Isoenzymes of wheat o-diphenolase revealed by column isoelectric focusing

Summary Common wheat o-diphenolase at different purification steps has been submitted to column isoelectric focusing electrophoresis. The fraction purified with calcium phosphate gel shows multiple forms focused at pI 3.60, 4.95, 6.80, and 9.60. The enzyme activity and the purification degree of the four enzymatic components obtained have been calculated. The major fraction recovered after purification by chromatography on DEAE-cellulose does not display other multiple forms; however, it has some enzymatically inactive proteins well separated in the pH-gradient. The enzymatic protein, focused at pI 9.60, thus achieves a 1753-fold purification over the crude extract. The application of the technique allows a characterization of the wheat o-diphenolase and its recovery in highly purified form.

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Isoenzyme von o-Diphenoloxydase aus Weichweizen, identifiziert durch isoelektrisches Fokussieren über Säule

Zusammenfassung Weichweizen-o-Diphenoloxydasen verschiedener Reinigungsstufen wurden über eine Säule isoelektrisch focussiert. Das durch Calciumphosphatgel gereinigte Enzym (Fraktion C) zeigt 4 multiple Formen, focussiert bei pI 3,60; 4,65; 6,80 und 9,60. Die Aktivität und der Reinheitszustand von diesen vier Formen wurden berechnet. Die Fraktion C wurde durch Chromatographie über Cellulose weiter gereinigt. Die in größeren Mengen gewonnene Fraktion zeigte beim isoelektrischen Focussieren keine multiple Formen mehr, nur eine Trennung von enzymatisch inaktiven Proteinen beim pH-Gradienten. Das enzymatisch aktive Protein, focussiert bei pI 9,60, zeigte einen 1753 mal besseren Reinheitszustand als das Rohenzym. Die Anwendung dieser Technik erlaubt die Charakterisierung der Weichweizen-o-Diphenoloxydasen und ihre Gewinnung in einem hochgereinigten Zustand.

     

Peptides from water buffalo cheese whey induced senescence cell death via ceramide secretion in human colon adenocarcinoma cell line

Scope: Milk proteins are a source of bioactive peptides. Recent studies have indicated that protein‐derived peptides released in buffalo cheese acid whey exert a cytomodulatory effect in human epithelial colon cancer (CaCo2) cells. The aim of the present study was to explain the molecular mechanism involved in the response of CaCo2 cells to oxidative stress in the presence of peptide fractions of buffalo cheese whey, purified and characterized by mass spectrometry. Methods and results: We demonstrated that treatment of CaCo2 treated with H₂O₂ (H‐CaCo2) cells with a partially purified peptide sub‐fraction (f3) from buffalo cheese acid whey induced a reduction of mitochondrial superoxide anion with subsequent decrease in heat shock protein 70 and 90 expression. Moreover, we observed a 5‐fold decrease in cyclin A expression and cell cycle arrest in G1/G0 phases. These responses were associated with increased activity of alkaline phosphatase and beta‐galactosidase, markers of differentiation and senescence respectively. Conclusions: The structural characterization of the active peptide fraction and the elucidation of the effects induced by its treatment on H‐CaCo2 cells in vitro demonstrated an activity of this peptide sub‐fraction in the modulation of cell cycle, thus suggesting potential application for the development of nutraceuticals as well as health‐promoting functional foods.     

Formation and Degradation of Beta-casomorphins in Dairy Processing

Milk proteins including casein are sources of peptides with bioactivity. One of these peptides is beta-casomorphin (BCM) which belongs to a group of opioid peptides formed from β-casein variants. Beta-casomorphin 7 (BCM7) has been demonstrated to be enzymatically released from the A1 or B β-casein variant. Epidemiological evidence suggests the peptide BCM 7 is a risk factor for development of human diseases, including increased risk of type 1 diabetes and cardiovascular diseases but this has not been thoroughly substantiated by research studies. High performance liquid chromatography coupled to UV-Vis and mass spectrometry detection as well as enzyme–linked immunosorbent assay (ELISA) has been used to analyze BCMs in dairy products. BCMs have been detected in raw cow's milk and human milk and a variety of commercial cheeses, but their presence has yet to be confirmed in commercial yoghurts. The finding that BCMs are present in cheese suggests they could also form in yoghurt, but be degraded during yoghurt processing. Whether BCMs do form in yoghurt and the amount of BCM forming or degrading at different processing steps needs further investigation and possibly will depend on the heat treatment and fermentation process used, but it remains an intriguing unknown.     

