Agraria

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The sensorial and physico-chemical characteristics described in the product specification for most PDO cheeses are inadequate to verify the compliance of cheeses on the market with the registered designation, particularly for grated products. During the past few years, much research has indicated the analytical parameters suitable for distinguishing Grana Padano (GP) from other similar hard cheeses. The characterization of grated GP is currently based on 3 analytical parameters, related to different aspects of cheese processing, which are: (i) the measurement of alkaline phosphatase (ALP) activity, a marker for possible heat treatment applied to milk, in the outermost layer of the cheese, just below the rind; (ii) the identification of specific peptides, that are identified only in the rind, due to the very slow progress of proteolysis in the rind during GP cheese ripening; and (iii) the free amino acid (FAA) composition. In the present study, we developed an extraction method, based on density gradient centrifugation of solubilized cheese, to separate the outermost layer of the cheeses from the rest in grated cheese, and we proposed a simplified criterion to evaluate the "typicalness" of the FAA pattern. The quality control scheme based on ALP activity, detection of specific peptides and FAA pattern was applied to more than 300 samples of marketed grated GP collected over three years, 10% of which were collected outside Italy, and ∼ 100 samples of grated generic ("Grana-type") hard cheeses. The results demonstrate that the simultaneous application of the three parameters allows one to distinguish grated GP from similar, non-PDO grated hard cheeses, and to recognize irregular GP cheeses.

The behavior of soluble nitrogen compounds during Grana Padano cheese-making was studied at eight dairies. Raw milk, skimmed milk, sweet whey and the derived natural whey culture, collected from 24 processes, were analyzed for soluble whey proteins (α-La and β-Lg), proteose-peptones (PP), small peptides (SP), caseinomacropeptides (CMPs), and free amino acids (FAA). The PP fraction increased during milk natural creaming, then part of it was selectively retained in the curd and the rest degraded in the first few hours of whey fermentation, together with α-La, CMPs and part of SP. Features outlined for the whey culture have been confirmed on 30 samples collected at six different dairies. A time course study of the whey fermentation showed that degradation of α-La begins when the pH drops below 4, whereas β-Lg content did not change. Uptake of specific FAA is shown to support the initial growth of lactic acid bacteria in whey.

The behavior of soluble nitrogen compounds during Grana Padano cheese-making was studied at eight dairies. Raw milk, skimmed milk, sweet whey and the derived natural whey culture, collected from 24 processes, were analyzed for soluble whey proteins (α-La and β-Lg), proteose-peptones (PP), small peptides (SP), caseinomacropeptides (CMPs), and free amino acids (FAA). The PP fraction increased during milk natural creaming, then part of it was selectively retained in the curd and the rest degraded in the first few hours of whey fermentation, together with α-La, CMPs and part of SP. Features outlined for the whey culture have been confirmed on 30 samples collected at six different dairies. A time course study of the whey fermentation showed that degradation of α-La begins when the pH drops below 4, whereas β-Lg content did not change. Uptake of specific FAA is shown to support the initial growth of lactic acid bacteria in whey.

The behavior of soluble nitrogen compounds during Grana Padano cheese-making was studied at eight dairies. Raw milk, skimmed milk, sweet whey and the derived natural whey culture, collected from 24 processes, were analyzed for soluble whey proteins (α-La and β-Lg), proteose-peptones (PP), small peptides (SP), caseinomacropeptides (CMPs), and free amino acids (FAA). The PP fraction increased during milk natural creaming, then part of it was selectively retained in the curd and the rest degraded in the first few hours of whey fermentation, together with α-La, CMPs and part of SP. Features outlined for the whey culture have been confirmed on 30 samples collected at six different dairies. A time course study of the whey fermentation showed that degradation of α-La begins when the pH drops below 4, whereas β-Lg content did not change. Uptake of specific FAA is shown to support the initial growth of lactic acid bacteria in whey.

Different chemical indices related to proteolysis and ripening time have been investigated in Grana Padano (n ¼ 155) and Parmigiano-Reggiano (n ¼ 27) cheese samples using ion-exchange chromatography and capillary electrophoresis. Levels of intact caseins, g-casein and free amino acids in cheese were not strictly related to the maturation period, varying among samples of the same age. A peptide, identified by HPLC/electrospray ionization-mass spectrometry as pyroglutamyl-g 3-casein, resulted from cyclisation of N-terminal glutamic acid into a pyroglutamic acid residue. Based on the peak area ratio of pyroglutamyl-g 3-casein and g 3-casein, an equation, suitable for determination of the cheese age, is proposed. The commercial classes of Grana Padano ripened over 16 and 20 months can be identified by adopting a minimum threshold value for this peak area ratio.

Molecular properties of proteins and starch were investigated in 2 accessions of Oryza glaberrima and Oryza sativa, and in one NERICA cross between the 2 species, to assess traits that could be relevant to transformation into specific foods. Protein nature and organization in O. glaberrima were different from those in O. sativa and in NERICA. Despite the similar cysteine content in all samples, thiol accessibility in O. glaberrima proteins was higher than in NERICA or in O. sativa. Inter-protein disulphide bonds were important for the formation of protein aggregates in O. glaberrima, whereas non-covalent protein-protein interactions were relevant in NERICA and O. sativa. DSC and NMR studies indicated only minor differences in the structure of starch in these species, as also made evident by their microstructural features. Nevertheless, starch gelatinization in O. glaberrima was very different from what was observed in O. sativa and NERICA. The content of soluble species in gelatiniz...

