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ADVISORY BOARDS
David Rodríguez-Lázaro
Loong-Tak Lim
Michael Eskin
Isabel Ferreira
Crispulo Gallegos
Se-Kwon Kim
Keizo Arihara
SERIES EDITORS
GEORGE F. STEWART (1948–1982)
EMIL M. MRAK (1948–1987)
C. O. CHICHESTER (1959–1988)
BERNARD S. SCHWEIGERT (1984–1988)
JOHN E. KINSELLA (1989–1993)
STEVE L. TAYLOR (1995–2011)
JEYAKUMAR HENRY (2011–2016)
FIDEL TOLDRÁ (2016– )
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Contributors
Alexios Alexopoulos
Laboratory of Agronomy, Department of Agriculture, University of the Peloponnese,
Kalamata, Messinia, Greece
Mikel Añibarro-Ortega
Centro de Investigação de Montanha (CIMO), Instituto Politecnico de Bragança, Bragança,
Portugal
Lillian Barros
Portugal
Edmundo Brito-de la Fuente
Product & Process Engineering Centre, Fresenius Kabi Deutschland GmbH, Bad Homburg,
Germany
Adriano Gomes da Cruz
Department of Food, Federal Institute of Education, Science and Technology of Rio de
Janeiro (IFRJ), Rio de Janeiro, RJ, Brazil
Isabel Diañez
Chemical Process and Product Technology Research Centre (Pro2TecS), Departamento de
Ingenierı́a Quı́mica, Universidad de Huelva, Huelva, Spain
N.A. Michael Eskin
Department of Food & Human Nutritional Sciences, University of Manitoba, Winnipeg,
MB, Canada
Isabel C.F.R. Ferreira
Portugal
Jose M. Franco
Crı́spulo Gallegos
Product & Process Engineering Centre, Fresenius Kabi Deutschland GmbH, Bad Homburg,
Germany
Loong-Tak Lim
Department of Food Science, University of Guelph, Guelph, ON, Canada
Bruna Marchesan Maran
Department of Chemical Engineering and Food Engineering, Federal University of Santa
Catarina, Technology Center, Florianópolis, Santa Catarina, Brazil
Emanueli Marchesan Maran
Department of Chemical Engineering and Food Engineering, Federal University of Santa
Catarina, Technology Center, Florianópolis, Santa Catarina, Brazil
ix
x Contributors
Inmaculada Martı́nez
Leticia Mora
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Paterna, Spain
Ruchira Nandasiri
Department of Food & Human Nutritional Sciences, University of Manitoba; Richardson
Centre for Functional Foods & Nutraceuticals, Winnipeg, MB, Canada
Spyridon A. Petropoulos
Department of Agriculture, Crop Production and Rural Environment, University of
Thessaly, Volos, Greece
Milena Dutra Pierezan
Department of Food Science and Technology, Agricultural Sciences Center, Federal
University of Santa Catarina, Florianópolis, Santa Catarina, Brazil
Tatiana Colombo Pimentel
Federal Institute of Paraná, Paranavaı́, Parana, Brazil
Jose Pinela
Portugal
David Rodrı́guez-Lázaro
Microbiology Division, Faculty of Sciences; Research Centre for Emerging Pathogens and
Global Health, University of Burgos, Burgos, Spain
Rachel Siqueira de Queiroz Simões
Institute of Technology in Immunobiologicals, Bio-Manguinhos, Oswaldo Cruz
Foundation, Fiocruz, Manguinhos, Rio de Janeiro, Brazil; Microbiology Division, Faculty of
Sciences; Research Centre for Emerging Pathogens and Global Health, University of Burgos,
Burgos, Spain
Fidel Toldrá
Silvani Verruck
Department of Food Science and Technology, Agricultural Sciences Center, Federal
University of Santa Catarina, Florianópolis, Santa Catarina, Brazil
Amr Zaitoon
Department of Food Science, University of Guelph, Guelph, ON, Canada
Hongfei Zhang
Department of Food Science and Technology, National University of Singapore, Singapore,
Singapore
Weibiao Zhou
Department of Food Science and Technology, National University of Singapore, Singapore,
Singapore
Preface
The series Advances in Food and Nutrition Research has reached Volume 100,
and this represents a significant milestone after more than 70 years of the
series’ history. This series was initially named Advances in Food Research,
and the first volume was published in 1948. It had 459 pages distributed
across 10 chapters and was edited by Emil M. Mrak (editor until 1987)
and George F. Stewart (editor till 1982). The link to this volume is
https://www.sciencedirect.com/bookseries/advances-in-food-research/
vol/1/suppl/C. A new editor, C.O. Chichester was appointed in 1959,
who remained until 1988. The name of the series was changed to Advances
in Food and Nutrition Research in 1989 with Volume 33 and John E. Kinsella
who was the editor until 1993. At this point, the series was not published
on a yearly basis; however, in 2001, it started with one or two volumes being
published every year. In 1995, Steve L. Taylor was the editor, who remained
until 2011, followed by Jeyakumar Henry from 2011 to 2016. Then, Fidel
Toldrá took over as the editor in 2016 with Volume 76. Due to its success,
the series moved up to three volumes a year in 2009, then four volumes a year
in 2018, and currently approved to reach five volumes a year in 2023 and suc-
cessive years. Success continues as ScienceDirect usage experienced
a noticeable increase of 68% in the period 2016–2020. The number of cita-
tions is also increasing, with a CiteScore of 4.7 in 2019 that increased up to 7.0
in 2020, for this series in Q1, ranking 32 out of 310 publications in Food
Science with 89th percentile based on CiteScore data in Scopus.
This volume (Volume 100) contains eight chapters written by an inter-
national board of authors—some of them were previous guest editors of
serial thematic volumes including this series’ editor—and reports the latest
developments in relevant and interesting topics such as the use of pepti-
domics tools and their applications in bioactive peptides, the controlled
release of bioactives in food, the role of canolol as an antioxidant and anti-
cancer agent, the applications of Solanaceae in foods and nutraceuticals,
the latest developments in 3D printing of foods, the safe use of raw milk
in the processing of dairy foods, the new threats of enteric viruses, and,
finally, the use of low-energy X-ray for innovative nonthermal food
processing.
The first chapter deals with the use of peptidomics and the methodolo-
gies involved in the study of bioactive peptides, including their identification
xi
xii Preface
and quantitation, and in silico approaches. An analysis of posttranslational

modifications in peptides that can modify their physical and chemical prop-
erties is also presented, and the specific applications of peptidomics in
bioactive peptides are finally reported. The second chapter provides an over-
view of the basic controlled and triggered release concepts relevant to food
and active packaging applications. It presents approaches to encapsulate bio-
active compounds, their mode of release, the mass transport processes, and
the interactive carriers that are responsive to certain specific stimuli. The
third chapter focuses on the nature of canolol and its applications in food
and medicine. Canolol is a unique phenol formed during processing that
has antioxidant and anticancer properties. Specific extractability require-
ments from oilseed using different processing conditions and canolol deriv-
atives, including dimers and trimers, are discussed. The fourth chapter
discusses the nutritional value of the most important Solanaceae species
commonly used for their edible fruit, as well as those used in the develop-
ment of functional foods and nutraceuticals due to their bioactive constitu-
ents. The toxic and poisonous effects as well as the sustainable management
practices implemented to increase the added value of such crops are also dis-
cussed. The fifth chapter reviews the current advances in extrusion-based
3D food printing systems, presenting innovative 3D food printing systems
based on gelling and/or mixing. The future of extrusion-based 3D food
printing and its applications in the production of customized foods for specific
needs such as controlled nutritional or rheological properties are also dis-
cussed. The sixth chapter reviews the contaminants that may be present in
raw milk, especially conventional and emerging pathogens, antimicrobial-
resistant pathogens and commensal strains found in milk and dairy products,
and biological toxic substances formed by microorganisms like biogenic
amines and mycotoxins. The chapter also reports other chemical contami-
nants such as antibiotic residues, heavy metals, pesticides, polycyclic aromatic
hydrocarbons, melamine, dioxins, polychlorinated biphenyls, plasticizers, and
additives. The seventh chapter deals with the threads of enteric viruses due to
their high capacity for infection and preservation in food environments and
their difficulty for correct and sensitive detection. The chapter discusses the
current efforts toward prioritizing different aspects of their detection, epide-
miology, and control as well as the need to calibrate the current disinfection
procedures in the food industry. The eight and last chapter focuses on the use
of low-energy X-ray irradiation of foods for nonthermal microbial inactiva-
tion. This technology provides a higher microbial inactivation efficacy than
other irradiation techniques, and, therefore, low-energy X-ray irradiation
Preface xiii
constitutes an attractive alternative food preservation technique. Furthermore,

the current applications of low-energy X-ray, consumer acceptance, and
its limitations are discussed in this chapter.
In summary, this volume presents the combined efforts of 27 profes-
sionals with different backgrounds who are developing their research in a
variety of countries like Canada, Brazil, Singapore, Portugal, Greece,
Germany, and Spain. The editor thanks Jhon Michael Peñano (develop-
mental editor), Sam Mahfoudh (acquisition editor), the production staff,
and all the contributors for sharing their experiences and for making this
book possible.
FIDEL TOLDRÁ
Editor
CHAPTER ONE
Peptidomics as a useful tool

