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.

Investigating Structural Defects in Extra Hard Cheese Produced from Low-Temperature Centrifugation of Milk.

The present study investigated some physico-chemical and microbiological traits of 20-month ripened hard cheeses produced from low-temperature high-speed centrifuged raw milk that developed a structural defect consisting of eyes or slits in the paste. Cheeses obtained using the same process and that did not develop the defect were used as controls. The colour, texture, moisture, water activity, proton molecular mobility, microstructure, extent of proteolysis, and viable microorganisms have been evaluated in all the cheese samples, and the significant differences between the defective and non-defective cheeses have been critically discussed. At a microstructural level, the defects caused fat coalescence and an unevenly organised protein matrix with small cracks in the proximity of the openings. The different fat organisation was correlated to a different transverse relaxation time of 1H population relaxing at higher times. The textural and colour features were not different from those of the control cheeses and were comparable with those reported in the literature for other long-ripened hard cheeses. On the other hand, the defective cheeses showed a higher moisture level and lower lactobacilli and total mesophilic bacteria concentrations, but the microbial origin of the defect remains an open hypothesis that deserves further investigation.

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.

Strategies for Exploiting Milk Protein Properties in Making Films and Coatings for Food Packaging: A Review.

Biopolymers of different natures (carbohydrates, proteins, etc.) recovered from by-products of industrial processes are increasingly being studied to obtain biomaterials as alternatives to conventional plastics, thus contributing to the implementation of a circular economy. The food industry generates huge amounts of by-products and waste, including unsold food products that reach the end of their shelf life and are no longer usable in the food chain. Milk proteins can be easily separated from dairy waste and adapted into effective bio-based polymeric materials. Firstly, this review describes the relevant properties of milk proteins and the approaches to modifying them for subsequent use. Then, we provide an overview of recent studies on the development of films and coatings based on milk proteins and, where available, their applications in food packaging. Comparisons among published studies were made based on the formulation as well as production conditions and technologies. The role of different additives and modifiers tested for the performances of films and coatings, such as water vapor permeability, tensile strength, and elongation at break, were reviewed. This review also outlines the limitations of milk-protein-based materials, such as moisture sensitivity and brittleness. Overall, milk proteins hold great potential as a sustainable alternative to petroleum-based polymers. However, their use in food packaging materials at an industrial level remains problematic.

Immunogold Labeling of Milk Proteins at Transmission Electron Microscopy.

Milk intended for human consumption is subjected to technological treatments to ensure its safety and storage stability. These treatments deeply modify some of its structural and nutritional characteristics. Principal modifications involve proteins that partly adsorb onto the membrane of milk fat globules upon homogenization or whey proteins that undergo denaturation and aggregation during thermal treatments. Transmission electron microscopy is a powerful approach to investigate milk ultrastructure, due to its high-resolution power. Immunogold labeling of ß-lactoglobulin and ß-casein proteins represents a sophisticated approach to examine their structure and localization following technological processes such as homogenization and UHT treatment. However, liquid milk is a very challenging matrix because of its complex multiphasic nature. To preserve both ultrastructure and antigenicity, and to obtain an efficient labelling in liquid milk samples, some precautions shall be adopted in fixation, embedding, and labeling steps as here reported.

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.

Vitamin D Fortification of Consumption Cow's Milk: Health, Nutritional and Technological Aspects. A Multidisciplinary Lecture of the Recent Scientific Evidence.

Vitamin D is essential in assuring bone health at all stages of life, but its non-skeletal effects are also essential: This vitamin impacts the physiology of the immune system, skeletal muscles and adipose tissue, glucose metabolism, skin, cardiovascular and reproductive systems, neuro-cognitive functions and cell division. The incidence of vitamin D deficiency is widespread worldwide, at any age, in young and healthy subjects, as well as in pregnant women and the elderly population, due to several factors, including inadequate sunlight exposure, skin pigmentation and coverage, adiposity, lifestyle and low dietary intakes. To overcome this problem, the fortification of foods that are consumed on a daily basis, such as milk, is strongly advisable. This opinion paper aims to discuss, in a multidisciplinary way, the current evidence supporting the importance of vitamin D in health and disease and the role of milk as an optimal carrier of this vitamin, to promote adequate intakes, highlighting its unique physico-chemical characteristics linked to both fat globule membrane and casein micelle structure. Moreover, it addresses the impact of industrial processing and storage of consumption milk on the stability of these structures, thus in determining vitamin D bioavailability and the achievement of adequate intakes.

Vitamin D Incorporation in Foods: Formulation Strategies, Stability, and Bioaccessibility as Affected by the Food Matrix.

Inadequate intake of vitamin D is a global health issue related to severe diseases, mainly involving subjects with dark skin pigmentation, patients affected by malnutrition, malabsorption syndromes, or obesity, and elderly people. Some foods fortified with vitamin D have been tested in vivo, but fortification strategies with a global outreach are still lacking. This review is focused on food fortification with vitamin D, with the aim to collect information on (a) formulation strategies; (b) stability during processing and storage; and (c) in vitro bioaccessibility. Approaches to add vitamin D to various foods were analyzed, including the use of free vitamin D, vitamin D loaded in simple and double nanoemulsions, liposomes, casein micelles, and protein nanocapsules. Numerous studies were reviewed to elucidate the impact of food technologies on vitamin D's stability, and mechanisms that lead to degradation were identified-namely, acid-catalyzed isomerization, radical-induced oxidation, and photo-oxidation. There is, however, a lack of kinetic data that allow for the prediction of vitamin D's stability under industrial processing conditions. The roles that lipids, proteins, fibers, and antioxidants play in vitamin bioaccessibility have been clarified in various studies, while future needs include the design of specific food matrices that simultaneously achieve a balance between the long-term stability, bioaccessibility and, ultimately, in vivo functionality of vitamin D.

