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Impact of a Shorter Brine Soaking Time on Nutrient Bioaccessibility and Peptide Formation in 30-Months-Ripened Parmigiano Reggiano Cheese

Authors:

Abstract

Reducing the salt content in food is an important nutritional strategy for decreasing the risk of diet-related diseases. This strategy is particularly effective when applied to highly appreciated food having good nutritional characteristics, if it does not impact either upon sensory or nutritional properties of the final product. This work aimed at evaluating if the reduction of salt content by decreasing the brine soaking time modifies fatty acid and protein bioaccessibility and bioactive peptide formation in a 30-month-ripened Parmigiano Reggiano cheese (PRC). Hence, conventional and hyposodic PRC underwent in vitro static gastrointestinal digestion, and fatty acid and protein bioaccessibility were assessed. The release of peptide sequences during digestion was followed by LC–HRMS, and bioactive peptides were identified using a bioinformatic approach. At the end of digestion, fatty acid and protein bioaccessibility were similar in conventional and hyposodic PRC, but most of the bioactive peptides, mainly the ACE-inhibitors, were present in higher concentrations in the low-salt cheese. Considering that the sensory profiles were already evaluated as remarkably similar in conventional and hyposodic PRC, our results confirmed that shortening brine soaking time represents a promising strategy to reduce salt content in PRC.
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Citation: Di Nunzio, M.; Loffi, C.;
Chiarello, E.; Dellafiora, L.; Picone,
G.; Antonelli, G.; Di Gregorio, C.;
Capozzi, F.; Tedeschi, T.; Galaverna,
G.; et al. Impact of a Shorter Brine
Soaking Time on Nutrient
Bioaccessibility and Peptide
Formation in 30-Months-Ripened
Parmigiano Reggiano Cheese.
Molecules 2022,27, 664. https://
doi.org/10.3390/molecules27030664
Academic Editors: Francesco Bonomi,
Stefania Iametti and
Pasquale Ferranti
Received: 6 December 2021
Accepted: 18 January 2022
Published: 20 January 2022
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molecules
Article
Impact of a Shorter Brine Soaking Time on Nutrient
Bioaccessibility and Peptide Formation in 30-Months-Ripened
Parmigiano Reggiano Cheese
Mattia Di Nunzio 1, *,† , Cecilia Loffi 2,† , Elena Chiarello 3, Luca Dellafiora 2, Gianfranco Picone 3,
Giorgia Antonelli 3, Clarissa Di Gregorio 4, Francesco Capozzi 3,4 , Tullia Tedeschi 2, Gianni Galaverna 2
and Alessandra Bordoni 3,4
1Department of Food, Environmental and Nutritional Sciences (Defens), University of Milan, Via Celoria 2,
20133 Milan, Italy
2Department of Food and Drugs, University of Parma, Parco Area delle Scienze 95/A, 43124 Parma, Italy;
cecilia.loffi@unipr.it (C.L.); luca.dellafiora@unipr.it (L.D.); tullia.tedeschi@unipr.it (T.T.);
gianni.galaverna@unipr.it (G.G.)
3Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Piazza Goidanich 60,
47521 Cesena, Italy; elena.chiarello2@unibo.it (E.C.); gianfranco.picone@unibo.it (G.P.);
giorgia.antonelli4@unibo.it (G.A.); francesco.capozzi@unibo.it (F.C.); alessandra.bordoni@unibo.it (A.B.)
4Interdepartmental Centre for Industrial Agri-Food Research (CIRI), University of Bologna,
Piazza Goidanich 60, 47521 Cesena, Italy; clarissadigregorio@gmail.com
*Correspondence: mattia.dinunzio@unimi.it; Tel.: +39-02-5031-6819
These authors contributed equally to this work.
Abstract:
Reducing the salt content in food is an important nutritional strategy for decreasing the
risk of diet-related diseases. This strategy is particularly effective when applied to highly appreciated
food having good nutritional characteristics, if it does not impact either upon sensory or nutritional
properties of the final product. This work aimed at evaluating if the reduction of salt content by
decreasing the brine soaking time modifies fatty acid and protein bioaccessibility and bioactive
peptide formation in a 30-month-ripened Parmigiano Reggiano cheese (PRC). Hence, conventional
and hyposodic PRC underwent
in vitro
static gastrointestinal digestion, and fatty acid and protein
bioaccessibility were assessed. The release of peptide sequences during digestion was followed by
LC–HRMS, and bioactive peptides were identified using a bioinformatic approach. At the end of
digestion, fatty acid and protein bioaccessibility were similar in conventional and hyposodic PRC,
but most of the bioactive peptides, mainly the ACE-inhibitors, were present in higher concentrations
in the low-salt cheese. Considering that the sensory profiles were already evaluated as remarkably
similar in conventional and hyposodic PRC, our results confirmed that shortening brine soaking time
represents a promising strategy to reduce salt content in PRC.
Keywords: Parmigiano Reggiano cheese; in vitro digestion; bioaccessibility; bioactive peptides
1. Introduction
High salt intake is a key contributing factor for the prevalence of non-communicable
diseases (NCDs) around the world. High-salt diets are linked to elevated blood pressure, a
major risk factor for heart diseases and stroke, which in turn are among the leading causes
of death worldwide [
1
,
2
]. Despite the current WHO recommendations for sodium (Na)
consumption by adults (<2 g per day), in Europe the overall sodium intake varies between
2.7 and 7.1 g per day (7 to 18 g salt per day) in most countries [
3
], and processed foods
might provide about 20% of the total Na intake [
4
]. Since salt reduction has been identified
as one of the five priority interventions in response to the global NCD crisis [
5
], there is a
growing interest in processing procedures lowering salt content, particularly when applied
to highly appreciated and nutritional valued foods.
Molecules 2022,27, 664. https://doi.org/10.3390/molecules27030664 https://www.mdpi.com/journal/molecules
Molecules 2022,27, 664 2 of 15
Parmigiano Reggiano cheese (PRC) is a long ripened, hard cheese made up of a com-
bination of partially skimmed and whole raw milk, with the addition of a natural whey
starter, principally consisting of thermophilic starting lactic acid bacteria [
6
]. The PRC is
included in the list of foods with a Protected Designation of Origin (PDO), according to
which the aging must last at least 12 months, starting from the forming of the cheese. The
most valuable cheeses are those that ripen from 24 to 36 months, during which hydrolytic
phenomena occur, and the organoleptic quality improves. EU regulation 1151/2012 ensures
the method of production, quality, and area of origin of PDO products. In particular,
PRC must be produced and transformed only in the Northern Italian provinces of Parma,
Reggio Emilia, Modena, and in some parts of Bologna and Mantova. PRC is highly ap-
preciated worldwide not only for its taste, but also for its nutritional characteristics [
7
].