in the follow-up of food bioactive
peptides
Fidel Toldrá∗ and Leticia Mora
∗
Corresponding author: e-mail address: ftoldra@iata.csic.es
Contents
1. Introduction 2
2. Peptidomics in the characterization of peptides 3
2.1 Identification of peptides 7
2.2 Quantification of peptides 15
3. In silico approaches 19
4. Mechanisms of enzymatic hydrolysis of food proteins 22
5. Release of bioactive peptides in foods by endogenous peptidases 24
6. Release of bioactive peptides through food proteins hydrolysis 30
7. Conclusions 39
References 39
Abstract
There is an intense research activity on bioactive peptides derived from food proteins in
view of their health benefits for consumers. However, their identification is quite chal-
lenging as a consequence of their small size and low abundance in complex matrices
such as foods or hydrolyzates. Recent advances in peptidomics and bioinformatics are
getting improved sensitivity and accuracy and therefore such tools are contributing to
the development of sophisticated methodologies for the identification and quantifica-
tion of peptides. These developments are very useful for the follow-up of peptides
released through proteolysis either in the food itself through the action of endogenous
peptidases during processing stages like fermentation, drying or ripening, or from food
proteins hydrolyzed by commercial peptidases or microorganisms with proteolytic
activity. This chapter is presenting the latest advances in peptidomics and its use for
the identification and quantification of peptides, and as a useful tool for controlling
the proteolysis phenomena in foods and protein hydrolyzates.
Advances in Food and Nutrition Research, Volume 100 Copyright # 2022 Elsevier Inc. 1
ISSN 1043-4526 All rights reserved.
https://doi.org/10.1016/bs.afnr.2022.03.001
2 Fidel Toldrá and Leticia Mora
1. Introduction
It has been widely reported that food proteins constitute a good source
of bioactive peptides that remain inactive while forming part of the parent
protein, although they may turn active if released through enzymatic hydro-
lysis. Food bioactive peptides may exert health promoting beneficial effects
such as antioxidant, antihypertensive, antiinflammatory, hypoglycemic,
hypocholesterolemic, antimicrobial and antitumor activities (Toldrá,
Reig, Aristoy, & Mora, 2018). Such bioactivity information is available
in open access databases that report data about chemical and structural char-
acteristics of bioactive peptides and protein of origin, its IC50, and references
(Minkiewicz, Iwaniak, & Darewicz, 2019). The beneficial effects of bioac-
tive peptides naturally generated in foods, preventing infection and diseases
is of great interest in an aging population. This makes such foods rich in bio-
active peptides an attractive option in daily diet. Furthermore, bioactive
peptides produced through enzymatic hydrolysis in large amounts make
them attractive as food supplements and as functional components to regu-
late health.
Peptidomics techniques are mainly focused on the study of peptides,
including their identification and quantitation. The target peptides may have
been generated in different ways specially when they come from food matri-
ces (see Fig. 1). So, bioactive peptides may be released during key stages in
FOOD
PEPTIDOMICS PEPTIDES
BIOLOGICAL
SYSTEM
PROTEOMICS Endogenous
PEPTIDOMICS
enzymes
PROTEINS Commercial
enzymes
Gastrointestinal
enzymes
Fig. 1 Scheme of peptides generation from food proteins and main disciplines for the
study of proteins and peptides.
Peptidomics as a useful tool 3
food processing like fermentation, drying, and ripening of meat and dairy
products, wine, sauces among other (Corr^ea et al., 2014; Mohanty,
Mohapatra, Misra, & Sahu, 2016; Mora et al., 2015). Bioactive peptides
may be also produced through controlled enzymatic hydrolysis, with com-
mercial peptidases or microorganisms, of proteins obtained from different
types of food waste and by-products resulting from slaughterhouses, fisher-
ies, whey, fruits peels, etc. (Mora, Reig, & Toldrá, 2014; Ryder, Bekhit,
McConnell, & Carne, 2016), and also hydrolyzates from egg, soybean
and peanut proteins, among other (De Oliveira et al., 2015; Ji, Sun,
Zhao, Xiong, & Sun, 2014). Commercial peptidases are adequate to be
used in food and can be obtained from different origins such as microorgan-
isms, vegetable sources or animal organs. Finally, gastrointestinal digestion
must be taken into account because pepsin in the gastric step and trypsin,
chymotrypsin and pancreatic enzymes in the intestinal step are also able
to release bioactive peptides from the ingested proteins or even inactivate
some peptides due to further hydrolysis (Capriotti et al., 2015; Pepe
et al., 2016). Furthermore, an intense proteolytic activity due to the action
of brush border intestinal epithelium proteases and blood stream enzymes
that complete protein digestion acting as exopeptidases and releasing
dipeptides and free amino acids.
The recent improvements in sensitivity and accuracy achieved in
peptidomics instrumentation and advances in bioinformatics tools are
allowing the development of more sophisticated methodologies for the
identification of peptides. The knowledge of structure and function of
bioactive peptides naturally generated in foods and in hydrolyzates to be
used in food supplements and nutraceuticals, is essential for the optimization
of processing and quality control.
This chapter is presenting the latest advances in peptidomics as a useful
tool for a better understanding and control of the proteolysis phenomena
and therefore as an adequate tool for following the generation of bioactive
peptides and its quantitation in foods and hydrolyzates.
2. Peptidomics in the characterization of peptides

Peptidomics techniques have been successfully used for a better
understanding of gastrointestinal digestion process, the contribution of
endogenous and microbial enzymes in different food processes such as
fermentation (Li & Wang, 2021; Yu, Yu, & Jin, 2021), curing (Gallego,
Processing
Color, texture, flavour,

Biomarkers Quality
taste peptides
Authentification, OGMs,
Safety allergens, pathogensor
toxins
Bioactivities: antioxidant, antihypertensive,

hypoglycemic, hypercholesterolemic, etc.
Bioactives
Structure-activity
Peptidomics prediction
Post-traductional Oxidation, deamidation,

modifications etc.
Gastrointestinal
Intestinal microbiota
digestion
Protein-peptide
interactions
Fig. 2 Major areas of peptidomics applications in foods.
Mora, & Toldrá, 2016), aging (Kominami, Hayashi, Tokihiro, & Ushio,
2021; Renzone, Novi, Scaloni, & Arena, 2021), the identification of bioac-
tive peptides, and the search of peptide markers in food systems (see
Fig. 2). A recent interest in peptidomics is growing in the area of food waste
and by-products valorization because such peptidomics tools contribute to
a better understanding of the proteolysis phenomena and therefore a
better control and steering of the processes (Martini, Solieri, Cattivelli,
Pizzamiglio, & Tagliazucchi, 2021; Martini, Solieri, & Tagliazucchi, 2021).
Peptidomics, in conjunction with other disciplines like proteomics,
genomics, transcriptomics, and metabolomics, contributes to a better
understanding of the food systems. However, there are some difficulties that
make the study of the proteome very complicated such as the low-
abundance of peptides, their heterogeneity and variety in size, charges,
characteristics, and the complexity of biological matrices when they are
distributed. For this reason, a high amount of extraction, separation, isola-
tion, identification, and quantitation techniques have been developed and
used in the last years.
The description of main steps used to simplify, reduce complexity and
eliminate potential interferences in food samples, before the identification
Pretreatment
Pulsed Electric Fields/High
Ultrasounds/Microwaves Acid/base
Hydrostatic Pressure
Extraction
Precipitation/
Solid phase cartridges Acid/water/organic Centrifugation deproteinization
Separation/fractionation
Size-exclusion
Electrophoresis Liquid chromatography
chromatography
Purification
Fractions collection Ultrafiltration
Fig. 3 Description of main steps used to simplify, reduce complexity, and eliminate
potential interferences in food samples before the identification and quantitation of
bioactive peptides.
and quantitation of bioactive peptides is shown in Fig. 3. The pretreatment

step is used to remove potential interferences, as well as to facilitate the
extraction and concentration of peptides. Current tendencies are the use
of green and cost-effective technologies such as ultrasounds, microwaves,
pulsed electric fields, or high hydrostatic pressure, although chemical
pretreatments have been also described to result very effective in the later
extraction of peptides or proteins (Franca-Oliveira, Fornari, & Hernández-
Ledesma, 2021). The extraction procedure of peptides is done using water
or organic solvents depending on the expected characteristics of the generated
peptides. Polar peptides are easily extracted using aqueous and slightly
acidic solvents whereas hydrophobic sequences are extracted with organic
phases. The extraction of peptides may also carry part of protein fraction,
especially when aqueous solvents are used, and therefore a later step of
deproteinization by precipitation and centrifugation is often needed. The next
step is the use of one or a combination of different separation techniques in
order to fractionate the peptide extract at the same time that concentrate
the biologically active fraction. The most used methodologies are Tricine
SDS-PAGE electrophoresis that allows the separation of oligopeptides and
long peptides up to 2500 Da (Sch€agger, 2006) although when the interest
is in the separation of smaller peptides, chromatography techniques are the
method of choice. Size-exclusion chromatography is based on the separation

of peptide fractions according to their molecular weight, and the resolution of
separation depends on particle size, pore size, flow rate, column length and
diameter, and sample volume. Generally, the highest possible resolution is
obtained using moderate flow rates, long, narrow columns, and small particle
size gels, using same eluent in sample and mobile phase. Molecular weight
standards are used in column calibration in order to know the molecular
weights of unknown peptides in the extract. Also high resolution liquid chro-
matography (HPLC) is a frequently used methodology in the separation of
peptides according to their amino acid composition and characteristics (charge,
hydrophobic interaction, and molecular size). In this sense, reversed-phase
followed by hydrophilic interaction chromatography, and ion-exchange chro-
matography are the most used methods for peptide mixtures separations.
Reversed-phase C18 stationary phases are the most popular, and the elution
occurs by increasing the hydrophobicity of the mobile phase. However, this
separation not always results adequate when highly polar and small peptides are
in the mixture, and hydrophilic interaction chromatography (HILIC) have
resulted in very good results to separate small polar compounds on polar
stationary phases. This method can be complementary to reversed-phase as
the order of elution of the peptides are opposite to that found in reversed-
phase. The use of ion-exchange chromatography in the analysis of peptides
is limited to those applications that permit salt elimination in later steps since
moderate concentrations of salts could inhibit the ionization using electrospray
ionization (ESI) prior mass spectrometry analysis. After the chromatographic
separation using HPLC, eluting fractions are collected for subsequent purifi-
cation steps such as ultrafiltration with different molecular cut-off membranes
or to be directly analyzed by mass spectrometry.
Mass spectrometry methodologies have been widely developed during
the last decade and, currently, they have become the best choice for the
identification and quantification of bioactive peptides (Panchaud, Affolter,
& Kussmann, 2012). In this context, the advances in mass spectrometry
applications and the progressive improvement in the resolution of the instru-
ments have increased the development of peptidomics applications in the
food field, especially regarding the identification and quantitation of bioac-
tive peptides. Also the latest developments in bioinformatics are necessary to
obtain the maximum benefit from the generated data, including the use of
in silico tools, structure–activity relationship models, chemometrics, protein
and peptide databases and search engines, and statistics to manage the
enormous amount of data generated.
2.1 Identification of peptides

