RAPID DETECTION OF SALMONELLA ENTERITIDIS AND ESCHERICHIA COLI USING SURFACE PLASMON RESONANCE BIOSENSOR

The Biacore surface plasmon resonance (SPR)‐based biosensor was used to detect Salmonella enterica serovar Enteritidis and Escherichia coli in spiked skim milk using specific antibodies. The optical SPR biosensor registers changes in the refractive index during binding of the analyte (bacteria) to the ligand (antibody) on the gold‐coated sensor chip surface. Immobilized protein A‐captured specific antibodies were used to detect the bacteria in less than an hour. The reaction was found to be specific, and the limits of detection of the assay were found to be 25 cfu/mL for E. coli and 23 cfu/mL for Salmonella. Because the assays were shown to be sensitive, reproducible and very specific, the SPR‐based Biacore biosensor has the potential for near real‐time, yet specific, detection and presumptive identification of bacteria in food and water.     

Kinetic models of non‐enzymatic browning in apple puree

The effects of thermal treatments on 11 °Brix apple puree were studied at temperatures from 80 to 98 °C. Colour changes (measured by reflectance spectroscopy, colour difference, L*, a* and b* and the evolution of 5‐hydroxymethylfurfural (HMF) and sugars (hexoses and sucrose) were used to evaluate non‐enzymatic browning. A kinetic model based on a two‐stage mechanism was applied to the evolution of colour difference and a*. A first‐order kinetic model was applied to L* evolution, while the evolution of absorbance at 420 nm (A₄₂₀) of the liquid fraction was described using a zero‐order kinetic model. Thermally treated samples became more reddish and suffered a slight loss of yellow hues. The effect of temperature on the kinetic constants was described by an Arrhenius‐type equation. The presence of pulp in the samples led to activation energies for A₄₂₀ and sucrose which were lower than those found previously for clarified juices with the same soluble solids content. © 2000 Society of Chemical Industry     

水产品过敏原的研究现状和展望

水产品作为人类食物的重要来源之一,其市场和消费群体不断扩大。与此同时,由水产品引发的食物过敏也日益增多,在联合国粮农组织公布的八大类过敏食物中,水产品就占了两大类。水产品过敏原有小清蛋白(Parvalbumin)、鱼卵蛋白(如鲑鱼的硫酸鱼精蛋白)和胶原蛋白(Collagen)、原肌球蛋白(Tropomyosin,TM)、精氨酸激酶(Arginine kinase)、肌球蛋白轻链(Myosin light chain)、肌钙结合蛋白(Sarcoplasmic calcium binding pro-tein)、血蓝蛋白亚基(Hemocyanin subunits)等。基于此,本文重点概述了国内外水产品过敏原及其加工脱敏技术的研究现状,以及今后水产品过敏原研究的发展趋势。     

Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N‐ and C‐terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N‐ and in the C‐terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N‐terminal and in the C‐terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS‐PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the K_m and k_cat values increased with increasing temperature (30 to 65 °C), although the k_cat/K_m values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the K_m and k_cat values increased with decreasing pH, and the k_cat/K_m values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.     

Kinetic Study of the Quenching Reaction of Singlet Oxygen by Common Synthetic Antioxidants (tert-Butylhydroxyanisol, tert-di-Butylhydroxytoluene, and tert-Butylhydroquinone) as Compared with α-Tocopherol

ABSTRACT: Effects of synthetic phenolic antioxidants (BHA, BHT, and TBHQ) on the methylene blue (MB) sensitized photooxidation of linoleic acid as compared with that of α‐tocopherol have been studied. Their antioxidative mechanism was studied by both ESR spectroscopy in a 2,2,6,6‐tetramethylpiperidone (TMPD)‐methylene blue (MB) system and spectroscopic analysis of rubrene oxidation induced by a chemical source of singlet oxygen. Total singlet oxygen quenching rate constants (k_ox?Q+k_q) were determined using a steady state kinetic equation. TBHQ showed the strongest protective activity against the MB sensitized photooxidation of linoleic acid, followed by BHA and BHT. TBHQ (1 × 10^?3 M) exhibited 86.5% and 71.4% inhibition of peroxide and conjugated diene formations, respectively, in linoleic acid photooxidation after 60‐min light illumination. The protective activity of TBHQ against the photosensitized oxidation of linoleic acid was almost comparable to that of α‐tocopherol. The data obtained from ESR and rubrene oxidation studies clearly showed the strong singlet oxygen quenching ability of TBHQ. The k_ox?Q+k_q of BHA, BHT, and TBHQ were determined to be 3.37 × 10⁷, 4.26 × 10⁶, and 1.67 × 10⁸ M^?1 s^?1, respectively. The k_ox?Q+k_q of TBHQ was within the same order of magnitude of that of α‐tocopherol, a known efficient singlet oxygen quencher. There was a high negative correlation (r² = ?0.991) between log (k_ox?Q+k_q) and reported oxidation potentials for the synthetic antioxidants, indicating their charge‐transfer mechanism for singlet oxygen quenching. This is the 1st report on the kinetic study on k_ox?Q+k_q of TBHQ in methanol as compared with other commonly used commercial synthetic antioxidants and α‐tocopherol.     

