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Twenty-four samples of differently produced commercial Italian cider were analyzed for their polyphenols profile and antioxidant activity. A partial least square regression was used to correlate the ciders’ antioxidant activity with their polyphenolic content. Statistical analyses revealed only a clustering pattern in the samples based on their geographical area of production (North-West, NW, vs. North-East, NE). Indeed, NW ciders, compared to NE ones, showed higher antioxidant activity and higher contents of procyanidin B1, catechin, chlorogenic acid, epicatechin, and total polyphenols. On the contrary, no clear-cut clustering pattern determined by cidermaking procedures (i.e., Charmat or Champenoise method) has been observed. These data suggest that the differences observed between NW and NE ciders might be accounted uniquely to the use of different apple varieties and/or the maturation status, as well as the pedoclimatic conditions of their production areas and not for cellar procedures’ specificity.

Grape pomace is a winery by-product that is more and more valorized as a source of healthy bioactive molecules such as polyphenols. In addition, it can be used to produce some alcoholic beverages, such as grappa, which is a typical distilled Italian alcoholic product. The spent grape pomace after grappa elaboration is mainly considered a food waste. The aim of this study was to reconsider and valorize red and white pomaces obtained after the production of grappa. The total phenolic content of both samples, as well as the antioxidant activity had a decrease after the distillation; however, it was not significant in the case of red pomace. Regarding the phenolic profile, the behavior during the distillation was different, according to the type of pomace. After the grappa production, catechins and epicatechins were the most significant phenolics in white and red pomace, respectively, demonstrating the remaining bioactivity of this by-product, which could be useful within the food industry.

BACKGROUND Grape pomace (GP), a wine-making by-product rich in dietary fiber (DF) and total phenolic compounds (TPC), is a potential functional ingredient in the fortification of baked goods. RESULTS In the present study, fortified breadsticks samples were obtained by replacing wheat flour with 0, 5 and 10 g 100 g-1 of powdered GP (GPP). The GPP inclusion affected the rheological properties of the doughs by increasing the water absorption and tenacity (P) at the same time as reducing the extensibility (L), with a significant increase in the P/L value and a decrease in the swelling index (G) value and deformation energy (W). Textural characteristics of breadsticks were influenced by the GPP addition, showing a reduction in hardness and fracturability as the amount of GPP increased in the recipe. The GPP fortified breadsticks exhibited decreased pH, volume and specific volume values compared to the control. The TPC and the antioxidant capacity increased in GPP fortified breadsticks, whereas the increased amount of DF allowed the products to benefit from the claim 'high fiber content' at the highest level of GPP inclusion. The sensory evaluation revealed that GPP addition increased wine odor, acidity, bitterness, astringency and hardness, and decreased the regularity of alveolation and friability. Finally, the GPP fortified products achieved good sensorial acceptability. CONCLUSION GPP improved the nutritional values of fortified breadsticks and changed the rheology of dough and breadsticks' technological properties without affecting sensory acceptability. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The concept of functional foods is gaining more importance due to its role in maintaining a healthy status and preventing some metabolic diseases. The control of diabetes, in particular type-2 (T2DM), could be considered a big challenge since it involves other factors such as eating habits. From the pharmacological point of view, inhibiting digestive enzymes, such as α-amylase and α-glucosidase, is one of the mechanisms mainly used by synthetic drugs to control this disease; however, several side effects are described. For that reason, using bioactive compounds may appear as an alternative without presenting the complications synthetic drugs available on the market have. The winemaking industry generates tons of waste annually, and grape pomace (GP) is the most important. GP is recognized for its nutritional value and as a source of bioactive compounds that are helpful for human health. This review highlights the importance of GP as a possible source of α-amylase and α-glucosidase inhibitors. Also, it is emphasized the components involved in this bioactivity and the possible interactions among them. Especially, some phenolic compounds and fiber of GP are the main ones responsible for interfering with the human digestive enzymes. Preliminary studies in vitro confirmed this bioactivity; however, further information is required to allow the specific use of GP as a functional ingredient inside the market of products recommended for people with diabetes. Graphical abstract

Wheat allergens are responsible for symptoms in 60–70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21–27 kDa that were recognized by the pooled baker’s IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.