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.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

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.

Exploiting Milling By-Products in Bread-Making: The Case of Sprouted Wheat.

This research investigated the effect of sprouting on wheat bran. Bran from un-sprouted (BUW) and sprouted (BSW) wheat were characterized in terms of chemical composition, enzymatic activities, and hydration properties. In addition, the rheological properties (using GlutoPeak, Farinograph, Extensograph, and Rheofermentometer tests) and bread-making performance (color, texture, volume of bread) of wheat doughs enriched in bran at 20% replacement level were assessed. Sprouting process caused a significant decrease in phytic acid (~20%), insoluble dietary fiber (~11%), and water holding capacity (~8%), whereas simple sugars (~133%) and enzymatic activities significantly increased after processing. As regards the gluten aggregation kinetics, the BSW-blend profile was more similar to wheat than BUW-blend, indicating changes in the fiber and gluten interactions. BSW led to a worsening of the mixing and leavening properties, instead, no significant changes in extensibility were observed. Finally, BSW improved bread volume (~10%) and crumb softness (~52%). Exploiting bran from sprouted wheat might be useful to produce bread rich in fiber with enhanced characteristics.

Sprouting improves the bread-making performance of whole wheat flour (Triticum aestivum L.).

BACKGROUND: Pre-harvest sprouting of wheat is viewed negatively because of the high level of enzymatic activity, which leads to a deterioration in the bread-making performance of the related flours. On the other hand, improvements in bread properties (i.e. volume and crumb softness) are reported when sprouted wheat under controlled conditions is used in mixtures with a conventional unsprouted flour. However, knowledge about the effects of sprouting on gluten functionality and its relationship with bread features is still limited, especially in the case of whole wheat flour. RESULTS: Under the conditions applied in this study (48 h, 20 °C and 90% relative humidity), proteins of sprouted wheat were still able to aggregate, even if changes in gluten aggregation kinetics suggested gluten weakening. On the other hand, sprouting led to an increase in gluten stretching ability, suggesting an increase in dough extensibility. In the dough system, sprouting was responsible for a decrease in water absorption, development time, and stability during mixing. However, when the values for development time and water absorption indicated by the Farinograph® were followed carefully, sprouting improved bread height (~20%), specific volume (~15%), and crumb softness (~200% after 24 h of storage), even when whole wheat flour was used. CONCLUSION: It is possible to produce bread with improved volume and crumb softness using whole wheat flour from sprouted kernels. Thus, sprouting can be exploited as a pre-treatment to improve the bread-making performance of fiber-enriched systems. © 2020 Society of Chemical Industry.

Antimicrobial activity of resveratrol-derived monomers and dimers against foodborne pathogens.

Plant polyphenolic compounds are considered a promising source for new antibacterial agents. In this study, we evaluated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers screened as single molecules against a panel of nine foodborne pathogens. The results demonstrated that two monomers (i.e., pterostilbene 2 and (E)-3-hydroxy-4',5-dimethoxystilbene 9) and three dimers (i.e., δ-viniferin 10, viniferifuran 14 and dehydro-δ-viniferin 15) were endowed with significant antibacterial activity against gram-positive bacteria. The exposure of gram-positive foodborne pathogens to 100 µg/mL of 2, 9 and 15 induced severe cell membrane damage, resulting in the disruption of the phospholipid bilayer. The most promising dimeric compound, dehydro-δ-viniferin 15, was tested against Listeria monocytogenes, resulting in a loss of cultivability, viability and cell membrane potential. TEM analysis revealed grave morphological modifications on the cell membrane and leakage of intracellular content, confirming that the cell membrane was the principal biological target of the tested derivative.

Impedance microbiology to speed up the screening of lactic acid bacteria exopolysaccharide production.

Bacterial production of exopolysaccharides (EPS) is of increasing interest near food manufacturers, biotechnology industries and nutritionists because of their different roles. Several analytical methods are available for recovery, quantification and characterization of EPS from lactic acid bacteria (LAB) in food. However, direct screening method for production of EPS is still based on the visual observation of filamentous texture of the colonies developed on supplemented solid growth media. To overcome weaknesses of many currently used screening methods, we propose adopting impedance microbiology to evaluate the EPS production from LAB in milk. In this work we have proven that the peculiar shape of capacitance curve of Lactobacillus delbrueckii subsp. bulgaricus 2214, measured in milk by means of a BacTrac 4300® system, is due to production of EPS. Besides the pH measurement, the amounts of EPS evaluated after 0, 8, 13 and 55 h of incubation in milk, were in agreement with the evaluation of gene expression and confirmed by the observations by confocal laser scanning microscopy and by transmission electron microscopy. With the aim to verify the applicability of the proposed method, the drop entity of the capacitance curve (ΔE%) of 22 EPS-producing (EPS+) LAB strains and one negative (EPS-) control was evaluated both in broth medium and in milk. The positive ΔE% value found for all of the strains cultivated in the clear broth medium allowed to confirm the EPS production, simply observing a strain-dependent amount of EPS on surface of the measurement electrodes of the device. When the same EPS+ strains were cultivated in milk, the obtained ΔE% values showed that only a few of them were able to produce EPS in this environment, supporting their diversified ability to utilize lactose for this purpose. Results obtained by this multidisciplinary study demonstrate that impedance microbiology represents a suitable method to overcome the limits of the most commonly used methods to screen LAB for EPS production in milk. Moreover, these results also open a door to the application to other food and beverages, in which the EPS produced in situ could be of great interest for food industry.

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.