Traditionally, proteomics has been used to identify and quantify proteins
from different types of food matrices, obtaining the characterization of
the protein profile of the sample. The most frequently used strategy has
been the separation of proteins using gel electrophoresis and the subsequent
digestion of the isolated proteins with proteases with cleavage sites included
in the databases such as trypsin, Arg-C or Asp-N. The obtained hydrolyzates
are analyzed by mass spectrometry using specialized tools to obtain a list of
peaks to be processed using protein databases. Main approaches for the iden-
tification of proteins are based on Peptide Mass Fingerprint (PMF), where
the experimental data are a list of peptide mass values from the digestion of a
protein by a specific enzyme as previously indicated, and on MS/MS ions
search, where the identification is based on raw MS/MS data from one
or more peptides. Despite these approaches are very useful for the identifi-
cation of the presence of certain proteins, the experimental design needs
to be adapted when the objective is to analyze bioactive peptides obtained
from either natural processes such as fermentation, curing, or gastrointes-
tinal digestion, or commercial hydrolysis such as the treatment of food
by-products.
Bioactive peptides can exert different type of bioactivities depending on
different factors such as their physico-chemical characteristics, amino
acids sequence, structural conformation, etc. In fact, bioactive peptides have
been described to be able to modify or modulate different physiological sys-
tems such as the cardiovascular, digestive, endocrine, immune, muscular,
nervous, renal, reproductive, respiratory, and skeletal systems, due to their
potential to exert antihypertensive, antidiabetic, anticholesterolemic, anti-
oxidant, immunomodulatory, among other activities. Fig. 4 shows the main
Others
Opioid ACE inhibitor
Celiac toxic
DPP IV
inhibitor
Antioxidant
Antimicrobial
Fig. 4 Diagram showing main percentages of the most reported bioactive peptides
available in BIOPEP database; accessed on Feb, 2022 (Minkiewicz et al., 2019).
bioactivities studied according to BIOPEP database (Minkiewicz et al.,

2019), showing ACE-inhibitory activity as the most reported biological
activity followed by antioxidant and antimicrobial activity.
These peptides are very different and can show different characteristics
such as charge state, hydrophobicity or length, including large, medium,
and small peptides. However, small bioactive peptides (from 2 to 10 amino
acids length) represent by far the largest category as indicated in Fig. 5
(obtained from BIOPEP bioactive peptides database), probably due to their
small size makes them more adequate to cross the intestinal barrier and
achieve intact the blood stream and target organs to develop their biological
activity. Thus, despite the numerous difficulties that involve the analysis of
small peptides, more than half of the peptides described in the literature
and included in the BIOPEP database (Minkiewicz et al., 2019) are smaller
than 6 amino acids length (see Fig. 5).
The strategy for the identification of bioactive peptides will be different
considering the different physical and chemical properties of target peptides.
In this sense, separation methods used in mass spectrometry instruments and
informatics analysis will depend on the characteristics and size of peptides,
being in the interface between proteomics and metabolomics approaches.
In this regards, data analysis results very challenging as there is an estimation
of false positives 100 times higher for peptidomics than for proteomics
because peptidomics relies on a single peptide identification and requires
very high quality spectra and frequent de novo identification.
2.1.1 Identification of di-, tri-, and tetrapeptides

Very small peptides are of particular interest to be used as bioactive peptides,
but the analytical strategy for their identification must be carefully designed
Fig. 5 Number of amino acids of the bioactive peptides available in BIOPEP database;
accessed on 17 Feb, 2022 (Minkiewicz et al., 2019).
considering that the analysis of these small peptides are between the analysis
of peptides and small molecules.
First steps of isolation and purification need to be planned in order to
avoid losses in part of the target peptides. In fact, whereas some of these
peptides contain hydrophobic amino acids and are properly retained in
C18 columns, others are mainly hydrophilic and need other types of station-
ary phases to be retained. Thus, liquid chromatographic separation of
peptides before their injection into the mass spectrometry analysis
should have taken into consideration the physico-chemical characteristics
of the expected peptides, the potential mixture of different small peptides
in the sample extract, and the choice of the most appropriate separation
techniques, for instance, a combination of different separation techniques
such as reversed phase and hydrophilic interaction chromatography.
Concentration steps are necessary to increase the abundance of these pep-
tides that frequently found at very low concentration, but this step requires
special attention because it may often lead to the loss of some of the
sequences in the washing/cleaning step.
Once retention and isolation is achieved, the analysis of peptides by
mass spectrometry is another very critical step. The selection of suitable mass
analyzer depends on the applications of interest. When analyzing samples
with complex matrices, a quadrupole would be useful to reduce the inter-
ferences from the matrices. For further quantitative analysis of trace compo-
nents in complex samples, QqQ or Q-Orbitrap would be a good choice due
to their high sensitivity and quantitative ability. Orbitrap is also used to
obtain the accurate mass of the targeted molecules, which reveals the
elemental compositions of the molecules (Chang et al., 2021). In the iden-
tification of di- and tripeptides, it is necessary to use high mass accuracy and
high resolution instruments such as Quadrupole Time-of-Flight (Q-ToF) or
Triple Quadrupole (QQQ) analyzers, although QTrap can realize MSn
analysis, which is beneficial to qualitative analysis. In this regards, the main
difficulty is the optimization of the collision energy necessary for the frag-
mentation of the di- and tripeptides. In longer peptides, it is possible to get
several ion fragments and differentiate the precursor, whereas in small
peptides the amounts and quality of fragments are lower.
In fact, in the identification of dipeptides by mass spectrometry it is nec-
essary to consider retention time, precursor mass, and ion fragments in order
to establish an adequate correlation between this information and the
sequence of the peptide. In this sense, most studies focused on the identifi-
cation of small di and tripeptides also cover the quantitation of the
molecules.
Dipeptide sequences can be repeated many times in protein sequences so

they are not unique. For this reason, dipeptides identification by matching
the obtained spectrum with protein databases such as UniProt and NCBInr
is not feasible. Furthermore, current search algorithms are often limited to
peptides containing 4 or more amino acids, as smaller peptides would result
in unacceptable scores because the search engines are not prepared for
this use (Tang, Li, Lin, & Li, 2014). Thus, de novo identification of the
sequences by highly-experienced personnel is frequently needed, which
can be difficult and time-consuming, mostly for untargeted analysis
(Mora et al., 2017). However, this has been the method of choice of many
researchers although a limited amount of spectra can be analyzed.
The main proteomic approach used for the identification and quantita-
tion of small peptides is the Multiple Reaction Monitoring (MRM) using
QQQ instruments. Quadrupole is often placed in front of other mass
analyzers to pre-select targeted ions. Interferences from matrix ions are
removed, significantly reducing chemical noises. In general, QqQ mass
spectrometer has the highest sensitivity. The QqQ is the most commonly
used mass analyzer for quantitative detection due to its higher accuracy.
MRM also presents certain difficulties, as it needs to be previously opti-
mized, and requires trained personnel and expensive instruments.
On the other hand, mass spectrometry approaches based on matrix-
assisted laser desorption/ionization (MALDI) mass spectrometry have the
advantage of convenience, speed, and accuracy, but also present good
resolution, robustness, and sensitivity. Relative and absolute quantification
can be performed using MALDI approaches based on mass spectrometry in
tandem (MS/MS) (Wang & Giese, 2017). However, this methodology is
not very popular for the identification of small peptides mainly due to the
potential interferences of low-molecular peptides with MALDI matrices
used for ionization although the high sensitivity of MALDI-MS and its
tolerance against interference by salts and other components allowed for
the detection of native peptides from complex biological mixtures with
reduced sample preparation efforts. Recently, a MALDI Time-of-Flight
(ToF) approach was developed to achieve the fast detection of dipeptides
in dry-cured ham peptide extracts with the advantage of using very low
amounts of sample, while reducing time and cost (Heres, Saldaña,
Toldrá, & Mora, 2021; Heres, Yokoyama, et al., 2021). The characteristic
matrix ions appearing between 200 and 300 m/z were eliminated by sub-
traction using bioinformatics tools, and an example of MALDI-ToF MS
spectra is shown in Fig. 6. Later, the identification of the dipeptides was
confirmed by QQQ (Fig. 7).
Standard Mix
35000
a.i.
Standard Mix
30000
25000
AH VF
DD
20000
AL
EV
15000
10000
5000
190 200 210 220 230 240 250 260 270 280
m/z
Fig. 6 MALDI-ToF MS spectra of peptide mixture (Mix). Peptide sequences are given as
amino acid one-letter code. Source: Reproduced from Alejandro Heres, Celia Saldaña, Fidel
Toldrá, Leticia Mora, (2021). Identification of dipeptides by MALDI-ToF mass spectrometry in
long-processing Spanish dry-cured ham, Food Chemistry: Molecular Sciences, 3, 100048,
with permission from Elsevier.
2.1.2 Pentapeptides or longer peptides identification

