Search

Growth-based impedometric analysis, applied to food microbiology, is a rapid method that enables the indirect tracing of microorganisms by measuring, during time, the changes of electrical conductivity in a matrix in which they grow. This method has been used over the years, to replace the conventional plate count method, to selectively detect the main food-borne diseases and spoilage bacteria. A new metabolic interpretation of the impedance data is the most promising application of this approach. In this review, the advantages of using this technique are discussed, together with the most interesting latest developments that involve the integration with biosensor technology. This method allows the detection of specific bacteria cells, based on their electrical properties, avoiding the culturing step and achieving results in a very short time.

Consumption of seaweeds (forms of algae), often categorized as a superfood, is becoming popular in western countries. Algae can be marketed fresh, but are usually sold dehydrated to ensure longer shelf life. Their consumption, often as ready-to-eat, opens up possible risks for public health because of foodborne pathogens that can contaminate the raw material during harvesting or manipulation. In this study, 14 ready-to-eat foods based on dehydrated algae, representative of the most consumed species, were considered. The microbial content, with a focus on Listeria monocytogenesand Bacillus cereus,was investigated by plate counts, and B. cereusstrains were isolated and identified by 16S rRNA gene sequencing. The microbiological quality was heterogeneous among the samples and, in particular, marine bacteria, Listeriaspp., B. cereus,and coliforms were detected. To contribute to related risk assessment, the ability of B. cereusto grow during refrigerated storage was evaluated, to our knowledge for the first time, by a microbiological challenge test on two ready-to-eat foods based on Undaria pinnatifidaand Palmaria palmata. Despite this study demonstrating the inability of B. cereusto proliferate in seaweed-based food, its presence in dehydrated foodstuffs cannot rule out replication after rehydration before consumption, making it necessary to elucidate the possible risks for consumers.

•Parmigiano Reggiano manufacturing involves a complex interplay of microbial species.•Raw cow’s milk and whey starter define the characteristics of microbial community.•The microbial populations are characterised by wide strain-level biodiversity.•Novel approaches have shed light on microbial dynamics during fermentation.•Cheesemaking process shapes microbiota composition.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37mutations. Modeling and functional analysis of CD37missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37mutations.

Sulforaphane is a natural product that is constantly under biological investigation for its unique biological properties. This naturally occurring isothiocyanate (ITC) and its analogs are the main components of cruciferous vegetables, such as cauliflower, watercress, broccoli, cabbage, Brussels sprouts, widely used as chemopreventive agents. Due to their interesting biological profiles, natural ITCs have been exploited as starting point to develop new synthetic analogs. The present mini-review briefly highlights the most important biological actions of selected new synthetic ITCs focusing on their structure-activity relationships and related synthetic strategies.

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1+ leukemias. Using CD34+ progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph+ B-ALL. Notably, XPO1 was also elevated in Ph− B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34+ progenitors, and increased survival of BCR-ABL1+ mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph+ leukemias.

Despite recent advances in treatment of B-acute lymphoblastic leukemia (B-ALL) there is still a need for novel targeted therapies. The tetraspanin CD9 is expressed in 60-80% of B-ALL and correlates with adverse prognosis. Recently, the mouse CD9 antibody ALB6 was shown to induce leukemia rejection in NOD/SCID mice. However, clinical development of ALB6 and other CD9-targeting antibodies was hampered by their CD9 mediated induction of platelet aggregation. It is known that CD9 is still expressed on tumor cells after treatment with chemotherapy or blinatumomab (Leung, 2019; Linder, 2016).

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)–activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase-dependent manner through the PI-3Kγ-PKC–induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2V617F also utilizes an alternative sphingosine kinase-1–mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET–mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2V617F MPNs.

