LWT - Food Science and Technology 130 (2020) 109616

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

An in silico model to predict and estimate digestion-resistant and bioactive T

peptide content of dairy products: A primarily study of a time-saving and
affordable method for practical research purposes
Meisam Baratia, Fardin Javanmardib, Masoumeh Jabbaric, Amin Mokari-Yamchic,
Fariba Farahmandd, Ismail Eşe, Hossein Farhadnejadf, Sayed Hossein Davoodii,∗,
Amin Mousavi Khaneghahj,∗∗
a
Student Research Committee, Department of Cellular and Molecular Nutrition, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
b
Department of Food Science and Technology, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
c
Department of Community Nutrition, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
d
Department of Community Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran
e
School of Chemical Engineering, Department of Material and Bioprocess Engineering, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
f
Nutrition and Endocrine Research Center, Research Institute for Endocrine Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
i
Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
j
Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), São Paulo, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The purpose of this study is to estimate the concentration of digestion-resistant and bioactive peptides in dairy
Food proteins products using an in silico method. The major contributors of milk protein sequences including αs1-casein, αs2-
Bioactive peptides casein, β-casein, k-casein, β-lactoglobulin, and α-lactalbumin were obtained from UniProt Knowledgebase
In silico (UniProtKB). In silico digestion and bioactive fragment, findings were analyzed using the BIOPEP tool. Bioactive
Functional food
peptide content of the dairy products was estimated based on molecular weight, percent of major proteins
Dairy products
existing in the food items, and the number of peptides obtained after in silico digestion from each protein. The
results showed that 100 g milk contains 6700.241 μmol digestion-resistant peptides; in which 1880.434 μmol out
of total peptides have anti-diabetic properties. Of all digestion-resistant peptides, 1978.24, 1955.024, 1700.907,
and 1066.07 μmol belong to very low, low, medium, and high bioactivity sub-groups, respectively. Using the
data introduced here, risk assessment could be done for dairy originated bioactive peptides and chronic disease.

1. Introduction
In the last few decades, natural ingredients in specific raw and
processed foods have been of great interest to the different fields of
science and industry (Li-Chan, 2015). These compounds have diverse
biological activities and are widely employed in the food and phar-
maceutical industry (Betoret, Betoret, Vidal, & Fito, 2011; Vieira da
Silva, Barreira, & Oliveira, 2016). Functional foods and nutraceuticals
are produced by components derived from plant or animal sources and
have attracted the attention of scientists in recent decades (Nazhand
et al., 2020). Nutraceuticals can play a role in preventing many diseases
including heart disease, stroke, and type 2 diabetes if their mechanism
of action is fully established and their safety is confirmed (Durazzo,
Lucarini, & Santini, 2020; Santini, Novellino, Armini, & Ritieni, 2013).
The molecules with amino acids as their building blocks such as food-
derived bioactive peptides (BPs) are one of the major components of
foods that affect the functional and biological activity of food products.
BPs usually consist of short chains of 2–20 amino acids and become
biologically active following the enzymatic or microbial hydrolysis of
their parent proteins where they are encrypted (Mohanty, Mohapatra,
Misra, & Sahu, 2016). The function of proteolytic enzymes is very im-
portant in the synthesis of BPs during gastrointestinal digestion or food
processing such as ripening and fermentation by breaking proteins into
shorter fragments (Toldrá, Reig, Aristoy, & Mora, 2018).
Several food products have been analyzed for their potential BP
content, but, milk and dairy products are one of the richest sources of

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: hdavoodi@sbmu.ac.ir, hdavoodi1345@gmail.com (S.H. Davoodi), mousavi@unicamp.br (A. Mousavi Khaneghah).

