Proteolysis in ultra‐heat‐treated skim milk after exposure to multispecies biofilms under conditions modelling a milk tanker

Bacteria from milk tankers can form multispecies biofilms and produce heat‐stable enzymes. In this study, milk was exposed to multispecies biofilms in stainless steel vessels and was then used to produce ultra‐heat‐treated (UHT) milk, which was stored for 5 months. The UHT milks were assessed for microbial counts, free peptide concentration and pH. The free peptide concentration, which indicated proteolysis, was higher in UHT milk that had been exposed to multispecies biofilms than in untreated UHT milk. Biofilm formation may be promoted in milk tankers that are not properly cleaned, which may compromise the quality of the final dairy product.     

Production and Characterization of Monoclonal Antibodies Specific to Pangasius Catfish, Basa, and Tra

ABSTRACT:  Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra ( Pangasius hypophthalmus ) and basa ( Pangasius bocourti ), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.     

杂交-杂交瘤技术制备BsMAb的研究进展及其在食品安全检测中的应用

双特异性单克隆抗体（bispecific monoclonal antibody,BsMAb）是指具有两个不同特异性抗原结合位点的单克隆抗体分子。杂交-杂交瘤技术因其简便易行、制备周期短,在制备BsMAb方面有较大优势。基于杂交-杂交瘤技术能制备出可以特异性识别两种或者两类小分子药物的BsMAb,在食品安全多残留免疫分析检测中具有较好的应用价值。本文综述了杂交-杂交瘤技术制备BsMAb的进展,探讨了基于该技术的BsMAb生成机制,讨论分析了杂交-杂交瘤及BsMAb筛选制备的关键技术要点,并归纳了BsMAb在检测食品中小分子污染物方面的应用进展,旨在为食品安全多残留免疫分析技术的发展应用提供参考。     

Monoclonal Antibodies to Porcine Thermal-Stable Muscle Protein for Detection of Pork in Raw and Cooked Meats

Four hybridoma cell lines secreting monoclonal antibodies (MAbs) specific to porcine thermal-stable muscle proteins (TSMPs) were developed. The MAbs reacted with three protein bands (20.5, 22 and 24 kD) from raw pork extract; the protein band (24 kD) which was also present in cooked pork was identified as porcine-specific TSMP. Epitope analysis indicated four MAbs recognized same or closely located antigenic sites on the pork TSMP. The developed MAb-based indirect enzyme-linked immunosorbent assay (ELISA) enabled the detection of pork in raw and cooked heterogeneous meat mixtures as low as 10 g/kg. The curvilinear relations of second-degree polynomial (r² > 0.995) between pork concentrations and ELISA responses enabled quantifying degree of adulteration of pork in meat products.     

Molecular structure and granule morphology of native and heat‐moisture‐treated pinhão starch

Pinhão seed is an unconventional source of starch and the pines grow up in native forests of southern Latin America. In this study, pinhão starch was adjusted at 15, 20 and 25% moisture content and heated to 100, 110 and 120 °C for 1 h. A decrease in λ max (starch/iodine complex) was observed as a result of increase in temperature and moisture content of HMT. The ratio of crystalline to amorphous phase in pinhão starch was determined via Fourier transform infra red by taking 1045/1022 band ratio. A decrease in crystallinity occurred as a result of HMT. Polarised light microscopy indicated a loss of birefringence of starch granules under 120 °C at 25% moisture content. Granule size distribution was further confirmed via scanning electron microscopy which showed the HMT effects. These results increased the understanding on molecular and structural properties of HMT pinhão starch and broadened its food and nonfood industrial applications.     

Lipid oxidation and colour in pressure‐ and heat‐treated minced chicken thighs

Lipid oxidation and colour in pressurised and heated chicken samples were evaluated. In a preliminary test, raw and overcooked (100 °C/60 min) minced chicken thighs were pressurised (500 MPa/50 °C/30 min). Samples were stored at 4 °C in contact with air. Thiobarbituric acid reactive substances (TBARS) were quantified at 1, 6 and 9 days. Pressure induced oxidation in chicken, but overcooking generated many more secondary oxidation compounds. In a second experiment, raw minced chicken thighs were pressurised (500 MPa/?10, 5, 20 and 50 °C/30 and 60 min) or cooked (90 °C/15 min). Samples were vacuum stored at 4 °C. TBARS were measured at 1 and 9 days, whereas colour parameters (L, a, b and ΔE) were determined at 1 day. No differences in TBARS values were observed between untreated and pressurised samples, whereas cooked samples presented the highest values. Pressurisation for 30 and 60 min generated similar TBARS contents. At 9 days, oxidation values did not increase. Pressurisation and cooking induced marked colour changes. Pressurised samples were lighter and less red than untreated ones. Samples pressurised at 50 °C were the palest and, together with cooked samples, presented the lowest a values. Therefore pressurised chicken thigh cannot be marketed as a fresh product but can be incorporated as an ingredient in ready‐to‐eat meals. Copyright © 2004 Society of Chemical Industry     