Bioactive peptides between 5 and 30 amino acids length can be generated
either naturally due to the action of endogenous/digestive enzymes or
due to the action of commercial enzymes. The extraction and purification
strategy will depend on the characteristics of the peptides of interest and it
would be optimized according to their physico-chemical profile although
the use of C18 reversed phase columns is the most popular separation
methodology used in this type of peptides (see an example in Fig. 8).
However, whereas peptides obtained from the hydrolysis with commer-
cial enzymes can be more abundant due to hydrolysis conditions and have
been optimized to obtain a high recovery in bioactive peptides, those pep-
tides naturally generated by endogenous or gastrointestinal enzymes will
be at very low concentrations within complex biological matrices, and
therefore the analytical methods used for their identification have to be very
sensitive and rely on a specific sample preparation strategy. Regarding the
identification by mass spectrometry in tandem, traditional proteomics pro-
cedures could be used since the size of the peptides is similar to those sizes
obtained in traditional proteomics protocols using trypsin but with a certain
Fig. 7 ESI-QQQ spectra of the dipeptides AH, AL, DD, EV and VF identified in 18 months
dry-cured ham extracts. Source: Reproduced from Alejandro Heres, Celia Saldaña, Fidel
Toldrá, Leticia Mora, (2021). Identification of dipeptides by MALDI-ToF mass spectrometry
in long-processing Spanish dry-cured ham, Food Chemistry: Molecular Sciences, 3, 100048,
with permission from Elsevier.
adjustment of the basic techniques in order to identify a larger number of

endogenous peptides. Best option for the identification of these peptides
is the use of tandem mass spectrometry instruments with high resolution.
Ionization sources used are MALDI or ESI that are usually coupled to tan-
dem mass spectrometers such as quadrupole-ion trap, commonly used to
Fig. 8 Reversed-phase chromatographic separation after in vitro gastrointestinal diges-

tion of an Alcalase hydrolysate of orange by-products for the production of bioactive
peptides after gastrointestinal digestion. The figure shows the fractions from 44 to
54 obtained after SEC separation. The fractions were automatically collected and
assayed for their DPPH scavenging activity (A), ferric reducing capacity (B), ACEI-
inhibitory activity (C) α-amylase inhibitory activity. Reproduced from Mazloomi, S.N.;
Mahoonak, A.S.; Mora, L.; Ghorbani, M.; Houshmand, G.; Toldrá, F. Pepsin Hydrolysis of
Orange By-Products for the Production of Bioactive Peptides with Gastrointestinal
Resistant Properties. Foods 2021, 10, 679.
conduct MSn fragmentation of the parent peptides, quadrupole/time-of-

flight (Q/ToF), or time-of-flight/ time-of-flight (ToF/ToF) (Panchaud
et al., 2012). In this regards, Fig. 9 shows the distribution of peptides iden-
tified in dry-cured ham before and after gastrointestinal digestion according
to their protein of origin. In this example, chromatographic and mass
A) B)
Fig. 9 Distribution of the peptides identified by nLC-MS/MS according to their origin proteins in (A) undigested and (B) digested dry-cured
ham samples simulating in vitro gastrointestinal digestion. The identification of peptides was performed by nanoliquid
chromatography-tandem mass spectrometry (nLC-MS/MS) using a Nano-LC Ultra 1D Plus system (Eksigent of AB Sciex, CA, USA) coupled
to the quadrupole/time-of-flight (Q/ToF) TripleTOF® 5600+ system (AB Sciex Instruments, MA, USA) with a nanoelectrospray ionization
source (nESI). Reproduced from Gallego, M., Mauri, L., Aristoy, MC., Toldrá, F., Mora, L. (2020). Antioxidant peptides profile in dry-cured ham
as affected by gastrointestinal digestion, Journal of Functional Foods, 69, 103956, with permission from Elsevier.
spectrometry analysis revealed changes in the peptide profiles and evidenced

the degradation of proteins, mainly titin, by the digestive enzymes (Gallego,
Mauri, Aristoy, Toldrá, & Mora, 2020).
The data analysis approach has to be adapted according to the origin of
the peptides. Current search engines such as Mascot has been designed to
work with spectra data corresponding to peptides of sizes longer than 4
amino acids. However, mass spectrometry instrument parameters, peak list
parameters, and database search parameters have to be carefully selected in
order to get the maximum amount of identifications with high score. The
cleavage sites of bioactive peptides are not included in any of the descriptions
included in the search engines so frequently the choose of no-enzyme is
the unique option. This fact increases the search times and decreases the
specificity in the identification but good results could be obtained if all
parameters are well adjusted. It is recommended a previous optimization
of the parameters using a controlled protein digestion such as BSA. In order
to validate automatically obtained results using a search engine like Mascot
(https://www.matrixscience.com/search_form_select.html), several tools
have been developed such as the repetition of the search using identical
search parameters, against a database in which the sequences have been
reversed or randomized. Then, it is not expected to get any true matches
from the “decoy” database so the number of matches that are found is an
excellent estimation of the number of false positives that are present in
the results from the real or “target” database (Chen, Zhang, Xing, &
Zhao, 2009; Elias, Haas, Faherty, & Gygi, 2005).
2.2 Quantification of peptides

There is a lack of information about the concentration of the bioactive pep-
tides generated during food processing, the amount of peptides bioavailable
in the bloodstream after gastrointestinal digestion, and even the necessary
concentrations to establish an adequate dose to be used as ingredients. In fact,
the quantification of bioactive peptides faces up to several difficulties due to
their small molecular size, that is between metabolomics and proteomics dis-
ciplines, and the complexity of food matrices (Fig. 10). Thus, the use of
advanced mass spectrometry techniques is critical to identify the sequence
of these small peptides and to develop fast, precise and sensitive metho-
dologies for their quantitation. In peptidomics, the study of peptides often
includes their relative quantification with labeled or label-free methods.
The relative quantification of peptides is focused on the assessment of
peptide amounts in two or more samples, and the results are given by the
comparison of these amounts between samples. These methodologies
Fig. 10 Proteomics vs peptidomics. Main differences are in the generation and identi-
fication of peptides. Different approaches are needed when objectives are the identi-
fication of protein biomarkers and the identification of protein-derived bioactive
peptides. Reproduced from Mora, L., Gallego, M., Reig, M., Toldrá, F. (2017). Challenges
in the quantitation of naturally generated bioactive peptides in processed meats, Trends
in Food Science & Technology, 69, Part B, 306–314, with permission from Elsevier.
provide a simple, reliable, versatile, and cost-effective quantification that can

be based on peak intensity measurements or spectral counting (Bantscheff,
Schirle, Sweetman, Rick, & Kuster, 2007; Zhu, Smith, & Huang, 2010).
These approaches result very interesting in the comparison of peptides
profiles between different hydrolyzates as well as to elucidate the main pro-
teins responsible for the generation of bioactive peptides. In this regards,
Fig. 11 shows the results of a PCA analysis of the peptides profile obtained
from dry-cured ham by-products cooked and digested, and this label-free
analysis allowed the identification and relative quantitation of those peptides
responsible for the antioxidant activity detected in samples after 1 h of
cooking, which were collagen peptides (Gallego, Mora, Hayes, Reig, &
Toldrá, 2017).
On the other hand, other approaches such as multiple reaction monitor-
ing (MRM) provide the most accurate quantitative values for bioactive pep-
tides, but they require complex experimental protocols. MRM is a highly
sensitive and selective method of directed MS that uses three quadrupoles
for the analysis of short sequences and low concentration peptides in com-
plex mixtures (Hu et al., 2011; Nakashima et al., 2011; Priyanto et al., 2015;
Rawendra et al., 2014). Main important challenge of this procedure is the
Fig. 11 (A) Principal Component Analysis (PCA) score plot to assess the variance among
all the peptides of digested samples H2O, 100 °C 20 min, and 100 °C 1 h in three repli-
cates (n ¼ 3). Discriminant component 1 (t[1]) and discriminant component 2 (t[2])
explained a 52.9% and 20% of variability in the dataset, respectively. (B) PCA loading
plot showing the proteins of origin of those peptides more responsible for main differ-
ences between the uncooked and cooked samples after digestion. Reproduced from
Gallego, Mora, Hayes, Reig, Toldrá, Effect of cooking and in vitro digestion on the antiox-
idant activity of dry-cured ham by-products, Food Research International, 97, 2017,
296–306, with permission from Elsevier.
Fig. 12 Graphical abstract including the whole procedure for the identification, quan-
titation and confirmation of Ala-Ala dipeptide as antihypertensive peptide. Reproduced
from Heres, Yokoyama, Gallego, Toldrá, Arihara, Mora, (2021) Antihypertensive potential
of sweet Ala-Ala dipeptide and its quantitation in dry-cured ham at different processing
conditions. Journal of Functional Foods, 87, 104818, with permission from Elsevier.
optimization of the MRM parameters of low abundant peptides or small

size peptides to get a final accurate and sensitive quantification without inter-
ferences from other peptides or signal suppression (Capriotti, Cavaliere,
Piovesana, Samperi, & Lagana, 2016). Fig. 12 includes main results obtained
after the quantification of the antihypertensive dipeptide AA at different

times of processing of dry-cured ham using a triple quadrupole instrument.
3. In silico approaches
An example of a classical empirical approach to identify and confirm
the presence of bioactive peptides is shown in Fig. 13. This includes the
hydrolysis step to obtain bioactive peptides followed by their extraction
and later chromatographic separation. Frequently, several chromatographic
techniques are used for a better isolation of the peptides of interest. In fact,
the previous step to mass spectrometry identification usually consists of
liquid chromatography separation of the already isolated peptides. After
identification by mass spectrometry, those peptide sequences apparently
responsible for the biological activities are synthesized and the bioactivity
is confirmed in vitro and in vivo.
Protein of origin Food matrix

Hydrolysis Extraction
Peptides
1st fractionation
Isolation of bioactive fractions (SEC, CE, LC, GF-IEF,...)
In vitro test
2nd fractionation
Purification of peptides of interest (HPLC)
In vitro test
Identification by MS/MS
In vitro test
Synthesis of peptides
Bioactivity test in vitro and in vivo