PRC contains high-value nutritional proteins that are also functionally and biologically
attractive, and their hydrolysates formed during ripening are supposed to be sources of
bioactive peptides [
8
,
9
]. Recently, we investigated the impact of reducing the brining time
in salt solution on PRC sensory properties and peptide content [
10
], but information is
still lacking about possible modification of the bioaccessibility of protein and fatty acids,
and on the release of biologically active peptides during digestion. Processing can deeply
modify both nutrient content and bioaccessibility, i.e., the quantity of a compound that is
released from the food matrix in the gastrointestinal tract, becoming available for intestinal
absorption [
11
]. Modification in bioaccessibility due to changes in the supramolecular orga-
nization and in the network of interactions between molecules, or to nutrient localization
within compartments may impact the nutritional value of food [
12
]. Indeed, in a previous
work we reported the impact of different ripening time (15 and 30 months) on the kinetics
of protein hydrolysis and the formation of peptides and small organic compounds during
PRC in vitro digestion [13].
The aim of the present work was to evaluate whether the reduction of brine soaking
time modifies fatty acid and protein bioaccessibility of a low-salt PRC produced and ripened
for 30 months according to the PDO specification. To this aim, conventional (C-PRC) and
hyposodic (Hypo-PRC) underwent
in vitro
static gastrointestinal digestion according to
the INFOGEST protocol [
14
]. Sampling was performed at the end of the gastric phase, in
the middle and at the end of the duodenal phase, and fatty acid and protein bioaccessibility
was assessed. Digested samples were also analyzed by nuclear magnetic resonance (NMR)
and liquid chromatography coupled with high resolution mass spectrometry (LC–HRMS),
and the release of bioactive peptide sequences was verified using a bioinformatic approach.
2. Results
2.1. Fatty Acid Bioaccessibility
In agreement with Malacarne et al. [
15
], the main fatty acids found in undigested
PRC were palmitic acid > oleic acid > myristic acid > stearic acid, which accounted for
approximately 85% of total fatty acid with no differences between C- and Hypo-PRC
(Supplementary Table S1). The release of fatty acids from the food matrix increased
over time, and no differences were detected between C-PRC and Hypo-PRC at the end
of digestion. However, the time course of fatty acid release was not the same in the
two samples,
bioaccessibility of myristic, stearic, oleic, total MUFA, and total fatty acids
being higher in Hypo-PRC at D60 (Table 1).
2.2. Protein Bioaccessibility
Protein bioaccessibility was evaluated by three different spectrophotometric methods
(OPA, Coomassie, absorbance at 280 nm), all evidencing a time-dependent release of
protein/peptides/amino acids from the matrix. Regardless of the analytical methodology,
no differences were detected between C- and Hypo-PRC at any digestion time, and maximal
bioaccessibility was already achieved at D60 (Figure 1).
Molecules 2022,27, 664 3 of 15
Molecules 2022, 27, x FOR PEER REVIEW 4 of 17
Figure 1. Protein bioaccessibility assessed by OPA (A) and Coomassie assay (B) and absorbance at
280 nm (C) in conventional Parmigiano Reggiano cheese (C-PRC) and hyposodic Parmigiano Reg-
giano cheese (Hypo-PRC) at different digestion times. Data are means ± SD of 3 independent in vitro
digestions, each analyzed in triplicate. Protein bioaccessibility is expressed as % and it was calcu-
lated as the ratio × 100 between the protein mass in the digestion fluid and the total protein mass in
the original undigested PRC. Statistical analysis was by the one-way ANOVA (A and C: p < 0.0001;
B: p < 0.005) with Tukey’s post-hoc test (different letters indicate significant differences). G120: end
of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase.
Figure 1.
Protein bioaccessibility assessed by OPA (
A
) and Coomassie assay (
B
) and absorbance
at 280 nm (
C
) in conventional Parmigiano Reggiano cheese (C-PRC) and hyposodic Parmigiano
Reggiano cheese (Hypo-PRC) at different digestion times. Data are means
±
SD of 3 independent
in vitro
digestions, each analyzed in triplicate. Protein bioaccessibility is expressed as % and it was
calculated as the ratio
×
100 between the protein mass in the digestion fluid and the total protein
mass in the original undigested PRC. Statistical analysis was by the one-way ANOVA (A and C:
p< 0.0001; B: p< 0.005) with Tukey’s post-hoc test (different letters indicate significant differences).
G120: end of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase.
2.3. HR-NMR Spectroscopy
In Figure 2, the NMR spectrum of the digestion fluids after the three digestion phases
is shown as traces with different colors, subdivided in three different spectral regions,
collecting signals from hydrogen atoms located on aromatic, alpha-carbon, and aliphatic
side chains of amino acids, respectively.
Molecules 2022,27, 664 4 of 15
More in detail, the progression of
in vitro
digestion of the two PRC evaluated by the
appearance of NMR signal in branched, aromatic, and
α
-amino acid proton spectral regions
is reported in Figure 3. The
α
-amino acid proton region includes the signals of all amino
acids, both in the free state and bound to peptides or in soluble proteins. In each spectral
region, the maximum release was already achieved at D60 with no significant difference
between C- and Hypo-PRC except for branched amino acids, the release of which was
higher in Hypo- than C-PRC at the end of the gastric digestion.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 17
2.3. HR-NMR Spectroscopy
In Figure 2, the NMR spectrum of the digestion fluids after the three digestion phases
is shown as traces with different colors, subdivided in three different spectral regions,
collecting signals from hydrogen atoms located on aromatic, alpha-carbon, and aliphatic
side chains of amino acids, respectively.
Figure 2. Proton HR-NMR spectrum of PRC digestion fluids at G120 (brown trace), D60 (green), and
D120 (blue) phases. The (top panel) shows the upfield region, where mainly aliphatic hydrogen
atoms of branched side chains of amino acids resonate. The (middle panel) shows the midfield
spectral region, where hydrogen atoms bound to amino acids alpha-carbon resonate. Finally, the
(bottom panel) shows the downfield spectral region, where hydrogen atoms belonging to aromatic
amino acids resonate.
Figure 2.
Proton HR-NMR spectrum of PRC digestion fluids at G120 (brown trace), D60 (green), and
D120 (blue) phases. The (
top panel
) shows the upfield region, where mainly aliphatic hydrogen
atoms of branched side chains of amino acids resonate. The (
middle panel
) shows the midfield
spectral region, where hydrogen atoms bound to amino acids alpha-carbon resonate. Finally, the
(
bottom panel
) shows the downfield spectral region, where hydrogen atoms belonging to aromatic
amino acids resonate.