A polyphasic approach was evaluated for the detection of eight staphylococcal enterotoxin (SE)-encoding genes (sea, sec, sed, seg, seh, sei, sej, sel)and the Escherichia coligenes most commonly associated with virulence factors (eae, elt, ipaH, stx)in traditional soft cheeses, manufactured artisanally from whole raw milk in the Lombardy region (northern Italy). To determine the presence of the target genes, two multiplex PCRs were performed on DNA extracted both directly from cheese samples (culture- independent approach) and from whole cultivable cells, formed by harvesting colonies from the first serial dilution agar plates of selective media, as representative of cultivable community (“bulk”). Genes associated with enteroinvasive E. coli, ipaH,and Shiga toxin-producing E. coli, stx,were detected in two of the bulk samples analyzed; no virulence genes were found by amplifying DNA directly extracted from cheeses. SE-encoding genes were found in three cheeses (seain all three samples, associated with sedand sejin two of these). More SE-encoding genes were detected by amplifying DNA obtained from bulk samples: sea, sed, sej, sec, seg, sel,and sei.No samples harbored the gene encoding for SE type H. The polyphasic approach followed has been useful in enhancing detection of target genes. Our results indicate that some of the artisanal cheeses examined may constitute a potential hazard for consumer health.

No Abstract

Licht bestimmt die Struktur: Eine photochemisch aktive Einheit macht die Selbstorganisation eines lipophilen Guanosinderivats mit Licht steuerbar: Die Bildung von G‐Quartetten lässt sich ein‐ und ausschalten (siehe Schema).

One hundred forty-one lactobacilli strains isolated from natural whey starter cultures and ripened Grana Padano and Parmigiano Reggiano cheeses were tested for their susceptibility to 13 antibiotics, in particular, penicillin G, ampicillin, amoxicillin, oxacillin, cephalotin, cefuroxime, vancomycin, gentamicin, tetracycline, erythromycin, clindamycin, co-trimoxazole, and nitrofurantoin. The strains belonged to the species Lactobacillus helveticus, L. delbrueckiisubsp. lactis, L. rhamnosus, and L. casei. The strains of the first two species were isolated from whey starter cultures, and the strains of the last two species were from the ripened cheeses. Significant differences among the strains in their antibiotic resistance were found in relation to the type of cheese and, especially, the strains from Parmigiano Reggiano were more resistant to gentamicin and penicillin G. The strains isolated in the ripened cheese were generally more resistant than those isolated from natural whey starter cultures; in particular, significant differences regarding oxacillin, vancomycin, cephalotin, and co-trimoxazole were observed. Finally, no significant difference in relation to the type of cheese was found among the thermophilic lactobacilli isolated from whey cultures, while the facultatively heterofermentative lactobacilli isolated from Parmigiano Reggiano showed higher resistance toward gentamicin and penicillin G than did the same species isolated from Grana Padano.

We showed that Eμ-MiR-155transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre–B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain–containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein β (C/EBPβ), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Shipand C/EBPβ, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABLtransformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3′ untranslated region (UTR). E2F3 levels were up-regulated in CML-BCCD34+ in a BCR/ABL kinase– and hnRNP-A1 shuttling–dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3−/− mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1–regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression.

