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Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese.

ABSTRACT: In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca²⁺ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1' subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.

SUBMITTER: Solieri L

PROVIDER: S-EPMC8773131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Peptide Profiling and Biological Activities of 12-Month Ripened Parmigiano Reggiano Cheese.

Project description:Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than samples having low-salt and low-fat content (LL). This positive correlation between salt/fat reduction and proteolysis was also confirmed by the analysis of biological activities, as the LL group showed higher average values of angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. UHPLC/HR-MS allowed the identification of 805 unique peptides: LL and HH groups shared 59.3% of these peptides, while 20.9% and 19.9% were LL and HH specific, respectively. Frequency analysis of peptides identified a core of 183 peptides typical of 12-month ripened PR cheeses (corresponding to the 22.7% of total peptides), but no significant differences were detected in peptide patterns between LL and HH groups. Forty bioactive peptides, including 18 ACE-inhibitors and 12 anti-microbial peptides, were identified, of which 25 firstly found in PR cheese. Globally, this work contributed to unraveling the potentially healthy benefits of peptides fraction in PR cheese and provided prior evidence that PR with reduced fat/salt content showed the highest antihypertensive and antioxidant activities.

| S-EPMC7408421 | biostudies-literature

Dynamics of whole and lysed bacterial cells during Parmigiano-Reggiano cheese production and ripening.

Project description:Microbial succession during Parmigiano-Reggiano cheesemaking was monitored by length heterogeneity PCR (LH-PCR), considering the intact and lysed cells at different stages of cheese production and ripening. When starter species underwent autolysis, species coming from milk were able to grow. For the first time, the LH-PCR technique was applied to study a fermented food.

| S-EPMC2565984 | biostudies-literature

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

Project description: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.

| S-EPMC7143483 | biostudies-literature

Characterization of Yeasts Isolated from Parmigiano Reggiano Cheese Natural Whey Starter: From Spoilage Agents to Potential Cell Factories for Whey Valorization.

Project description:Whey is the main byproduct of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast population inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48 °C. In whey fermentation trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

| S-EPMC8623691 | biostudies-literature

The Interrelationship Between Microbiota and Peptides During Ripening as a Driver for Parmigiano Reggiano Cheese Quality.

Project description:Cheese microbiota contribute significantly to the final characteristics of cheeses due to the growth and interaction between cheese microorganisms during processing and ripening. For raw milk cheeses, such as Parmigiano Reggiano (PR), the microbiota derive from the raw milk itself, the dairy environment, and the starter. The process of cheese making and time of ripening shape this complex ecosystem through the selection of different species and biotypes that will drive the quality of the final product by performing functions of their metabolism such as proteolysis. The diversity in the final peptide and amino acid composition of the cheese is thus mostly linked to the diversity of this microbiota. The purpose of this study was to get more insight into the factors affecting PR cheese diversity and, more specifically, to evaluate whether the composition of the bacterial community of cheeses along with the specific peptide composition are more affected by the ripening times or by the cheese making process. To this end, the microbiota and the peptide fractions of 69 cheese samples (from curd to cheese ripened 24 months) were analyzed during 6 complete PR production cycles, which were performed in six different dairies located in the PR production area. The relation among microbial dynamics, peptide evolution, and ripening times were investigated in this unique and tightly controlled production and sampling set up. The study of microbial and peptide moieties in products from different dairies - from curd to at least 12 months, the earliest time from which the cheese can be sold, and up to a maximum of 24 months of ripening - highlighted the presence of differences between samples coming from different dairies, probably due to small differences in the cheese making process. Besides these differences, however, ripening time had by far the greatest impact on microbial dynamics and, consequently, on peptide composition.

| S-EPMC7561718 | biostudies-literature

Peptide profile of Parmigiano Reggiano cheese after simulated gastrointestinal digestion: From quality drivers to functional compounds.

Project description:Time of ripening has a strong impact on shaping the valuable and recognizable characteristics of long-ripened types of cheese such as Parmigiano Reggiano (PR) due to the interrelationship between microbiota and proteolysis that occurs during ripening. The derived peptide profile is linked to cheese quality and represents the canvas for enzymes upon digestion, which could be responsible for the release of potentially bioactive peptides (BPs). In this study, we aimed at investigating the presence of BP in 72 PR cheese samples of different ripening times, from curd to 24 months of ripening, produced in six different dairies, and following their fate after simulated gastrointestinal digestion. A small number of peptide sequences sharing 100% similarity with known antimicrobial, antioxidant, and ACE-inhibitor sequences were found in PR cheeses, while a higher number of potential BPs were found after their simulated gastrointestinal digestion, in different amounts according to ripening time. Taking advantage of the complex organization of the sampling plan, we were able to follow the fate of peptides considered quality drivers during cheese ripening to their release as functional compounds upon digestion.

| S-EPMC9445588 | biostudies-literature

Quantification of Cheese Yield Reduction in Manufacturing Parmigiano Reggiano from Milk with Non-Compliant Somatic Cells Count.