https://doi.org/10.1016/j.lwt.2020.109616
Received 7 April 2020; Received in revised form 8 May 2020; Accepted 15 May 2020
Available online 04 June 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
M. Barati, et al.
BPs and many studies have been published about the functional prop-
erties of dairy product-derived BPs (Basilicata et al., 2018; Georgalaki
et al., 2017). These BPs possess very essential biological activities and
functionalities including antimicrobial, antihypertensive, antioxidative,
anticytotoxic, immunomodulatory, opioid, and mineral-carrying activ-
ities (Korhonen & Pihlanto–Leppälä, 2016; Lopez-Exposito & Recio,
2008; Mada, Ugwu, & Abarshi, 2019). Numerous studies have shown
that α-, β-, and κ-casein (Bessette et al., 2016) and whey proteins such
as α-lactalbumin, β-lactoglobulin, and glycomacropeptide (GMP)
(Arrutia, Rubio, & Riera, 2016) are prominent parent proteins which
are major sources for releasing a number of BPs. Despite their pro-
mising benefits, unfortunately, scale-up production of BPs is mainly
hindered by bioprocess challenges related to purification and quanti-
fication (Agyei, Ongkudon, Wei, Chan, & Danquah, 2016).
An efficient quantification of the beneficial effects of BPs is highly
required to better understand their role in our health. Although in vitro
(Kwon et al., 2011), in vivo (Carrizzo et al., 2019) and meta-analysis of
randomized control trial (Fekete, Givens, & Lovegrove, 2015) studies
have been done to explore the short-term beneficial effects of BPs
(especially dairy originated ones) on human health, investigations on
the long-term beneficial effects of these BPs are still at unsatisfactory
levels. This is mostly due to the general lack of consistent methods to
better understand their synthesis from different food products
(Chakrabarti, Guha, & Majumder, 2018). Cohort and case-control stu-
dies are commonly used to assess the long-term effects of food-in-
gredients (Song & Chung, 2010). To the best of our knowledge, due to
the lack of quantitative measurement of BPs in food items, no case-
control or cohort studies have been done to reveal the association of
BPs intake and reduction in chronic diseases. The major reason why BPs
content of food items have not been measured so far is the high cost of
BPs measurement and characterization. Therefore, if we successfully
measure the BP content of food items included in food frequency
questionnaires (FFQ) used in cohort or case-control studies, the asso-
ciation between BPs intake and their effect on chronic diseases such as
cancer, cardiovascular disease, and other diseases can be effectively
assessed.
The rapid promotion of bioinformatics leads to the establishment of
integrated biological knowledge databases such as PepBank, BioPD,
BIOPEP, EROP-Moscow, and SwePep. Among all, BIOPEP focuses
mainly on peptides of food origins (Iwaniak, Minkiewicz, Darewicz,
Sieniawski, & Starowicz, 2016; Kęska, Stadnik, Kononiuk, Libera, &
Wójciak, 2018). In the food sciences, computer simulation (in silico) can
be used as a rapid and low-cost primary screening tool for sequencing
bioactive peptides analysis and their biological effects in various foods
(Sayd et al., 2018). In silico studies allow the creation of profiles for
potential biological activities of proteins, as well as all activities which
may be found in a protein sequence (Carrasco-Castilla, Hernández-
Álvarez, Jiménez-Martínez, Gutiérrez-López, & Dávila-Ortiz, 2012).
Also, in silico proteolysis can help us to select the appropriate enzyme
for hydrolysis in the simulation of gastrointestinal digestion and is
useful for identifying the relationship between peptide structure and its
function (Lin et al., 2018).
Research on BPs has shown that they have great potential to im-
prove human health and prevent chronic diseases by exerting positive
effects on the body's digestive, cardiovascular, immune, and nervous
systems, but the beneficial effects of them should also be proved in long
term studies. In vitro and/or in vivo measurement and characterization
of dairy originated BPs are time-consuming and expensive. Therefore,
the objective of the present study is to estimate the amount of BPs in
dairy products using an in silico method.
2. Methods
2.1. Bovine milk proteins sequences
According to the literature, the initial sequence of six types of food
2
LWT - Food Science and Technology 130 (2020) 109616
protein derived from cow's milk was used in this study. The major
contributors of milk protein sequences are αs1-casein (UniProtKB -
P02662), αs2-casein (UniProtKB - P02663), β-casein (UniProtKB -
P02666), k-casein (UniProtKB - P02668), β-lactoglobulin (UniProtKB -
P02754) and α-lactalbumin (UniProtKB - P00711) obtained from
UniProt Knowledgebase (UniProtKB) (https://www.uniprot.org) and
re-checked in The National Center for Biotechnology Information
(NCBI) database (https://www.ncbi.nlm.nih.gov). The mass of the
mature proteins was obtained by subtracting the signal peptides from
the total mass of the pre-proteins. The signal peptide mass was calcu-
lated by the Expasy peptide mass calculator (https://web.expasy.org/
peptide_mass/). Pre-protein mass and variables related to post-transla-
tional modifications such as numbers of disulfide bonds, phosphory-
lated residues, glycosylated residues were extracted from UniProtKB.
2.2. Food item selection
This study was performed within the framework of the Tehran Lipid
and Glucose Study (TLGS). For estimation of dietary intake of BPs
originated from dairy products, all of the dairy-related food items from
the FFQ list of TLGS including fat-free milk, low-fat milk, high-fat milk,
cacao milk, strained yogurt, low-fat yogurt, high-fat yogurt, doogh,
cheese, ice cream (traditional), ice cream (industrial), butter, cream
and kashk were included (Mirmiran, Esfahani, Mehrabi, Hedayati, &
Azizi, 2010). The characteristics of the food items in terms of protein
composition were abstracted in Table 1.
2.3. In silico digestion and function prediction
The extracted protein sequences (without signal peptide) of bovine
milk protein were separately used for in silico digestion. Theoretical
prediction of released peptide sequences in the result of the application
of proteolytic enzymes was performed using the BIOPEP tool
(Minkiewicz, Dziuba, Iwaniak, Dziuba, & Darewicz, 2008). The pepsin
(pH: 1.3), chymotrypsin A and trypsin were used for the in silico pro-
teolysis. Furthermore, theoretically released peptides derived from
bovine milk proteins using the proteases were submitted to the BIOPEP
tool for search of active fragments option. The in silico digestion was
separately done for each milk proteins and the number of (1) total
peptides, (2) peptides with biological function, (3) di, tri, tetra, penta,
hexa, heptapeptide, and peptides with more residues, (4) anti-diabetic
peptides, (5) anti-hypertensive peptides, and (6) antioxidant peptides
were calculated.
2.4. Peptide ranking
PeptideRanker tool (http://bioware.ucd.ie/compass/biowareweb/)
was used for the prediction of the potential of peptides derived from the
bovine dairy proteins (Mooney, Haslam, Pollastri, & Shields, 2012).
PeptideRanker is a server for the prediction of bioactive peptides and
gives a score in the range of 0–1 for each peptide. The maximum score
represents the most potent active peptides and the minimum score
denotes the peptides with the lowest bioactivity. In the current study,
the obtained peptides were divided into multiple subgroups based on
their bioactivity scores. The peptides classified as number (1) had < 0.1
scores, (2) 0.1 ≤ X < 0.3 scores, (3) 0.3 ≤ X < 0.7 scores, and (4)
had 0.7≤ scores and above made the mentioned sub-groups.
2.5. Toxicity and physicochemical assessment
The toxicity and physicochemical properties of the obtained pep-
tides were assessed using the ToxinPred online tool (Gupta et al., 2013).
(https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php). The
support vector machine (SVM)-based prediction method with a
threshold value of 0.0 was chosen for the toxicity prediction. Several
parameters including peptide charge, hydrophobicity, and
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Table 1
The protein composition (per 100 g) of the included food items.
Proteins

Food Item Total αs1-casein αs2-casein k-casein β-casein α-lactalbumin β-lactoglobulin Ref.
protein

