Kvantitativno određivanje lipaze pomoću fluoresceina

An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.U radu je opisana jednostavna i brza metoda određivanja i praćenja aktivnosti lipaze pomoću fluoresceina. Mjerenjem intenziteta fluorescencije fluoresceina može se dobiti kalibracijska krivulja s koeficijentom regresije boljim od onog dobivenog mjerenjem promjera fluorescentne zone. Kvantitativnim određivanjem intenziteta fluorescencije fluoresceina, oslobođenog hidrolizom estera fluorescein dibutirata na podlozi s agarom, ispitana je aktivnost komercijalne lipaze, te one izolirane iz obranog mlijeka. Ovom se metodom može pratiti aktivnost lipaze u reakcijskoj smjesi koja sadržava spojeve što uzrokuju zamućenje ili interferiraju s uzorkom pri mjerenju UV-spektrofotometrom i fluorimetrom. U istraživanju je upotrijebljena reakcijska smjesa od prokuhanog obranog mlijeka raspršenog na agar gelu s fluorescein dibutiratom. Daljnim bi se razvojem ova metoda mogla primijeniti u farmaceutskoj i prehrambenoj industriji, zaštiti okoliša i sl

A Novel Method for the Quantification of White Wine Mannoproteins by a Competitive Indirect Enzyme-Linked Lectin Sorbent Assay (CI-ELLSA)

Mannoproteins (MPs) are cell wall proteoglycans released in wine by yeast during fermentation and ageing on lees, a procedure used for the production of several wines to enrich them in these components with consequences from both a technological and sensory point of view. Given the significance that wine MPs have for wine quality, winemakers would welcome a simple and accurate method for their quantification, as this would allow them to have a better control of this aspect at different winemaking stages. This study develops and validates a novel, simple and accurate method for MPs quantification in white wines based on a competitive indirect enzyme-linked lectin sorbent assay (CI-ELLSA), using the highly mannosylated yeast invertase as the standard. The method utilizes the lectin concanavalin A (ConA) as the immobilized ligand for MPs, and peroxidase, an enzyme rich in mannose, as the competitor for ConA. After addition of the peroxidase substrate, the intensity of the signal produced by the activity of this enzyme (absorbance at 450 nm) is inversely proportional to the amount of mannosylated proteins in the sample. Results have been validated on several wine styles including still, sparkling and sweet wines

Current and future strategies for wine yeast lees valorization

Wine lees is a sludge material mainly composed of dead yeast precipitated at the bottom of wine tanks. Along with grape pomace and grape stalks, it is one of the main by-products of the winemaking industry. Given that wine lees are considered a soil pollutant, their disposal represents a cost for wineries. Numerous wine lees recovery and valorization strategies have been proposed, with a particularly steep increase in published research in recent years. This attention is strictly linked to the concepts of circular economy and environmental sustainability that are attracting the interest of the scientific community. In this review, an overview on the available wine lees recovery and valorization strategies is reported. Additionally, the methods for the extraction and valorization of yeast's cell wall polysaccharides (\u3b2-glucans and mannoproteins) are discussed. Finally, current and future innovative applications in different sectors of yeast \u3b2-glucans and mannoproteins are described and critically discussed

Garlic (Allium sativum L.) fed to dairy cows does not modify the cheese-making properties of milk but affects the color, texture, and flavor of ripened cheese

Garlic and garlic components have recently been proposed as ruminal activity modulators to reduce the enteric methane emissions of ruminants, but little is known of their influence on milk coagulation proper- ties, nutrient recovery, cheese yield, and sensorial and rheological characteristics of milk and cheese. The pres- ent study assessed the effects of garlic and diallyl sul- fide supplements on dry matter intake (DMI), produc- tive performance, milk coagulation properties, cheese yield, milk and cheese sensory profiles, and rheological characteristics. Four dairy cows were fed a total mixed ration either alone (control) or supplemented with 100 or 400 g/d of garlic cloves or 2 g/d of diallyl sulfide in 4 consecutive experimental periods in a 4 7 4 Latin square design. The diallyl sulfide dose was established to provide approximately the same amount of allyl thiosulfinate compounds as 100 g of fresh garlic cloves. The total mixed ration was composed of 0.29 corn si- lage, 0.23 corn-barley mixture, 0.17 sunflower-soybean mixture, 0.12 alfalfa hay, 0.12 grass hay, 0.04 sugar beet pulp, and 0.02 other additives, and contained 0.253 starch, 0.130 crude protein, and 0.375 neutral detergent fiber, on a dry matter basis. Each experimental period consisted of 7 d of transition and 14 d of treatment. On d 18 and 21 of each period, milk samples (10 L) were collected from each cow for chemical analysis and cheese-making. The organoleptic properties of the milk and 63-d-ripened cheeses were assessed by a panel of 7 trained sensory evaluators. The experimental treat- ments had no effects on DMI, milk yield, feed efficiency (milk yield/DMI), milk coagulation properties, nutrient recovery, or cheese yield. Garlic-like aroma, taste, and flavor of milk and cheese were significantly influenced by the treatments, particularly the highest dose of gar- lic cloves, and we found close exponential relationships between milk and cheese for garlic-like aroma (R2 = 0.87) and garlic-like flavor (R2 = 0.79). Diallyl sulfide and 400 g/d of garlic cloves resulted in lower pH, shear force, and shear work of ripened cheeses compared with the other treatments. Garlic cloves and diallyl sulfide had opposite effects on cheese color indices. We conclude that adding 400 g/d of garlic to the feed of lactating dairy cows highly influences the sensory and rheological characteristics of cheese