Confirmation
Fig. 13 Scheme of the traditional empirical procedure for the identification and confir-
mation of bioactive peptides from food matrices. SEC, size-exclusion chromatography;
CE, capillary electrophoresis; LC, liquid chromatography; IEF, isoelectric focusing; HPLC,
high-performance liquid chromatography; MS/MS, mass spectrometry in tandem.
Reproduced from Mora, Gallego, Toldrá, F. (2018) ACEI-Inhibitory Peptides Naturally
Generated in Meat and Meat Products and Their Health Relevance. Nutrients, 10, 1259.
Despite this procedure is very effective, it results very expensive and

time-consuming. For this reason, modern in silico strategies based on simu-
lation using bioinformatics tools in most of the steps and peptide databases
are of high interest nowadays (see Fig. 14).
There are different tools that comprise from the selection of the protein
by determining the occurrence frequency of bioactive sequences in the pro-
tein in order to get bioactive peptide sequences after hydrolysis such as
PATTINPROT from PBIL server (https://npsa-prabi.ibcp.fr/cgi-bin/) or
BIOPEP (https://biochemia.uwm.edu.pl/). In this sense, BIOPEP database
contains a tool that permits the scanning of a protein sequence searching for
one or several patterns previously established to show the potential of the
protein to be a source of bioactive peptides (A) and the calculation of
the potential biological activity of each protein (B). The frequency of bio-
active fragments occurrence in protein sequence (A) is calculated using the
formula:
a
A¼
N:
where a is the number of fragments with given activity in a protein sequence,

and N is the number of amino acid residues of protein.
Fig. 14 Main steps of in silico approaches and open access databases for the selection of
the protein, hydrolysis simulation and bioactivity prediction. Reproduced from Mora,
Gallego, Toldrá, F. (2018) ACEI-Inhibitory Peptides Naturally Generated in Meat and
Meat Products and Their Health Relevance. Nutrients, 10, 1259.
The potential biological activity of protein (B) [μM1] is calculated with

the formula:
i
Σki¼1 ECa 50i
B¼
N
where ai is the number of repetitions of i-th bioactive fragment in protein

sequence, EC50i is the concentration of i-th bioactive peptide corresponding
to its half-maximal activity [μM] or half-maximal inhibition (IC50) in case of
peptides with inhibitory activity, k is the number of different fragments
with given activity, and N is the number of amino acid residues.
On the other hand, there are different tools designed to obtain theoret-
ical fragments from protein sequences by using enzymes with known
cleavage specificities. The hydrolysis of the selected protein is simulated
to generate in silico peptide profiles using bioinformatics tools such as
BIOPEP, where it is possible to simulate the digestion with up to three dif-
ferent enzymes simultaneously; PeptideMass from ExPASy (Gasteiger et al.,
2005; Wilkins et al., 1997), very intuitive and extended; or PoPS (Boyd,
Garcia de la Banda, Pike, Whisstock, & Rudy, 2004), a tool that allows
the prediction of substrate cleavage using protease specificity models that
have to be uploaded by the user.
However, the prediction when using commercial enzymes such as
Alcalase, Protamex, Neutrase, etc. is more complicated because they are
broad-spectrum endopeptidases. In this cases, the hydrolysis pattern of these
enzyme needs to be characterized in the laboratory to determine the poten-
tial fragments that would be obtained after optimal conditions of hydrolysis.
The online tool EnzymePredictor (http://bioware.ucd.ie/enzpred/
Enzpred.php) evaluates the evidence for which enzymes are most likely
to have cleaved a sample containing peptides from hydrolyzed proteins,
which would result very useful to drive hydrolysis processes toward the gen-
eration of certain specific bioactive peptides (Dalkiran et al., 2018). Also the
tool Proteasix is a web-based, peptide-centric tool (http://proteasix.cs.man.
ac.uk/index.html) dedicated to proteolytic events involved in naturally
occurring peptide generation. Proteasix takes a peptide list from the user
and then automatically reconstructs the N- and C-terminal cleavage sites
and identifies the observed and predicted proteases involved in the proteol-
ysis of these cleavage sites in three main species: Homo sapiens, Mus musculus
and Rattus norvegicus. This tool will allow to determine the potential enzymes
that are responsible for the generation of certain cleavage sites and results
very useful in the characterization of commercial enzymes main actions.
Once the theoretical peptide sequences are known, they can be analyzed
to identify desirable amino acids at certain position or interesting character-
istics that make them potential candidates to exert bioactivity by using
the software PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/),
which gives a list of scores that identifies those peptides that may be more
likely to be bioactive. This predictive tool is based on such general shared
features of bioactive peptides across different functional classes and aids in
the improved design of existing bioactive peptides (Mooney, Haslam,
Pollastri, & Shields, 2012).
Also there are many tools that predict structure and potential biological
activities. In this sense, some authors have reported the optimization of
Quantitative Structure–Activity Relationship (QSAR) models for revealing
relationships between structural properties of chemical compounds and bio-
logical activities. SAR modeling is essential for drug discovery but it is also
being used in bioactive peptides characterization (Kwon, Bae, Jo, & Yoon,
2019). Finally, the prediction of the three-dimensional structure of peptides
from their amino acid sequence and post-transductional modifications is
very important in peptidomics because the structure of the peptide can affect
its functionality. Thus, on-line tools such as PEPstrMOD (http://osddlinux.
osdd.net/raghava/pepstrmod/) (Singh et al., 2015) or PEP-FOLD (http://
bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/) for long peptides,
allow the de novo prediction of multiple peptide structures for linear and
cyclic peptides (Lamiable et al., 2016; Shen, Maupetit, Derreumaux, &
Tuffery, 2014; Thevenet et al., 2012).
4. Mechanisms of enzymatic hydrolysis of food proteins

Proteolysis phenomena occurs in proteins that are hydrolyzed during
food fermentation and/or ripening. Endo- and exo-peptidases are the main
enzymes responsible for proteolysis and can be present in the food as either
endogenous enzymes or as microbial enzymes from the microorganisms
responsible for fermentation.
Proteins are first broken down by endopeptidases releasing protein frag-
ments and polypeptides. When such polypeptides are further fragmented
into smaller peptides, they act as substrates for further hydrolytic action
by exo-peptidases that will generate smaller tri- and dipeptides and free
amino acids (Toldrá et al., 2018). In this sense, peptidomics constitutes a
basic tool for the identification and quantification of the released peptides
and therefore the follow-up of the resulting peptide profiles (Mora
et al., 2017).
A scheme of how peptidases act on proteins is shown in Fig. 15. In such
example, endo-peptidases act on an internal linkage Phe-Pro (Mora,
Gallego, & Toldrá, 2019). The exo-peptidases may act on either the amino
or carboxy terminal. If a tripeptide is released, then they are named
tripeptidylpeptidases (TPP) and if it is a dipeptide, dipeptidylpeptidases
(DPP). For instance, X-prolyl dipeptidyl peptidase (PepX) releases dipep-
tides X-proline from the amino terminal. Tripeptidases may hydrolyze a
tripeptide into a dipeptide and a single amino acid while dipeptidases hydro-
lyze dipeptides into its two single amino acids (Toldrá, Gallego, Reig,
Aristoy, & Mora, 2020a). However, the major release of free amino acids
is caused by aminopeptidases (i.e., Pep N, Pep A, Pep C, Pep P or others)
acting on the amino terminal or by carboxypeptidases (A or B) acting on the
carboxy terminal (Mora, Gallego, Aristoy, & Toldrá, 2015).
The generation of bioactive peptides can take place in the food itself as
a consequence of the action of endogenous enzymes and autochthon-
ous microorganisms, and also during gastrointestinal digestion due to brush
A D E C C P C C
Thr Val Lys Glu Asp Gln Val Phe Pro Met Asn Pro Pro Lys Phe Asp Lys Ile Glu Asp
TVKEDQVFPMNPPKFDKIED
PPKFDKIED
TVKEDQVFPMNPPKFDKIED
VKEDQVFPMNPPKFDKIED
EDQVFPMNPPKFDKIED
VKEDQVFPMNPPKFDKIED
VKEDQVFPMNPPKFDKIE
VKEDQVFPMNPPKFDKI
TVKEDQVFPMNPPKFD
TVKEDQVFPMNPPK
TVKEDQVFPMNPP
Fig. 15 Scheme of food protein hydrolysis and enzymes involved. The

amino acids sequence is a fragment belonging to myosin heavy chain.
Aminopeptidase (A), Dipeptidylpeptidase (D), Endopeptidase (E), Carboxypeptidase
(C) and Peptidylpeptidase (P). Reproduced from Toldrá, F., Gallego, M., Reig, M.,
Aristoy, M.-C., Mora, L. (2020a). Recent progress in enzymatic release of food-derived
peptides and assessment of bioactivity. Journal of Agricultural & Food Chemistry, 68,
12842–12855, with permission from ACS.
Proteins in foods Isolated food proteins
Endogenous peptidases Microbial peptidases Peptidases Microbial peptidases
Ripening Fermentation Reactor Fermenter
Small amounts of bioactive Large amounts of bioactive

peptides within the food peptides as extracts
Gastrointestinal digestion
Absorption in the organism

Resistance to serum peptidases
Bioactive peptides
Physiological functions
Fig. 16 Scheme of the generation of bioactive peptides from protein hydrolysis in foods
and/or the hydrolysis of isolated food proteins. Reproduced from Toldrá, F., Reig, M.,
Aristoy, M.C., Mora, L. (2018). Generation of bioactive peptides during food processing.
Food Chemistry, 267, 395–404, with permission from Elsevier.
border intestinal epithelium proteases and blood stream enzymes that

complete protein digestion acting as exopeptidases. It can also be carried
out on food proteins previously extracted from foods or food by-products
and hydrolyzed with commercial peptidases (see Fig. 16). Both cases are
described below.
5. Release of bioactive peptides in foods by endogenous

peptidases
Food proteins may be hydrolyzed by endogenous or microbial pepti-
dases during ripening and/or fermentation (see Fig. 16). The result of such
proteolysis is the generation of numerous peptides and some of them may be
bioactive. Of course, such peptides must keep the bioactivity after the gas-
trointestinal digestion and absorption through the intestinal membrane in
order to exert its physiological benefit in the body (Gallego, Grootaert,
et al., 2016).
The action of endogenous peptidases is typical in long ripened foods like
meat and dairy products. For instance, in the case of meat products there
are several muscle endopeptidases, such as calpains and cathepsins, able to
hydrolyze myofibrillar and sarcoplasmic proteins into large protein

