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Peptidomics: new trends in food science
Serena Martini, Lisa Solieri and Davide Tagliazucchi
In recent years, peptidomics is gaining ever-growing relevance
in food science. The emerging field of food peptidomics is
defined as the whole pool of peptides existing in food products
or generated during food processing, storage or digestion. The
recent advancement in high-resolution mass spectrometry
techniques provides a more detailed picture, although not yet
exhaustive, of the peptides present or derived from food. Food
peptidomics techniques have been successfully applied in
understanding food protein digestion, in the study of the
microbial contribution to food protein hydrolysis, in the
identification of food-derived bioactive peptides and peptide
biomarkers. Ad-hoc efforts are still needed to improve the
identification of short peptides and the analysis of the large
data set generated.
Address
Department of Life Sciences, University of Modena and Reggio Emilia,
Via Amendola 2, 42100 Reggio Emilia, Italy
Corresponding author:
Tagliazucchi, Davide (davide.tagliazucchi@unimore.it)
Current Opinion in Food Science 2021, 39:51–59
This review comes from a themed issue on Foodomics technologies
Edited by Alexandre Lamas
For complete overview of the section, please refer to the article col-
lection, “Foodomics technologies”
Available online 25th December 2020
https://doi.org/10.1016/j.cofs.2020.12.016
2214-7993/ã 2020 Elsevier Ltd. All rights reserved.
Introduction
Peptidomics was defined at the beginning of 2000 by
Schulz-Knappe et al. [1] to indicate the pool of all the
peptides existing in a biological sample (i.e. the pepti-
dome). Peptidomics can be considered as a branch of
proteomics, which apply the fundamental techniques
developed in the shot-gun proteomics approach but with
a different target, that is, the identification of the peptides
in specific samples rather than the intact proteins [2].
Originally, peptidomics techniques were essentially
applied in the medical field, focusing on the subject area
of neuroendocrine research, disease biomarker discovery
or drug development. Eventually, peptidomics methods
were successfully widened to other areas including food
and nutrition. The first paper reporting the term ‘food
peptidomics’ was published in 2008 by Minkiewicz et al.
www.sciencedirect.com
[3]. Since 2008, 343 articles have been published with the
words ‘food peptidomics’ in the title, abstract or key-
words, with a growing trend over the years (Figure 1).
This review describes the new trends of applications of
peptidomics techniques in food research with special
emphasis on food digestion (identification and discovery
of bioactive peptides), fermented food analysis, bio-
marker discovery (giving information on product origin
and authenticity as well as in food adulteration) and
recent application in food waste exploitation.
Digestomics: peptidomics application in food
digestion
Gastro-intestinal digestion of dietary proteins is a crucial
step both to supply the organism of essential and non-
essential amino acids and for the release of medium-size
and short-size peptides with potential biological activity.
Peptidomics approach has demonstrated to be a suitable
choice to monitor hydrolysis during digestion of food
proteins [4,5–8]. The recent development of high-
throughput peptidomics techniques by the application
of high-resolution mass spectrometry (MS) allowed the
identification of many, though not all, of the peptides
released during the digestion of different foods [4].
Major limitations are related to the identification of very
short peptides (<5 amino acids), large-sized polypeptides
and disulfide cross-linked hetero-oligomers [4].
The release of bioactive peptides from different food
sources has been followed in a number of studies by using
in vitro digestion models. Dairy products are the most
studied food class regarding the release of bioactive
peptides during digestion. Peptidomics analysis was
employed to study the differences in protein hydrolysis
and bioactive peptide release after in vitro gastro-intesti-
nal digestion of cow, goat, sheep and camel milk and
yogurt [5,6]. A wide range of bioactive peptides was
identified with angiotensin-converting enzyme (ACE)-
inhibitory, antioxidant and dipeptidyl peptidase-IV
(DPP-IV)-inhibitory activities. Likewise, Basilicata
et al. [7] applied peptidomics techniques to study differ-
ences in the release of bioactive peptides after in vitro
gastro-intestinal digestion of buffalo-milk dairy products,
such as cheeses, yogurt and ice cream. They evidenced
that the specific technological process influenced the
release of bioactive peptides during in vitro digestion.
Furthermore, Martini et al. [8] compared the peptidomics
profile of in vitro digested cooked beef, pork, chicken and
turkey meat. They found that about 30% of identified
Current Opinion in Food Science 2021, 39:51–59
52 Foodomics technologies