Molecules 2022,27, 664 5 of 15
Molecules 2022, 27, x FOR PEER REVIEW 6 of 17
More in detail, the progression of in vitro digestion of the two PRC evaluated by the
appearance of NMR signal in branched, aromatic, and α-amino acid proton spectral re-
gions is reported in Figure 3. The α-amino acid proton region includes the signals of all
amino acids, both in the free state and bound to peptides or in soluble proteins. In each
spectral region, the maximum release was already achieved at D60 with no significant
difference between C- and Hypo-PRC except for branched amino acids, the release of
which was higher in Hypo- than C-PRC at the end of the gastric digestion.
Figure 3. Integral area of branched (A), aromatic (B), and α-proton (C) amino acid region in conven-
tional Parmigiano Reggiano cheese (C-PRC) and hyposodic Parmigiano Reggiano cheese (Hypo-
PRC) at different digestion times. Data are means ± SD of 3 independent in vitro digestions, each
one in triplicate. Integrals are expressed as arbitrary units. Statistical analysis was by the one-way
Figure 3.
Integral area of branched (
A
), aromatic (
B
), and
α
-proton (
C
) amino acid region in conven-
tional Parmigiano Reggiano cheese (C-PRC) and hyposodic Parmigiano Reggiano cheese (Hypo-PRC)
at different digestion times. Data are means
±
SD of 3 independent
in vitro
digestions, each one in
triplicate. Integrals are expressed as arbitrary units. Statistical analysis was by the one-way ANOVA
(all integrals p< 0.0001) with Tukey’s post-hoc test (different letters indicate significant differences).
G120: end of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase.
2.4. Peptide Formation
The total number of peptides generated at different digestion times from parent
proteins was evaluated by LC–HRMS (Table 2). On average, fewer than 80 peptides were
found in not-digested PRC, and
in vitro
digestion increased their number to >85 at G120 and
>100 at D60 and D120. In both C- and Hypo-PRC, most of the peptides were from
β
-casein
(60%) and
α
S1-casein (20%), whereas less than 10% of the identified sequences came from
α
S2-casein and
κ
-casein. The number of peptides from
β
-casein increased mainly during
Molecules 2022,27, 664 6 of 15
the duodenal phase, while those from
κ
-casein were mainly released during the gastric
phase. Regarding whey proteins, a small number of peptides from
β
-lactoglobulin was
released during the duodenal digestion. Overall, no significant differences were detected
between C- and Hypo-PRC.
Table 1.
Fatty acid bioaccessibility in conventional Parmigiano Reggiano cheese (C-PRC) and hy-
posodic Parmigiano Reggiano cheese (Hypo-PRC) at different digestion times.
G120 D60 D120
C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC
C8:0 1.53 ±0.58 b 1.87 ±0.12 b 11.70 ±3.73 ab 1.08 ±1.53 b 16.32 ±6.09 a 16.69 ±1.82 a
C10:0 1.24 ±0.34 c 2.05 ±0.16 c 22.18 ±3.55 ab 16.76 ±6.50 b 31.36 ±4.48 a 32.10 ±3.63 a
C12:0 1.55 ±0.17 c 2.51 ±0.11 c 27.32 ±4.78 b 35.60 ±3.62 ab 42.51 ±4.30 a 38.21 ±1.20 a
C14:0 1.48 ±0.23 c 3.00 ±0.05 c 26.30 ±4.92 b 45.17 ±1.24 a 43.99 ±9.63 ab 34.19 ±5.46 ab
C16:0 1.52 ±0.38 b 2.89 ±0.02 b 22.32 ±4.81 ab 43.83 ±5.05 a 39.80 ±11.00 a 28.24 ±4.79 a
C16:1 n-7 0.00 ±0.00 c 1.32 ±1.86 c 33.92 ±5.47 b 49.02 ±3.33 ab 53.47 ±9.05 a 44.45 ±4.37 ab
C18:0 1.84 ±0.37 c 2.88 ±0.08 c 20.68 ±5.26 bc 43.23 ±5.89 a 37.24 ±10.12 a 25.30 ±5.6 ab
C18:1 n-9 0.96 ±0.74 c 2.03 ±0.15 c 27.99 ±6.26 b 45.29 ±6.01 a 47.79 ±5.10 a 47.86 ±3.24 a
C18:2 n-6 0.53 ±0.92 c 1.07 ±1.52 c 34.01 ±5.20 b 47.58 ±7.64 ab 56.43 ±9.48 a 48.29 ±4.37 ab
ΣSFA 1.47 ±0.30 b 2.69 ±0.05 b 21.72 ±4.69 a 39.12 ±2.00 a 37.58 ±8.64 a 27.95 ±4.27 a
ΣMUFA 0.88 ±0.68 c 1.98 ±0.00 c 28.40 ±6.24 b 45.60 ±5.76 a 48.22 ±5.38 a 47.60 ±2.69 a
ΣPUFA 0.53 ±0.92 c 1.07 ±1.52 c 34.01 ±5.20 b 47.58 ±7.64 ab 56.43 ±9.48 a 48.29 ±4.37 ab
Total 1.28 ±0.40 c 2.45 ±0.07 c 23.95 ±5.13 b 41.13 ±3.15 a 40.97 ±7.59 a 33.90 ±2.48 ab
Data are means
±
SD of three independent
in vitro
digestions, each one in duplicate. Fatty acid bioaccessibility is
expressed as % and was calculated as fatty acid methyl ester concentration in digested/fatty acid methyl ester
concentration in PRC before digestion
×
100. Statistical analysis was by one-way ANOVA (C8:0: p= 0.0017; C16:0:
p= 0.0002; C10:0, C12:0, C14:0, C16:1 n-7, C18:0, C18:1 n-9, C18:2 n-6,
Σ
SFA,
Σ
MUFA,
Σ
PUFA and total fatty acids:
p< 0.0001) with Tukey’s post-hoc test (different letters indicate significant differences). G120: end of gastric phase;
D60: 60 min of duodenal phase; D120: end of duodenal phase.
2.5. Bioinformatic Analysis
Bioactive sequences detected in not digested PRC and at different time points of
in vitro
digestion, their semiquantitative content, and their putative biological activity
are reported in Table 3. Seven peptides with a documented bioactive sequence were
found in not digested PRC, with no difference in their semiquantitative content between
C- and Hypo-PRC. At the end of the gastric phase, bioactive peptides originally found in
not digested samples were no longer detectable, or their concentration was significantly
reduced, while new sequences were detected in both C- and Hypo-PRC, although in
different amounts. All bioactive peptides detected at the end of the gastric phase were
further hydrolyzed during the duodenal phase, and 15 peptides were detected in both C-
and Hypo-PRC at the end of
in vitro
digestion. Among them, four (PQNIPPL, VYPFPGPI,
LHLPLPL, PGPIPN) were more abundant in Hypo-PRC than C-PRC.