Grape pomace (GP), is the main winemaking by-product and could represent a valuable functional food ingredient being a source of bioactive compounds, like polyphenols. Polyphenols prevent many non-communicable diseases and could contrast the oxidation reaction in foods. However, the high content in polyunsaturated fatty acid, the described pro-oxidant potential action of some polyphenols and the complex interactions with other components of matrices during food processing must be considered. Indeed, all these factors could promote oxidative reactions and require focused and specific assay. The aims of this study were to evaluate the effects of GP powder (GPP) addition (at 0%, 5% and 10% concentrations) in breadsticks formulations both on the antioxidant activity at room temperature during storage and on the shelf-life by the OXITEST predictive approach. GPP fortification increased the total polyphenols content and the antioxidant activities of breadsticks. FRAP reduced during the first two days of storage at room temperature, TPC increased during the 75 days, while ABTS showed a slight progressive decrease. However, GP negatively influenced OXITEST estimated shelf-life of breadsticks, incrementing the oxidation rate. In conclusion, even if GP fortification of breadsticks could improve the nutritional value of the products, the increased commercial perishability represents a drawback that must be considered.

Breads were prepared by substituting common wheat flour with 0 (GP0), 5 (GP5) and 10 (GP10) g/100 g (w/w) of grape pomace powder (GPP) and were analyzed for the phenolic profile bioaccessibility as well as the in vitro starch digestion during simulated digestion. The free and bound phenolic composition of native GPP and resulting breads were profiled using ultra-high-performance chromatography-quadrupole-time-of-flight (UHPLC-QTOF). The raw GPP was characterized by 190 polyphenols with the anthocyanins representing the most abundant class, accounting for 11.60 mg/g of cyanidin equivalents. Regarding the fortified bread, the greatest (p &lt, 0.05) content in phenolic compounds was recorded for the GP10 sample (considering both bound and free fractions) being 127.76 mg/100 g dry matter (DM), followed by the GP5 (106.96 mg/100 g DM), and GP0 (63.76 mg/100 g DM). The use of GPP determined an increase of anthocyanins (considered the markers of the GPP inclusion), recording 20.98 mg/100 g DM in GP5 and 35.82 mg/100 g DM in GP10. The bioaccessibility of anthocyanins increased in both GP5 and GP10 breads when moving from the gastric to the small intestine in vitro digestion phase with an average value of 24%. Both the starch hydrolysis and the predicted glycemic index decreased with the progressive inclusion of GPP in bread. Present findings showed that GPP in bread could promote an antioxidant environment in the digestive tract and influence the in vitro starch digestion.

Background: The by-products of red chicory leaves are a valuable source of bioactive compounds that can be exploited in the development of functional foods. Objective: This work aimed to combine healthy properties of red chicory by-products with other ingredients in the formulation of a functional jam, which is easy and safe to swallow, especially for people suffering from dysphagia. Methods: The physicochemical parameters, as well as the total polyphenols content (TPC), was assessed in the obtained product. Results: The TPC (549.44 mg GAE/100 g) was higher than the values reported in other jams, and it remained stable along with the colour during six weeks of storage. Within the carbohydrates, 0.4% of the prebiotic fibre inulin has been detected, suggesting that this jam formulation is a promising delivery system of phenols and fibre. From the sensorial point of view, the functional jam obtained an overall good acceptability judgment. The bitterness of the red chicory is persistent, which helps people with dysphagia swallow more easily. Conclusion: The functional jam, based on chicory by-products, could be a good source of bioactive compounds, which are helpful even in the disabled subjects’ diet.

The grape pomace, the main by-product from the winemaking industry, contains many bioactive substances that must be valorized. The aim of this study was to assess the total phenolic content (TPC), phenolics profile by using HPLC and the antioxidant activity (AOA). The results showed a TPC of 38.86 ± 5.22 g gallic acid equivalent (GAE)/kg while an AOA of 247.84 ± 18.65 µmol Trolox equivalent (TE)/g. Epicatechins were the most representative phenolic compound, according to the HPLC analysis. Then, the grape pomace powder (GPP) was tested in the Rancimat equipment as a natural antioxidant for delaying the corn oil oxidation. Results showed statistically significant differences between the corn oil treated with GP and the control, so the GPP could be a promising natural antioxidant to tackle the oxidation vulnerability of corn oil.