Figure 1

(a)

(b)

Current Opinion in Food Science

Number of published articles in the scientific literature in the field of “food peptidomics” from 2008 to date.
The search was carried out with Web of Science (http://apps.webofknowledge.com/) by using the words ‘food peptidomic’ or ‘food peptidomics’.
(a) Number of published articles per year. (b) Number of published papers per research area.

bioactive peptides were commonly released after in vitro
digestion of the four types of meat. In the case of meat
products, peptidomics analysis was also applied to study
the effect of meat cooking and processing on protein
digestibility and peptide profile after in vitro digestion
[9,10]. Once again, the method of processing as well as the
cooking time and temperature affected the peptidomics
profile of the in vitro digested meat. Understanding the
relationships between food preparation and the digestive
release of bioactive peptides may help to design the most
appropriate process to maximize and modulate their
release and to better understand the healthiest diet
feature.
An integrated approach combining in vitro digestion and
peptidomics was successfully applied to study the effect
of the ripening time on the peptidome and bioactive
peptide profile of dry-cured ham and cheese after diges-
tion [11,12]. With both matrices, substantial differences
were found between the peptides released after digestion
as a function of the ripening time.
Several papers explored the release of bioactive peptides
from marine products during in vitro digestion by apply-
ing peptidomics techniques. For example, Ling et al. [13]
identified two novel ACE-inhibitory peptides after diges-
tion of tilapia skin gelatin, whereas Zhao et al. [14]