Fat free Milk 3.4 1.08 0.27 0.32 1.22 0.08 0.39 (Kukovics & Németh, 2013)
Low fat milk 3.4 1.08 0.27 0.32 1.22 0.08 0.39 (Kukovics & Németh, 2013)
Full fat milk 3.4 1.08 0.27 0.32 1.22 0.08 0.39 (Kukovics & Németh, 2013)
Cacao milk 3.4 1.08 0.27 0.32 1.22 0.08 0.39 (Kukovics & Németh, 2013)
Strained yogurt 9 2.88 0.72 0.86 3.24 0.23 1.04 (Ozer, Stenning, Grandison, & Robinson, 1999; Trachoo,
2002)
Low fat yogurt 4.5 1.44 0.36 0.43 1.62 0.52 0.11 (Puvanenthiran, Williams, & Augustin, 2002)
High fat yogurt 3.9 1.24 0.31 0.37 1.40 0.1 0.45 (Jahanbakhsh Oskouie, Hesari, Azadmard Damirchi, Rafat,
& Rezaei Kouchemeshki, 2016)
Doogh 1.9 0.61 0.15 0.20 0.68 0.04 0.22 (Kiani, Mousavi, & Emam-Djomeh, 2008)
Cheese 25 6 1.50 2.75 6.25 0.97 3 (Wedholm, Larsen, Lindmark-Månsson, Karlsson, &
Andrén, 2006)
Cream cheese 8 3.2 0.8 0.96 3.6 0 0 (Jeon, Lee, Ganesan, & Kwak, 2012)
Ice cream (traditional) 3 0.96 0.24 0.29 1.08 0.07 0.34 (Kukovics & Németh, 2013)
Ice cream (industrial) 4 1.28 0.32 0.38 1.44 0.10 0.46 (Kukovics & Németh, 2013)
Butter 1.16 0.37 0.09 0.11 0.41 0.03 0.13 (Holzmüller & Kulozik, 2016)
Cream 2.67 0.85 0.21 0.25 0.95 0.06 0.30 (Holzmüller & Kulozik, 2016)
Kashk 8 2.56 0.64 0.76 2.88 0.20 0.92 (Mashak, Sodagari, Mashak, & Niknafs, 2014)

Data are presented as percent.

hydrophilicity were also extracted. Based on hydrophobicity and hy-

drophilicity scores the peptides were divided into high (0≤) or low
(( 1.08
22956 ) ) ((
× 106 × 14 +
0.27
24331 ) )
× 106 × 22 +

(0 >) tendency to dissolve in water. (( 1.22

23565
× 10 ) × 17) + ( (
6 0.32
18956
× 106) × 14) +
2.6. Estimation of digestion-resistant and bioactive peptide content of dairy 25.14μmol/g =
(( 0.39
18263
× 10 ) × 20) + ( (
6 0.08
14168
× 10 ) × 12)
6

100
food items

For estimation of digestion resistant-peptides, first, the molar con- 2.7. The validation
centration of each dairy protein is calculated by dividing the amount (g)
of each protein (per 100 g food item) by its molecular weight multiplied The validation was performed according to the results reported by
by 106 (first calculation) and then the number of digestion resistant- Barbé et al. (2014). They reported about 16000 digestion-resistant
peptides obtained from each protein was multiplied with the value peptides in an experimental model for the digestion of dairy products.
obtained in the first calculation (second calculation). The result of the From the identified peptides, 3297 of them were related to the digestion
sum of the second calculations for each dairy protein is the digestion- of raw milk. After ignoring duplicates, 867 peptides were enrolled for
resistant content of each food item per 100 g. Digestion-resistant and the validity assessment of our study. All of the enrolled peptides have
bioactive peptide content of the food items were estimated using the more than 5 residues. From the peptides obtained from our in silico
following equation: study, only peptides with more than 5 residues were enrolled. There-
fore, the similarity of the in silico identified peptides with the peptides
Digestion − resistant peptide content reported by Barbé et al. (867 peptides) was assessed using bioinfor-
matics. The similarity assessment was done using The Basic Local
(μmol/g)
Alignment Search Tool (BLAST) for proteins in NCBI (https://blast.ncbi.
(( A
A1 ) ) ((
× 106 × a +
B
B1 ) ) ((
× 106 × b +
C
C1 ) )
× 106 × c + nlm.nih.gov/Blast.cgi#Query_47856). Each of the in silico identified
peptides was individually aligned to all of the experimentally char-

=
(( D
D1
× 10 ) × d ) + ( (
6 E
E1
× 10 ) × e ) + ( (
6 F
F1
× 10 ) × f )
6
acterized peptides and the number of similar structures for each of our
100 peptides was recorded. The similarity criterion in this study was E-
value < 0.05 and 100% identity. The similarity was also checked
Where A is αs1-casein content per 100 g food item, B is αs2-casein within the in silico derived peptides.
content per 100 g food item, C is β-casein content per 100 g food item,
D is κ-casein content per 100 g food item, E is β-lactoglobulin content 3. Results
per 100 g food item, F is α-lactalbumin content per 100 g food item, A1
is αs1-casein molecular weight (MW), B1 is αs2-casein MW, C1 is β- In the present study, six different proteins derived from dairy pro-
casein MW; D1 is κ-casein MW, E1 is β-lactoglobulin MW, F1 is α-lac- ducts were included for in situ analysis. The mass of the pre-proteins,
talbumin MW, a is the number of peptides obtained after in-silico di- signal peptides, and mature proteins are reported in Table 2. Among the
gestion of αs1-casein, b is the number of peptides obtained after in silico proteins, αs2-casein (Mass: 24,331.07 Da) and α-lactalbumin (Mass:
digestion of αs2-casein, c is the number of peptides obtained after in- 14,168.88 Da) have the highest and lowest masses, respectively. As
silico digestion of β-casein, d is the number of peptides obtained after in reported in Table 3, after in silico digestion of each molecule of αs1-
silico digestion of κ-casein, e is the number of peptides obtained after in casein, αs2-casein, β-casein, k-casein, α-Lactalbumin, and β-lactoglo-
silico digestion of β-lactoglobulin, and f is the number of peptides ob- bulin, the number of obtained peptides were 44, 46, 49, 35, 32 and 38,
tained after in silico digestion of α-lactalbumin. For instance, the esti- respectively. Therefore, β-casein, compared to other proteins, produces
mation of milk-derived di-peptides could be calculated by the following more fragments per molecule. Among the obtained fragments, 77 of all
equation: identified peptides were known for their biological functions. The