Background Pasta is a staple food that is consumed worldwide and is an excellent product for the addition of ingredients rich in bioactive compounds. The fortification of pasta with such compounds could represent a healthy choice for consumers. Results In this study, fresh pasta was formulated by replacing durum wheat semolina with 0, 5, 10, and 15 g 100 g-1 of dried Moringa oleifera leaf powder (MOLP), rich in fibers, minerals, and antioxidant compounds. Increasing levels of MOLP influenced the technological and nutritional properties of wheat-based fresh pasta. Moringa oleifera reduced the optimum cooking time, the swelling index and firmness, while increasing the cooking loss and adhesiveness. From a nutritional viewpoint, the inclusion of MOLP enhanced the phenol content, the antioxidant activity, and the mineral content of fresh pasta. The products obtained had good sensorial acceptability and can make several nutritional claims due to MOLP richness minerals. Conclusions The fortification of fresh pasta with MOLP could represent a valuable strategy to increase the nutritional value of the product, preserving pasta technological properties without affecting sensory acceptability. © 2020 Society of Chemical Industry.

Fresh pasta was formulated by replacing wheat semolina with 0, 5, 10, and 15 g/100 g (w/w) of Moringa oleifera L. leaf powder (MOLP). The samples (i.e., M0, M5, M10, and M15 as a function of the substitution level) were cooked by boiling. The changes in the phenolic bioaccessibility and the in vitro starch digestibility were considered. On the cooked-to-optimum samples, by means of ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry, 152 polyphenols were putatively annotated with the greatest content recorded for M15 pasta, being 2.19 mg/g dry matter (p < 0.05). Multivariate statistics showed that stigmastanol ferulate (VIP score = 1.22) followed by isomeric forms of kaempferol (VIP scores = 1.19) and other phenolic acids (i.e., schottenol/sitosterol ferulate and 24-methylcholestanol ferulate) were the most affected compounds through the in vitro static digestion process. The inclusion of different levels of MOLP in the recipe increased the slowly digestible starch fractions and decreased the rapidly digestible starch fractions and the starch hydrolysis index of the cooked-to-optimum samples. The present results showed that MOLP could be considered a promising ingredient in fresh pasta formulation.

The wine industry produces tons of pomace that, in a circular economy model, should be valorised. The phenols fraction of grape pomace represents a source of food colorants, but also of natural food preservatives. Furthermore, the pomace contains dietary fibre, unsaturated lipids, antioxidants and minerals that have been recognised as wholesome and technologically useful. The main drawback of using grape pomace is represented by its chemical and microbiological instability. Different authors proposed strategies to overcome these limitations, and different approaches for the use of grape pomace as an ingredient have been described. Here, we sum up the chemical and microbiological solutions for the stabilisation of grape pomace, describing the approaches used to transform this waste in a functional ingredient, and comparing the physiochemical characteristics of the resulting products. We further describe the studies concerning sensory aspects of the products by enriching animal fleshes with different pomace powder preparations.

Fresh pasta was formulated by replacing wheat semolina with 0, 5, 10, and 15 g/100 g (w/w) of Moringa oleifera L. leaf powder (MOLP). The samples (i.e., M0, M5, M10, and M15 as a function of the substitution level) were cooked by boiling. The changes in the phenolic bioaccessibility and the in vitro starch digestibility were considered. On the cooked-to-optimum samples, by means of ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry, 152 polyphenols were putatively annotated with the greatest content recorded for M15 pasta, being 2.19 mg/g dry matter (p &lt, 0.05). Multivariate statistics showed that stigmastanol ferulate (VIP score = 1.22) followed by isomeric forms of kaempferol (VIP scores = 1.19) and other phenolic acids (i.e., schottenol/sitosterol ferulate and 24-methylcholestanol ferulate) were the most affected compounds through the in vitro static digestion process. The inclusion of different levels of MOLP in the recipe increased the slowly digestible starch fractions and decreased the rapidly digestible starch fractions and the starch hydrolysis index of the cooked-to-optimum samples. The present results showed that MOLP could be considered a promising ingredient in fresh pasta formulation.

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.

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.