The inability of myeloid chronic myelogenous leukemia blast crisis (CML-BC) progenitors to undergo neutrophil differentiation depends on suppression of C/EBPα expression through the translation inhibitory activity of the RNA-binding protein hnRNP-E2. Here we show that “oncogene dosage” is a determinant factor for suppression of differentiation in CML-BC. In fact, high levels of p210-BCR/ABL are required for enhanced hnRNP-E2 expression, which depends on phosphorylation of hnRNP-E2 serines 173, 189, and 272 and threonine 213 by the BCR/ABL-activated MAPKERK1/2. Serine/threonine to alanine substitution abolishes hnRNP-E2 phosphorylation and markedly decreases its stability in BCR/ABL-expressing myeloid precursors. Similarly, pharmacologic inhibition of MAPKERK1/2activity decreases hnRNP-E2 binding to the 5′UTR of C/EBPα mRNA by impairing hnRNP-E2 phosphorylation and stability. This, in turn, restores in vitro and/or in vivo C/EBPα expression and G-CSF–driven neutrophilic maturation of differentiation-arrested BCR/ABL+cell lines, primary CML-BCCD34+patient cells and lineage-negative mouse bone marrow cells expressing high levels of p210-BCR/ABL. Thus, increased BCR/ABL oncogenic tyrosine kinase activity is essential for suppression of myeloid differentiation of CML-BC progenitors as it is required for sustained activation of the MAPKERK1/2-hnRNP-E2-C/EBPα differentiation-inhibitory pathway. Furthermore, these findings suggest the inclusion of clinically relevant MAPK inhibitors in the therapy of CML-BC.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting class I histone deacetylase (HDAC)-containing repressor complex to the promoter of AML1 target genes. Valproic acid (VPA), a commonly used antiseizure and mood stabilizer drug, has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition. VPA causes selective proteasomal degradation of HDAC2 but not other class I HDACs (i.e., HDAC 1, 3, and 8). Therefore, we raised the question of whether this drug can effectively target the leukemogenic activity of the AML1/ETO fusion protein that also recruits HDAC1, a key regulator of normal and aberrant histone acetylation. We report here that VPA treatment disrupts the AML1/ETO-HDAC1 physical interaction, stimulates the global dissociation of AML1/ETO-HDAC1 complex from the promoter of AML1/ETO target genes, and induces relocation of both AML1/ETO and HDAC1 protein from nuclear to perinuclear region. Furthermore, we show that mechanistically these effects associate with a significant inhibition of HDAC activity, histone H3 and H4 hyperacetylation, and recruitment of RNA polymerase II, leading to transcriptional reactivation of target genes (i.e., IL-3) otherwise silenced by AML1/ETO fusion protein. Ultimately, these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis. Taken together, these data support the notion that VPA might effectively target AML1/ETO-driven leukemogenesis through disruption of aberrant HDAC1 function and that VPA should be integrated in novel therapeutic approaches for AML1/ETO-positive AML.

Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebpβ mRNA. Constitutive expression or conditional activation of wild-type CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because expression of CEBPB (and CEBPA) is low in the blast crisis (BC) stage of chronic myelogenous leukemia (CML) and is inversely correlated with BCR/ABL tyrosine kinase levels, these findings point to the therapeutic potential of restoring C/EBP activity in CML-BC and, perhaps, other types of acute leukemia.

Altered mRNA translation is one of the effects exerted by the BCR/ABL oncoprotein in the blast crisis phase of chronic myelogenous leukemia (CML). Here, we report that in BCR/ABL+ cell lines and in patient-derived CML blast crisis mononuclear and CD34+ cells, p210BCR/ABL increases expression and activity of the transcriptional-inducer and translational-regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K or HNRPK) in a dose- and kinase-dependent manner through the activation of the MAPKERK1/2 pathway. Furthermore, HNRPK down-regulation and interference with HNRPK translation-but not transcription-regulatory activity impairs cytokine-independent proliferation, clonogenic potential, and in vivo leukemogenic activity of BCR/ABL-expressing myeloid 32Dcl3 and/or primary CD34+ CML-BC patient cells. Mechanistically, we demonstrate that decreased internal ribosome entry site (IRES)-dependent Myc mRNA translation accounts for the phenotypic changes induced by inhibition of the BCR/ABL-ERK-dependent HNRPK translation-regulatory function. Accordingly, MYC protein but not mRNA levels are increased in the CD34+ fraction of patients with CML in accelerated and blastic phase but not in chronic phase CML patients and in the CD34+ fraction of marrow cells from healthy donors. Thus, BCR/ABL-dependent enhancement of HNRPK translation-regulation is important for BCR/ABL leukemogenesis and, perhaps, it might contribute to blast crisis transformation. (Blood. 2006;107:2507-2516)