SEAFOOD ALLERGY: PREVALENCE AND TREATMENT

Seafood is a common cause of food allergy. Allergic reactions are reported by consumers following ingestion of seafood meat and by processing workers after occupational exposure to seafood by inhalation of vapors generated during cooking. Although seafood allergy is commonly observed in clinical practice, its precise prevalence is not established. Based on our estimates, approximately 100,000 to 250,000 Americans are at risk of developing allergic reactions to seafood products. In this study, skin testing, in vitro assays and double-blind, placebo-controlled food challenge were employed to investigate seafood allergy in shrimp-allergic individuals. As in most food allergy studies only 1/3 of the alleged shrimp-sensitive subjects had a positive shrimp challenge test. The combination of a positive shrimp skin test and shrimp RAST (>11% bound) had the best predictive value (87%) for a positive challenge response. Although occupational seafood allergy is not well-studied, based on a Canadian investigation, it can be estimated that 57,000 American seafood workers are at risk of developing work-related allergic reactions. Since seafood is a major food allergen in consumers and industrial workers, further studies are necessary. Despite developments of new antiallergic therapies, avoidance continues to be the best "treatment"for allergic ingestive, inhalative and occupational disease.     

Isolation,cloning, and characterization of the 2S albumin: A new allergen from hazelnut

Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP‐HPLC. After N‐terminal sequencing, degenerated and poly‐d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut‐allergic patients. Results: N‐terminal sequencing of a ～10 kDa peak from size exclusion chromatography/RP‐HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N‐terminal and a 10aa internal peptide. The obtained clone (441 bp) encoded a 147aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.     

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.     

Microbial Inactivation and Electron Penetration in Surimi Seafood During Electron Beam Processing

Electron penetration and microbial inactivation by electron beam (e‐beam) in surimi seafood were investigated. Dose map revealed that 1‐ and 2‐sided e‐beam could efficiently penetrate 33‐ and 82‐mm thick surimi seafood, respectively. Modeling of microbial inactivation by e‐beam demonstrated that 2‐sided e‐beam may control Staphylococcus aureus if the surimi seafood package is thinner than 82 mm. The D_e‐beam value for S. aureus was 0.34 kGy. An e‐beam dose of 4 kGy resulted in a minimum of a 7‐log and most likely a 12‐log reduction of S. aureus. Microbial inactivation was slower when frozen samples were subjected to e‐beam.     

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen

BACKGROUND: Although crustaceans have been reported to be one of the most common causes of IgE‐mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS: The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX‐4T‐3 and expressed in Escherichia coli BL 21. 2‐D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION: Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies. Copyright © 2011 Society of Chemical Industry     

Fish Allergy: Fish and Products Thereof

ABSTRACT: Allergy to fish is a common cause of IgE‐mediated food allergic reactions especially in geographic regions where fish is an important dietary component. Fish allergy is estimated to affect 0.4% of the total population in the United States. All species of fish are believed to be allergenic, but allergic reactions to fish reported in the medical literature are most commonly caused by cod and salmon. The major allergen in fish is a naturally occurring muscle protein called parvalbumin. Some evidence exists of allergic reactions to other fish proteins including collagen. This review addresses fish allergy and fish‐derived ingredients, namely gelatin, isinglass, fish maws, ice‐structuring protein, fish oil, and Worcestershire sauce.     

Two-Site Antibody Immunoanalytical Detection of Food Allergens by Surface Plasmon Resonance

Bovine protein β-lactoglobulin (βLG) and peanut protein Ara h1 are considered good targets for detecting milk and peanut allergen, respectively. We used surface plasmon resonance (SPR) to detect βLG and Ara h1 by immobilizing the affinity-purified monoclonal antibodies on the biosensor chip. Proteins that bound to the antibody surface were detected by a shift in resonance angle. Adding polyclonal antibodies in the sandwich assay enhanced the sensitivity. βLG and Ara h1 were detected at 5.54 and 0.77 ng/mL, respectively, by SPR, and the results demonstrated good linear relation with relatively low concentrations of protein. The limit of detection with SPR was comparable to that with sandwich enzyme-linked immunosorbent assay (S-ELISA) with the same polyclonal and monoclonal antibodies. Use of the SPR biosensor is a simple, fast, and reliable way to detect βLG and Ara h1.     