The late blowing defect still represents a problem for hard cheeses. Thus, the behaviour of the cheese spoiling bacterium C. tyrobutyricum was studied throughout the cheesemaking and ripening of Grana Padano using an innovative approach. Cells and spores, independently sealed within dialysis tubes, were kept in the vat during the entire cheesemaking and then into cheese until 6-2 month ripening. At each sampling step, morphological changes of cells and spores were monitored by electron microscopy and supported with plate counts. Vegetative cells died during curd cooking and then were no longer cultivable. However, 2 x 10 2 spores appeared at the end of this stage, likely triggered by the exponential growth phase, and were present until 6-month ripening. In cheese, C. tyrobutyricum UC7086 proved to convert free arginine to citrulline and then to ornithine, and to produce γ-aminobutyric acid by glutamate transamination rather than by decarboxylation. Compartmentalization of vegetative cells and spores into dialysis tubes was effective in studying their respective behaviour in a real cheesemaking. This approach allowed to demonstrate that the number of vegetative cells in milk in addition to that of spores should be considered for the eradication of the late blowing defect.

Although free amino acids (FAAs) represent a significant component of ripened cheeses and can provide useful information for their characterization, no inter-laboratory validated analytical method exists which allows a reliable comparison of data obtained by different laboratories and the adoption of quality control schemes based on FAA pattern. The objective of the present work was to test the effectiveness of an analytical protocol for the determination of the FAA composition of cheese and to verify the adequateness of this type of analysis for quality control procedures of Grana Padano PDO cheese as well as for research purposes. After an initial test to compare performances of ion-exchange chromatography (IEC) and HPLC techniques, an inter-laboratory collaborative study (seven laboratories, four samples) was organized to validate an IEC method with post-column ninhydrin derivatization and using L-norleucine as an internal standard. Determined amounts of individual FAA ranged from 8 to over 1380 mg/100 g cheese, with relative standard deviation of repeatability (RSD r) ranging from 0.5 to 4.6%, and relative standard deviation of reproducibility (RSD R) ranging from 1.3 to 9.9% for FAA concentrations over 100 mg/100 g. For lower concentrations, RSD r and RSD R were about thrice as high. On the basis of the results of this investigation, at present, the validated method is adopted as the official method for the determination of FAA patterns in the quality control of Grana Padano PDO cheese.

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 i...

Cheese Whey Permeate (CWP) is the by-product of whey ultrafiltration for protein recovery. It is highly perishable with substantial disposal costs and has serious environmental impact. The aim of the present study was to develop a novel and cheap CWP-based culture medium for Lactobacillus sakei to produce the food-grade sakacin A, a bacteriocin exhibiting a specific antilisterial activity. Growth conditions, nutrient supplementation and bacteriocin yield were optimized through an experimental design in which the standard medium de Man, Rogosa and Sharpe (MRS) was taken as benchmark. The most convenient formulation was liquid CWP supplemented with meat extract (4 g/L) and yeast extract (8 g/L). Although, arginine (0.5 g/L) among free amino acids was depleted in all conditions, its supplementation did not increase process yield. The results demonstrate the feasibility of producing sakacin A from CWP. Cost of the novel medium was 1.53 €/L and that of obtaining sakacin A 5.67 €/106 AU, ...

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 β-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.

– The sensorial and physico-chemical characteristics described in the product specifica-tion for most PDO cheeses are inadequate to verify the compliance of cheeses on the market with the registered designation, particularly for grated products. During the past few years, much research has indicated the analytical parameters suitable for distinguishing Grana Padano (GP) from other similar hard cheeses. The characterization of grated GP is currently based on 3 analytical param-eters, related to different aspects of cheese processing, which are: (i) the measurement of alkaline phosphatase (ALP) activity, a marker for possible heat treatment applied to milk, in the outermost layer of the cheese, just below the rind; (ii) the identification of specific peptides, that are iden-tified only in the rind, due to the very slow progress of proteolysis in the rind during GP cheese ripening; and (iii) the free amino acid (FAA) composition. In the present study, we developed an extraction method,...

Lysozyme (LZ) is used in several cheese varieties to prevent late blowing which results from fermentation of lactate by Clostridium tyrobutyricum. Side effects of LZ on lactic acid bacteria population and free amino acid pattern were studied in 16 raw-milk hard cheeses produced in eight parallel cheese makings conducted at four different dairies using the same milk with (LZ+) or without (LZ-) addition of LZ. The LZ-cheeses were characterized by higher numbers of cultivable microbial population and lower amount of DNA arising from lysed bacterial cells with respect to LZ+ cheeses. At both 9 and 16 months of ripening, L. delbrueckii and L. fermentum proved to be the species mostly affected by LZ. The total content of free amino acids indicated the proteolysis extent to be characteristic of the dairy, regardless to the presence of LZ. In contrast, the relative patterns showed the microbial degradation of arginine to be promoted in LZ+ cheeses. The data demonstrated that the arginine-deiminase pathway was only partially adopted since citrulline represented the main product and only trace levels of ornithine were found. Differences in arginine degradation were considered for starter and non-starter lactic acid bacteria, at different cheese ripening stages.

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 tha...

Chemical composition and structure of different types of macroparticles (specks, spots) and microparticles (microcrystals) present in hard and extra-hard cheeses were investigated. Light microscopy revealed that the small hard specks had the structure of crystalline tyrosine, as confirmed by amino acid analysis. Spots showed a complex structure, including several curd granules, cavities, and microcrystals, and were delimited by a dense protein layer. Spots contained less moisture and ash than the adjacent cheese area, and more protein, including significantly higher contents of valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine. Microcrystals were observed by light and electron microscopy and analyzed by confocal micro-Raman. Among others, calcium phosphate crystals appeared to consist of a central star-shaped structure immersed in a matrix of free fatty acids plus leucine and phenylalanine in free form or in small peptides. A hypothetical mechanism for the formation of these structures has been formulated. Key words: hard and extra hard cheeses, cheese ultrastructure, calcium phosphate crystal, free amino acids, electron microscopy