fragments and polypeptides. Such polypeptides constitute the substrates
for further hydrolysis by muscle exopeptidases such as tri- and
dipeptidylpeptidases, amino and carboxypeptidases (Mora, Escudero,
Arihara, & Toldrá, 2015; Mora, Sentandreu, & Toldrá, 2011; Toldrá,
Aristoy, & Flores, 2000) although such enzymes may be partly inhibited
by salt added during processing (Toldrá, Cerveró, & Part, 1993).
DPP I and II that are active at pH 5.5–6.5, near the pH found in most
meat products, can release dipeptides such as AG, AQ, RG, NP, PA, IL, SG,
SQ, among other from the N-terminal. TPP I, also active at pH 5.5–6.5,
releases specific tripeptides like RGA, IIP, GNP, GAG, GPG from the
N-terminal. As a result of dipeptidylpeptidases action during the processing
of dry-cured ham, dipeptides PA, GA, VG, EE, ES, DA, and DG were
reported to be generated (see Table 1), most of them with ACE inhibitory
activity and released in amounts ranging from 2 to 45 μg/g in dry-cured
ham (Heres et al., 2022). Dipeptides have important advantages for
biodisponibility and therefore are able to reach target organs because they
have small size and good resistance to further peptidase hydrolysis during
gastrointestinal digestion, intestinal absorption and circulation in the blood-
stream (Pentzien & Meisel, 2008).
Meat products like dry-cured ham, that have long processing times
like 9–12 months or longer, present numerous bioactive peptides
(Gallego et al., 2019; Toldrá, Gallego, Reig, Aristoy, & Mora, 2020b).
So, several ACE inhibitory peptides like AAATP and TLKRVP were iso-
lated from Spanish ham (Escudero et al., 2013; Gallego, Mora, & Toldrá,
2018), while peptides LGL, GVVPL and SFVIT were isolated from
Parma ham (Dellafiora et al., 2015). Numerous antioxidant peptides have
been also reported like GKFNV, FLKMN, and LPGGGHGNL in Jinhua
ham (Zhu et al., 2016), DLEE in Xuanwei ham (Xing et al., 2016),
AEEEYPDL and SNAAC in Spanish ham (Gallego, Mora, Reig, &
Toldrá, 2018; Gallego, Mora, & Toldrá, 2018). It is also noteworthy
the DPP IV inhibitory activity of some peptides isolated from Spanish
ham such as AAAAG, ALGGA and LVSGM (Gallego et al., 2014). An
extract with 36 peptide sequences from Xuanwei dry-cured ham was
reported exerted anti-inflammatory effect in RAW264.7 macrophage
cells, suppressing the secretion of nitric oxide, interleukin-6 and tumor
necrosis factor-α in the lipopolysaccharide peptides GPAGPL and
GPPGAP were those exhibiting the highest anti-inflammatory activity
(Fu et al., 2021).
Table 1 Examples of peptides released in foods during processing through the action of endogenous enzymes and microorganisms.
Food Type/Fermentation Peptide sequences Potential activity References
Dry-cured Chinese Jinhua FLKMN, GKFNV, Antioxidant Zhu, Zhang, Zhou, and Xu
ham LPGGGHGDL (2016)
Chinese Xuanwei DLEE Antioxidant Xing et al. (2016)
GPAGPL, GPPGAP Antiinflammatory Fu et al. (2021)
Italian Parma GVVPL, LGL, SFVIT ACE inhibitory Dellafiora et al. (2015)
Spanish SNAAC, AEEEYPDL Antioxidant Gallego, Mora, Reig, and
Toldrá (2018) and Gallego,
Mora, and Toldrá (2018)
FNMPLTIRITPGSKA Antiinflammatory, Gallego, Mora, and Toldrá
ACE inhibitory (2019)
HCNKKYRSEM, Antimicrobial, Castellano et al. (2016), Gallego
MDPKYR antiinflammatory, et al. (2019)
antioxidant, ACE
inhibitory
TKYRVP, Antiinflammatory, Gallego et al. (2019)
TSNRYHSYPWG antioxidant, ACE
inhibitory
AAATP ACE inhibitory Escudero et al. (2013)
AAAAG, ALGGA, LVSGM DPP IV inhibitory Gallego, Aristoy, and Toldrá
(2014)
PA, GA, VG, ES, DA ACE inhibitory Heres, Saldaña, Toldrá, and
Mora (2022)
Mutton ham MWTD, APYMM, FWIIE Antioxidant Wang, Lu, Li, Wang, and Huang
(2020)
Fermented Koji (Aspergillus, 10%, 30 °C, 24w) QYP Antioxidant Ohata, Uchida, Zhou, and
meat sauce Arihara (2016)
Cheese Italian Stracchino AVPYPQ, EAMAPK Antioxidant Pepe et al. (2016)
Parma cheese VPP, IPP, LHLPLP, HLPLP, ACE inhibitory Solieri et al. (2020)
ENLLRF
HLPLP, LHLPLP ACE inhibitory Basiricò et al. (2015)
APFPE DPP IV inhibitory Martini, Solieri, Cattivelli, et al.
(2021) and Martini, Solieri, and
Tagliazucchi (2021)
VPP, IPP, RYLGY, RYLG, ACE inhibitory Basiricò et al. (2015)
AYFYPEL, AYFYPE,
LHLPLP, and HLPLP
Brazilian Canastra artisanal Minas RPKHPIKHQG, Antimicrobial Fialho et al. (2018)
RPKHPIKHQ
Hard cow milk cheese EIVPN Antioxidant Timon, Andres, Otte, and
Petron (2019)
DKIHPF Antioxidant Timon et al. (2019)
VAPFPQ Antioxidant Timon et al. (2019)
Brazilian Prato/Lactobacillus helveticus QEPVLGPVRGPFPIIV ACE inhibitory Baptista et al. (2018)
(10%, 40 °C, 18 h)
YQEPVLGPVRGPFP ACE inhibitory Baptista et al. (2018)
Yoghurt Chinese Feng Wei Suan Ru/ FVAPFPEVF, PPFLQPEVM Antidiabetic, ACE Jin et al. (2016)
Streptococcus thermophellolus inhibitory
+ Lactobacilus bulgaricus
QEPVLGPVRGPFPIIV ACE inhibitory Jin et al. (2016)
Continued
Table 1 Examples of peptides released in foods during processing through the action of endogenous enzymes and microorganisms.—cont’d
Food Type/Fermentation Peptide sequences Potential activity References
Probiotic yoghurt with pineapple SLPQNIPPLTQTPVVVPPF Antioxidant, anticancer Sah, Vasiljevic, McKechnie, and
peel/S. thermophilus + L. bulgaricus Donkor (2016)
+ L. acidophilus + L. casei + L. paracasei
YQEPVLGPVRGPFPIIV Antioxidant, anticancer Sah et al. (2016)
(1%, 42 °C, pH 4.5)
Fermented Lactobacillus, Saccharomyces IPP, VPP ACE inhibitory Fekete, Givens, and Lovegrove
milk (2015)
Antiinflammatory, Chakrabarti and Wu (2015)
adipogneic
Antidiabetic Chakrabarti, Jahandideh,
Davidge, and Wu (2018)
Kluyveromyces marxianus (6%, 32 °C, LRFF, VLSRYP ACE inhibitory Li, Sadiq, Liu, Chen, and He
pH 6.5, 48 h) (2015)
Kombucha culture (1%, 37 °C, 72 h) FVAPEPFVFGKEK, ACE inhibitory Elkhtab, El-Alfy, Shenana,
LVYPFPGPLH, Mohamed, and Yousef (2017)
VAPFPEVFGK
L. actobacillus casei (1%, 37 °C, 72 h) LVESPPELNTVQ, ACE inhibitory Elkhtab et al. (2017)
VLESPPELN,
WGYLAYGLD
Fermented Lactobacillus pentosus (28 °C, 43d) IPP, KP, LPP, VPP ACE inhibitory Fideler, Johanningsmeier,
cucumber Ekel€
of, and Muddiman, 2019)
pickles
Fermented fish Malaysian pekasam/Lactobacillus AIPPHPYP, IAEVFLITDPK Antioxidant Najafian and Babji (2018)
plantarum (27 °C, 15d)
Fermented Thai Kapi Ta Dam IF, SV ACE inhibitory Kleekayai et al. (2015)
shrimp pastes
Thai Kapi Ta Dam, Kapi Ta Deang WP Antioxidant Kleekayai et al. (2015)
The use of starter cultures is an extended practice today for most fer-
mented foods that contributes to improve their safety and quality. The
microorganisms used as starter cultures contain a complex enzyme system
that may include peptidases (Flores & Toldrá, 2011) and their action may
result in the generation of bioactive peptides with beneficial health effects
(Martı́nez-Villaluenga, Peñas, & Frı́as, 2017). Particular attention is given
to lactic acid bacteria (LAB), microorganisms that have a high proteolytic
activity due to their content in extra and intracellular peptidases, that are
typically used for food fermentation. So, PepX, tripeptidase, dipeptidase,
PepN, PepA, PepC and other aminopeptidases have been reported in
LAB (González, Sacristán, Arenas, Fresno, & Tornadijo, 2010, Sinz &
Schwab, 2012; Stressler, Eisele, Kranz, & Fischer, 2014; Stressler et al.,
2016). Depending on the type and balance of such peptidases, different
peptide patterns are obtained in fermented foods and this may give
different bioactivity (Martı́nez-Villaluenga et al., 2017). For instance, cell
free extracts of Lactobacillus paracasei subsp. paracasei demonstrated X-prolyl
dipeptidylpeptidase, aminopeptidase, dipeptidase and carboxypeptidase
activities while Leuconostoc mesenteroides subsp. mesenteroides exhibited
endopeptidase, aminopeptidase, dipeptidase and carboxypeptidase activities
(Macedo, Vieira, Poças, & Malcata, 2010). Therefore, large amounts of
bioactive peptides may be expected in fermented foods. For instance, the
use of Lactobacillus pentosus and Staphylococcus carnosus in dry-fermented sau-
sages containing sodium caseinate as ingredient contributed to large amounts
of peptides with ACE inhibitory activity (Mora, Gallego, Escudero, et al.,
2015). Both microorganisms, Lactobacillus pentosus and Staphylococcus carnosus
are used for fermenting milk because both are able to hydrolyze casein
through their extracellular proteinase and the generated oligopeptides are
hydrolyzed by intracellular peptidases into smaller peptides once transported
into the cell (Chaves-López et al., 2014). Two antioxidant peptides were
reported after the simulated gastrointestinal digestion of soft cheese (Pepe
et al., 2016). Lactobacillus helveticus and Lactobacillus acidophilus are able to
hydrolyze Ƙ-casein and release short peptides, some with ACE inhibitory
activity (Ali et al., 2019). Other microorganisms of interest, typically used
for food fermentation and able to hydrolyze proteins, are yeasts (Santos
et al., 2001). So, peptidases such as PepX, leucine aminopeptidase, and
DPP IV and V have been reported in Aspergillus oryzae
(Matsushita-Morita et al., 2011), while proteinases A and D, and prolyl
and arginyl aminopeptidases were reported in Debaryomices hansenii
(Santos et al., 2001).
The presence of dipeptides have been reported in fermented foods as a