Project description:The mammary gland inflammation process is responsible for an increased number of somatic cells in milk, and transfers into the milk of some blood components; this causes alterations in the chemical composition and physico-chemical properties of milk. For this reason, somatic cell count (SCC) is one of the most important parameters of milk quality; therefore, European Union (EU) Regulation no 853/2004 has stated that it must not exceed the limit value of 400,000 cells/mL. The research aimed to compare chemical composition, cheese yield, and cheesemaking losses of two groups of vat milks used for Parmigiano Reggiano production, characterized by different SCC levels. During two years, ten cheesemaking trials were performed in ten different cheese factories. In each trial, two cheesemaking processes were conducted in parallel: one with low SCC milk (below 400,000 cells/mL; Low Cell Count (LCC)) and the other with high SCC milk (400,000-1,000,000 cells/mL; High Cell Count (HCC)). For each trial, vat milk and cooked whey were analyzed; after 24 months of ripening, cheeses were weighed to calculate cheese yield. The HCC group had lower casein content (2.43 vs. 2.57 g/100 g; p ? 0.05) and number (77.03% vs. 77.80%; p ? 0.05), lower phosphorus (88.37 vs. 92.46 mg/100g; p ? 0.05) and titratable acidity (3.16 vs. 3.34 °SH/50 mL; p ? 0.05) compared to LCC. However, chloride (111.88 vs. 104.12 mg/100 g; p ? 0.05) and pH (6.77 vs. 6.71; p ? 0.05) were higher. Fat losses during cheesemaking were higher (20.16 vs. 16.13%). After 24 months of ripening, cheese yield was 8.79% lower for HCC milk than LCC (6.74 vs. 7.39 kg/100 kg; p ? 0.05).

| S-EPMC7074290 | biostudies-literature

An Integrated Peptidomics and In Silico Approach to Identify Novel Anti-Diabetic Peptides in Parmigiano-Reggiano Cheese.

Project description:Inhibition of key metabolic enzymes linked to type-2-diabetes (T2D) by food-derived compounds is a preventive emerging strategy in the management of T2D. Here, the impact of Parmigiano-Reggiano (PR) cheese peptide fractions, at four different ripening times (12, 18, 24, and 30 months), on the enzymatic activity of α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPP-IV) as well as on the formation of fluorescent advanced glycation end-products (fAGEs) was assessed. The PR peptide fractions were able to inhibit the selected enzymes and fAGEs formation. The 12-month-ripening PR sample was the most active against the three enzymes and fAGEs. Mass spectrometry analysis enabled the identification of 415 unique peptides, 54.9% of them common to the four PR samples. Forty-nine previously identified bioactive peptides were found, mostly characterized as angiotensin-converting enzyme-inhibitors. The application of an integrated approach that combined peptidomics, in silico analysis, and a structure-activity relationship led to an efficient selection of 6 peptides with potential DPP-IV and α-glucosidase inhibitory activities. Peptide APFPE was identified as a potent novel DPP-IV inhibitor (IC50 = 49.5 ± 0.5 μmol/L). In addition, the well-known anti-hypertensive tripeptide, IPP, was the only one able to inhibit the three digestive enzymes, highlighting its possible new and pivotal role in diabetes management.

| S-EPMC8234620 | biostudies-literature

Reduction in the Brining Time in Parmigiano Reggiano Cheese Production Minimally Affects Proteolysis, with No Effect on Sensory Properties.

Project description:Brine soaking is one of the most important steps in the production of Parmigiano Reggiano cheese, since it determines the amount of salt in the final product. Reduction in salt in Parmigiano Reggiano cheese might be important for improving its nutritional profile, but it could affect the manufacturing processes by altering proteolysis and consequently the product quality. In this study, for the first time, salt reduction was explored at the industrial level on real cheese samples manufactured in a local dairy. In particular, 20 wheels were produced with conventional (18 days, 10 wheels) and shorter (12 days, 10 wheels) brining steps. In every group, wheels were studied at two different ripening times, 15 and 30 months. A shorter brining time resulted in an average 12% decrease in salt content. A full characterization of free amino acids and peptides was performed by LC-MS on all samples. Free amino acids and peptides, as expected, increased with ripening, due to proteolysis, with samples having low salt content showing a slightly faster increase when compared to standard ones, hinting to a slightly accelerated proteolytic process. Nonetheless, low-salt and conventional cheeses shared similar sensory profiles at both ripening times.

| S-EPMC8066690 | biostudies-literature

Validation of Parmigiano Reggiano Cheese Aroma Authenticity, Categorized through the Use of an Array of Semiconductors Nanowire Device (S3).

Project description:Parmigiano Reggiano (PR) cheese is one of the most important Italian Protected Designation of Origin (PDO) cheeses and it is exported worldwide. As a PDO, the product is supposed to have distinctive sensory characteristics. In this work we present the use of the Small Gas Sensor System (S3) device for the identification of specific PR markers, as compared to classical chemical techniques, such as Gas chromatography-mass spectrometry solid-phase microextraction (SPME-GC-MS). Markers are used to determine the percent of grated pulp and rind commercially utilized. The S3 device is equipped with an array of six metal oxide semiconductor (MOX) gas sensors, three of them with a nanowire (NW) morphology and the other three in the form of thin films. PDO can cover grated PR cheese as well, but only if made with whole cheese. Grated PR cheese must be characterized by the absence of additives and no more than 18% crust. The achieved results strongly encourage the use of S3 for a rapid identification of the percentage of grated PR.

| S-EPMC5456476 | biostudies-other

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