A survey of the physicochemical and microbiological quality of ultra‐heat‐treated whole milk in Brazil during their shelf life

The objective was to evaluate the quality of important brands of ultra‐heat‐treated (UHT) milk marketed in the northern of Parana State, Brazil, during the shelf life of the product. Five brands were analysed at 30, 60, 90 and 120 days after UHT milk production. The physicochemical quality of the milk was assessed in terms of fat content, titratable acidity, density, freezing point depression, total solids, solids‐not‐fat, proteolysis and alcohol stability. Microbiological quality was assessed by the enumeration of aerobic mesophilic micro‐organisms. None of the brands complied with all the physicochemical standards. Two brands met all applicable microbiological requirements. Gelation was observed from 90 days of storage onwards in all brands.     

Immunological determination of gliadin 33‐mer equivalent peptides in beers as a specific and practical analytical method to assess safety for celiac patients

BACKGROUND: Cereals used for beer manufacturing contain gluten, which is immunotoxic for celiac patients. The gluten remaining after processes of malting and brewing is mostly hydrolyzed, which makes practical evaluation of the immunotoxicity of the gluten pools challenging. RESULTS: We analyzed the presence of gluten peptides equivalent to the major immunotoxic protease‐resistant gliadin 33‐mer in 100 Belgium beers, using monoclonal antibodies (G12/A1). Immunochromatographic strips and enzyme‐linked immonosorbent assay G12/A1 methods estimated at least 20 ppm gluten equivalents in 90 beers and gluten‐free in 10 beers. The G12/A1 reactivity of beer high‐performance liquid chromatographic fractions correlated to the presence of T‐cell‐reactive epitopes identified by peptide sequencing. CONCLUSION: The determination of equivalent gliadin 33‐mer epitopes in beers has been shown to be practical, specific, and sensitive for the measurement of potential immunotoxicity for celiac patients. Copyright © 2012 Society of Chemical Industry     

Characterization of a 12 kDa thermal-stable antigenic protein in bovine blood

We have previously developed an immunoassay based on monoclonal antibody (MAb) Bb1H9 for quantitative detection of ruminant blood in processed food and feedstuffs. The purpose of this study was to characterize the unknown 12 kDa thermal-stable ruminant-specific antigenic protein recognized by MAb Bb1H9 in order to better define the application scope of the developed assay. Extracts obtained from raw and heat-treated bovine blood-derived products were analyzed with indirect ELISA and Western blot. Target proteins resolved by 2D electrophoresis were subjected to N-terminal sequencing. Results indicated that the 12 kDa protein is a monomer of the tetrameric hemoglobin molecule (64.5 kDa) and that the heme group is not required for its binding with MAb Bb1H9. This MAb can be utilized as a probe for red blood cell derived products of ruminant origin in raw or processed food and feedstuffs to enforce labeling regulations and to address consumer concerns. PRACTICAL APPLICATION: MAb Bb1H9 represents the first antibody with the capacity to recognize bovine hemoglobin both in the absence and presence of the heme group, regardless of the heat treatment. MAb Bb1H9 can therefore be utilized in immunoassays by manufacturers and regulators to detect any ingredients containing hemoglobin or globin (hemoglobin without the heme group) in both raw and processed food and feed materials for product quality control and labeling law enforcement.     

Thermal and Pasting Properties of Acid‐treated Rice Starches

The physicochemical properties of acid‐treated rice starches were investigated. Rice starches were treated with hydrochloric acid at different acid concentrations and hydrolysis times. The pasting properties were tested using a Rapid Visco Analyser, and gelatinization and retrogradation properties using a differential scanning calorimeter. The results showed that acid concentration had a more pronounced effect on degree of polymerization (DP) and viscosity than hydrolysis time. The onset, peak and conclusion temperatures of gelatinization were increased significantly with hydrolysis time, while the gelatinization enthalpy (ΔH_G) was decreased. In addition, there was an increase in the gelatinization temperature range with longer hydrolysis time. After storing gelatinized starches at 4°C for 7 days, the transition temperature and enthalpy (ΔH_R) to melt retrograded amylopectin did not change significantly. Additionally, the temperature and enthalpy transition for melting amylose‐lipid complex of all gelatinized and retrograded starches were in the same range.     

l‐histidine improves water retention of heat‐induced gel of chicken breast myofibrillar proteins in low ionic strength solution

The effects of 5 mm l ‐histidine (l ‐His) on water‐binding capacity, gel strength, thermal gelling properties of chicken breast myofibrillar proteins (MPs) in 1 mm NaCl or 0.6 m NaCl solutions (pH 7.0) were investigated. l ‐His could significantly increase the solubility and thermal gelling ability of MPs in 1 mm NaCl. l ‐His at 1 mm NaCl shortened the water relaxation time and decreased the water mobility of MPs gel. l ‐His promoted the formation of MPs gel structure with small pores and thin strands at 1 mm NaCl. These resulted in the enhanced water retention and weak gel strength of MPs in low ionic strength solution. The water‐binding capacity of MPs gels formed in 1 mm NaCl containing 5 mm l ‐His was equivalent to that with 0.6 m NaCl. The information could offer certain theoretical foundation to apply l ‐His as sodium salt substitute for developing low‐salt meat gelling product with high yield.     