3
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Table 2 casein made four QK, three TK, two AL, two PQY, and two PW frag-
Dairy proteins mass after subtracting the mass of signal peptides. ments; however, no similar fragments were obtained from αs1-casein
Protein name Pre-protein Mass Signal peptide Mass Mature Protein Mass digestion. On the other hand, some peptides were found to have several
origins; for instance, in this study, EL was obtained after digestion of
αs1-casein 24529 1571.962 22956.46 αs1-casein, β-casein, and β-lactoglobulin.
αs2-casein 26019 1687.931 24331.07
The final part of the current study was the estimation of BPs origi-
β-casein 25107 1541.952 23565.05
k-casein 21269 2312.316 18956.68
nated from dairy products. In supplementary Tables (S2, S3, S4, S5, S6,
α- lactalbumin 19883 1619.868 14168.88 S7, S8, S9, S10, S11, S12, and S13), the amount of different BPs in terms
β-lactoglobulin 16247 2087.117 18263.13 of molecular weight (MW), charge, bioactivity, hydrophobicity, and
function are summarized. One of the most important food items in this
Data are presented as Dalton.
study was milk. The results showed that 100 g milk contains
6700.241 μmol digestion-resistant peptides; in which, 1880.434 μmol
number of anti-diabetic molecules originated from αs1-casein, αs2- out of total peptides have anti-diabetic properties. Of 6700.241 μmol
casein, β-casein, κ-casein, α-Lactalbumin, and β-lactoglobulin were 10, digestion-resistant peptides obtained from 100 g milk, 1978.24,
14, 14, 11, 8, and 14, respectively. The results showed that κ-casein is 1955.024, 1700.907, and 1066.07 μmol belong to very low, low,
very diverse in terms of post-translational modifications compared to medium, and high bioactivity sub-groups, respectively (Table S2). For
other proteins due to the presence of three disulfide bonds, eight gly- each protein, it was observed that the amount of peptide (μmol) was
cosylated residues, four phosphorylated residues, and one pyrrolidone strongly associated with its bioactivity. The smaller peptides were
carboxylic acid-modified residue, which are highly active molecules in found to show more bioactivity than bigger ones. This finding was
protein modification (Lodish et al., 2000). Proteolysis of each molecule previously demonstrated for different bioactive peptides (Ajibola,
of α-Lactalbumin produces 3, 13, 8, and 5 peptides with very low, low, Fashakin, Fagbemi, & Aluko, 2011; Cheung, Cheung, Tan, & Li-Chan,
medium, and high bioactivity, respectively. 2012). Estimation of digestion-resistant peptides and BPs were also
In Table S1, the physicochemical and functional properties of the performed for strained yogurt (Table S3), low-fat yogurt (Table S4),
obtained peptides after in silico proteolysis are reported. The highest high-fat yogurt (Table S5), Doogh (Table S6), cheese (Table S7), cream
peptide in terms of bioactivity for αs1-casein (Fig. 1), αs2-casein (Fig. 2), cheese (Table S8), ice cream (industrial) (Table S9), ice cream (tradi-
β-casein (Fig. 3), κ-casein (Fig. 4), α-Lactalbumin (Fig. 5), and β-lac- tional) (Table S10), butter (Table S11), cream (Table S12) and kashk
toglobulin (Fig. 6) was GF, PW, PF, SF, IW, and CM, respectively. (Table S13). Among all analyzed food items, the cheese was found to
EAESISSSEEIVPN is the heaviest fragment obtained from αs1-casein. contain the highest BP content (40842.02 μmol), while the butter has
SIGSSSEESAEVATEEVK, QSEEQQQTEDEL, TIASGEPTSTPTTEAVESTV- the lowest BP content (2273.194 μmol).
ATL, DTQAIVQN, and TPEVDDEAL are the heaviest fragments obtained Among 226 types of peptide obtained during the in silico digestion,
from αs2-casein, β-casein, κ-casein, α-Lactalbumin, and β-lactoglobulin, 76 peptides with known biological properties could be identified.
respectively. The in silico digestion did not make necessarily different Among the peptides with known biological activity, 12 and 33 peptides
peptides in terms of amino-acid residues. For instance, digestion of αs2- possess only anti-hypertensive and anti-diabetic properties,

Table 3
Number of peptides obtained from in silico digestion of dairy proteins with respect to physico-chemical and functional properties.
Variables The dairy proteins