Molecules 2022,27, 664 7 of 15
Table 2.
Number of different peptides released in conventional Parmigiano Reggiano cheese (C-PRC) and hyposodic Parmigiano Reggiano cheese (Hypo-PRC) at
different digestion times.
Protein Source Not digested aG120 D60 D120 ANOVA
C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC
β-casein 41.0 ±0.0 c 41.0 ±0.0 c 46.33 ±2.52 c 50.0 ±2.0 bc 66.0 ±5.2 a 60.0 ±3.46 ab 65.33 ±9.45 a 66.0 ±2.0 a p< 0.0001
αS1-casein 25.0 ±0.0 a 25.0 ±0.0 a 19.67 ±0.58 a 21.67 ±1.15 a 25.0 ±3.61 a 23.3 ±2.61 a 21.67 ±3.79 a 24.33 ±3.06 a p= 0.0992
αS2-casein 9.0 ±0.0 a 9.0 ±0.0 a 7.0 ±1.0 ab 7.33 ±1.15 ab 6.67 ±0.58 b 8.33 ±0.58 ab 7.33 ±1.53 ab 8.0 ±0.0 ab p= 0.0167
κ-casein 3.0 ±0.0 c 3.0 ±0.0 c 9.33 ±0.58 a 9.67 ±1.15 a 3.33 ±0.58 c 6.0 ±1.0 b 3.33 ±1.15 c 3.67 ±0.58 c p< 0.0001
β-LG(A and B isoforms) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 1.0 ±0.0 a 0.67 ±0.58 ab 1.0 ±0.0 a 0.67 ±0.58 ab p= 0.0004
Total 78.0 ±0.0 c 78.0 ±0.0 c
82.33
±
2.31 bc
88.67 ±4.16 ABC 102.0 ±8.72 a 98.0 ±6.56 ab 98.67 ±12.50 ab
102.67
±
4.93 a
p= 0.0002
a
[
10
]. Data are means
±
SD of three different PRC and independent
in vitro
digestions. Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (different
letters indicate significant differences). G120: end of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase; LG: lactoglobulin.
Table 3.
Relative bioactive peptide abundance in conventional Parmigiano Reggiano cheese (C-PRC) and hyposodic Parmigiano Reggiano cheese (Hypo-PRC) at
different digestion times.
Peptide Sequence Protein Source Not Digested G120 D60 D120 Reported Activity (uM IC50)
C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC
LHLPLP β-CN (133–138) 62 ±3.8 a 64.7 ±7.8 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition (5.5)
RPKH αS1-CN (1–4) 0.9 ±0.1 a 0.9 ±0.1 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition (>1863)
YKVPQL αS1-CN (104–109) 0.6 ±0.1 a 0.6 ±0.1 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition (>22)
NLHLPLPLL β-CN (132–140) 25.2 ±1.9 a 25.5 ±2.7 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition (15)
RELEEL β-CN (1–6) 1.5 ±0.4 ab 2.0 ±0.5 a 0.9 ±0.1 b 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c ACE inhibition (1000)
LLYQEPVLGPVRGPFPIIV β-CN (191–209) 2.6 ±0.3 a 2.7 ±0.3 a 0.3 ±0.0 b 0.3 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition
andimmunomodulating (nr)
RPKHPIKHQGLPQEVLNENLLRF αS1-CN (1–23) 4.7 ±1.3 a 5.22 ±1.2 a 0.3 ±0.1 b 0.3 ±0.1 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b Antibacterial (nr)
AYFYPEL αS1-CN (143–149) 0.0 ±0.0 c 0.0 ±0.0 c 0.26 ±0.0 a 0.22 ±0.04 b 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c ACE inhibition (6.6)
DAYPSGAW αS1-CN (157–164) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.05 ±0.01 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition (98)
LKKISQRYQKFALPQYLKT αS2-CN (164–182) 0.0 ±0.0 c 0.0 ±0.0 c 1.5 ±0.0 a 1.4 ±0.06 b 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c Hemolytic (nr)
PLW αS1-CN (196–199) 0.0 ±0.0 b 0.0 ±0.0 b 0.1 ±0.01 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b ACE inhibition (36)
VENLHLPLPLL β-CN (129–139) 0.0 ±0.0 c 0.0 ±0.0 c 0.4 ±0.01 a 0.35 ±0.03 b 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c Anticancer (nr)
VYQHQKAMKPWIQPKTKVIPYVRYL αS2-CN (183–207) 0.0 ±0.0 b 0.0 ±0.0 b 2.3 ±0.4 a 2.4 ±0.1 a 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b Antibacterial (nr)
YQKFPQY αS2-CN (89–95) 0.0 ±0.0 a 0.0 ±0.0 a 0.8 ±0.08 a 0.7 ±0.0 b 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c ACE inhibition (20.1)
TPEVDDEALEK β-Lg (125–135) 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.4 ±0.05 a 0.2 ±0.0 b 0.0 ±0.0 c 0.0 ±0.0 c DPP-IV inhibition (319.5)
VPYPQ β-CN (177–181) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.1 ±0.01 a 0.1 ±0.01 a 0.0 ±0.0 b 0.0 ±0.0 b Antioxidant (nr)
FYPEL αS1-CN (145–149) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.7 ±0.0 a 0.0 ±0.0 b 0.0 ±0.0 b Antioxidant (nr)
HLPLP β-CN (134–138) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.5 ±0.2 b 1.7 ±0.8 a 0.4 ±0.08 b 0.4 ±0.1 b ACE inhibition (41)
EMPFPK β-CN (108–113) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 2.5 ±0.4 a 2.8 ±0.4 a 2.7 ±0.1 a 2.4 ±0.1 a ACE inhibition (423)
HLPLPL β-CN (134–139) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.5 ±0.2 b 1.7 ±0.8 a 0.4 ±0.08 b 0.4 ±0.1 b Antiamnestic (10)
KEDVPSE αS1-CN (83–89) 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.06 ±0.0 b 0.1 ±0.04 a 0.1 ±0.0 c 0.1 ±0.0 c ACE inhibition (41)
LPLPL β-CN (135–139) 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.8 ±0.3 b 1.8 ±0.3 a 1.1 ±0.2 b 1.1 ±0.3 b DPP-IV inhibition (325)
LPYP k-CN (56–59) 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 1.1 ±0.3 ab 1.5 ±0.1 a 1.1 ±0.2 ab 0.9 ±0.2 b ACE inhibition (5)
NIPPLTQTPV β-CN (73–82) 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.9 ±0.2 ab 0.5 ±0.09 bc 1.3 ±0.6 a 0.9 ±0.2 ab ACE inhibition (173)
Molecules 2022,27, 664 8 of 15
Table 3. Cont.
