow trout after cooking: A (170 kDa), B

Allergy to all mammalian Bovidae proteins but cow's milk in a child

Cross reactivity between mammalian proteins (cow, goat, ewe, buffalo) has been previously described in both in vitro and in vivo studies. The highest homologies are observed between the milk proteins from cows and other Bovidae, being on average 96.1% for buffalo, 91.1% for ewe, and 87.6% for goat. Lower sequence similarities are associated with proteins contained in milks from Suidae (pigs and boars, 64.2%), Equidae (horse 62.4% and donkey 62.8%), Camelidae (dromedary 60.0%) and humans (58.4%). Despite the high degree of homology with cow’s milk, allergy to buffalo’s milk is poorly investigated and so far only one case of severe allergy to buffalo’s milk has been shown in a 70-year-old German patient who indeed tolerated cow’s milk and had negative goat milk skin tests. We report the case of a six-year-old boy with a past medical history of atopic dermatitis. At the age of two years, he developed asthma. The skin tests with common inhalants (Alk-Abelló extracts) resulted positive to grass, dust mite, cypressus, cat and mugworth. The child was eating regular cow’s milk proteins from the first year of life. At the age of three years he had vomit immediately after the intake of ewe cheese. Prick-to-prick with ewe’s and goat’s milks resulted positive as well as skin prick tests with goat’s milk and ewe’s milk caseins (Alk-Abello, Milan, Italy). Cow’s milk prick-to-prick and skin prick test (SPT) with purified casein, -lactalbumin and -lactoglobulin (AlkAbello, Milan, Italy) from cow’s milk were negative. He was advised to eat only cow’s milk and cow’s milk products. At the age of four years after the intake of cheese made with cow’s and goat’s milk (the last ingredient was not declared) he developed asthma plus vomit. The in vitro diagnostic test confirmed the presence of specific IgE to goat and ewe milk: >100 KUA/L and 78.9 KuA/L respectively, and the absence of specific IgE to cow’s milk, bovine casein and whey proteins -lactalbumin and -lactoglobulin (ImmunoCAP system, Phadia Diagnostics, Milan Italy). The total IgE level was 100 KUA/L. At the age of five years he had an anaphylactic reaction (asthma, vomit, generalised urticaria) immediately after the intake of buffalo’s mozzarella cheese. At the time of anaphylaxis the child could eat cow’s milk proteins without adverse reactions. Wheal and flare (W/F) reactions by means of prick-to-prick tests with buffalo’s milk, buffalo’s mozzarella and histamine at 10 mg/ml were 10/20, 10/20 and 4/8, respectively. Prick-to-prick with buffalo’s mozzarella was performed in three non-allergic children as controls. Under double-blinded, placebo-controlled oral provocation with buffalo’s fresh untreated milk, the patient developed urticaria and rhinitis (1 mL = single provoking dose). The reaction was treated with oral corticosteroids and anti-histamines. This study was performed with the approval of the ethics committee, and both parents gave written consent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with different mammalian milks and with purified proteins from cow’s milk ( e -casein, -lactalbumin, lactoglobulin and bovine serum albumin) was performed. As shown in Fig. 1, different electrophoretic patterns were obtained according to the mammalian species considered. Cow’s milk contains four different bands from top to bottom: and -casein, -lactoglobulin and -lactalbumin. In the goat’s milk sample (both fresh and commercial), the -casein amount is lower than that in cow’s milk according to previous evidence. Ewe’s milk electrophoretic profile is slightly different in terms of electrophoretic mobilities of casein and whey proteins. Buffalo’s milk shows an electrophoretic profile overlapping that of cow’s milk. In the buffalo’s mozzarella both -casein and -casein are easily identified, whereas whey proteins are less clearly visible (only a weak band corresponding to -lactoglobulin). After transfer of proteins from gel to PVDF membrane, incubation with the serum of the allergic patient was performed. The immunoblotting showed specific IgE reactivity against -casein in goat’s milk and ewe’s milk; a lower but still significant reaction was observed versus buffalo’s milk and mozzarella. A marked IgE-mediated reaction with another protein having an electrophoretic run higher than -casein, can be observed in commercial goat’s milk. This is probably due to the presence of -caseins (a fraction of -casein) coming from a partial proteolysis. The same band is also present in fresh goat’s milk but in traces; in fact caseins are fragments due to proteolysis whose abundance increases progressively from fresh milk to cheese. No reactivity with cow’s milk proteins can be detected (Fig. 2). Cross reactivity between goat or ewe’s milk and cow’s milk has been frequently described. Moreover, allergy to milk of goat and ewe, two phylogenetically closely related mammalians, without allergy to cow’s milk has also been reported. Again, despite the close phylogenetic relationship between buffalo and cow, an isolated buffalo’s milk allergy has been described. In this case, specific IgE against two bands were demonstrated and in immunoblot experiments, specific IgE reactivity could be reproduced against non-glycosylated and glycosylated bufaline K-caseins, corresponding to the 17-kDa band.

Effect of Dry Heat Puffing on Nutritional Composition, Fatty Acid, Amino Acid and Phenolic Profiles of Pseudocereals Grains

Abstract The impact of puffing on nutritional composition and phenolic profiles of kiwicha (Amaranthus caudatus L.) and quinoa (Chenopodium quinoa Willd.) was investigated. Popped kiwicha showed increased protein and lipid contents and lower contents of carbohydrates compared to the untreated grains. Higher lipid, ash and carbohydrates contents and a decreased protein content were observed after puffing of quinoa. Fatty acid profile and ω-6/ω-3 ratio was not affected by puffing, although it was observed a healthier ratio in quinoa (7:1) compared to kiwicha (65:1). Thermal treatment reduced essential amino acid contents and protein quality of both grains, although amino acids content remained adequate according to FAO/WHO requirements for adults. Puffing decreased hydroxybenzoic and hydroxycinnamic acids content of both pseudocereals. Flavonoid levels were negatively affected by puffing in kiwicha while a noticeable increase was observed in popped quinoa. In summary, puffing of kiwicha and quinoa grains is an alternative processing method to obtain expanded products or precooked flours of adequate nutritional value.