Proteomics Parameters for Assessing Authenticity of Grated Grana Padano PDO Cheese: Results from a Three-Year Survey.

Assessing the authenticity of PDO cheeses is an important task because it allows consumer expectations to be fulfilled and guarantees fair competition for manufacturers. A 3-year survey was carried out, analyzing 271 samples of grated Grana Padano (GP) PDO cheese collected on the European market. Previously developed analytical methods based on proteomics approaches were adopted to evaluate the compliance of market samples with selected legal requirements provided by the specification for this cheese. Proteolysis follows highly repeatable pathways in GP cheese due to the usage of raw milk, natural whey starter, and consistent manufacturing and ripening conditions. From selected casein breakdown products, it is possible to calculate the actual cheese age (should be >9 months) and detect the presence of excess rind (should be <18%). Furthermore, due to the characteristic pattern of free amino acids established for GP, distinguishing it from closely related cheese varieties is feasible. Cheese age ranged from 9 to 25 months and was correctly claimed on the label. Based on the amino acid pattern, three samples probably contained defective cheese and there was only one imitation cheese. Few samples (9%) were proven to contain some excess rind. Overall, this survey highlighted that the adopted control parameters can assure the quality of grated GP.

High-speed cold centrifugation of milk modifies the microbiota, the ripening process and the sensory characteristics of raw-milk hard cheeses.

The microbial population of raw milk plays a crucial role in the development of distinctive traits of raw-milk cheeses particularly appreciated by consumers. It was previously demonstrated that the microbial population of raw milk is modified by a high-speed centrifugation (also called bactofugation) conducted at 39 °C. The aim of the present study was to evaluate the effects of this process, performed once or twice, on the microbial, compositional, biochemical, and sensory characteristics of the derived hard cheeses. Experimental and control cheesemaking were conducted in parallel at a cheese factory during a 13-month period. Cheeses were analysed after 9, 15 and 20 months of ripening for microbial count, composition, proteolysis extent, volatile compounds, and sensory profile. Results evidenced that experimental cheeses were characterized by lower numbers of viable lactobacilli respect to control. Experimental cheeses also showed differences in the progress of primary and secondary proteolysis which, in turn, caused different patterns of free amino acids at all ripening times. Experimental cheeses had significantly lower content of esters and were differentiated from control for some traits by assessors. In conclusion, use of high-speed centrifugation of milk shall be discouraged if characteristic traits of raw-milk cheeses, particularly PDO cheeses, want to be retained.

Focus on the Protein Fraction of Sports Nutrition Supplements.

Increasing awareness of balanced diet benefits is boosting the demand for high-protein food and beverages. Sports supplements are often preferred over traditional protein sources to meet the appropriate dietary intake since they are widely available on the market as stable ready-to-eat products. However, the protein components may vary depending on both sources and processing conditions. The protein fraction of five commercial sports supplements was characterized and compared with that of typical industrial ingredients, i.e., whey protein concentrates and isolates and whey powder. The capillary electrophoresis profiles and the amino acid patterns indicated that, in some cases, the protein was extensively glycosylated and the supplemented amino acids did not correspond to those declared on the label by manufacturers. The evaluation by confocal laser scanning microscopy evidenced the presence of large aggregates mainly enforced by covalent crosslinks. The obtained findings suggest that, beside composition figures, provisions regarding sports supplements should also consider quality aspects, and mandatory batch testing of these products would provide more reliable information to sport dieticians.

Effects of microbial coagulants from Rhyzomucor miehei on composition, sensory and textural characteristics of long-ripened hard cheeses.

The increasing use of rennet substitutes entails evaluating their performances on different types of cheese. The production of hard cheese using either microbial coagulants from Rhyzomucor miehei (MC) or calf rennet (CR) from different manufacturers was investigated in parallel cheese makings at three industrial dairies. Cheeses were analysed after 9, 12, 16 and 18 months of ripening. Minor differences in cheese composition were found between treatments, principally related to fat content. Cheeses produced with one out of the three MC showed slower primary proteolysis on both αs1- and αs0-casein, compared to the corresponding CR cheeses, indicating a different activity of this coagulant. The same cheeses also had significantly different sensory profiles at 9 months of ripening. Treatments did not differ in free amino acid composition nor in rheological parameters, regardless of ripening period. The long ripening of hard cheeses thus smooths possible differences attributable to MC.

Evaluating the Authenticity of the Raw-Milk Cheese Fontina (PDO) with Respect to Similar Cheeses.

The implementation of quality assurance schemes for the assessment of PDO food authenticity is an issue involving manufacturers, traders, retailers and consumers. In this respect, reliable analytical methods are needed to integrate paper-trailing information. The feasibility of distinguishing the Italian Fontina PDO cheese from the generic Fontal cheese was preliminarily evaluated on a set of commercial samples by measuring selected parameters (pH, alkaline phosphatase activity, content of copper, volatiles, extent of proteolysis) related to the different manufacturing processes. The relative profile of free amino acids proved to be a promising tool. A new set of 41 samples of Fontina PDO cheese was collected at representative dairies within the recognized production area and analyzed for free amino acids. A chemometric model of Fontina PDO cheese was built based on the mean content and standard deviation of 15 free amino acids. On this basis, all of the PDO samples were correctly identified, whereas all of the Fontal cheeses were recognized as different cheeses.

Storage of pasteurized milk in clear PET bottles combined with light exposure on a retail display case: A possible strategy to define the shelf life and support a recyclable packaging.