Wheat kernel albumins/globulins (A/G) and gluten proteins are responsible for baker's asthma and food allergy in atopic subjects. Although no commercial genetically modified wheats are currently being grown, they are under study and the allergenicity of GM products is a major concern. In order to establish the expected and unexpected effects of genetic transformation on allergenicity and also to carry out a safety assessment of genetic transformation, two GM wheat lines (bread and pasta wheat) transformed with endogenous genes were compared to their untransformed counterparts (wt), first by an allergenomic approach, and second, using ELISA with sera from patients suffering from food allergy to wheat and baker's asthma. The 2D immunoblots performed on sera from patients suffering from food allergy and baker's asthma on the A/G fraction of the four lines (two GM and two wt) revealed comparable IgE-binding profiles. A total of 109 IgE-binding spots were analyzed by mass spectrometry, and most of the proteins identified had already been described as allergens or potential allergens. Only two IgE-binding proteins were specific to one GM line. The concentration of specific IgE against the A/G fractions of GM wheat lines and their wt genotypes differed for some sera. Biological significance The originality of our paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera, such focus has never been done before in wheat and should be of interest to the researches working in this field. Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy. In this paper we used a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis. Concerning the LC–MS/MS analyses classical software and parameters were used as described in Material and methods . We worked on wheat which is actually not fully sequenced that was a difficulty; we therefore searched against two databanks (proteins and ESTs) in order to compare the results. Moreover all proteins reported in our paper were identified with at least three unique peptides. The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP). This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity.

Food allergy represents an increasing public health issue, and a large number of food control authorities have provided regulations aimed to minimize the risk of allergic reaction for sensitized consumers. The Food and Agriculture Organization of the United Nations together with the World Health Organization established the Codex Alimentarius Commission whose main goal is to protect the consumers’ health. To purse this task the Commission listed the foods and ingredients causing the most severe allergic reactions that should be labelled. It has been reported that some cases of specific foods hypersensitivity display a different prevalence among different Countries. Thus, the European Union drew up a list of mandatory allergens (that must be labelled) longer than that provided by Codex Alimentarius. As a consequence of the complexity of the legal phraseology of the European Union (EU) and/or European Community (EC) the Regulations and/or Directives were differently translated in all EC/EU official languages determining possible misinterpretations of the legislation. Moreover, food labelling regulations were also established with the goal to promote the consumers’ conscious choice about what they eat. Starting from the case of the fermented beverages, we analysed the European legislative scenario concerning the allergen labelling of the last fifteen years, highlighting that mistranslations, misinterpretations and lack of information in the EU Regulations might lead to health and ethical issues.

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 Ca 2+ -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 52 weeks, the microbeads showed a total-astaxanthin retention of 94.1 ± 4.1% (+4 °C/−light/+O 2 ), 83.1 ± 3.2% (RT/−light/−O 2 ), 38.3 ± 2.2% (RT/−light/+O2), and 57.0 ± 0.4% (RT/+light/+O 2 ), with different degradation kinetics. Refrigeration, therefore, resulted the optimal storage condition to preserve astaxanthin stability.

The aim of the work was to characterize the expression of various α-amylase inhibitors (αAIs), well known anti-nutritional compounds, for the development of healthier diploid wheat-based functional foods. The salt-soluble protein fractions from the seeds of 53 accessions among Triticum monococcum subsp. monococcum (T.m.), T. monococcum subsp. boeoticum (T.b.) and Triticum urartu (T.u.) were analyzed by immunoblotting after SDS-PAGE and Urea-PAGE using polyclonal antibodies (PABs) raised against 0.19 and 0.28 αAIs expressed in bread-wheat. Reverse zymography with human saliva and Tenebrio molitor α-amylases was used to assay inhibition activity. A great variability of the expression of αAI-related proteins was observed among T.b. and T.u. PABs, and reverse zymography revealed different bands, often not correlating with those present in bread-wheat. Two-dimensional electrophoresis followed by immunoblotting and mass spectrometric analysis identified these proteins as αAIs. Interestingly, no signal was observed within T.m. accessions. This makes T.m. an important candidate for the production of novel functional foods.

Wheat α-amylase inhibitors (α-AIs) are known anti-nutritional factors, respiratory allergens, and they can sporadically cause food allergy. α-AIs are therefore expected to reach the enteric mucosae in an immunologically active form, but information on their stability to gastric digestion is not available. Resistance to pepsinolysis is nonetheless a key factor for any food allergen. We therefore investigated whether α-AIs could resist pepsin digestion in simulated gastric fluids (SGF) and in emulsified SGF, the latter simulating more realistically the multi-phase nature of stomach bolus. Since α-AIs comprise a huge family of proteins, we investigated 0.19 α-AI as a prominent member. The digestion patterns were analyzed by immunoblotting using anti-0.19 polyclonal antibodies and sera from wheat allergic patients sensitized to 0.19 α-AI. The results show that the immune epitopes of α-AI are detectable up to 120 min of digestion in emulsifying conditions. Intra-molecular disulfide bonds and, in particular, emulsification were found to be crucial factors for protein stability. The results show that 0.19 α-AI must be considered a potential food allergen.