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 Peptidomics: new trends in food science Martini, Solieri and Tagliazucchi 53
characterized some new antioxidant peptides released
from Spanish mackerel following in vitro digestion.
Less attention has been paid to vegetables as a source of
bioactive peptides following in vitro gastro-intestinal
digestion [15].
Released peptides in the gastro-intestinal tract are not
invariably healthful. Recently, a peptidomics approach
was applied to characterize peptides released from
extracted wheat proteins and whole wheat flours after
in vitro gastro-intestinal digestion [16]. Some gluten pep-
tides, which were found to be able to survive gastro-
intestinal digestion, are involved in triggering the adap-
tive or the innate immune response typical of the celiac
disease.
Despite efforts made to develop an in vitro digestion
protocol that mirrors the in vivo scenario (such as the
INFOGEST harmonized in vitro digestion protocol), it is
still uncertain whether the in vitro data may be physio-
logically relevant. However, two recently published
papers addressed the question by comparing the in vitro
and in vivo data on the release of peptides from milk
during digestion in pigs and humans [17,18]. The major
conclusion of both works was that the in vitro harmonized
digestion protocol closely emulated the physiological
conditions. This conclusion was supported by the similar
pattern of released peptides identified in in vivo and in
vitro experiments and by the presence of milk proteins
common regions, resistant to digestion.
Until now, only few studies have been carried out in vivo
to track the release of bioactive peptides. The peptido-
mics analysis of human jejunum fluids after ingestion of
casein or whey proteins revealed the presence of 356 and
146 peptides released during digestion, some of them
potentially bioactives [19]. Recently, Beverly et al. [20]
applied peptidomics to compare the gastric release of
milk peptides in pre-term and term infants. Besides the
obvious differences in milk proteins digestibility between
these infants, they identified an anti-microbial peptide
released from the C-terminus of b-casein, which was
more abundant in pre-term infants and may protect their
intestinal tract from bacterial infections.
Peptidomics in fermented food
Dairy fermented food
Fermented foods have been considered fundamental
components of the human diet since ancient times and
among them, dairy fermented food (DFF) is the most
widely consumed category worldwide. A plethora of
reviews summarized the peptidome of fermented foods.
Here, we focused on studies that exploited peptidomics
methods to highlight the microbial contribution to protein
hydrolysis and the role of microbial proteolytic system to
enrich food in bioactive peptides (Figure 2).
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Lactic acid bacteria (LAB), the workhouse in the dairy
industry, convert lactose into lactic acid and significantly
affect DFF sensorial and textural properties through
lipolysis and proteolysis. To complement their amino
acid auxotrophies, LAB possess a three-component sys-
tem, consisting in cell-envelope proteinases (CEP)
encoded by prt genes, peptide transport systems and
intracellular peptidases ( pep genes), which are responsi-
ble for the milk proteins breakage (Figure 2) [21,22].
Especially a-caseins and b-caseins are susceptible to LAB
proteolysis due to their unstructured, flexible and open
structure accessible for CEP [22]. The specificities of
these enzymes towards the substrates are variable at inter
and intra-species level and affect peptides composition in
the final product [23]. Thus, large-scale MS techniques
were indispensable tools to determine the peptidome
footprinting of LAB microbiota in different DFF, like
kefir [22], yogurts [24] and cheese [25].
Nowadays, growing perception about diet in relation to
health has extended the necessity to explore the biologi-
cally active components in DFF. In this context, pepti-
domics studies burst into the scientific arena also to
explore the functional potential of LAB to improve bio-
active peptides content in DFF. Non-starter LAB from
Parmigiano Reggiano cheese were found to release ACE-
inhibitory peptides in milk [26] and were used to enrich
yogurt in the anti-hypertensive lactotripeptides VPP and
IPP [27]. Gu et al. [28] successfully used UPLC-MS/MS
to evaluate how multiple-species starters affected peptide
profiles in yogurts and identified a novel peptide
(NENLLRFF) with multiple healthy actions.
Advances of proteolytic systems genetics were usefully
combined with LC–MS/MS peptidomics analysis to dis-
sect the mechanisms underpinning the bioactive peptides
production in LAB. Ravenchot et al. [29] recently
exploited multiparametric analysis to identify the best
bioactive peptides-producers among 120 LAB strains
isolated from different Mongolian DFF. The authors
showed that b-casein-derived peptide profiles were cor-
related with the distribution of prt genes in the LAB
candidate genomes: differently from Lactobacillus del-
brueckii, Lactobacillus helveticus strains harboring prtH2
and prtH3 genes showed preferential cleavage for hydro-
phobic b-casein regions, which are less accessible than
hydrophilic regions when b-casein is in a micellar form.
The ability to preferentially cleave these regions could
confer a selective growth advantage to L. helveticus. Huang
and Kok [30] combined deletion of pep genes and
nanoLC–MS/MS to identify novel bioactive peptides
released from b-casein by Lactococcus lactis pep mutants.
Fermented sausages
Fermented sausages (FS) are meat products consisting of
a mixture of mainly lean meat, fat, NaCl, curing agents
and spices stuffed in casings, fermented and ripened.
Current Opinion in Food Science 2021, 39:51–59
54 Foodomics technologies

Figure 2

1. Source of proteins

(a) CEP-mediated breakage

(b) Uptake

(c) Oligopeptide and aa release

2. Whole-cell proteolysis

3. Functional FF enriched in BP
Current Opinion in Food Science

Overview of proteolytic activities of lactic acid bacteria that contribute to the conversion of dairy, meat and plant-derived proteins into bioactive
peptides during food fermentation.
Cell-wall bound enzymes encoded by prt genes are responsible for protein breakdown into long oligopeptides, which are intracellularly
accumulated via highly specialized transport systems. Here, long oligopeptides are converted into short oligopeptides and amino acids by a
plethora of different peptidases. A balance between production and consumption of oligopeptides and amino acids is critical for determining the
peptide profiles of fermented food and the enrichment in bioactive peptides. Abbreviation are: BP, bioactive peptides; CEP, cell-envelope
proteinase; FF, fermented food; aa, amino acids.