Characterisation of Macedonian white‐brined cheese: Effect of raw or heat‐treated caprine milk

This research analysed the use of raw and pasteurised milk in the production of Macedonian white caprine milk cheese. Pasteurisation resulted in a considerable decrease in the nitrogen fractions, urea‐polyacrylamide gel electrophoresis of caseins, peptide profiles and volatiles during the maturation period. Forty‐five volatile components were detected, including twelve acids, fourteen esters, six ketones, three alcohols, four terpenes and six other compounds. It was deduced that pasteurisation of milk for the manufacture of artisanal white‐brined cheese delayed the evolution of volatiles, which are related to the unique flavour of the cheese.     

Physicochemical,crystallinity, pasting and thermal properties of heat‐moisture‐treated pinhão starch

The effect of heat‐moisture treatment (HMT) on the properties of pinhão starches under different moisture and heat conditions was investigated. The starches were adjusted to 15, 20 and 25% moisture levels and heated to 100, 110 and 120°C for 1 h. The X‐ray diffractograms, swelling power, solubility, gel hardness, pasting properties and thermal properties of the native and HMT pinhão starches were evaluated. Compared to native starch, there was an increase in the X‐ray intensity and gel hardness of HMT starches, with the exception of the 25% moisture‐treated and 120°C heat‐treated starch. HMT reduced the swelling power and solubility of the pinhão starches when compared to native starch. There was an increase in the pasting temperature, final viscosity and setback and a decrease in the peak viscosity and breakdown of HMT pinhão starches compared to native starch. HMT increases the gelatinisation temperature of native pinhão starch and reduces gelatinisation enthalpy.     

A novel conformation-dependent monoclonal antibody specific to the native structure of beta-lactoglobulin and its application

Molten globules are thought to be general intermediates in protein folding and unfolding. β-lactoglobulin (β-LG) is one of the major bovine whey proteins, constituting ∼10 to 15% of total milk proteins. We have recently identified β-LG as a superior marker for evaluating thermally processed milk. Strand D of β-LG participates in irreversible thermal unfolding as probed by a monoclonal antibody (mAb) specific to thermally denatured β-LG. In the present study, we used native β-LG as an immunogen to test the hypothesis that a specific mAb against the native β-LG could be established. As result, a mAb (4H11E8) directed against the native structure of β-LG was made. The antibody did not recognize the heat-denatured form of β-LG, such as its dimer and aggregates. Immunoassay using this “native” mAb showed that the stability of β-LG was at temperatures ≤70°C. β-Lactoglobulin began to deteriorate between 70 and 80°C over time. The denaturation was correlated with the transition temperature of β-LG. Further chemical modification of Cys (carboxymethylation) or positively charged residues (acetylation) of β-LG totally abolished its immunoreactivity, confirming the conformation-dependent nature of this mAb. Using competitive ELISA, the 4H11E8 mAb could determine the native β-LG content in commercially processed milks. Concentrations of native β-LG varied significantly among the local brands tested. From a technological standpoint, the mAb prepared in this study is relevant to the design and operation of appropriate processes for thermal sanitation of milk and of other dairy products.     

Decreased fat accumulation in 3T3‐L1 pre‐adipocytes treated with extracts of heat‐processed soy flour and breads

The potential anti‐adipogenic effects of gluten‐free soy breads made from germinated soybean (GS), steamed soybean (SS) or roasted soybean (RS) were evaluated in an in vitro adipocyte cell model. GS and RS increased the total phenolic (TP) and total flavonoid (TF) contents of flours compared with the raw soybean (NS) flour. RS and GS had the highest TP (1.04 GAE mg g^?1) and TF (0.92 CAE mg g^?1) contents. Baking increased the TP content of breads, 0.09–0.26 GAE mg g^?1, compared with the flours. Fermentation during breadmaking increased the DPPH scavenging activities compared with the flours. The ABTS scavenging exhibited similar patterns to those of DPPH. Lipid accumulation in 3T3‐L1 cells shows that the alcoholic extracts (100 μg mL^?1) of SS flour and bread decreased adipocyte differentiation by 1.6‐ and 2.1‐fold, respectively, compared with control. SS bread extract substantially downregulated the adipogenesis‐related genes such as acetyl‐CoA carboxylase and glycerol‐3‐phosphate dehydrogenase.