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.

Lipid transfer proteins (LTPs) are a family of low molecular mass (7-9 kDa) polypeptides, the members of which share 35-95% sequence homology. These proteins are widely distributed throughout the plant kingdom and are receiving attention for their biochemical characteristics and biological activity. LTPs are indeed studied in different research fields varying from allergy to food technology, and numerous molecules belonging to this class are progressively being identified and investigated. Proteins from pomegranate juice were fractioned by cation exchange chromatography and analyzed by SDS-PAGE. Two proteins were identified as putative LTPs on the basis of their molecular weights and their electrophoretic behaviors under reducing and nonreducing conditions. Finally, proteins were purified and characterized by mass spectrometry. This analysis confirmed that the two polypeptides are LTPs on the basis of an amino acid sequence common to LTPs from other plant sources and cysteine content. The two proteins, named LTP1a and LTP1b, showed similar molecular masses but different immunological profiles when immunodetected with rabbit antibodies specific for Pru p 3 and human IgE from a patient suffering from pomegranate allergy. The demonstration of the existence of two immunologically unrelated LTPs in pomegranate confirms the variability and the complexity of the plant LTP family. This should be taken into account when the role of these proteins as elicitors of allergies to fruits is investigated and could help to explain the contradictory literature data on pomegranate allergy.

A phenolic fraction purified form defatted seeds of Oenothera biennis promoted selective apoptosis of human and mouse bone marrow-derived cell lines following first-order kinetics through a caspase-dependent pathway. In non-leukemia tumour cell lines, such as human colon carcinoma CaCo(2) cells and mouse fibrosarcoma WEHI164 cells, this fraction inhibited (3)H-thymidine incorporation but not cell death or cell cycle arrest. Human peripheral blood mononuclear cells showed low sensitivity to treatment. Single bolus injection of the phenolic fraction could delay the growth of established myeloma tumours in syngeneic animals. HPLC and mass spectrometry analysis revealed that the fraction contains gallic acid. However, the biological activity of the fraction differs from the activity of this phenol and hence it should be attributed to other co-purified molecules which remain still unidentified.

A recently-described immunoenzymatic (ELISA) method for the quantitative determination of biologically-active wheat germ agglutinin (WGA) in unknown samples has been applied to measure the concentration of active WGA in raw and cooked wheat-derived foodstuffs. The method exploits the binding specificity of WGA to ovalbumin as a first step followed by identification of bound lectin with polyclonal antibodies. Purified WGA was used to obtain calibration curves. Detectable amounts of WGA were found in raw foodstuffs and wheat flours, whilst variable amounts of agglutinin were found in wholemeal pasta probably as a consequence of thermal inactivation during food processing. The thermal gradient gel electrophoresis (TGGE) technique was therefore applied to analyse the thermal stability of WGA. The biological activity of WGA decreased as a function of heating temperatures and time of exposure to thermal treatment in an S-shaped fashion with an inflection point around 65 °C. As a consequence, WGA might represent a biochemical “indicator” allowing one to determine the thermal treatment undergone by wheat-derived foods during processing.

Headspace solid phase micro extraction (HS–SPME) was used for extraction of aroma compounds characterizing a Piedmont wine ( Ruche ) derived from a non aromatic vine. Extracted compounds were identified by ion trap mass spectrometry (ITMS) after gas-chromatographic analysis. In this way a selection of 59 identified primary aromatic compounds, related to the typical flavour of Ruche was made possible. The SPME technique showed peculiar behaviour in that 23 of the 59 compounds identified were not detected by liquid-liquid solvent extraction of the same samples. Subsequent comparison with the aromatic profiles of different wine samples obtained by microvinification from different grape varieties showed similarities between Ruche and the wines, Brachetto and Malvasia , originating from aromatic vines. SPME analysis proved to be useful in understanding aroma compositions of all samples examined, establishing bases for further investigations on the chemical and biochemical mechanisms underlying wine aroma development.