result of microbial dipeptidyl peptidases (DPP) hydrolysis on the amino
terminal. So, this is the case of dipeptides AF, PL, KL, LG and KF released
in Feta cheese by Lactobacillus paracasei (Bintsis, Vafopoulou-Mastrojiannaki,
Litopoulou-Tzanetaki, & Robinson, 2003), proline-containing dipeptides
released in sourdoughs by Leuconostoc mesenteroides and Lactobacillus curvatus
(Zotta, Ricciardi, & Parente, 2007) and by Lactobacillus helveticus in casein
hydrolysates (Stressler, Eisele, & Fischer, 2013), RP and GF by
Leuconostoc mesenteroides subsp. mesenteroides, and GP by Lactobacillus paracasei
subsp. paracasei in Serra da Estrela cheese (Macedo et al., 2010).
The release of bioactive peptides during food processing has been
widely documented, especially in milk, cheese and dairy foods, meat and
meat products, wine, fish and seafood, eggs, and other foods as shown in
Table 1. A large number of short peptides (>100) were identified, as endog-
enously released, in European sea bass and most of them were bioactive
(Cerrato et al., 2021). ACE inhibitory peptides VPP and IPP were reported
to increase with the ripening time in semi-hard cheeses (Meyer, Butikofer,
Walther, Wechsler, & Sieber, 2009). Two hexapeptides with relevant
antioxidant activity were isolated and identified after the simulated gastro-
intestinal digestion of Stracchino which is a soft cheese produced in the
Northern Italy (Pepe et al., 2016). More than 800 peptides were reported
to be released during the 12 months processing of Parma cheese. From them,
18 were reported as ACE inhibitors like VPP, IPP, LHLPLP, HLPLP, or
ENLLRF and 12 longer peptides (>12 amino acids) as antimicrobial
(Solieri et al., 2020). ACE inhibitory peptides VPP and IPP were observed
to increase up to 18 months of ripening of Parma cheese and then decreased
at 24 months of ripening (Martini, Conte, & Tagliazucchi, 2020). Six pep-
tides were selected for DPP-IV and α-glucosidase inhibitory activity by
combining peptidomics, in silico analysis, and a structure–activity relation-
ship, outstanding peptide APFPE as a powerful DPP IV inhibitor
(Martini, Solieri, Cattivelli, et al., 2021; Martini, Solieri, & Tagliazucchi,
2021). Furthermore, ACE inhibitory peptides HLPLP and LHLPLP were
demonstrated to be resistant to simulated gastrointestinal digestion and able
to be partially absorbed during transepithelial transport in a Caco-2 cell
monolayer (Basiricò et al., 2015).
6. Release of bioactive peptides through food proteins

hydrolysis
The activity of endogenous enzymes is quite limited and depends on
the time of processing and conditions found in the food (pH, moisture
content, temperature, redox potential) during the process. This usually