αs1-casein αs2-casein β-casein k-casein α-Lactalbumin β-lactoglobulin

Total Peptides 44 46 49 35 32 38
di-peptides 14 22 17 14 12 20
Tri-peptides 11 8 11 8 8 1
Tetra-peptides 8 8 6 2 6 5
Penta-peptides 6 1 7 4 5 5
Hexa-peptides 2 4 4 2 0 2
Hepta-peptides 1 0 0 1 0 3
Peptides with more than 7 residues 2 3 4 4 1 2
Antidiabetic peptide 10 14 14 11 8 14
Hypotensive peptide 7 7 6 8 6 9
Antioxidant peptide 4 2 2 0 2 2
DPP-III inhibitor 1 0 0 0 0 1
Vasoactive peptide 1 0 0 0 0 0
Disulfide bond 0 0 0 3 0 3
Glycosylated residue 0 0 0 8 0 0
Phosphorylated residue 9 13 5 4 9 0
PCA modified residue 0 0 0 1 0 0
Immunomodulatory peptide 0 0 1 0 0 0
CaMPDE inhibitor peptide 0 0 0 0 0 1
Peptide bioactivity Very Low 15 22 11 10 3 12
Low 12 10 15 9 13 13
Medium 12 9 13 10 8 7
High 5 5 10 6 5 6
Peptide charge Zero 19 20 26 20 16 21
Negative 19 12 13 5 11 13
Positive 6 14 10 10 5 4
hydrophobicity Low 33 34 26 26 17 19
High 11 12 23 9 15 19
hydrophilicity Low 20 19 28 19 15 19
High 24 27 21 16 17 19
PCA: Pyrrolidone carboxylic acid; CaMPDE: Calmodulin-dependent cyclic nucleotide phosphodiesterase.

4
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Fig. 1. Sequences and functional properties

of peptides derived from in-silico digestion
of αs1-casein. ACE-I: Angiotensin con-
verting enzyme inhibitor; DPP: dipeptidyl
peptidase; A: Alanine; N: Asparagine; E:
Glutamic acid; Q: Glutamine; G: Glycine; L:
Leucine; K: Lysine; M: Methionine; P:
Proline; S: Serine; T: Threonine; Y:
Tyrosine; V: Valine.

Fig. 2. Sequences and functional properties

of peptides derived from in-silico digestion
of αs2-casein. ACE-I: Angiotensin con-
verting enzyme inhibitor; DPP: dipeptidyl
peptidase; A: Alanine; R: Arginine; E:
Glutamic acid; Q: Glutamine; H: Histidine;
L: Leucine; K: Lysine; M: Methionine; F:
Phenylalanine; P: Proline; S: Serine; T:
Threonine; W: Tryptophan; Y: Tyrosine; V:
Valine.

5
M. Barati, et al.
Fig. 3. Sequences and functional properties of peptides derived from in-silico digestion of β-casein. ACE-I: Angiotensin converting enzyme inhibitor; DPP: dipeptidyl
peptidase; R: Arginine; N: Asparagine; D: Aspartic acid; C: Cysteine; E: Glutamic acid; Q: Glutamine; G: Glycine; H: Histidine; I: Isoleucine; L: Leucine; K: Lysine; M:
Methionine; F: Phenylalanine; P: Proline; S: Serine; T: Threonine; Y: Tyrosine; V: Valine.
respectively, while 26 peptides have both anti-hypertensive and anti-
diabetic properties. The β-casein-originated peptide PGPIPN is the only
immunomodulatory peptide that was characterized in all of the dairy
proteins analyzed in the current in silico study. The peptides with pre-
viously reported biological activity were summarized in Table 4.
The data reported by Barbé et al. study (Barbé et al., 2014) were
used for the validation of our results. From the in-silico identified
peptides only 36 peptides have more than 5 residues. The validation
results were reported in Table 5. In model 1 of the validation, in which
peptides with E-value less than 0.05 were considered as similar pep-
tides, the structure of 44.5 percent of the experimental results was
predicted. While in the second model, 79.46 percent of the experi-
mentally characterized peptides were estimated. In model 2, the simi-
larity was considered for E-value < 0.05 or 100% identity. No sig-
nificant similarity was seen between the in silico characterized peptides.
Among the assessed peptides, the maximum similarity percentage be-
longed to QSEEQQQTEDEL. QSEEQQQTEDEL predict 5.99 and 6.68
percent of the experimentally identified peptides in model 1 and 2,
respectively. Also, TEEEK predicts 4.15 percent of the results in both
models as reported by Barbé et al.
4. Discussion
The digestion-resistant and bioactive peptide content of yogurt, low-
6
LWT - Food Science and Technology 130 (2020) 109616
fat yogurt, high-fat yogurt, Doogh, cheese, cream cheese, ice cream
(industrial), ice cream (traditional), butter, cream and kashk (kashk as a
fermented dairy product is produced either in dry or liquid form) were
estimated based on MW, percent of major proteins existing in the food
items and a number of peptides obtained after in silico digestion from
each protein. The selection of proteolytic enzymes is also essential for a
more efficient quantification. In this study, we used pepsin (pH: 1.3),
chymotrypsin A and trypsin. These proteolytic enzymes are excellent
models as they facilitate the cleavage of numerous peptide bonds via
hydrolysis in the digestive systems (S. Wang, Li, Zhang, Wang, &
Copeland, 2017).
As previously stated, the quantitative measurement of BP content of
food items is of extreme importance to understand their potential
benefits on our health. Recent immunological studies have shown that
BPs exert potent immunomodulatory effects by induction of oral tol-
erance elements (Moronta, Smaldini, Fossati, Añon, & Docena, 2016;
Nishikimi et al., 2018; Thota, Ponnusamy, Philip, Lu, & Mundkur,
2017). Although the elements of oral tolerance have a positive role in
cardiovascular disease (CVD) and allergy treatment, it was shown to
increase the risk of cancer (Barth et al., 2015). In fact, the lack of BPs
content of food items has limited cohort and retrospective studies to
evaluate the long-term effects of digestion-resistant and bioactive
peptides.
In the present in-silico study, 226 types of peptide were
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Fig. 4. Sequences and functional properties

of peptides derived from in-silico digestion
of K-casein. ACE-I: Angiotensin converting
enzyme inhibitor; DPP: dipeptidyl pepti-
dase; A: Alanine; R: Arginine; N:
Asparagine; Q: Glutamine; G: Glycine; H:
Histidine; I: Isoleucine; L: Leucine; K:
Lysine; F: Phenylalanine; P: Proline; S:
Serine; T: Threonine; W: Tryptophan; Y:
Tyrosine; V: Valine.