Kinetic Study of the Radical Scavenging of Capsaicin in Homogeneous Solutions and Aqueous Triton X‐100 Micellar Suspensions

A kinetic study of capsaicin (CAP) toward radicals has been performed using stopped‐flow spectrophotometry in detail. The second‐order rate constants (k₂) for the reaction of CAP toward 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and galvinoxyl have been measured in methanol, ethanol, 2‐propanol/water (5:1, v/v), and aqueous micellar suspensions containing 5% Triton X‐100 (pH 4.0 to 10.0), respectively. The decay rates of DPPH and galvinoxyl for the reaction with CAP increased linearly in a concentration‐dependent manner in homogeneous solutions and aqueous micellar suspensions. However, the k₂ for CAP obtained in an aqueous micellar suspension showed notable pH dependence; that is, the reactivity of CAP increased with an increasing pH value from 4 to 10. In addition, a good correlation between the k₂ value and the molar fraction of CAP (phenolate anion (CAP‐O^?)/undeprotonated form (CAP‐OH)) was observed. These properties are associated with the pKa of CAP. Furthermore, it was found that the CAP‐O^? reacts with galvinoxyl about 6 times as fast as the CAP‐OH. These results indicate that sequential proton loss electron transfer from the phenolic hydrogen of CAP may be responsible for the scavenging of radicals in an aqueous micellar suspensions.     

Kinetic Study of Sasa veitchii Extract as a Radical Scavenger and an Antioxidant

Abstract: We examined the free radical scavenging activity of Sasa veitchii extract (Hoshi's Striped Bamboo Extract^®, HSBE), well known in folk medicine as an efficient drug and antioxidant in detail. To evaluate the free radical scavenging activity of HSBE, its reactivity as hydrogen atom donor toward the 1, 1‐diphenyl‐2‐picrylhydrazyl has been measured using stopped‐flow spectrophotometry. It was found that the second‐order rate constant, k₂, obtained at 25 °C was 1.4 (g/L)^?1s^?1 for HSBE. To compare different chain‐breaking antioxidants quantitatively, we obtained the second‐order rate constant, k₂′, on the molar basis of active hydroxyl groups in the tested substances. As a result, the k₂′ values for HSBE, 6‐hydroxy‐2, 5, 7, 8‐tetramethylchroman‐2‐carboxylic acid (Trolox), caffeic acid, and (+)‐catechin were 2.6 × 10³, 2.0 × 10³, 2.3 × 10², and 6.0 × 10² M^?1s^?1, respectively. These results show that HSBE and Trolox exerted the same free radical scavenging activity under these conditions. In addition, HSBE significantly inhibited the oxidation of methyl linoleate micelles in aqueous dispersions at 30 °C and its antioxidant activity (k_inh) was more effective than those of caffeic acid and (+)‐catechin. This is the first study on bamboo extracts in the context of radical scavenging activity that reports kinetic results. Practical Application: We determined the stoichiometric number and the rate constants of bamboo extracts using the method that was devised in determining the antioxidant activity of mixtures. This is the first study of bamboo extracts as an antioxidant that reports the stoichiometric and kinetic results.     

Kinetic analysis of the degradation and its color change of cyanidin-3-glucoside exposed to pulsed electric field

The degradation and its corresponding color change of cyaniding-3-glucoside (Cy-3-glu) exposed to pulsed electric field (PEF) was investigated in the study. In an aqueous-methanol solution of H₂O (0.5% TFA) and methanol (MeOH) (7:3, v/v), the degradation of Cy-3-glu, which was exposed to PEF at 1.2, 2.2 and 3.3 kV/cm, was increased as the electric field intensity and the treatment time increased (p<0.05), the degradation kinetics was well fitted to a first order reaction. The degradation kinetic constant k _A increased while the half-lives (t _1/2) and decimal reduction time (D values) decreased with an increment of the electric field intensity. PEF had a greater effect on the visual color, lightness L ^* value and redness a ^* value had a slight increase (changing towards the positive direction) while yellowness b ^* value and hue angle H value presented a significant decrease (changing towards the negative direction) (p<0.05), the total color difference (ΔE) showed an increase with an increase of the treatment time and the electric field intensity, the change in b ^* value and H was well fitted to the first-order reaction. Furthermore, a good positive linear correlation between b ^* value and Cy-3-glu content, their regression coefficients were 0.994, 0.999, and 0.997 at 1.2, 2.2, and 3.0 kV/cm, respectively.     