Due to its high sensitivity, silver staining is a widely popular method for the revelation of biopolymers separated by both native and denaturing electrophoresis. A step-by-step method for the destaining and restaining of overdeveloped/overloaded silver-stained bands is described that is applicable to both proteins and nucleic acids. The procedure significantly improves densitometric analysis of gels that have been silver stained with either commercial kits or solutions made in-house. The method permits reproducible densitometry of silver-stained gels and allows quantification of both main and minor components in complex mixture of molecules resolved on the same gel slab. All steps may be interrupted and are readily reversible, allowing for facile densitometric analyses and photographic recording under optimized conditions. Furthermore, common artifacts such as differential staining of the two gel surfaces, localized uneven yellow-ochre background, and the presence of fold marks and fingerprints can be easily removed.

Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freund's adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis.

A previously-developed method for protein recovery from wine has been applied to beer and beer foam samples. The method involves the complexation of proteins with dodecyl sulfate (added as sodium salts) and subsequently the insolubilization of the protein-detergent complexes by addition of potassium ions (added as KCl). The protocol allows preparation of proteins from a few hundred microliters of beverage in a few minutes. The precipitated proteins are free from interfering materials and are directly utilizable for quantitative and electrophoretic assays.

The visualisation of wine yeast esterase (carboxylesterase EC 3.1.1.1) activity was performed by using different chromogenic and fluorescent substrates. The zymograms revealed the presence of several isoforms in yeast extracts, differing in sub-cellular location, substrate specificity and electrophoretic mobility, indicating a high level of polymorphism.

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.

Xylanase from Aspergillus niger (ANX) is widely used in bakeries as a processing aid since it stabilises and improves dough quality. An association between allergic symptoms among bakery workers and sensitisation to ANX has been reported, indicating that this enzyme is an occupational allergen. The presence of ANX in dough improvers and semi-finished goods is often hidden due to incomplete and unclear labelling. The quantification of microbial enzymes in these products is necessary and the determination of the actual concentration of ANX in workplaces is therefore essential to assess the occupational risk. To this purpose we have developed and characterised monoclonal antibodies to ANX. The monoclonal antibodies do not show any cross-reaction with other commonly used microbial enzymes, and they allow the detection of ANX in complex mixtures by ELISA inhibition assays down to the concentration limit of approximately 10 µg kg−1. These mAbs are a valuable tool to detect and quantify ANX and to investigate its allergenic potential in the workplace.

A two-step method to recover separately wine proteins and glycoproteins, useful to quantify and to analyse them by electrophoresis, has been presented. Five different unclarified wines from North of Italy were utilised. The results indicate that in our samples protein and glycoprotein concentrations are different while their electrophoretic patterns are almost superimposable. Finally, relations between protein and glycoprotein concentrations and haze tendency of the different wines were investigated. The results confirm the existence of a straight correlation between haze tendency and both protein concentration and protein/glycoprotein ratio.

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only approximately 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

Wheat proteinaceous alpha-amylase inhibitors (alpha-AIs) are increasingly investigated for their agronomical role as natural defence molecules of plants against the attack of insects and pests, but also for their effects on human health. The wheat genomes code for several bioactive alpha-AIs that share sequence homology, but differ in their specificity against alpha-amylases from different species and for their aggregation states. Wheat alpha-AIs are traditionally classified as belonging to the three classes of tetrameric, homodimeric and monomeric forms, each class being constituted by a number of polypeptides that display different electrophoretic mobilities. Here we describe a proteomic approach for the identification of bioactive alpha-AIs from wheat and, in particular, a 3-D technique that allows to best identify and characterize the dimeric fraction. The technique takes advantage of the thermal resistance of alpha-AIs (resistant to T > 70 degrees C) and consists in the separation of protein mixtures by 2-D polyacrylamide/starch electrophoresis under nondissociating PAGE (ND-PAGE, first dimension) and dissociating (urea-PAGE or U-PAGE second dimension) conditions, followed by in-gel spontaneous reaggregation of protein complexes and identification of the alpha-amylase inhibitory activity (antizymogram, third dimension) using enzymes from human salivary glands and from the larvae of Tenebrio molitor coleopter (yellow mealworm). Dimeric alpha-AIs from Triticum aestivum (bread wheat) were observed to exist as heterodimers. The formation of heterodimeric complexes was also confirmed by in vitro reaggregation assays carried out on RP-HPLC purified wheat dimeric alpha-AIs, and their bioactivity assayed by antizymogram analysis. The present 3-D analytical technique can be exploited for fast, full-fledged identification and characterization of wheat alpha-AIs.