The stability of whole pasteurized milk packaged in clear PET bottles was studied throughout 13-days storage in the dark, but including, at specific times, light exposure of 6, 12 or 18 h to simulate conditions potentially occurring in refrigerated display counters. The aim was to investigate the effects of light exposure when overlapping the unavoidable endogenous modifications in pasteurized milk during storage. Dissolved oxygen, riboflavin and other flavins, proteolysis products, volatile compounds, and sensory characteristics were evaluated. Besides the expected progress of proteolysis occurring during storage, light negatively affected milk flavour especially after longer exposure times. The development of "mushroom" flavor related to the increase of volatile 2,3 octanedione was the most characterizing modification. Gathered data were considered in view of providing the background knowledge for the control of light exposure conditions on a retail display, thus supporting the shelf life extension of pasteurized milk in a fully recyclable packaging.

Impact of Extending Hard-Cheese Ripening: A Multiparameter Characterization of Parmigiano Reggiano Cheese Ripened up to 50 Months.

Extending ripening of hard cheeses well beyond the traditional ripening period is becoming increasingly popular, although little is known about the actual evolution of their characteristics. The present work aimed at investigating selected traits of Parmigiano Reggiano cheese ripened for 12, 18, 24, 30, 40 and 50 months. Two cheeses per each ripening period were sampled. Although moisture constantly decreased and was close to 25% in 50-month cheeses, with a parallel increase in cheese hardness, several biochemical changes occurred involving the activity of both native and microbial enzymes. Capillary electrophoresis demonstrated degradation of αs1- and ß-casein, indicating residual activity of both chymosin and plasmin. Similarly, continuous release of free amino acids supported the activity of peptidases deriving from lysed bacterial cells. Volatile flavor compounds, such as short-chain fatty acids and some derived ketones, alcohols and esters, evaluated by gas chromatography with solid-phase micro-extraction, accumulated as well. Cheese microstructure was characterized by free fat trapped in irregularly shaped areas within a protein network, with native fat globules being no longer visible. This study showed for the first time that numerous biochemical and structural variations still occur in a hard cheese at up to 50 months of aging, proving that the ripening extension deserves to be highlighted to the consumer and may justify a premium price.

Bacterial proteolysis of casein leading to UHT milk gelation: An applicative study.

Heat-stable peptidases released in refrigerated raw milk by psychrotrophic bacteria are responsible for UHT milk gelation. K-casein-derived caseinomacropeptides, identified by mass spectrometry, were constantly detected in gelled milk by capillary electrophoresis. Strains of Pseudomonas fluorescens, Ps. poae and Chryseobacterium joostei, selected among aprX-positive strains from raw milk, were incubated in milk up to 6 days at 4 °C before sterilization (98 °C/4 min). Samples were then stored at 25 or 40 °C, visually observed for gelation, and analysed for presence of caseinomacropeptides throughout 90 days of storage. Depending on cold pre-incubation time, caseinomacropeptides accumulated well before gelation onset in milk stored at 25 °C. Caseinomacropeptides were successively degraded, especially in milk stored at 40 °C, due to extensive proteolysis, and an abundant sediment developed instead of a gel. The caseinomacropeptides are here presented as an early indicator of UHT milk gelation and a mechanism explaining this phenomenon is proposed.

Proteolytic Activity and Production of γ-Aminobutyric Acid by Streptococcus thermophilus Cultivated in Microfiltered Pasteurized Milk.

A set of 191 strains of Streptococcus thermophilus were preliminarily screened for the presence of the genes codifying for cell envelope-associated proteinase (prtS) and for glutamate decarboxylase (gadB) responsible for γ-aminobutyric acid (GABA) production. The growth and proteolytic activity of the gadB-positive strains (9 presenting the prtS gene and 11 lacking it) were studied in microfiltered pasteurized milk. Degradation of both caseins (capillary electrophoresis) and soluble nitrogen fractions (HPLC) and changes in the profile of free amino acids (FAAs; ion-exchange chromatography) were evaluated at inoculation and after 6 and 24 h of incubation at 41 °C. None of the strains was capable of hydrolyzing caseins and ß-lactoglobulin, and only two hydrolyzed part of α-lactalbumin, these proteins being present in their native states in pasteurized milk. Contrarily, most strains were able to hydrolyze peptones and peptides. For initial growth, most strains relied on the FAAs present in milk, whereas, after 6 h, prtS+ strains released variable amounts of FAA. One prtS+ strain expressed a PrtS- phenotype, and two prtS- strains showed a rather intense proteolytic activity. Only five strains (all prtS+) produced GABA, in variable quantities (up to 100 mg/L) and at different rates, depending on the acidification strength. Addition of glutamate did not induce production of GABA in nonproducing strains that, however, unexpectedly were shown to adopt the degradation of arginine into citrulline and ornithine as an alternative acid resistance system and likely as a source of ATP.

Different analytical approaches in assessing antibacterial activity and the purity of commercial lysozyme preparations for dairy application.

Hen egg-white lysozyme (LSZ) is currently used in the food industry to limit the proliferation of lactic acid bacteria spoilage in the production of wine and beer, and to inhibit butyric acid fermentation in hard and extra hard cheeses (late blowing) caused by the outgrowth of clostridial spores. The aim of this work was to evaluate how the enzyme activity in commercial preparations correlates to the enzyme concentration and can be affected by the presence of process-related impurities. Different analytical approaches, including turbidimetric assay, SDS-PAGE and HPLC were used to analyse 17 commercial preparations of LSZ marketed in different countries. The HPLC method adopted by ISO allowed the true LSZ concentration to be determined with accuracy. The turbidimetric assay was the most suitable method to evaluate LSZ activity, whereas SDS-PAGE allowed the presence of other egg proteins, which are potential allergens, to be detected. The analytical results showed that the purity of commercially available enzyme preparations can vary significantly, and evidenced the effectiveness of combining different analytical approaches in this type of control.