Francesca Lambertini

Cheese peptidomics: A detailed study on the evolution of the oligopeptide fraction in Parmigiano-Reggiano cheese from curd to 24 months of aging

In this work, we performed a detailed evaluation of the evolution of the oligopeptide fractions in samples of Parmigiano-Reggiano cheese from the curd up to 24 mo of aging. The samples were taken from wheels produced the same day, in the same factory, from the same milk, during the same caseification process, thus simplifying the natural variability of a whey-based starter fermentation. This unique and homogeneous sampling plan, never reported before in the literature, provided a detailed study of the peptides produced by enzymatic events during Parmigiano-Reggiano aging. Given the large dimensions of the 35-kg wheels of Parmigiano-Reggiano, samples were taken from both the internal and external parts of the cheese, to evidence eventual differences in the oligopeptide composition of the different parts. Fifty-seven peptides were considered, being among the most abundant during at least one of the periods of ripening considered, and their semiquantification indicated that the peptide fraction of Parmigiano-Reggiano cheese constantly evolves during the aging period. Five trends in its evolution were outlined, which could be clearly correlated to the enzymatic activities present in the cheese, making it possible to discriminate cheeses according to their aging time. Several known bioactive peptides were also found to be present in Parmigiano-Reggiano cheese samples, and for the first time, the age at which they are most abundant has been identified. Aged cheeses have been shown to be dominated by nonproteolytic aminoacyl derivatives, a new class of peptide-like molecules recently reported. Finally, the changing peptide pattern may be related to the changing enzymatic activities occurring inside the cheeses during the aging period, which, in turn, are also related to the microbiological composition.

Tolerability of a Fully Maturated Cheese in Cow’s Milk Allergic Children: Biochemical, Immunochemical, and Clinical Aspects

Background From patients’ reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow’s milk (CM) allergic patients. Objective and Methods To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), β-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. Results The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly αS1-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. Conclusions 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.

Microbial origin of non proteolytic aminoacyl derivatives in long ripened cheeses

Cheese ripening involves a complex series of biochemical events that contribute to the development of each cheese characteristic taste, aroma and texture. Proteolysis, which has been the subject of active research in the last decade, is the most complex of these biochemical events. However, also aminoacyl derivates of non-proteolytic origin (γ-glutamyl-amino acids and lactoyl-amino acids) with interesting sensory properties have been identified in cheeses. In the present work, an enzymatic activity producing γ-glutamyl-phenylalanine in Parmigiano-Reggiano water soluble extracts was observed. It was hypothesized that γ-glutamyl-amino acids and lactoyl-amino acids could be originated by enzymes of bacterial origin. In order to confirm this hypothesis, Lactobacillus helveticus and Lactobacillus rhamnosus were chosen as representative of starter and non starter microbiota of Parmigiano Reggiano cheese. They were used as model bacteria, in the presence of suitable precursors, to verify their ability to produce γ-glutamyl-phenylalanine and lactoyl-phenylalanine. The eventual abilities of these strains were tested both during growth and after cell lyses. While γ-glutamyl-phenylalanine was produced only by lysed cells, lactoyl-phenylalanine was produced either by growing or lysed cells in different amount depending on the species, the cells condition and the time of incubation.

Migration of selected hydrocarbon contaminants into dry pasta packaged in direct contact with recycled paperboard.

This paper deals with the migration of selected hydrocarbon contaminants, namely mineral oil hydrocarbons (MOH), diisopropyl naphthalenes (DIPN) and polyalphaolefins (PAO) from adhesives into dry semolina and egg pasta packaged in direct contact with recycled paperboard. Migration was monitored during its shelf life (for up to two years) simulating storage in a supermarket (packs on shelves) and conditions preventing exchange with the surrounding environment (packs wrapped in aluminium foil). Migration from the secondary packaging (transport boxes of corrugated board) was also studied for semolina pasta. After 24 months of exposure, semolina pasta stored on shelves reached 3.2 and 0.6 mg kg−1 of MOSH and MOAH, respectively, Migration from the adhesives used to close the boxes and from the transport boxes contributed about 30% and 25% of the total contamination, respectively. The highest contamination levels (14.5 and 2.0 mg kg−1 of MOSH and MOAH, respectively, after 24 months) were found in egg pasta stored on shelves (no adhesives), and seemed due to the highest contribution from the external environment. Graphical Abstract

Antioxidant capacity of water soluble extracts from Parmigiano-Reggiano cheese.