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed also by gastrointestinal epithelial cells. WGA is currently investigated as an anti-tumor drug and as a carrier for oral drugs. Information on whether it can cross the gastrointestinal epithelium and on its possible effects on the integrity of the epithelial layer is however scanty or lacking, and herein we address these issues. Differentiated Caco2 cells have been used as a model of polarized intestinal epithelium. WGA concentration at both the apical and the basolateral side of the epithelium has been quantified using a sensitive ELISA assay (sensitivity threshold 0.84 nM). Trans epithelial electrical resistance (TEER) has been measured to evaluate the integrity of the epithelium upon treatments with WGA. (3)H-Mannitol (182.2 Da) and FITC-dextran (3000 Da) have been used to measure the permeability of the epithelium. Cell viability has been measured by the MTT, by 7-AAD uptake, and Annexin-V binding assays. Up to a concentration of 5.6 microM, approximately 0.1% of intact WGA molecules only could cross the epithelial layer. WGA perturbed the integrity of the epithelium and increased the permeability of the tissue in a dose- and time-dependent manner. WGA did not induce cell death but increased the permeability of individual cells to 7-AAD which is normally not uptaken by viable cells. These data allowed us to define a toxicity threshold for WGA on epithelial cells. WGA suitability as a carrier for oral drugs can therefore be evaluated on a rational basis.

Wheat Germ Agglutinin (WGA) cytotoxicity has been studied using two human leukemia cell lines, Molt3 and K562, and human peripheral blood mononuclear cells (PBMC). In spite of similar binding at the cell surface, WGA was found to promote cell death to a different extent in Molt3, K562 and PBMC and to induce different death events leading to apoptosis in Molt3 and either apoptosis and necrosis in K562 cells and PBMC. In Molt3 but not in K562 cells, WGA cytotoxicity could be potentiated 66-200 fold by 50 nM monensin, a carboxylic ionophore that perturbs the intracellular trafficking of endocytosed molecules. Synergism between the cytotoxic activities of WGA and monensin was demonstrated in Molt3 cells by comparing non toxic, or slightly toxic, doses of WGA and monensin alone or in combination. These results show that the cytotoxic effect of WGA is dependent on internalisation events which may differ among the cell lines used. WGA and monensin can enter the human diet being a component of wheat germ and an antibiotic used for zootechnic reasons in the bioindustry, respectively. These data reveal the synergistic effect between two dietary molecules, otherwise per se toxic at much higher concentrations, with possible implications for human and animal health.

A recently described immunoenzymatic method for the quantitative determination of biologically active lectins in unknown samples has been adapted to measure the concentration of active soybean lectin (SBA) in foodstuffs. The method was developed by using purified SBA to build up reference standard curves and to determine the specificity and the sensitivity of the assay. Detectable amounts of soybean lectin were found in soy-based edible products such as hamburger, milk and sprouts and they have been compared to those determined in standard hemagglutination tests. Particular attention has been given to SBA quantitation in soy sprouts as a function of germination time. SBA was not degraded during germination although the hamagglutination activity of sprout extracts rapidly decreased. SBA expression was modulated during the first 10 days of germination, a time period spanning that used to produce soy-sprouts for alimentary purposes.

Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.

Due to the importance of esters in determining wine aroma, the presence of different esterases in soluble and insoluble cell fractions of an oenological strain of Saccharomyces cerevisiae was studied. Cells of an oenological yeast strain were separated into three fractions corresponding to the cytoplasm, periplasm and plasma membrane, all showing esterase activity. The conditions for optimal solubilization of the membrane-bound esterase were found, as well as those allowing its electrophoretic analysis, using the detergent disodium-n-lauryl-β-iminodipropionate (Deriphat) in the cathodic buffer. Comparison of the activity bands detected on gels revealed the presence of different esterase iso-forms in the three sub-cellular fractions. The method can also be used as the first electrophoretic step in two-dimensional PAGE. Therefore, the procedures described are also a promising tool for the study of the yeast enzymes in relation to their effects during winemaking.