Peptide Sequence Protein Source Not Digested G120 D60 D120 Reported Activity (uM IC50)
C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC C-PRC Hypo-PRC
PQNIPPL β-CN (71–77) 0.0 ±0.0 d 0.0 ±0.0 d 0.0 ±0.0 d 0.0 ±0.0 d 0.02 ±0.0 c 0.05 ±0.01 b 0.025 ±0.0 c 0.1 ±0.01 a DPP-IV inhibition (1500)
VVPPFLQPE β-CN (83–91) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 1.5 ±0.1 a 1.6 ±0.6 a 1.5 ±0.5 a 2.0 ±0.2 a Antioxidant (nr)
VYPFPGPI β-CN (59–66) 0.0±0.0 d 0.0 ±0.0 d 0.0 ±0.0 d 0.0 ±0.0 d 2.9 ±0.2 c 4.0 ±0.2 b 4.2 ±0.6 b 5.2 ±0.4 a Antiamnestic (110)
YPEL αS1–CN (146-149) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 1.4 ±0.2 a 1.5 ±0.2 a 1.2 ±0.1 a 1.3 ±0.1 a Antioxidant (nr)
YPFPGPI β-CN (60–66) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.8 ±0.06 a 1.1 ±0.4 a 0.9 ±0.1 a 0.8 ±0.007 a ACE inhibition (500)
YPVEPF β-CN (114–119) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 7.6 ±1.0 a 9.0 ±1.8 a 7.7 ±0.8 a 8.7 ±0.8 a Opioid (nr)
LHLPLPL β-CN (133–139) 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.0 ±0.0 c 0.2 ±0.09 b 0.3 ±0.03 a ACE inhibition (432.7)
PGPIPN β-CN (63–68) 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.0 ±0.0 b 0.09 ±0.0 a Immunomodulating (nr)
Data are means
±
SD of three samples. Semiquantitative content was performed normalizing single peptide area against the sum of all peptide areas. Statistical analysis was performed
by one-way ANOVA (all peptides p< 0.0001) with Tukey’s post-hoc test (different letters indicate significant differences). ACE: angiotensin converting enzyme; DPP-IV: dipeptidyl
peptidase 4; G120: end of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase.
Molecules 2022,27, 664 9 of 15
3. Discussion
Sodium chloride (NaCl) is one of the most widely used additives in food processing,
and the adverse effects of high sodium (Na) consumption on blood pressure and NCD
have been well documented [
16
,
17
]. The overall concern about Na dietary intake has
boosted the development of new approaches to decrease the amount of salt added to food
products [
18
,
19
]. In this study, we investigated the possible impact of an approximately
20% salt reduction (2.32
±
0.06% dry matter (DM) and 1.90
±
0.20% DM in C-PRC and
Hypo-PRC, respectively) on the nutritional value of 30-months-ripened PRC. Indeed, the
nutritional value of food does not exclusively depend on its chemical composition, as the
bioaccessibility of nutrients is also a critical aspect to consider. The release/production of
bioactive compounds, including bioactive peptides, upon digestion of protein-rich food
is crucial as well. Since both bioaccessibility and release/generation of bioactive com-
pounds can be affected by food processing [
12
,
20
,
21
], we evaluated fatty acids and protein
bioaccessibility, as well as peptide formation in C-PCR and experimental Hypo-PCR.
The different salt concentrations modulated the kinetics of fatty acid release from the
food matrix during
in vitro
digestion. This effect could reflect a higher hydrolysis degree
of triacylglycerol during ripening. Salaberría et al. [
22
] already evidenced an accelerate
lipolysis process in low-salt PRC. The faster lipolysis could be due to the decrease in the
NaCl:moisture ratio, which could increase the amount of water available for triglyceride
hydrolysis. However, at the end of
in vitro
digestion, fatty acid bioaccessibility was similar
in C- and po-PRC.
Like fatty acids, protein bioaccessibility increased over time during digestion. Since
our aim was to verify the time course and extent of protein hydrolysis during digestion,
the use of the three spectrophotometric methods was mandatory since they selectively
quantify proteins with different molecular weights. The evident disparity between the
three absolute values of bioaccessibility reflected the powerlessness of the Coomassie assay
to detect small peptides and amino acids [
23
], the latter instead detected by OPA assay [
24
].
Comparing results from the three methods, we assume that most proteins were hydrolyzed
to fragments between 3000 Dalton and peptides bigger than five amino acids in length,
which are detected by the absorbance in UV spectra only. The highest release of protein
fragments below 3000 Dalton, a dimension compatible with peptide formation, was already
reached in the middle of intestinal digestion (D60). The kinetics of protein hydrolysis and
peptide/amino acid release was similar in C- and Hypo-PRC protein, which evidenced the
same protein bioaccessibility at the end of the in vitro digestion.
Since the coexistence of soluble protein fragments larger than 3000 Dalton was still
compatible with the above data, NMR spectroscopy was applied to verify their pres-
ence in the digested fractions. High resolution NMR spectroscopy provides information
about the whole set of molecules present in solution if they have hydrogen atoms in-
cluded in their structure, practically all organic molecules, including amino acids and their
oligo-/polymers. A simple inspection of the resulting spectra provides quantitative and
qualitative information such as (i) the solute concentrations, which are linearly correlated
to the signal area independently from the molecule the hydrogen atom belongs to, (ii) the
type of functional group hosting the identified atoms, for instance aromatic or aliphatic
moieties, and (iii) the size or flexibility of the molecules which the atom is bound to. The
latter property is revealed by the signal width: the broader the signal the larger and more
rigid the molecule is, as expected by their shorter relaxation times, in turn reflected in
the signal width [
25
]. The clear increment in the area of NMR signals demonstrated that
digestion was hydrolyzing soluble fragments from insoluble proteins. It is worth noting
that even in the last phase of digestion there were both narrow and broad signals, evi-
dencing the co-presence of small peptides, with MW < 3 kDa and larger protein fragments
with MW > 3 kDa. In not digested samples, the same bioactive peptides were present at
similar concentration in both C- and Hypo-PRC. Among them, the sequences NLHLPLPLL
(from
β
-CN, residues 132–140), LHLPLP (from
β
-CN, residues 133–138), and YKVPQL
Molecules 2022,27, 664 10 of 15
(from
α
S1-CN, residues 104–109) were already identified as ACE-inhibitory peptides in
12-months-ripened PRC [
9
], while the ACE-inhibitory and immunomodulating peptide
LLYQEPVLGPVRGPFPIIV (
β
-CN 191–209) was detected in 24-months-ripened PRC ex-
tracts [
26
]. All peptides detected in not digested PRC were almost completely degraded
during gastric digestion, and new bioactive sequences were released. In digested samples,
most identified sequences derived from the hydrolysis of high molecular weight peptides
already identified in undigested cheese [
10
] and from the cleavage of intact
α
- and
β
-casein
and whey proteins. At the end of gastric digestion, a total of 10 bioactive peptides origi-
nated from
β
-casein,
α
S1-casein, and
α
S2-casein, confirming the high affinity of pepsin for
these proteins [
27
,
28
]. Again, all peptides originated during gastric digestion were further
hydrolyzed during the duodenal phase, which generated most of the bioactive peptides.