Abstract In this work the antioxidant capacity of water soluble extracts of Parmigiano-Reggiano cheese (Water Soluble Extracts – WSEs) at different aging time was studied, by measuring their radical scavenging capacity with a standard ABTS assay. The WSEs were also fractionated by semi-preparative HPLC-UV and for each fraction the antioxidant capacity and the molecular composition was determined by LC/ESI-MS, in order to identify the most active antioxidant compounds. The antioxidant capacity was also determined after simulated in vitro gastrointestinal digestion of WSEs. The data indicated that antioxidant capacity in WSE from Parmigiano-Reggiano cheese, quite unaffected by ripening time and gastrointestinal digestion, is mostly due to free amino acids, mainly tyrosine, methionine and tryptophan, and only in minimal part to antioxidant peptides.

Migration of selected hydrocarbon contaminants into dry semolina and egg pasta packed in direct contact with virgin paperboard and polypropylene film

Migration of mineral oil saturated hydrocarbons (MOSH), polyolefin oligomeric saturated hydrocarbons (POSH), and polyalphaolefins (PAO from hot melts) into dry semolina and egg pasta packed in direct contact with virgin paperboard or polypropylene (PP) flexible film was studied. Migration was monitored during shelf life (up to 24 months), through storage in a real supermarket (packs kept on shelves), conditions preventing exchange with the surrounding environment (packs wrapped in aluminium foil), and storage in a warehouse (packs inside of the transport box of corrugated board). Semolina pasta packed in virgin paperboard (without hot melts) had a MOSH content lower than 1.0 mg kg−1. An increasing contamination with PAO belonging to the adhesives used to close the boxes was detected in egg pasta, wrapped in aluminium (1.5 and 5 mg kg−1 after 3 and 24 months, respectively). An environmental contribution to total hydrocarbon contamination was observed in egg pasta kept on shelves that, after 3 and 24 months, showed levels of PAO/MOSH < C25 around 3 and 10 mg kg−1, respectively. The migration of POSH from PP film into egg pasta wrapped in aluminium was around 0.6 mg kg−1 after 3 months of contact and reached 1.7 mg kg−1 after 24 months of contact. After 9 months of contact, semolina pasta packed in PP film and stored in the transport box showed that some MOSH migrated into the pasta from the board of the transport box (through the plastic film). Graphical Abstract

Identification and quantification of different species in animal fibres by LC/ESI‐MS analysis of keratin‐derived proteolytic peptides

In the present paper, a proteomic method for species determination in fibres has been developed. Keratin was extracted from yak, wool and cashmere fibres and digested by trypsin, providing peptide mixtures that were analyzed by liquid chromatography coupled with electrospray mass spectrometry (LC/ESI-MS) in order to identify peptidic species-specific markers able to differentiate the fibres. Several suitable peptide markers were identified and validated in different fibres of different origin and having undergone different technological treatments, showing 100% specificity and 100% selectivity. Most of the peptide markers were also identified by means of high-resolution mass spectrometry, confirming the origin from species-specific keratin sequences. Some peptides were also used for the quantification of the different species in mixed fibres by LC/ESI-MS. Validation experiments and blind tests confirmed their ability to act as very specific quantitative and qualitative markers. The method here developed is a valid complement to the standard benchmark methods for fibre identification and quantification and will be very useful for assessing the authenticity of textile products.

Reliable liquid chromatography-mass spectrometry method for investigation of primary aromatic amines migration from food packaging and during industrial curing of multilayer plastic laminates.

Primary aromatic amines (PAAs) can migrate from packaging into food from different sources such as polyurethanic adhesives used for the manufacture of multilayer films, which may contain residual aromatic isocyanates, or recycled paperboard, because of the presence of azo dyes in the printed paper massively used in the recycling process. In the present work, a reliable analytical method, exploiting a conventional high-performance liquid chromatography-(selected ion monitoring)-mass spectrometry system, for PAAs compliance assessment in food contact materials was developed as an effective alternative to the current standard spectrophotometric one, moving in this way from the screening to the accurate and selective quantitation perspective for the analysis of PAAs both in aqueous and acidic food simulants. The main validation parameters were verified achieving very satisfactory results in terms of linearity range, limit of detection (ranging from 0.1 to 1.0 µg kg(-1)) and quantitation (ranging from 0.1 to 3.6 µg kg(-1)), repeatability and accuracy. Suitability of the method was demonstrated for a wide range of commercial samples, chosen among different producers of the most common used food packaging plastic and paperboard categories and then analyzed to assess the risk related to PAAs migration. Finally, the method was also successfully exploited to monitor the evolution of potential PAAs migration during the industrial curing process of multilayer plastic laminates, prior to their release for delivery to the food industry end user.