Weissella cibaria short-fermented liquid sourdoughs based on quinoa or amaranth flours as fat replacer in focaccia bread formulation

Production of the traditional Italian Focaccia bread (FcBread) requires reformulation to meet the new rules for healthier foods. We tested the applicability of a short-fermented (15 h) liquid sourdough (LS) (dough yield, DY, 250), obtained using quinoa (Q) or amaranth (Am) flour fermented with a Weissella cibaria strain (C43-11) producing exopolysaccharides (EPS) as fat replacer in yeast-leavened FcBread. The LSs were applied during the FcBread-making process at industrial pilot plant, reducing the added fat amount by 20% (FcBread-QLS and FcBread-AmLS) and products were compared with reference samples not containing EPS. All the products were analysed for physico-chemical properties (pH, TTA, organic acids, protein content and profile, instrumental colour), textural properties, sensory quality and glycemic index. The application of both pseudo-cereal-based W. cibaria LSs in FcBread allowed obtaining products with a reduced amount of fat, increased protein amount, mainly due to the albumin and glutenin/glutelin fractions, reduced glycemic index, improved texture and preserved in the traditional sensory profile of ‘focaccia’.     

Lycopene-rich cream obtained via high-pressure homogenisation of tomato processing residues in a water–oil mixture

Tomato processing residues are still rich in bioactive compounds that may be recovered and reused, with environmental and economic benefits. This short communication discloses for the first time that the high-pressure homogenisation (HPH) treatment of tomato residues in the presence of water and sunflower oil is able to promote the extraction of bioactive compounds concurrently to the formation of an oil-in-water emulsion stabilised by the micronized residues. The mechanical disruption effect of HPH improved the mass transfer of lycopene into the oil phase, and formed fine fibrous debris, improving stabilisation and visual appearance of the emulsion. Results showed a progressive increase of lycopene concentration in the cream phase up to 5 HPH passes and a concurrent reduction of its content in the pellet. Total polyphenols content and antioxidant activity in the aqueous phase gradually decreased when increasing the number of passes, suggesting their progressive transfer in the cream phase. The proposed process that relies on a purely physical treatment and uses only water/sunflower oil as extraction media resulted in the production of a lycopene-rich cream of potential use as a functional food ingredient.     

The effects of extrusion conditions and the addition of volatile compounds and flavour enhancers to corn grits on the retention of the volatile compounds and texture of the extrudates

Corn grits that were supplemented with isovaleraldehyde, ethyl butyrate, butyric acid and flavour enhancers were extruded under different processing conditions. Volatile compounds retained in the extrudates were isolated by dynamic headspace and analysed using gas chromatography–mass spectrometry. The expansion ratio, density and cut force to break down the extrudates were evaluated and aroma intensity was assessed using a multisample difference test. Butyric acid showed the greatest retention (96.4%), regardless of the extrusion conditions. All compounds were better retained when samples were extruded at 20% feed moisture and 90 °C processing temperature (2.9–81.0%), conditions that also resulted in greater aromatic intensity (moderate to moderate‐strong intensity). The addition of volatile compounds reduced the expansion ratio and cut force, whereas the addition of flavour enhancers increased the expansion ratio but reduced ethyl butyrate and butyric acid retention.     

Validation by a Collaborative Interlaboratory Study of an ELISA Method for the Detection of Caseinate Used as a Fining Agent in Wine

The clarification or fining of wine removes undesirable substances such as proteins, phenols, and tannin compounds that would cloud the wine and cause bitterness and astringency. Caseinates have useful fining properties, but their residues could present a risk for allergic subjects. A commercial kit that was developed to detect caseinates in food has been examined for its applicability to a wine matrix; it is sensitive to caseinates at concentrations as low as 1 ppm. The general characteristics of the caseinate assay, described below in detail, are as follows. It is a sandwich enzyme-linked immunosorbent assay (ELISA): the microplate is first coated with a specific anti-casein antibody; and after incubation with the wine sample, a secondary anti-casein antibody conjugated with horseradish peroxidase is added to form a sandwich. The antibodies have been tested for their immunoreactivity and the reproducibility of antigen recognition has been measured. An interlaboratory collaborative trial was organized to evaluate the performance of this ELISA method and its ease of use by laboratories routinely dealing with wine/food analyses. The results satisfy the criteria established by the International Organisation of Vine and Wine in the Compendium of International Methods of Analysis MA-EAS1-07-ETCOL.