Among these sequences, many fragments contained a PXP motif or a proline residue near to
the carboxylic terminus. These structural motifs increase the resistance to the hydrolysis by
digestive enzymes, which do not readily hydrolyze proline-containing peptides [
29
]. Most
of the bioactive sequences detected at the end of the
in vitro
digestion were ACE-inhibitory.
Among them, HLPLP (
β
-CN 134–138) and LHLPLPL (
β
-CN 133–139) originated from the
hydrolysis of NLHLPLPLL (
β
-CN 132–140) and VENLHLPLPLL (
β
-CN 130–140) identified
in not digested samples. Among bioactive peptides formed during duodenal digestion, and
thus theoretically absorbed through the intestinal barrier, the sequences HLPLP, KEDVPSE,
and LHLPLPL could elicit ACE-inhibitory activity.
Although the same bioactive peptides were detected in C- and Hypo-PRC, semi-
quantitative analysis revealed that most of them were present at higher concentration in
Hypo-PRC at the end of digestion. A previous
in vitro
study performed on myoglobin
evidenced that NaCl reduced protein digestibility, promoting the exposure of hydrophobic
amino acids, making the binding of phenylalanine with its surrounding environment within
the protein core stronger, and eventually resulting in a protein organization less prone to
undergoing enzyme-dependent hydrolysis [
30
]. However, it is not easy to explain how salt
concentration selectively modulated the formation of biologically active peptides during
gastrointestinal digestion, and further studies are needed to clarify this important aspect.
To the best of our knowledge, the present study is the first one investigating the kinetics of
bioactive fragment formation during the different phases of digestion. Indeed, although
the evolution of bioactive peptide formation with ripening was already addressed [
26
],
and several studies reported bioactive peptides released from PRC after
in vitro
diges-
tion [
26
,
31
,
32
], exhaustive information regarding the fate of these peptides was still lacking.
Our results represent a further step to uncover the hidden functionality of foods that is
linked to bioactive peptide formation.
Based on the herein reported results, we can conclude that reduction of salt content in
ripened PRC did not significantly affect fatty acid and protein bioaccessibility and led to the
formation of a higher number of bioactive peptides after gastrointestinal digestion. Of note,
ACE-inhibitors peptides were more abundant in Hypo- than C-PRC. The presence of these
peptides has been reported in various fermented milk products, the antihypertensive effect
of which has been proved by various
in vitro
and
in vivo
(animal and human trials) experi-
ments [
33
]. Considering that in a previous study we evidenced that the sensory profiles
were remarkably similar in C- and Hypo-PRC, without any off-flavor development [
10
],
our results confirmed that reduction of salt content in ripened PRC represents a promising
strategy to reduce Na intake, potentially protecting consumers’ health. In this light, the
evaluation of the actual ACE-inhibitory activity of digested C- and Hypo-PRC deserves
further studies in biological systems.
4. Materials and Methods
4.1. Materials
Unless specified, chemicals and solvents were of the highest analytical grade and
purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA).
Molecules 2022,27, 664 11 of 15
4.2. Parmigiano Reggiano Cheese (PRC)
PRC was produced and ripened according to the Protection Designation of Origin spec-
ification, which includes restrictions to its geographical area of production, cow feeding, and
cheese manufacturing. PRC was produced in a local dairy by using conventional (
18 days
,
3 samples from 3 different wheels) and reduced (12 days, 3 samples from
3 different
wheels)
brine soaking times in a saturated sodium chloride solution (about 36% w/w). Average
salt content was 2.32
±
0.06% dry matter (DM) and 1.90
±
0.20% DM in C-PRC and short
brine soaking time Hypo-PRC, respectively (p= 0.0253). A detailed description of PRC
production and proximate analysis of C-PRC and Hypo-PRC is reported in a previous
work [10].
4.3. In Vitro Digestion
In vitro
digestion was performed in triplicate according to the INFOGEST protocol [
14
].
To simulate chewing, PRC was chopped before starting oral digestion.
In vitro
digestion
lasted for 242 min (2 min of oral digestion, 120 min of gastric digestion, and 120 min
of intestinal digestion) at 37
C. During the process, several consecutive enzymatic reac-
tions took place by the addition of simulated saliva, simulated gastric juice (containing
2000 U/mL
pepsin) at pH 3, and simulated pancreatic juice (containing 10 mM bile and
100 U/mL
pancreatin) at pH 7. Samples were taken at the end of the gastric phase (G120),
after 60 min (D60), and at the end of the duodenal phase (D120). Digested samples were
centrifuged at 50,000
×
gfor 15 min. To remove any turbidity, supernatants were filtered
with 0.2 µm membranes and stored at 80 C until further analysis.
4.4. Fatty Acid Bioaccessibility
Total lipids were extracted according to Bligh and Dyer [
34
], with slight modifications.
Briefly, 6 mL of methanol, 3 mL of chloroform, and 2.4 mL of distilled water were sequen-
tially added to 0.1 g of food or 0.8 mL of digested sample followed by thorough mixing with
magnetic stirring at the maximum intensity for 3 min. Successively, 3 mL of chloroform
and 3 mL of distilled water, each one followed by thorough mixing with magnetic stirring
for 1 min, were added. The solution was kept overnight at 4
C, and then the upper layer
was removed by suction, and the lower chloroform layer was transferred to a test tube.
Pentadecanoic acid (1 mg) was added as internal standard, and the chloroform layer was
dried under nitrogen infusion. After methylation [
35
], the quantitative and qualitative
content of fatty acids (as methyl esters—FAMEs) was determined by fast-GC (GC-2030AF;
Shimadzu, Kyoto, Japan) using a capillary column (30 mt, 0.2
µ
m film thickness) with
a programmed temperature gradient (50–250
C, 10
C/min). The gas chromatographic
peaks were identified based on their retention time ratios relative to methyl stearate and
predetermined by use of authentic samples [
36
]. Gas chromatographic traces and quanti-
tative evaluations were obtained using Lab Solution software (Shimadzu, Kyoto, Japan)
and normalized for the dilution factor due to the addition of digestive fluids. FAMEs from
chemicals added during
in vitro
digestion system were subtracted, and bioaccessibility
was calculated as FAME concentration in digested sample/FAME concentration in PRC
before digestion ×100 [37].