results in low yields of peptides for much foods. In this way, when the objec-
tive is the large production of bioactive peptides, the procedure is to hydro-
lyze food proteins with exogenous peptidases or proteolytic microorganisms
(see Fig. 16). Numerous peptidases are commercially available as shown in
Table 2. Mostly used peptidases in commercial production are Alcalase from
Bacillus licheniformis, Protamex from Bacillus sp., Thermolysin from Bacillus
stearothermophilus, Flavorzyme from Aspergillus oryzae, Prolidase from
Lactobacillus casei, Neutrase from Bacillus subtilis or Bacillus amyloliquefaciens,
among other (Toldrá et al., 2020a). Each enzyme has its specific active site
and mode of action and therefore the selected enzyme, the protein source
used as substrate and the degree of hydrolysis will determine the obtained
yield of peptides and their bioactivity (Xing, Li, Toldrá, & Zhang, 2021).
Some variations in protein hydrolysis may be due to altered enzyme
reproducibility and stability as a consequence of batch to batch variability
(Merz, Apple, et al., 2016; Toldrá et al., 2018).
Commercial peptidases may exert some side activities in addition to the
main enzyme activity shown in the manufacturers specifications and
this may affect the peptides pattern as well as the hydrolysis degree. For
instance, it has been reported that Flavourzyme, an enzyme extracted from
Aspergillus oryzae, was characterized to have a wide activity spectrum con-
sisting of three endopeptidases, two dipeptidylpeptidases, two aminopepti-
dases and one amylase (Merz et al., 2015; Merz, Apple, et al., 2016).
Furthermore, aminopeptidase and carboxypeptidase activity, based on the
release of free amino acids, was reported for other enzymes like Alcalase
2.4 L (Novozymes), Maxazyme NNP DS (DSM), Flavourzyme 1000 L
(Novozymes) and Protease AN (Amano Enzyme Inc.) while a major endo-
peptidase activity was reported for Bioprase SP-20FG (Nagase), Collupulin
200 L (DSM), Corolase2TS (AB Enzymes), Promod 439 L (Biocatalysts
Ltd.), Proteinase T (DuPont) and Protin SD-AY10 (Amano Enzyme
Inc.) (Merz, Claaßena, et al., 2016).
A large number of bioactive peptides generated through enzymatic
hydrolysis of food proteins, usually isolated from food waste and food
co-products have been reported in the literature and some examples are
given in Table 3. For instance, protein rich extract from almond flour
was hydrolyzed with a neutral protease from Bacillus subtilis and 208 small
bioactive peptides with 2–4 amino acids sequences were identified
(Huang et al., 2022). Some of the most relevant were reported to be VY
and FY, both dipeptides as ACE inhibitors with in vivo hypotensive activity
in animal assays (Suetsuna, Maekawa, & Chen, 2004), FK has also antioxi-
dant activity (Huang et al., 2022). Dipeptides GW, VF and YP found in
Table 2 Commercial enzyme preparations with specific characteristics and some relevant applications.
Commercial
preparation Origin Manufacturers Activity Cleavage sites Application References
Flavourzyme Aspergillus oryzae 3 endopeptidases Cereals Huang et al. (2015),
1000 M 2 aminopeptidases Calcium chelating Meinlschmidt,
2 peptides, Soy Sussmann,
dipeptidylpeptidases Schweiggert-Weisz,
1 α-amylase and Eisner (2016),
Merz et al. (2015)
Valkerase Bacillus licheniformis Bri Enzymes Keratinase, Serin Non especific Feather meal –
Endopeptidase
Prolidase L-lactis cremoris Dipeptidase Bonds including proline Cheese making Kitchener and Grunden
Other many sources or hydroxiproline (2012)
Bioprase Bacillus sp. Subtilisin Merz et al. (2015)
SP-20FG Endo
metalloprotease
Aminopeptidase
Neutrase Bacillus subtilis Novozymes Metalloprotease Collagen Ou et al. (2010),
B. amyloliquefaciens Calcium- and Meinlschmidt et al.,
iron-chelating 2016
peptides Soy
Alcalase 2.4 L Bacillus licheniformis Novozymes Subtilisin Non especific Calcium-chelating Choi, Lee, Chun, and
Alkaline serin peptides Song (2012),
endopeptidase Charoenphun,
Extracellular neutral Cheirsilp, Sirinupong,
metallo protease and Youravong (2013)
Aminopeptidase
Savinase Bacillus lentus. Novozymes Subtilisin Higher affinity by Phe Lentil proteins Garcı́a-Mora, Peñas,
Alkaline serin and Leu Frias, and
endopeptidase Martı́nez-Villaluenga
(2014)
Esperase Bacillus lentus. Novozymes Subtilisin Broad especificity Georgieva, Stoeva,
Alkaline serin Voelter, Genov, and
endopeptidase Betzel (2001)
Protamex Bacillus licheniformis Novozymes Subtilisin Calcium- chelating Sung et al. (2014),
Bacillus Serin peptides Slizyte et al. (2016),
amyloliquefaciens endopeptidase, Bioactivity in Fish, Meinlschmidt et al.
Metallo Antiallergenic in (2016)
endopeptidase cereals
Neutral protease Soy
Protex 6 L Bacillus licheniformis Genencor Alcaline serine Bioactivity in Fish Slizyte et al. (2016)
endopeptidase
Protease N-01 Bacillus subtilis ASA Endoprotease
Spezialenzyme
GmbH
Protease M Endo & Exo Calcium chelating Lv, Bao, Liu, Ren, and
peptides in soybean Guo (2013)
Promod 439 L Bacillus licheniformis Biocatalysts Subtilisin Generation of flavor Mihulová, Vejlupková,
in animal, vegetable Hanušová, Štětina, and
and fish proteins. Panovská (2013)
Pronase Streptomyces griseus Sigma-Aldrich Endo & Exo Yoshida et al. (1988)
Corolase 7089 Bacillus Subtilis AB Enzymes Neutral Bioactivity in Fish Garcı́a-Mora, Peñas,
GmbH Endopeptidase Frias, and
Martı́nez-Villaluenga
(2014), Slizyte et al.
(2016)
Continued
Table 2 Commercial enzyme preparations with specific characteristics and some relevant applications.—cont’d
Commercial
preparation Origin Manufacturers Activity Cleavage sites Application References
Corolase PP Porcine pancreatic AB Enzymes Endopeptidase, Bioactivity in Fish Slizyte et al. (2016)
gland GmbH Amino- &
carboxypeptidase
Corolase 2TS Bacillus AB Enzymes Endopeptidase Cereals, soy Merz et al. (2015),
thermoproteolyticus Meinlschmidt et al.
Bacillus (2016)
stearothermophylus
GC710 Genencor Neutral proteinase Cereals
Biotech
GC106 Aspergillus niger Genencor Acid proteinase Antiallergenic in Sung et al. (2014)
Biotech Aspartic-type cereals
peptidase
Pancreatic Porcine pancreatic Novozymes Endopeptidase Meinlschmidt et al.
trypsin Novo glands (2016)
Trypsin Bovine pancreas Sigma-Aldrich Endopeptidase Broad specificity Calcium-chelating Guo et al. (2015)
Fluka Arg-j-Xaa, Lys-j-Xaa peptides from shrimp
by-products, Tilapia
or Alaska Pollock skin
Chymotrypsin A Bovine pancreas Sigma-Aldrich Endopeptidase Tyr-j-Xaa, Trp-j-Xaa,
Fluka Phe-j-Xaa, Leu-j-Xaa
Chymotrypsin C Bovine pancreas Sigma-Aldrich Endopeptidase Leu-j-Xaa, Tyr-j-Xaa,
Fluka Phe-j-Xaa, Met-j-Xaa,
Trp-j-Xaa, Gln-j-Xaa,
Asn-j-Xaa
Pepsin Porcine Gastric Sigma-Aldrich Hydrophobic,
mucosa Fluka preferably aromatic,
residues in P1 and P1’
positions
Alkaline protease Bacillus licheniformis Genencor Serine-type Aromatic or Antiallergenic in Sung et al. (2014)
peptidase hydrophobic residues cereals
Seabzyme L 200 Carica papaya Speciality Endoprotease Hydrolysis of proteins
Enzymes & with broad specificity
Biotechnologies for peptide bonds, but
preference for an amino
acid bearing a large
hydrophobic side chain
at the P2 position
Bromelain Pineapple stem Great food Cysteine-type Broad especificity Antiallergenic in Sung et al. (2014)
(Biochem) peptidase cereals
Collupulin Carica papaya Gist-brocades Cysteine-type Aromatic or Antiallergenic in Sung et al. (2014)
peptidase hydrophobic residues cereals
Papain Carica papaya Sigma-Aldrich Cysteine-type Broad especificity Collagen Sung et al. (2014)
Fluka peptidase Antiallergenic in
cereals
Ficain (ficin) Figs latex Sigma-Aldrich Cysteine-type Broad especificity
Fluka peptidase
Xaa: chain with amino acids at the C-terminal.
Reproduced from Toldrá, F., Reig, M., Aristoy, M.C., Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395–404. Generation of bioactive
peptides during food processing. Food Chemistry, 267, 395–404, with permission from Elsevier.
Table 3 Examples of bioactive peptides released by hydrolysis with commercial peptidases under controlled conditions.
Food Type Treatment hydrolysis Peptide sequence Potential activity References
Almond Flour Neutral protease Bacillus subtilis VY, FY ACE inhibitory Huang, Goncalves
(0.5%, 50 °C, pH 9, 2 h) Dias, de Moura, Bell,
and Barile (2022)
Milk Goat (Capra Trypsin (3%, 37 °C, pH 8, 3 h) INNQFLPYPY, Antidiabetic Zhang, Chen, Ma, and
hircus) milk SPTVMFPPQSVL Chen (2015)
MHQPPQPL
Algae Gracilariopsis Trypsin (2%, 2 h) FQIN[M(O)]CILR, TGAPCR ACE inhibitory Deng et al. (2018)
lemaneiformis
(Rhodophyta)
Palmaria palmata Corolase PP (2%, 50 °C, pH 7, 4 h) SDITRPGGQM Antioxidant Harnedy, O’Keeffe,
and FitzGerald (2017)
Red seeweed Pepsin (1%, 37 °C, pH 2, 3 h) GGSK, ELS Antidiabetic Admassu, Gasmalla,
(Porphyra spp) Yang, and Zhao
(2018)
Spirulina platensis Pepsin (6%, 37 °C, pH 2, 10 h) CANPHELPNK, NPVWKRK, Anti-obesity Fan, Cui, Zhang, and
NALKCCHSCPA, Zhang (2018)
LNNPSVCDCDCMMKAAR
Fish Atlantic salmon Corolase PP (1%, 50 °C, pH 7, 1 h) GPAV, FF ACE inhibitory Neves, Harnedy,
(Salmo salar) Antidiabetic O’Keeffe, and
Antioxidant FitzGerald (2017)
Cuttlefish (Sepia Bacillus mojavensis (3 U/mg, 50 °C, AFVGYVLP ACE inhibitory Balti et al. (2015)
officinalis) pH 10)
Cuttlefish hepatopancreas enzymes EKSYELP, VELYP ACE inhibitory Balti et al. (2015)
(3 U/mg, 50 °C, pH 8)
Leatherjacket Insoluble bromelain (0.5%, 50 °C, AER, EQIDNLQ ACE inhibitory Salampessy, Reddy,
(Meuchenia sp.) 2 h) Phillips, and
Kailasapathy (2017)
Insoluble papain (0.5%, 50 °C, 6 h) DPHI, EPLYV ACE inhibitory Salampessy et al.
(2017)
Insoluble flavourzyme (1.25%, WDDME ACE inhibitory Salampessy et al.
50 °C, 2 h) (2017)
Sardinelle Bacillus amyloliquefaciens (4%, 37 °C, ITALAPSTM, SLEAQAEKY ACE inhibitory, Jemil et al. (2016)
(Sardinella aurita) 24 h) Antioxidant
GTEDELDKY Antioxidant Jemil et al. (2016)
Bacillus subtilis (4%, 37 °C, 24 h) NVPVYEGY Antihypertensive, Jemil et al. (2016)
Antioxidant
Smooth hound Esperase (50 °C, pH 9, 9.5 h) IAGPPGSAGPAG, ACE inhibitory Abdelhedi et al. (2016)
viscera VVPFEGAV, PLPKRE
Pacific herring Trypsin (1.39 U/Kg, 32.06 °C, pH KEEKFE, LHDELT Antioxidant Wang et al. (2019)
(Clupea pallasii) 6.78, 7 h)
Legumes Soy Alkaline proteinase (6 U/Kg, 50 °C, LLPLPVLK, SWLRL, WLRL Antidiabetic Wang et al. (2019)
pH 9)
Erythrina edulis Alcalase (0.5%, 50 °C, pH 8.3, 2 h) CCGDYY, DGLGYY, Antioxidant Intiquilla et al. (2018)
(pajuro) GESWCR, YDLHGY,
SQLPGW, WAL
FLWGKSY, WPW, Antiinflammatory Intiquilla et al. (2018)
SFMNVKHWPW
Duck (Anas Protamex (0.75%, 50 °C, pH 6, 4 h) AGRDLTDYLMKIL Antioxidant
platyrhynchos) GYDLGEAEFARIM
Continued
Table 3 Examples of bioactive peptides released by hydrolysis with commercial peptidases under controlled conditions.—cont’d
Food Type Treatment hydrolysis Peptide sequence Potential activity References
LQAEVEELRAALE Wang, Huang, Chen,
NWDDMEK Huang, and Zhou
IEDPFDQDDWGAWKK (2015)
Kacang goat Protamex + Flavourzyme (0.5%, FQPS ACE inhibitory Mirdhayati,
(Capra aegagrus 50 °C, pH 7, 4 h) Hermanianto, Wijaya,
hircus) Sajuthi, and Arihara
(2016)
Pork loin Thermolysin (0.008%, 5 °C, 24 h) LVGRPRHGQ, VFPS ACE inhibitory Choe et al. (2019)
By- Chicken combs Alcalase (5%, 4 h) APGLPGPR, FPGPPGP, Piro- ACE inhibitory Bezerra et al. (2019)
products and wattles GPPGPT
Bovine gelatin Thermolysin (65 °C, pH 8, 6 h) AG, AGP, VGP, PY, QY, DY, ACE inhibitory Herregods et al. (2011)
IY
Oil palm (Elaeis Alcalase (0.5%, 45 °C, pH 8.5, 2 h) ADVFNPR, VIEPR, LPILR, ACE inhibitory Zheng, Li, Zhang,
guineensis Jacq) + flavourzyme (0.5%, 50 °C, pH 7, VVLYK Ruan, and Zhang
kernel expeller 2 h) + pepsin (0.3%, 37 °C, pH 2, (2017)
1 h) + trypsin (0.3%, 37 °C, pH 7,
1 h)
Tomato seeds Bacillus subtilis (2%, 37 °C, 24 h) DGVVYY ACE inhibitory Moayedi et al. (2018)
GQVPP Antioxidant Moayedi et al. (2018)
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Advances in Food and Nutrition Research 1St Edition Fidel Toldra Full Chapter

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and quantitation, and in silico approaches. An analysis of posttranslational

constitutes an attractive alternative food preservation technique. Furthermore,

Peptidomics as a useful tool

2. Peptidomics in the characterization of peptides

Color, texture, flavour,

Bioactivities: antioxidant, antihypertensive,

Post-traductional Oxidation, deamidation,

Fig. 2 Major areas of peptidomics applications in foods.

and quantitation of bioactive peptides is shown in Fig. 3. The pretreatment

method of choice. Size-exclusion chromatography is based on the separation

2.1 Identification of peptides

bioactivities studied according to BIOPEP database (Minkiewicz et al.,

2.1.1 Identification of di-, tri-, and tetrapeptides

Dipeptide sequences can be repeated many times in protein sequences so

2.1.2 Pentapeptides or longer peptides identification

adjustment of the basic techniques in order to identify a larger number of

Fig. 8 Reversed-phase chromatographic separation after in vitro gastrointestinal diges-

conduct MSn fragmentation of the parent peptides, quadrupole/time-of-

spectrometry analysis revealed changes in the peptide profiles and evidenced

2.2 Quantification of peptides

provide a simple, reliable, versatile, and cost-effective quantification that can

optimization of the MRM parameters of low abundant peptides or small

after the quantification of the antihypertensive dipeptide AA at different

Protein of origin Food matrix

Bioactivity test in vitro and in vivo

Despite this procedure is very effective, it results very expensive and

where a is the number of fragments with given activity in a protein sequence,

The potential biological activity of protein (B) [μM1] is calculated with

where ai is the number of repetitions of i-th bioactive fragment in protein

4. Mechanisms of enzymatic hydrolysis of food proteins

Fig. 15 Scheme of food protein hydrolysis and enzymes involved. The

Proteins in foods Isolated food proteins

Endogenous peptidases Microbial peptidases Peptidases Microbial peptidases

Ripening Fermentation Reactor Fermenter

Small amounts of bioactive Large amounts of bioactive

Absorption in the organism

border intestinal epithelium proteases and blood stream enzymes that

5. Release of bioactive peptides in foods by endogenous

hydrolyze myofibrillar and sarcoplasmic proteins into large protein

The presence of dipeptides have been reported in fermented foods as a

6. Release of bioactive peptides through food proteins

content, temperature, redox potential) during the process. This usually

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