characterized. Almost all of the obtained peptides (the intact or similar activity (n = 76) and (2) peptides with un-known biological activity
structure) were previously reported by in vitro or in vivo studies. As (n = 150); in other words, %33.6 percent of the in-silico analyzed
shown in Tables 4 and 6, the peptides obtained in the current in silico peptides were previously characterized either in-vitro or in-vivo and
study were divided into two groups: (1) peptides with known biological submitted to BIOPEP-UWM database as functional peptides. Among the

Fig. 5. Sequences and functional properties of peptides derived from in-silico digestion of α-Lactalbumin. ACE-I: Angiotensin converting enzyme inhibitor; DPP:
dipeptidyl peptidase; A: Alanine; G: Glycine; H: Histidine; I: Isoleucine; L: Leucine; K: Lysine; P: Proline; T: Threonine; W: Tryptophan; Y: Tyrosine.

7
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Fig. 6. Sequences and functional properties

of peptides derived from in-silico digestion
of β-lactoglobulin. ACE-I: Angiotensin con-
verting enzyme inhibitor; CaMPDE:
Calmodulin-dependent cyclic nucleotide
phosphodiesterase; DPP: dipeptidyl pepti-
dase; A: Alanine; R: Arginine; E: Glutamic
acid; Q: Glutamine; G: Glycine; I: Isoleucine;
L: Leucine; K: Lysine; M: Methionine; F:
Phenylalanine; P: Proline; S: Serine; T:
Threonine; W: Tryptophan; Y: Tyrosine; V:
Valine.

Table 4
Peptides with known biologically function obtained after the in-silico digestion.
Protein Hypotensive BPs Anti-diabetic BPs hypotensive and anti-diabetic BPs Antioxidant BPs Immunomodulatory BPs

αs1-casein GK, GTQY PK, QL, SK, VN, VPL QF, PL, GY, AY, EK EL, PEL, AY, GTQY –
αs2-casein QK, TVY AL, SK, TK, TM, QH, EH, QY, PW QF, VR PW –
β-casein PQR, DM SL, TL, PK, IN, VL, PF, IH VY, VK, PL EL, VY PGPIPN
k-casein PSY, IAK, AR IPIQY, QW, PY, IN, VL QF, SF, GL, PH – –
α-Lactalbumin – AL, TK IL, GL, GY, PH, IW, AH EL, AH –
β-lactoglobulin IIAEK, VAGTW, QK VL, AL, SL, PM, TK, IPAVF SF, GL, VR, EK, IR, VY IR, VY –

Biological activity of digestion resistance peptides were retrieved from BIOPEP database (Minkiewicz et al., 2008; Søren Drud Nielsen, Robert L. Beverly, Yunyao Qu,
& David C. Dal as, 2017l).
A: Alanine; R: Arginine; N: Asparagine; D: Aspartic acid; C: Cysteine; E: Glutamic acid; Q: Glutamine; G: Glycine; H: Histidine; I: Isoleucine; L: Leucine; K: Lysine; M:
Methionine; F: Phenylalanine; P: Proline; S: Serine; T: Threonine; W: Tryptophan; Y: Tyrosine; V: Valine.

obtained peptides in the current study, only PGPIPN has im-
munomodulatory effects. The immunomodulatory effects of PGPIPN
were examined in many different in vivo and in vitro studies (Qi et al.,
2017). Although the BIOPEP-UWM database introduces PGPIPN as an
anti-inflammatory peptide, recent studies showed anti-tumor effects of
PGPIPN (W. Wang et al., 2013; Zhao et al., 2016).
Based on the results of this study, 150 of the characterized peptides
sequences have an unknown biological function. To the best of our
knowledge, only 4 in vivo studies have been done to characterize milk-
derived digestion resistant peptides (Barbé et al., 2014; Bouzerzour
et al., 2012; Tu et al., 2019; Wada, Phinney, Weber, & Lonnerdal,
2017). In most of the experimental studies, after dairy consumption,
small intestine content was aspirated and dairy digestion-resistant
peptides were characterized. Bouzerzour et al. (2012) and Barbé et al.
(2014) used piglet and mini-pig models, respectively (Barbé et al.,
2014; Bouzerzour et al., 2012). On the other hand, the suckling rat pup
and mice models have been used by Wada et al. (2017) and Tu et al.
(2019), respectively (Tu et al., 2019; Wada et al., 2017). Peptides with
unknown physiological function obtained in the current study and si-
milar structures reported by the mentioned in vivo studies were
summarized in Table 6. Among the peptides with unknown function,
neither exact nor similar structures for EEEY, EVVR, and CVK were
reported by the in vitro or in vivo studies.
Table 6 shows that the results of in silico studies are slightly different
from in vitro and in vivo due to the different conditions of in vitro and in
vivo environments. Thus, in silico studies will help us to identify these
differences and move the conditions toward the production of the de-
sired BPs, which will be crucial to predicting biotechnological treat-
ments to develop functional foods with specific health effects.
Among the assessed peptides, the maximum similarity percentage
belonged to QSEEQQQTEDEL. QSEEQQQTEDEL predicts 5.99 and 6.68
percent of the experimentally identified peptides in models 1 and 2,
respectively. Also, TEEEK predicts 4.15 percent of the results obtained
by Barbé et al., 2014. The most abundant residues in both peptides are
glutamine (Q) and glutamate (E) and it seems these residues have a
critical role in digestion resistance.
To the best of our knowledge, there was no study trying to quantify
digestion resistance and bioactive peptides in foodstuffs. For the first
time in the current in-silico study, the concentration of dairy items'
peptides was estimated; however, this method has also its limitations.