Different LAB species, present in raw meat as autoch-
thonous microbial cells or intentionally added as starter
cultures, are primarily responsible for sausage fermenta-
tion. In addition to the acid-induced increase of cathepsin
activity, LAB directly complement meat endogenous
proteolytic machinery to achieve sarcoplasmic and myo-
fibrillar protein hydrolysis during fermentation and
curing, with significant impact on the final characteristics
of FS. However, peptidome has been investigated to a
lesser extent in FS compared with DFF. In a pioneering
work [31], peptides with size lower than 3 kDa were
characterized as quality biomarkers for FS. Latilactoba-
cillus curvatus (formerly known as Lactobacillus curvatus)
starter culture CRL705 highly enriched the FS in both

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 Peptidomics: new trends in food science Martini, Solieri and Tagliazucchi 55
peptide and amino acid concentrations. Since Latl. cur-
vatus genome does not have CEP-encoding genes, intra-
cellular peptidases released after cell lysis were supposed
to be responsible for the breakage of a-actin (susceptible
to LAB-mediated degradation in three different regions),
myoglobin and creatine kinase M-type.
In addition to taste and flavor contribution, LAB signifi-
cantly improved the release of bioactive peptides in
different FS [32]. Although the detection of bioactive
peptides is still challenging in FS, dry-fermented sau-
sages produced with a mixed starter culture of Lactiplan-
tibacillus pentosus (formerly known as Lactobacillus pento-
sus) and Staphylococcus carnosus were found to possess b
casein-derived bioactive peptides, such as the ACE-
inhibitory peptides YQEPLV, YQEPVLGPVR and
YQEPVLGPVRGPFPI, when sodium caseinate was
added as emulsifying agent [33].
Most recently, fermented meat technology has been
faced with the challenge to decrease the NaCl content.
Starter cultures with proteolytic activities have been
proposed as suitable solutions to attenuate sensorial
defects arisen from the usage of NaCl substituents, like
magnesium, calcium or potassium chloride. In low-
sodium beaker sausage models, Latilactobacillus sakei
CRL1862 (formerly known as Lactobacillus sakei) and
Enterococcus mundtii CRL35 were proposed as starter
cultures in combination with Staphylococcus vitulinus: Latl.
sakei + S. vitulinus co-culture produced significant incre-
ments in free amino acids, while E. mundtii + S. vitulinus
co-culture was able to release the highest quantity of low
molecular weight peptides mainly originated from myo-
fibrillar proteins, in particular a-actin [34].
Plant-derived fermented food
Identification of bioactive peptides from plant food pro-
teins has gained increasing interest in the scientific com-
munity due to the public opinion that plant-based foods
are higher sustainable than animal-based foods. Tradi-
tional vegetable fermented food, consumed in either
Eastern or Western countries, were proved to be relevant
dietary source of bioactive peptides [15], but poor infor-
mation is still available on how lactic microbiota or
inoculated starter cultures affect the development of
specific peptide patterns. In fermented wholegrain oats,
co-culture of Lactoplantibacillus plantarum (formerly
known as Lactobacillus plantarum) and Rhizopus oryzae
released peptides from a- subunits and b-subunits of
12S oat globulins with ACE-inhibitory activities [35].
Antioxidant and anti-inflammatory properties of sour-
doughs containing selected Companilactobacillus farciminis
(formerly known as Lactobacillus farciminis) strains H3 and
A11 and Fructilactobacillus sanfranciscensis (formerly
known as Lactobacillus sanfranciscensis) strain I4 have been
recently investigated [36]. Low molecular weight pep-
tides from these breads suppressed the NFkB pathway as
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well as reduced the intracellular ROS levels, suggesting
that these strains could be exploited to develop functional
bread. The combination of functional starter LAB and
whole grain flour has been also considered to optimize the
bioactive peptides content in healthy breads [37]. Several
evidences showed that sourdough fermentation reduced
overall gliadin allergenicity [38]. Recently mass spec-
trometry was useful to demonstrate that Bacillus strains
from wheat had proteolytic activities against the celiac
epitopes, particularly 33-mer peptide from gliadin [39],
whereas sourdough-related lactobacilli also degraded
amylase-trypsin inhibitors in addition to gliadins [40].
The a-amylase/trypsin inhibitors have recently been
identified as inducers of an innate immune response
via toll-like receptor 4 in celiac disease and non-celiac
wheat sensitivity [41]. Mass spectrometry will be essen-
tial in the next future to test the sourdough matrix effect