Microbial inactivation of raw chicken meat by supercritical carbon dioxide treatment alone and in combination with fresh culinary herbs

The objective of the present study was to assess the potential synergistic effect between supercritical carbon dioxide (SC-CO2) and fresh culinary herbs (Coriandrum sativum and Rosmarinus officinalis) on the microbial inactivation of raw chicken meat. The microbiological inactivation was performed on Escherichia coli and natural flora (total mesophilic bacteria, yeasts, and molds). High pressure treatments were carried out at 40\ub0C, 80 or 140 bar from 15 to 45 min. Microbial inactivation had a strong dependence on treatment time, achieving 1.4 log CFU/g reduction of E. coli after 15 min, and up to 5 log after 45 min, while a pressure increase from 80 up to 140 bar was not significant on the microbial inactivation. Mesophilic microorganisms were strongly reduced (>2.6 log CFU/g) after 45 min, and yeasts and molds were below the detection limits of the technique (<100 CFU/g) in most cases. The combination of fresh herbs together with SC-CO2 treatment did not significantly increase the inactivation of either E. coli or natural flora, which was similar to the SC-CO2 alone. The synergistic effect was obtained on the inactivation of E. coli using a proper concentration of coriander essential oil (EO) (0.5% v/w), while rosemary EO did not show a significant effect. Color analysis after the treatment showed an increment of lightness (L*), and a decrease of redness (a*) on the surface of the sample, making the product visually similar to cooked meat. Texture analysis demonstrated the modification of the texture parameters as a function of the process pressure making the meat more similar to the cooked one

Studio di enzimi in saccharomyces cerevisiae con tecniche di biochimica analitica e biologia molecolare

Dottorato di ricerca in biocatalisi applicata e fermentazioni industriali. 12. ciclo. Coordinatore Pier Giorgio Pifferi. Tutore Paolo Spettoli.Consiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Sparkling Cider Paired with Italian Cheese: Sensory Analysis and Consumer Assessment

Cider is a beverage belonging to the tradition of many European rural areas. Pairing beverages and cheeses, even if it is part of an ancient tradition, is gaining more and more interest from the consumer. For this reason, in this research, we wanted to conduct a preliminary study on the combination of cider and cheese. In particular, six Italian sparkling ciders were selected, obtained through the Charmat and Champenoise method, and four types of Italian cheeses, from the Veneto region: Casatella&nbsp;Trevigiana, Fienil, Morlacco and Ubriaco, with very different sensory characteristics. The cider-cheese pairing test, conducted by a panel of experts, revealed how some cider parameters are reduced in intensity, such as astringency, while others are enhanced, such as fruitiness and persistence taste aroma. The hedonic test, conducted on the matching by 90 consumers, promoted some combinations while others were rejected. The sensory parameters associated with liking were fruity and taste aroma persistence, particularly expressed in some cider-cheese pairings

Protein haze formation in white wines: effect of Saccharomyces cerevisiae cell wall components prepared with different procedures.

Cell wall material was extracted by five different methods from an oenological strain of Saccharomyces cerevisiae. Enzyme preparations containing beta-glucanase activity (Zymolyase, Glucanex, and Finizym 250L) allowed a better extraction yield compared to that of dithiothreitol (DTT) and EDTA. The yeast extracts were only soluble in part in wine. The wine-soluble fraction (WSF) of the five extracts, differing in both protein and sugar contents, when added in increasing amounts to white wine differently affected protein haze formation, as determined by the heat test, giving dose/response curves of different shapes. The curves obtained with the WSF derived from DDT and Zymolyase extracts showed a plateau value corresponding to 90% and 80% of wine haze reduction, respectively. In contrast, addition of the WSF derived from the other extracts resulted in increased turbidity with respect to the original wine. The mannoproteins (MP), isolated from each yeast extract by Concanavalin-A chromatography, gave dose/response curves showing shapes more similar among them than those obtained with the whole WSFs. The best wine stabilization was obtained with the MP of the DTT and Zymolyase extracts. The supernatants obtained after heating the MP of the different extracts were also tested. The stabilizing effect of the heat-stable MP (HSMP) was always larger than that of the corresponding total (un-heated) MP. The HSMP obtained starting from the DTT and Zymolyase extracts showed the best haze-protecting effect, which was, however, lower than that obtained with their corresponding WSF. This result suggests that wine protein stabilization by compounds of the yeast cell wall could be related, in addition to the action of the MP, also to the presence of other substances of different nature

Characterization of crude esterase activity from two plants used in cheese making: Cynara cardunculus L. and ficus carica L

For centuries, extracts of Cynara cardunculus and the latex of Ficus carica, have been used in cheesemaking, thanks to their proteases that clot milk casein. Since esterase activity is very important to release odorous compounds in cheese, in this study we characterized the esterase activity of an extract of Cynara cardunculus and the latex of Ficus carica. The results, obtained by titration, spectrophotometric techniques, zymograms and tests on agar plates, show the presence of esterase activity in the samples tested, with affinity towards the esters of short chain fatty acids, such as acetic, propionic and butyric acid. In conclusion, this study provides a background information on esterases of two plants used in cheesemaking and represents a starting point for further investigations of their applications, not only in dairying but also in other industries involved in the processing and transformation of fats