Federica Mainente

Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine.

Recently, wheat gluten has been proposed as technological adjuvant in order to clarify wines. However, the possibility that residual gluten proteins remain in treated wines cannot be excluded, representing a hazard for wheat allergic or celiac disease patients. In this work, commercial wheat glutens, in both partially hydrolyzed (GBS-P51) and nonhydrolyzed (Gluvital 21000) forms, were used as fining agents in red wine at different concentrations. Beside immunoenzymatic analyses using anti-gliadin, anti-prolamin antibodies and pooled sera of wheat allergic patients, a method based on liquid chromatography coupled to mass spectrometry has been proposed to detect residues of gluten proteins. Residual gluten proteins were detected by anti-prolamin antibodies, anti-gliadin antibodies and sera-IgE only in the wine treated with GBS-P51 at concentration 50, 150, and 300 g/hL, respectively, whereas no residual proteins were detected by these systems in the wine treated with Gluvital 21000. In contrast liquid chromatography-mass spectrometry analyses allowed the detection of proteins in red wines fined down to 1 g/hL of Gluvital 21000 and GBS-P51. Our results indicate that MS methods are superior to immunochemical methods in detecting gluten proteins in wines and that adverse reactions against gluten treated wines cannot be excluded.

Post-harvest proteomics of grapes infected by Penicillium during withering to produce Amarone wine

The study of withered grape infection by Penicillium, a potentially toxigenic fungus, is relevant to preserve grape quality during the post-harvest dehydration process. This report describes the first proteomic analysis of Amarone wine grapes, infected by two strains of Penicillium expansum (Pe1) and Penicillium crustosum (Pc4). Protein identification by MS analysis allowed a better understanding of physiological mechanisms underlying the pathogen attack. The Pe1 strain had a major impact on Vitis vinifera protein expression inducing pathogenesis-related proteins and other protein species involved in energy metabolism. A greater expression of new Penicillium proteins involved in energy metabolism and some protein species related to redox homeostasis has been observed on grapes infected by Pc4 strain. Moreover, the new induced proteins in infected grapes could represent potential markers in withered grapes, thus creating the chance to develop case-sensitive prevention strategies to inhibit fungal growth.

Production of stable food-grade microencapsulated astaxanthin by vibrating nozzle technology

Astaxanthin is a carotenoid known for its strong antioxidant and health-promoting characteristics, but it is also highly degradable and thus unsuited for several applications. We developed a sustainable method for the extraction and the production of stable astaxanthin microencapsulates. Nearly 2% astaxanthin was extracted by high-pressure homogenization of dried Haematococcus pluvialis cells in soybean oil. Astaxanthin-enriched oil was encapsulated in alginate and low-methoxyl pectin by Ca2+-mediated vibrating-nozzle extrusion technology. The 3% pectin microbeads resulted the best compromise between sphericity and oil retention upon drying. We monitored the stability of these astaxanthin beads under four different conditions of light, temperature and oxygen exposition. After 52weeks, the microbeads showed a total-astaxanthin retention of 94.1±4.1% (+4°C/-light/+O2), 83.1±3.2% (RT/-light/-O2), 38.3±2.2% (RT/-light/+O2), and 57.0±0.4% (RT/+light/+O2), with different degradation kinetics. Refrigeration, therefore, resulted the optimal storage condition to preserve astaxanthin stability.

Hidden Exogenous Proteins in Wine: Problems, Methods of Detection and Related Legislation - a Review

Rizzi C., Mainente F., Pasini G., Simonato B. (2016): Hidden exogenous proteins in wine: problems, methods of detection and related legislation – a review. Czech J. Food Sci., 34: 93–104. Fining agents are commonly used in the winemaking process to clarify and stabilise wines. They have different origins (animal, vegetal or mineral) and are added to wines in order to remove specifically undesirable compounds that are discarded. Fining agents should not be present in the final product but their possible persistence, as well as other exogenous residual proteins such as the enzymes utilised in winemaking, cannot be excluded for sure. The principal concern about the presence of exogenous residual proteins is the health of allergic subjects. Nevertheless, the respect of religious creed or other practice of living of the consumer must be considered as well. In the present review we itemise the proteins used in winemaking and possible drawbacks of their permanence in the final products and the related risks, depict the status of the art of the studies performed about the detection of exogenous proteins, and describe the wine labelling laws adopted in different countries to avoid the drawbacks associated with these hidden substances.