on these beneficial bacteria and to unravel the synergic
effects of different LAB in vivo.
Peptidomics for biomarker discovery
Nowadays, food safety has gained even more importance
at a global level, and the prevention of health-related
problems caused by contaminated food or false nutrition
is a strongly felt topic with social, economic and health
implications. In this context, the most recent and sophis-
ticated techniques have been developed to improve the
field of food analysis, becoming the major scientific area
in applied analytical chemistry. In this framework, pep-
tidomics represents a new specific and sensitive tech-
nique, with the ability to track the peptide composition of
foods and its changes during the production process. The
identification of specific biomarkers is fundamental to
standardize and facilitate the diagnosis of food authenti-
cation as well as monitoring the presence of harmful or
dangerous agents and food quality.
The major bottlenecks in the biomarker discovery are
related to the identification of a significant number of
biomarker candidates and the following development of
targeted techniques to quantify one or more candidates.
High-resolution MS-based peptidomics techniques are
appealing tools to overcome these bottlenecks (Figure 3).
Adulteration of food with less expensive ingredients is a
hot research matter in food science and foodomics. Sev-
eral studies, focused on MS-advanced techniques, were
addressed to the peptide biomarkers identification for the
research of fraudulent manipulation in the dairy industry.
For example, the addition of cheap milk (cow milk,
caseins and b-lactoglobulin) to buffalo, donkey, goat
and sheep milk at levels below 0.5% was thoroughly
reported [42].
Indeed, peptidomics provides a valid strategy for meat
speciation, differentiating between meat origins. Apply-
ing UPLC–MS/MS analysis, Li et al. [43] detected and
Current Opinion in Food Science 2021, 39:51–59
56 Foodomics technologies
Figure 3
Current Opinion in Food Science
Typical workflow for the application of peptidomics in the discovery of
food peptide biomarkers.
The basic procedure involves the application of an untargeted
approach for de novo identification of hundreds or thousands
biomarker peptide candidates followed by the development of a
targeted method to quantify the selected peptide(s). Candidate
peptide biomarkers in food should be specific for the food under
study, present in amount easily measurable, stable in replicate
samples and to the food processing and insensitive to amino acid
modifications.
identified 25 heat stable peptide biomarkers unique to
muscle and species protein, capable of distinguishing
between cooked chicken, pork, beef, lamb and duck in
concentrations below 0.5% (w/w) of meat mixtures. Von
Bargen et al. [44] also choose a peptidomics approach to
select meat biomarkers specific for beef, pork, and horse
meat. They also implemented multiple reaction moni-
toring (MRM) methods able to prove the fraudulent
adulteration of beef with pork or horse meat at sub-1%
levels, in raw or cooked meat. Another interesting and
promising peptidomics study was performed by Marbaix
et al. [45], where their MS-based method was optimized to
identify prohibited animal feedstuff adulteration using
Current Opinion in Food Science 2021, 39:51–59
24 bovine-specific, 15 porcine-specific, and 5 ovine-spe-
cific peptide biomarkers present in processed animal
proteins.
Concerning allergenic foodstuff, many efforts have
addressed to the identification of the specific peptide
biomarkers both in raw or processed foodstuff. Based on
UPLC–MS/MS, Planque et al. [46] were able to identify
and quantify target allergens for milk, soy, peanut and
tree nuts (almond, cashew, hazelnut, pecan nut, pistachio
and walnut). In a series of recent papers, several peptide
biomarkers were identified for the detection of allergenic
peanut, hazelnut and egg in processed food products
[47,48].
Recently, UPLC-high resolution MS-based peptidomics
approach was used for the identification of novel peptide
biomarkers to authenticate high-value mountain-culti-
vated ginseng [49]. Peptidomics techniques have also
been applied in food process control, through the identi-
fication of peptide as biomarkers of the time of proces-
sing, with the intention to unmask fraud and mislabeling
in seasoned food such as ham [50].
The optimization and development of this new inte-
grated and analytical approach based on specific, robust
and discriminant biomarkers at a molecular level may
allow to determine a ‘food fingerprint’, useful to evaluate
quality, safety, security, authenticity and nutritional value
of foods.
Peptidomics in agro-food waste and by-
products valorization
A large amount of agricultural and food waste and by-
products are generated annually in the agro-food produc-