4.5. Protein Bioaccessibility
Protein concentration was determined spectrophotometrically by o-phthaldialdehyde
(OPA) [
38
] and Coomassie assay [
39
], and by measuring the absorbance at 280 nm [
40
]
using L-isoleucine, bovine serum albumin, and non-fat dry milk as standards, respectively.
Values were normalized for the dilution factor due to the addition of digestive fluids, and
protein content from enzymes added during
in vitro
digestion was subtracted. Bioacces-
sibility was calculated as protein mass in digested sample/protein mass in PRC before
digestion ×100 [37].
Molecules 2022,27, 664 12 of 15
4.6. HR-NMR Spectroscopy
HR-NMR analysis was performed in digested sample as previously reported [
41
], with
slight modification. Samples were thawed, centrifuged first at 2300
×
gfor 5 min at 4
C to
eliminate the coarser particles, and then at 50,000
×
gfor 5 min at 4
C to eliminate the fine
particulates. Afterward, each sample was vortexed for 30 s, then 750
µ
L of supernatant was
taken and added to 120
µ
L of 100 mM phosphate buffer with 10 mM trimethylsilylpropanoic
acid (TSP), molecular weight (MW) 172.27 g/mol (Cambridge Isotope Lab Inc., Tewksbury,
MA, USA) and brought to pH 7.0. HR-NMR spectra were recorded at 298 K on a Bruker US+
Avance III spectrometer operating a proton frequency of 600.13 MHz, equipped with a BBI-z
probe and a SampleCase
sampler for automation. The spectra were collected with a 90
pulse of 13.1
µ
s with 10 W of power, a relaxation delay of 5 s, and an acquisition time of
2.3 s
;
for each sample 256 scans were obtained, resulting in 32 K data points over
7,194,245 Hz
encompassing a spectral width of 12 ppm. The residual signal of monodeuterated water
(HOD) was suppressed using the NOESYGPPR1D sequence (a standard pulse sequence
provided in the Bruker library), which incorporated the first increment of the NOESY pulse
sequence plus a spoil gradient. A Fourier transform was exploited to extract the frequency–
domain spectrum of 64 K data points from the raw time–domain FID. TopSpin version 3.5.6
was used to automatically execute phase and base line fixes (Bruker BioSpin, Karlsruhe,
Germany). The chemical shifts were internally referenced to the DSS at
0.000 ppm
. Data
are expressed as arbitrary units [12].
4.7. Bioactive Peptide Release and Identification
Digested samples were centrifuged at 14,000 rpm for 40 min at 4
C with a 5810 R
centrifuge (Eppendorf s.r.l, Milan, Italy) to remove any particulate matter formed during
freezing/thawing. The supernatants were filtered through 0.45
µ
m PTFE filters and directly
injected in the UPLC–MS system. Samples were separated by a reverse-phase Acquity
UPLC Peptide BEH 300 C18 column (1.7
µ
m, 2.1
×
150 mm, Waters, Milford, MA, USA) in
a UPLC system coupled with ESI and MS (UPLC Acquity I-class, with a Vion IMS QTof
Mass Spectrometer, Waters, Milford, MA, USA). Gradient elution was set as follows with
eluent A (H
2
O with 0.2% CH
3
CN and 0.1% HCOOH) and eluent B (CH
3
CN with 0.1%
HCOOH)): 0 to 7 min, isocratic 100% A; 7 to 50 min, linear gradient from 100% A to 50% A;
50–52.6 min 50% A, 52.6–53 min from 50% A to 0% A, 53–58.2 min 0% A, 58.2–59 min from
0% A to
100% A
, 59–72 min 100% A and reconditioning. Flow rate was set at 0.20 mL min
–1
,
injection volume 1
µ
L, column temperature 35
C, sampler temperature 18
C, and the total
run time was 72 min. Detection was performed using a Vion IMS QTof mass spectrometer
(Waters, Milford, MA, USA) with the following parameters. Experiment type: peptide map
(IMS), experiment type: MSe, source type: ESI, polarity: positive, analyzer mode: sensitivity,
mode: standard transmission, capillary: 3.00 kV, sample cone voltage: 40 V, source offset
voltage: 80 V, source temperature: 120
C, desolvation temperature: 450
C, cone gas flow:
50 L/h, desolvation gas flow: 800 L/h. MSe mode: high definition MSe, acquisition time:
0–58.2 min, scan range: 100–2000 m/z, scan time: 0.4 s, low collision energy: 6 V, high
collision energy ramp: 20 to 45 V, automatic lock correction (leucine enkephaline). The
software used for data processing was UNIFI (Waters Corporation, Milford MA, USA).
The expected component list comprised the following protein Uniprot accession numbers:
P02666 (
β
-casein), P02662 (
α
S1-casein), P02663 (
α
S2-casein), P02668 (
κ
-casein), P02754 (
β
-
lactoglobulin), P00711 (
α
-lactalbumin). Variable amino acid modifications were included
as deamidation (N, Q), pyroglutamic acid N-term (E, Q), oxidation (single or double, M or
W), phosphorylation (S, T, Y). Peptide semiquantitative data were obtained normalizing
integral areas.
Released bioactive peptides were identified using a bioinformatic approach. The
whole set of peptide sequences under analysis was searched into two benchmark databases
of peptide bioactivity, namely BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/
en/biopep, (accessed on 9 July 2021)) [
42
] and AHTPDB (http://crdd.osdd.net/raghava/
Molecules 2022,27, 664 13 of 15
ahtpdb/, (accessed on 9 July 2021)) [
43
], which were queried automatically and systemati-
cally employing a script pipeline developed “in-house”.
4.8. Statistical Analysis
Statistical differences were determined by the one-way analysis of variance (ANOVA)
followed by Tukey’s post hoc test using Prism software ver. 7.0 (GraphPad, San Diego, CA,
USA). Different letters indicate significant differences (at least p< 0.05).
Supplementary Materials:
The following supporting information can be downloaded. Table S1:
Fatty acid methyl ester content in conventional Parmigiano Reggiano cheese (C-PRC) and hyposodic
Parmigiano Reggiano cheese (Hypo-PRC).
