Advances in Food and Nutrition Research 1St Edition Fidel Toldra Full Chapter
Advances in Food and Nutrition Research 1St Edition Fidel Toldra Full Chapter
Advances in Food and Nutrition Research 1St Edition Fidel Toldra Full Chapter
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– )
Academic Press is an imprint of Elsevier
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
525 B Street, Suite 1650, San Diego, CA 92101, United States
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
125 London Wall, London, EC2Y 5AS, United Kingdom
No part of this publication may be reproduced or transmitted in any form or by any means, electronic
or mechanical, including photocopying, recording, or any information storage and retrieval system,
without permission in writing from the publisher. Details on how to seek permission, further
information about the Publisher’s permissions policies and our arrangements with organizations such
as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website:
www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the
Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical
treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating
and using any information, methods, compounds, or experiments described herein. In using such
information or methods they should be mindful of their own safety and the safety of others, including
parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume
any liability for any injury and/or damage to persons or property as a matter of products liability,
negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas
contained in the material herein.
ISBN: 978-0-323-99082-0
ISSN: 1043-4526
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
Centro de Investigação de Montanha (CIMO), Instituto Politecnico de Bragança, Bragança,
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
Centro de Investigação de Montanha (CIMO), Instituto Politecnico de Bragança, Bragança,
Portugal
Jose M. Franco
Chemical Process and Product Technology Research Centre (Pro2TecS), Departamento de
Ingenierı́a Quı́mica, Universidad de Huelva, Huelva, Spain
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
Chemical Process and Product Technology Research Centre (Pro2TecS), Departamento de
Ingenierı́a Quı́mica, Universidad de Huelva, Huelva, Spain
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
Centro de Investigação de Montanha (CIMO), Instituto Politecnico de Bragança, Bragança,
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
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Paterna, Spain
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
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.
Processing
Authentification, OGMs,
Safety allergens, pathogensor
toxins
Gastrointestinal
Intestinal microbiota
digestion
Protein-peptide
interactions
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
Peptidomics as a useful tool 5
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.
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).
8 Fidel Toldrá and Leticia Mora
Fig. 5 Number of amino acids of the bioactive peptides available in BIOPEP database;
accessed on 17 Feb, 2022 (Minkiewicz et al., 2019).
Peptidomics as a useful tool 9
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.
10 Fidel Toldrá and Leticia Mora
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.
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.
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.
Peptidomics as a useful tool 15
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.
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.
18 Fidel Toldrá and Leticia Mora
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.
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.
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
a
A¼
N:
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.
Peptidomics as a useful tool 21
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).
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
Gastrointestinal digestion
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.
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)
Peptidomics as a useful tool 29
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).
30 Fidel Toldrá and Leticia Mora
1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside the
United States, check the laws of your country in addition to the terms
of this agreement before downloading, copying, displaying,
performing, distributing or creating derivative works based on this
work or any other Project Gutenberg™ work. The Foundation makes
no representations concerning the copyright status of any work in
any country other than the United States.
• You pay a royalty fee of 20% of the gross profits you derive from
the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
about donations to the Project Gutenberg Literary Archive
Foundation.”
• You comply with all other terms of this agreement for free
distribution of Project Gutenberg™ works.
1.F.
1.F.4. Except for the limited right of replacement or refund set forth in
paragraph 1.F.3, this work is provided to you ‘AS-IS’, WITH NO
OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED,
INCLUDING BUT NOT LIMITED TO WARRANTIES OF
MERCHANTABILITY OR FITNESS FOR ANY PURPOSE.
Please check the Project Gutenberg web pages for current donation
methods and addresses. Donations are accepted in a number of
other ways including checks, online payments and credit card
donations. To donate, please visit: www.gutenberg.org/donate.
Most people start at our website which has the main PG search
facility: www.gutenberg.org.