tion and processing chains, with a direct consequence on
the country’s economy and environmental pollution. The
whole exploitation of these waste and by-products with
innovative industrial processes and technologies is a
global challenge of paramount economic and environ-
mental importance. Actually, valorization of agro-food by-
products goes in the direction of their incorporation in
food formulation as a source of biological active com-
pounds in the added-value chain.
The production of protein hydrolysates has been exten-
sively exploited in the last years as a cheap and environ-
mental friendly process to increase the added-value of
waste and by-products. In this context, peptidomics
techniques have been applied to profile the bioactive
peptides released after hydrolysis of protein-rich food
waste and by-products [51–54]. For instance, whey and
‘scotta’ are highly pollutant dairy liquids resulting from
cheese-making and Ricotta cheese-making processes,
respectively [55,56]. These pollutants are rich in b-lacto-
globulin (50–60%), a-lactalbumin (15–25%), bovine
serum albumin (6%), lactoferrin (<3%) and several
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 Peptidomics: new trends in food science Martini, Solieri and Tagliazucchi 57
immunoglobulins and, thus, can be valorized as a source
of bioactive peptides through either LAB whole-cell or
enzymatic proteolysis [56]. Ali et al. [57] identified two
Lactobacillus helveticus and Lactobacillus acidophilus strains
able to release bioactive peptides from whey isolates with
antimicrobial activities against Salmonella typhimurium. In
another study, L. delbrueckii subsp. bulgaricus strain was
proved to cleave allergenic peptides of b-lactoglobulin in
whey and release antioxidant and ACE-inhibitory pep-
tides [58]. Similarly, Enterococcus fecalis strain 2/28 was
able to produce ACE-inhibitory, antimicrobial, DPP-IV-
inhibitory, proliferation stimulating and cytotoxic pep-
tides from whey proteins [59]. Indeed, Monari et al. used
nine different proteases to obtain bioactive peptides from
‘scotta’ by-products [51]. Recently, peptidomics analysis
was coupled to bioactivity assays to demonstrate the
ability of whey peptides to modify the lipid accumulation
and metabolism in adipocytes and skeletal muscle cells
[52]. Like milk-derived whey, soy whey, a by-product of
tofu fermentation, was recently proven to be a suitable
substrate for producing drinks enriched in bioactive pep-
tides via a kefir consortium-driven fermentation [60].
Peptidomics approaches have been also utilized to profile
bioactive peptides released after hydrolysis of food by-
products from animal or vegetable sources [53,54].
To this purpose, peptides with antioxidant, ACE-inhibi-
tory and DPP-IV-inhibitory activities were identified
after cooking and in vitro digestion of dry-cured ham
by-products [61,62].
Marine food processing by-products represent a great
source of high quality proteins that may be an important
fount of bioactive peptides after hydrolysis. As an exam-
ple, Rivero-Pino et al. [63] identified DPP-IV inhibitory
peptides after hydrolysis of discarded Sardine pilchardus
proteins whereas ACE-inhibitory peptides were found
following hydrolysis of pearl oyster shell proteins [64].
Conclusions
Nowadays, peptidomics represent a robust and large-scale
tool to identify food peptides offering a crucial contribu-
tion in quality control in the food processing chain, in food
safety as well as in nutrition and healthy food designing.
Future progress in high-resolution MS apparatus will
increase specificity and sensitivity in the identification
and quantification of peptides. However, more thorough
research is needed to fill the major gaps especially related
to the identification of short peptides and the bioinfor-
matic and statistical analysis of the large data set
generated.
Financial support
The present study was founded by FAR-2019 from
Department of Life Sciences, University of Modena
and Reggio Emilia, Italy. SM was supported by a grant
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under the frameshift ‘Programma Operativo Regionale
POR FSE 2014/2020 Obiettivo Tematico 10 - Bando
Regione Emilia Romagna Alte competenze per nuove
imprese: Laboratorio regionale per l’imprenditorialità’
from Regione Emilia Romagna, Italy.
Conflict of interest statement
None.
CRediT authorship contribution statement
Serena Martini: Writing - original draft, Writing - review
& editing. Lisa Solieri: Writing - original draft, Writing -
review & editing. Davide Tagliazucchi: Conceptualiza-
tion, Writing - original draft, Writing - review & editing.
References and recommended reading
Papers of particular interest, published within the period of review,
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 of special interest
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