Red wine proteins: two dimensional (2-D) electrophoresis and mass spectrometry analysis.

The aim of the present study was to optimize protein extraction from red wine (cv. Cabernet) in order to obtain a separation by two-dimensional electrophoresis (2-DE) compatible with mass spectrometry identification. Proteins were denatured by sodium dodecyl-sulphate (SDS) and precipitated as potassium salts. The potassium-DS (KDS) protein complexes obtained were treated with different solutions in order to remove the detergent. Proteins were solubilized with different buffers and separated by different electrophoretic approaches [native, urea, acid urea PAGEs and isoelectric focusing (IEF)] as the first-dimension (1-DE). The best 2D separation was achieved by using 10% saccharose in the DS removal step, and 6-cyclohexylhexyl β-d-maltoside detergent in the solubilisation buffer combined with the IEF approach. Several well focalized protein spots were obtained and analyzed through mass-spectrometry.

Setup of a procedure for cider proteins recovery and quantification

Cider contains low amount of proteins that, nonetheless, can affect its stability, foam formation and potential allergenicity. At present, scarce information is available on cider proteins, probably due to the lack of methods for their recovery and analysis. The aim of the present study was to set up a method for recovering and quantifying cider proteins. To this purpose, the proteins from 13 Italian commercial ciders were recovered by dialysis, gel filtration, trichloroacetic acid/acetone (TCA/acetone) and potassium dodecyl sulfate (KDS) precipitation. The protein content of the samples was then determined by bicinchoninic acid (BCA), Bradford and o-phthaldialdehyde (OPA) assays. The results were compared to quantitative data obtained by densitometry of electrophoretic gels. The most reliable protocol resulted in the KDS method followed by OPA assay. KDS, in addition, allowed also to separate proteins from glycocompounds. KDS/OPA is the method of choice for cider proteins precipitation and quantification.

Hen egg white lysozyme is a hidden allergen in Italian commercial ciders

ABSTRACT Hen egg white lysozyme (HEWL) is an enzyme used in alcoholic fermentation for its ability to control the growth of Gram-positive and spoilage bacteria, without inhibiting yeast growth, and it allows a reduction in the use of sulphur dioxide. Nevertheless, considering the potential allergenicity of this protein, the presence of HEWL should be declared on the label of the final product. In this work, we analysed 18 commercial Italian ciders by LC-MS/MS and found traces of HEWL in 12 samples without label declaration. We used Western blot and enzyme-linked immunosorbent assay (ELISA) to verify the immunological activity of HEWL, and to quantify its content in the ciders. Two out of 18 samples were found to be positive both by immunoblot and ELISA. The results indicate the requirement of a more stringent control of commercial ciders and the need of label declaration for ciders treated with such compounds. GRAPHICAL ABSTRACT

Effects of microencapsulation by ionic gelation on the oxidative stability of flaxseed oil

Flaxseed oil is a major source of omega-3 polyunsaturated fatty acids (PUFAs), as it contains nearly 50% of alpha-linolenic acid. For this reason it is highly susceptible to auto-oxidation. The aim of the work was to increase the stability of flaxseed oil by a microencapsulation process based on ionic gelation through vibrating-nozzle extrusion technology, using pectin as shell material. Two different drying systems, passive air drying (AD) and fluid bed (FB), were compared. The results show that the encapsulation efficiency is very high (up to 98%). Besides being approximately 20-fold faster, FB gives beads showing on average higher payload (76% vs 68%) and lower peroxide value (9.64 vs 21.33) than the AD. An accelerated test carried out on FB-dried beads shows that the oxidative stability of encapsulated oil is 13-fold higher than bulk oil (PV FB: 20 vs PV oil: 260), demonstrating the protecting effect of microencapsulation.