Altered mRNA translation is one of the effects exerted by the BCR/ABL oncoprotein in the blast crisis phase of chronic myelogenous leukemia (CML). Here, we report that in BCR/ABL+cell lines and in patient-derived CML blast crisis mononuclear and CD34+cells, p210BCR/ABLincreases expression and activity of the transcriptional-inducer and translational-regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K or HNRPK) in a dose- and kinase-dependent manner through the activation of the MAPKERK1/2pathway. Furthermore, HNRPK down-regulation and interference with HNRPK translation-but not transcription-regulatory activity impairs cytokine-independent proliferation, clonogenic potential, and in vivo leukemogenic activity of BCR/ABL-expressing myeloid 32Dcl3 and/or primary CD34+CML-BC patient cells. Mechanistically, we demonstrate that decreased internal ribosome entry site (IRES)-dependent Myc mRNA translation accounts for the phenotypic changes induced by inhibition of the BCR/ABL-ERK-dependent HNRPK translation-regulatory function. Accordingly, MYC protein but not mRNA levels are increased in the CD34+fraction of patients with CML in accelerated and blastic phase but not in chronic phase CML patients and in the CD34+fraction of marrow cells from healthy donors. Thus, BCR/ABL-dependent enhancement of HNRPK translation-regulation is important for BCR/ABL leukemogenesis and, perhaps, it might contribute to blast crisis transformation. (Blood. 2006;107:2507-2516)

The presence of Listeria monocytogeneson the rind of Gorgonzola cheese is difficult to avoid. This contamination can easily occur as a consequence of handling during ripening. The aims of this study were to determine the efficiency of high-pressure processing (HPP) for inactivation of L. monocytogeneson cheese rind and to evaluate the influence of HPP treatments on sensory characteristics. Gorgonzola cheese rinds, after removal, were inoculated (about 7.0 log CFU/g) with L. monocytogenesstrains previously isolated from other Gorgonzola cheeses. The inoculated cheese rinds were processed with an HPP apparatus under conditions of pressure and time ranging from 400 to 700 MPa for 1 to 15 min. Pressures higher than 600 MPa for 10 min or 700 MPa for 5 min reduced L. monocytogenesmore than 99%. A reduction higher than 99.999% was achieved pressurizing cheese rinds at 700 MPa for 15 min. Lower pressure or time treatments were less effective and varied in effectiveness with the cheese sample. Changes in sensory properties possibly induced by the HPP were evaluated on four different Gorgonzola cheeses. A panel of 18 members judged the treated and untreated cheeses in a triangle test. Only one of the four pressurized cheeses was evaluated as different from the untreated sample. HPP was effective in the reduction of L. monocytogeneson Gorgonzola cheese rinds without significantly changing its sensory properties. High-pressure technology is a useful tool to improve the safety of this type of cheese.

The oligopeptide fractions of Grana Padano and Parmigiano Reggiano cheese samples, at various stages of ageing (6, 12, 18 and 24 months), were analysed by means of high-performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MS). The main oligopeptides (< 5000 Da) present in the samples were extracted by using an originally developed method which allowed good enrichment of the oligopeptide fractions and identified according to their molecular weights. The casein sequences compatible with the found molecular weights were determined and the exact oligopeptide sequences were identified by using the fragmentation peaks originated by in-source collisionally-induced dissociation (CID). The ESI-MS method reported here allowed the sequence identification of oligopeptides very quickly with a single liquid chromatography-mass spectrometry run. Oligopeptides were also semi-quantified by comparison with a suitable internal standard (the dipeptide phenylalanyl–phenylalanine, Phe–Phe). This methodology demonstrated that, as far as the main oligopeptides are concerned, Grana Padano and Parmigiano Reggiano presented very similar composition. Anyway, the evolution of the fractions during ageing time was characteristically distinct among the two cheeses: in Grana Padano cheeses most of the oligopeptides reached a maximum at 12 months of ageing and then decreased, whereas in Parmigiano Reggiano cheeses the oligopeptide amounts were usually lower and had a less regular trend. The reason for this different behaviour may be ascribed to the different production techniques.