高静压对水产品加工及其致敏性影响的研究进展

水产品过敏已成为当今世界性的重大卫生和食品安全问题之一。高静压作为一种新的非热加工技术, 在水产品中具有潜在的应用前景。本文介绍了高静压加工的基本原理、高静压加工在水产品中的应用、水产品过敏的流行性, 重点阐述了水产品过敏原蛋白类型及特性、高静压对过敏原蛋白结构及其致敏性影响的研究概况, 提出了今后高静压水产品加工和过敏原蛋白研究的方向和建议。     

Food allergy,a summary of eight cases in the UK criminal and civil courts: effective last resort for vulnerable consumers?

Food allergy has a forensic context. The authors describe eight cases in the UK courts involving fatalities, personal injury or criminal non‐compliance with food law from mainly ‘grey’ literature sources. The potentially severe consequences for people with food allergy of contraventions of labelling law have led to enforcement action up to criminal prosecution for what might otherwise be regarded as ‘trivial’ non‐compliance. The authors suggest there should be central collation of such cases. Non‐compliances should be followed up in a more rapid and robust manner. Evidence of fraud in the catering supply chain supports recent calls for zero tolerance of food fraud. Businesses must guard against gaps in allergen management, for which there are readily available sources of training and guidance, but also against fraudulent substitution in the supply chain, about which training and guidance should be developed. New allergen labelling legislation and case law appear to place responsibility on food businesses even for the forensically problematic area of allergen cross‐contamination. The courts can be an effective last resort for vulnerable consumers; however, there is evidence of knowledge and skill gaps in both the investigation and prosecution of potentially serious incidents of food allergen mismanagement and mislabelling. Thorough investigation of food allergy deaths is required with a tenacious and skilled approach, including early realisation that samples of the food and/or stomach contents from a post mortem examination should be retained and analysed. The supply chain must be rigorously examined to find out where adulteration or contamination with the fatal allergen occurred. © 2014 Society of Chemical Industry     

Effect of Heat Processing on IgE Reactivity and Cross‐Reactivity of Tropomyosin and Other Allergens of Asia‐Pacific Mollusc Species: Identification of Novel Sydney Rock Oyster Tropomyosin Sac g 1

1 Scope

Shellfish allergy is an increasing global health priority, frequently affecting adults. Molluscs are an important shellfish group causing food allergy but knowledge of their allergens and cross‐reactivity is limited. Optimal diagnosis of mollusc allergy enabling accurate advice on food avoidance is difficult. Allergens of four frequently ingested Asia‐Pacific molluscs are characterized: Sydney rock oyster (Saccostrea glomerata), blue mussel (Mytilus edulis), saucer scallop (Amusium balloti), and southern calamari (Sepioteuthis australis), examining cross‐reactivity between species and with blue swimmer crab tropomyosin, Por p 1.

2 Methods and results

IgE ELISA showed that cooking increased IgE reactivity of mollusc extracts and basophil activation confirmed biologically relevant IgE reactivity. Immunoblotting demonstrated strong IgE reactivity of several proteins including one corresponding to heat‐stable tropomyosin in all species (37–40 kDa). IgE‐reactive Sydney rock oyster proteins were identified by mass spectrometry, and the novel major oyster tropomyosin allergen was cloned, sequenced, and designated Sac g 1 by the IUIS. Oyster extracts showed highest IgE cross‐reactivity with other molluscs, while mussel cross‐reactivity was weakest. Inhibition immunoblotting demonstrated high cross‐reactivity between tropomyosins of mollusc and crustacean species.

3 Conclusion

These findings inform novel approaches for reliable diagnosis and improved management of mollusc allergy.     

Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance

An assay method using a surface plasmon resonance (SPR) biosensor has been developed that allows quantitative measurement of the specific antibody concentration in crude materials. By injecting non-labeled antibody samples onto a biosensor surface on which antigen was immobilized at high densities, the concentration of active antibodies can be accurately measured. To clarify applicability of this method to pharmacokinetic studies, the concentration of active antibodies in mouse plasma was measured for 4 h after injection of antibodies in mice. Although this period of measurement might be insufficient for determining the pharmacokinetics of blood pool clearance, this method has some advantages over conventional methods in measurement of single-chain antibody fragment (scFv) concentrations. Using the SPR biosensor, scFv and antibodies without epitope tag peptides were easily detected in real time, requiring as little as 20 mul of blood sample. Moreover, from the apparent dissociation rate in the dissociation phase of the sensorgrams, we could identify whether the antibody fragments existed as bivalent or monovalent in animal blood. We also evaluated the antigen binding activity of the scFvs against human CD47 and found scFvs had slightly weak affinity to their antigen (K(D), about 10 nM) compared with F(ab')2 and Fab' fragments (K(D), about 3-4 nM). This assay method promises to be a convenient tool for quality control, screening, and simple pharmacokinetic analysis of antibody fragments and other recombinant proteins not having epitope tags.