8
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Table 5
The validation of in-silico obtained results using data reported by Barbé et al. study (Barbé et al., 2014).
In-silico obtained peptides Number and percent of similar peptides reported by Barbé et al. study.

Model 1 Model 2

Origin Sequence Number Percent Number Percent

k-casein TIASGEPTSTPTTEAVESTVATL 4 0.461 5 0.576

αs2-casein SIGSSSEESAEVATEEVK 3 0.346 7 0.807
αs2-casein VSSSEESIISQETY 6 0.692 6 0.692
k-casein EDSPEVIESPPEIN 4 0.461 4 0.461
αs1-casein EAESISSSEEIVPN 7 0.807 61 7.03
β-casein QSEEQQQTEDEL 52 5.99 58 6.68
αs1-casein DIGSESTEDQAM 13 1.49 71 8.18
β-casein TQTPVVVPPF 15 1.73 40 4.61
β-casein SSSEESITR 6 0.692 6 0.692
β-lactoglobulin TPEVDDEAL 8 0.922 9 1.03
k-casein SCQAQPTTM 1 0.115 1 0.115
β-casein VPGEIVESL 7 0.807 8 0.922
k-casein TVQVTSTAV 1 0.115 1 0.115
α-Lactalbumin DTQAIVQN 1 0.115 1 0.115
β-lactoglobulin SAEPEQSL 1 0.115 1 0.115
αs1-casein EDVPSER 13 1.49 13 1.499
αs2-casein AVPITPTL 9 1.03 11 1.26
k-casein TEIPTIN 7 0.807 7 0.807
k-casein QEQPIR 1 0.115 1 0.115
β-lactoglobulin PTPEGDL 20 2.30 25 2.88
αs2-casein ESTEVF 7 0.807 26 2.99
β-lactoglobulin DAQSAPL 18 2.07 63 7.26
β-lactoglobulin IVTQTM 11 1.26 11 1.26
αs2-casein ITVDDK 26 2.99 57 6.57
β-lactoglobulin AASDISL 1 0.115 1 0.115
αs2-casein STSEEN 9 1.03 9 1.03
β-casein TESQSL 10 1.15 12 1.38
β-casein AQTQSL 19 2.19 43 4.95
β-casein PPQSVL 4 0.461 11 1.26
αs1-casein TDAPSF 9 1.03 10 1.15
β-lactoglobulin GECAQK 1 0.115 1 0.115
αs2-casein TEEEK 36 4.15 36 4.15
k-casein SPAQIL 11 1.26 14 1.61
αs2-casein QGPIVL 8 0.922 9 1.03
αs1-casein PIGSEN 34 3.92 47 5.42
β-casein PGPIPN 3 0.346 3 0.346
Total 386 44.5 689 79.46

Model 1: peptides with E-value less than 0.05 were considered as similar peptides.
Model 2: peptides with E-value less than 0.05 or 100% identity were considered as similar.
* Percent was calculated as (number of similar peptides/867) × 100.

The lack of validation experiments was the main limitation of the Disclosure statement
present study. Although lack of validation experiments may call our
results into question, using current in silico study, for the first time, we No potential conflict of interest was reported by the authors.
tried to reveal that determination of digestion resistance and bioactive
peptide content of foodstuffs is a very important matter. Without ret-
Author contributions
rospective and/or prospective studies we could not consider food-de-
rived peptides as bioactive. In other words, although in vitro, in vivo,
Conceptualization, M.B; Data collection and analysis, M.B, F.J, M.J;
and RCTs showed that most food-derived peptides are bioactive, but
writing—original draft preparation, A.M.Y, FF, F.J, M.B.; Software,
beneficial or detrimental effects of these peptides on health and disease
M.B, H.F; review and editing, A.M.K and I.E, S.H.D. All authors have
should be confirmed in long-term studies. Determination of food-de-
read and agreed to the published version of the manuscript.
rived peptides can help researchers to perform long-term studies.

CRediT authorship contribution statement

5. Conclusion
In this in silico study, we reported the amount of BPs content of dairy
products from FFQ used in the TLGS, a large-scale community-based
prospective study. Using the results obtained from the current study
risk assessment could be performed on whether dietary intake of di-
gestion-resistant peptides decreases the risk of chronic diseases such as
CVD and cancer or not. The validity of the data reported in this study
should be evaluated by in vitro (enzymatic digestion) and in vivo (sta-
bility of the peptides to gastrointestinal digestion) studies.
Meisam Barati: Software, Writing - original draft, Formal analysis,
Data curation, Conceptualization. Fardin Javanmardi: Writing - ori-
ginal draft, Formal analysis, Data curation. Masoumeh Jabbari:
Formal analysis, Data curation, Resources, Visualization. Amin
Mokari-Yamchi: Writing - original draft, Methodology, Validation.
Fariba Farahmand: Investigation, Resources. Ismail Eş: Writing - re-
view & editing. Hossein Farhadnejad: Software. Sayed Hossein
Davoodi: Project administration, Funding acquisition, Supervision.
Amin Mousavi Khaneghah: Writing - review & editing, Project ad-
ministration, Supervision.