Author Contributions:
Conceptualization, F.C., T.T. and A.B.; Data curation, M.D.N., C.L., E.C., L.D.,
G.P., G.A. and C.D.G.; Funding acquisition, A.B. and G.G.; Investigation, M.D.N., C.L., E.C., L.D.,
G.P., G.A. and C.D.G.; Methodology, M.D.N., C.L., E.C., L.D., G.P., G.A. and C.D.G.; Supervision,
F.C., T.T. and A.B.; Validation, M.D.N., C.L., E.C., L.D., G.P., G.A. and C.D.G.; Writing—original draft,
M.D.N. and C.L.; Writing—review and editing F.C., T.T. and A.B. All authors have read and agreed to
the published version of the manuscript.
Funding:
This study was funded by Italian POR-FESR 2014-2020 (Innovative Milk and Meat products
for Consumer’s Health—MiMe4Health). Call for strategic industrial research projects aimed at the pri-
ority areas of the Intelligent Specialization Strategy (DGR n. 986/2018). Project nr. PG/2018/632065.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No applicable.
Acknowledgments:
The authors are grateful to the Parmigiano Reggiano Consortium and Elena
Bortolazzo (Centro Ricerche Produzioni Animali S.p.A, Italy) for supplying Parmigiano Reggiano
cheese and to Simona Vita for her helpful technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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... Among all these processes, ripening is regarded as the most important and despite the static appearance of the cheese wheels in the ripening chamber, it is a deeply active period of radical biochemical changes in the matrix. In particular, proteolysis is recognized as one of the most important events during cheese ripening, and the interrelationship between cheese microbiota and aging results in a specific peptide profile (Bottari et al., 2020), which leads to the valuable and recognizable characteristics of long-ripened types of cheese, and where bioactive peptides (BPs) have frequently been found (Coppola et al., 2000;Ardö et al., 2009;López-Expósito et al., 2017;Di Nunzio et al., 2022). BPs are protein fragments produced from parent proteins, involved in carrying out various physiological functions, such as antimicrobial, antioxidant, antihypertensive, and ACE-inhibitory activities, mainly described for BPs identified in various types of cheese (Sforza et al., 2012;Sultan et al., 2018;Martini et al., 2020;Solieri et al., 2020). ...
... BPs are protein fragments produced from parent proteins, involved in carrying out various physiological functions, such as antimicrobial, antioxidant, antihypertensive, and ACE-inhibitory activities, mainly described for BPs identified in various types of cheese (Sforza et al., 2012;Sultan et al., 2018;Martini et al., 2020;Solieri et al., 2020). Recently, some authors have investigated the presence of BPs in Parmigiano Reggiano (PR) cheese (Basiricò et al., 2015;Martini et al., 2020Martini et al., , 2021Solieri et al., 2020;Tagliazucchi et al., 2020;Di Nunzio et al., 2022). PR is a protected designation of origin (PDO), raw milk, and hard-cooked cheese, with a minimum ripening time of 12 months (Gatti et al., 2014). ...
... In our experiments, only four potentially BPs and three NPADs were detected in undigested cheese samples, while among the 105 different peptides revealed by the analysis of Gamma-Glu-Leu DPP-IV Inhibitory, potential functional ingredients in type 2 diabetic diet Lu et al., 2021 digested cheese samples, 21 peptides and 3 NPADs showed at least one bioactivity as reported by the Milk Bioactive Peptides Database (MBDP). This is in agreement with the literature that reports a higher number of BP released after digestion than undigested food (Egger and Ménard, 2017;Giromini et al., 2019;Fernández-Tomé and Hernández-Ledesma, 2020;Di Nunzio et al., 2022). Digestion is a key step to freeing BPs from their cryptic form and increasing the probability of having a positive effect exerted by the food components. ...
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Background The relationship between dietary sodium intake and blood pressure (BP) has been tested in clinical trials and nonexperimental human studies, indicating a direct association. The exact shape of the dose–response relationship has been difficult to assess in clinical trials because of the lack of random-effects dose–response statistical models that can include 2-arm comparisons. Methods After performing a comprehensive literature search for experimental studies that investigated the BP effects of changes in dietary sodium intake, we conducted a dose–response meta-analysis using the new 1-stage cubic spline mixed-effects model. We included trials with at least 4 weeks of follow-up; 24-hour urinary sodium excretion measurements; sodium manipulation through dietary change or supplementation, or both; and measurements of systolic and diastolic BP at the beginning and end of treatment. Results We identified 85 eligible trials with sodium intake ranging from 0.4 to 7.6 g/d and follow-up from 4 weeks to 36 months. The trials were conducted in participants with hypertension (n=65), without hypertension (n=11), or a combination (n=9). Overall, the pooled data were compatible with an approximately linear relationship between achieved sodium intake and mean systolic as well as diastolic BP, with no indication of a flattening of the curve at either the lowest or highest levels of sodium exposure. Results were similar for participants with or without hypertension, but the former group showed a steeper decrease in BP after sodium reduction. Intervention duration (≥12 weeks versus 4 to 11 weeks), type of study design (parallel or crossover), use of antihypertensive medication, and participants’ sex had little influence on the BP effects of sodium reduction. Additional analyses based on the BP effect of difference in sodium exposure between study arms at the end of the trial confirmed the results on the basis of achieved sodium intake. Conclusions In this dose–response analysis of sodium reduction in clinical trials, we identified an approximately linear relationship between sodium intake and reduction in both systolic and diastolic BP across the entire range of dietary sodium exposure. Although this occurred independently of baseline BP, the effect of sodium reduction on level of BP was more pronounced in participants with a higher BP level.
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Background Although phenolic compounds have a role in the health benefits of fruit juice consumption, little is known about the effect of processing on their bioaccessibility. The release of phenolic compounds from the food matrix during digestion is an important pre‐requisite for their effectiveness within the human body, so it is fundamental to identify technological treatments able to preserve not only the concentration of phytochemicals but also their bioaccessibility. In this study we investigated the impact of high‐pressure homogenization (HPH), alone and in the presence of 100 g kg⁻¹ trehalose or Lactobacillus salivarius, on bioaccessibility of flavonoids in mandarin juice. In addition, digested mandarin juices were supplemented to liver cultured cells in basal and stressed condition to evaluate their protective effect in a biological system. Results HPH reduced the concentration of total phenolics and main flavonoids but increased their bioaccessibility after in vitro digestion (p < 0.001). In basal condition, supplementation with all digested juices significantly reduced intracellular reactive oxygen species (ROS) concentration (p < 0.001). Thiobarbituric acid reactive substances concentration in the medium was also reduced by supplementation with HPH‐treated juices. Although pre‐treatment with juices did not completely counteract the applied oxidative stress it preserved cell viability, and cells pre‐treated with juices submitted to HPH in the presence of probiotics showed the lowest ROS concentration. Conclusion Our study represents an important step ahead in the evaluation of the impact of processing on the nutritional and functional value of food, which cannot simply be assessed based on chemical composition. This article is protected by copyright. All rights reserved.