9
M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Table 6
The obtained peptides with un-known biological function and similar structures which have been reported by in-vivo studies.
αs1-casein
In-silico peptide Similar peptides characterized in-vivo In-silico peptide Similar peptides characterized in-vivo
Sequence Sequence
PSGAW QLDAYPSGA (Wada et al., 2017) IGVN IGVNQE (Wada et al., 2017), QQKEPMIGVN (Barbé et al., 2014)
EN EGIH EGIHAQQKEPMIG (Wada et al., 2017), HSMKEGIHA (Barbé et al.,
2014)
SEK PIGSEN PNPIGSENSEK (Barbé et al., 2014)
SVEQK SVEQKHIQKEDVPSER (Barbé et al., 2014) PQEVL HQGLPQEVL (Barbé et al., 2014)
EDIK AMEDIK (Bouzerzour et al., 2012), DAY QLDAYPSGA (Wada et al., 2017)
DQAMEDIKQ (Wada et al., 2017), EDIKQMEAE
(Barbé et al., 2014)
EQL YLGYLEQLLR (Barbé et al., 2014) VPQL KYKVPQL (Wada et al., 2017), KYKVPQL (Barbé et al., 2014)
SAEER EIVPNSAEER (Barbé et al., 2014) PIK HPIKH (Wada et al., 2017), HPIKHQGLPQEV (Barbé et al., 2014)
QEL QQKEPMIGVNQEL (Barbé et al., 2014) SDIPN FSDIPNG (Bouzerzour et al., 2012), FSDIPNPI (Wada et al., 2017),
SDIPNPIGSENSEK (Barbé et al., 2014), SDIPNPI (Tu et al., 2019)
IQK IQKEDVPSER (Barbé et al., 2014), HIQKE PEVF APFPEVFG (Wada et al., 2017), PFPEVFGK (Barbé et al., 2014)
(Wada et al., 2017)
AQQK GIHAQQKEPMIG (Barbé et al., 2014), TDAPSF TDAPSFS (Wada et al., 2017), TDAPSFSDIPNPIGSENSEKT (Barbé
IHAQQKEPMIG (Wada et al., 2017) et al., 2014)
EDVPSER EDVPSERY (Barbé et al., 2014) EPM GIHAQQKEPMIG (Barbé et al., 2014), IHAQQKEPMIG (Wada et al.,
2017)
EAESISSSEEIVPN SSEEIVPN, SEEIVPN, ISSSEEI (Bouzerzour QGL HQGLPQEVL (Barbé et al., 2014), HPIKHQGLPQEV (Barbé et al.,
et al., 2012), ISSSEEIVPNS (Wada et al., 2017) 2014)
EIVPN SEEIVPNS (Wada et al., 2017), EIVPNSAEER QM EDIKQMEAE (Barbé et al., 2014)
(Barbé et al., 2014)
DIGSESTEDQAM TEDQAMEDIK (Bouzerzour et al., 2012), SM HSMKEGIHA (Barbé et al., 2014)
DIGSESTEDQAMEDIK (Barbé et al., 2014)
TTM VAPF FVAPFPE (Tu et al., 2019), FFVAPFPE (Wada et al., 2017),
FVAPFPEVFGK (Barbé et al., 2014)
αs2-casein
CSTF NLCSTFC (Wada et al., 2017) TVDM KTVDMESTEVFTKKT (Barbé et al., 2014)
AM ESTEVF TVDMESTEVFTK
QGPIVL YQGPIVLNPWDQVK (Barbé et al., 2014) SIGSSSEESAEVATEEVK SIGSSSEESAE (Wada et al., 2017), TEEVKITVDDKHYQ (Barbé et al.,
2014)
PQY FALPQYLKT (Wada et al., 2017) VSSSEESIISQETY KNTMEHVSSSEESIISQETYKQE (Barbé et al., 2014)
CK EQL
AVPITPTL VPITPTL (Bouzerzour et al., 2012), EIN ALNEINQFYQKFPQ (Barbé et al., 2014)
NAVPITPTLN (Barbé et al., 2014)
VIPY TKVIPYVRY (Barbé et al., 2014) DQVK NPWDQVKR (Barbé et al., 2014)
IQPK KPWIQPK (Tu et al., 2019), AMKPWIQPK ITVDDK ITVDDKHYQ (Barbé et al., 2014)
(Barbé et al., 2014)
PSK NPSKENLCSTFC (Barbé et al., 2014), STSEEN NREQLSTSEENSKK (Barbé et al., 2014)
NPSKENLC (Wada et al., 2017),
AIN AINPSKENLCSTFCKE (Barbé et al., 2014) EN
ISQR LKKISQRYQK (Barbé et al., 2014) QEK KNTMEHVSSSEESIISQETYKQEK (Barbé et al., 2014)
AN EEEY –
EVVR – TEEEK LTEEEKNRL (Barbé et al., 2014)
Beta-casein
GPF GPFPIIV (Tu et al., 2019), PVRGPFPII (Barbé EM
et al., 2014), RGPFP (Wada et al., 2017)
IPPL NIPPLT (Bouzerzour et al., 2012), QEPVL LLYQEPVLGPVRGPF (Barbé et al., 2014), QEPVLGPV (Wada et al.,
SLPQNIPPLTQ (Barbé et al., 2014), IPPL 2017)
(Nongonierma et al., 2017), PQNIPPLTQT
(Barbé et al., 2014)
PIQAF DMPIQAF (Nongonierma et al., 2017), EAM VKEAMAPK (Barbé et al., 2014)
SQSKVLPVPQKAVPYPQRDMPIQAF (Barbé
et al., 2014)
QSW QSWMHQPHQPLPPTV (Barbé et al., 2014), SSSEESITR GEIVESLSSSEESITRINKKIEKFQSEKQQQTEDELQDKIHPF (Barbé
LQSWMHQPH (Nongonierma et al., 2017) et al., 2014)
QPL HQPLPPT (Tu et al., 2019), QPLPPT VPGEIVESL NVPGEIV (Bouzerzour et al., 2012), LNVPGEIVESL (Barbé et al.,
(Nongonierma et al., 2017), HQPLPP (Wada 2014)
et al., 2017), MHQPHQPLPPT (Barbé et al.,
2014)
PVEPF PKYPVEPF (Tu et al., 2019), YPVEPF AQTQSL IHPFAQTQSL (Barbé et al., 2014)
(Nongonierma et al., 2017), PVEPFT (Wada
et al., 2017), PVEPFTERQ (Barbé et al., 2014)
PPTVM LPPTVMFPPQ (Tu et al., 2019), PLPPTVMFPP SQSK QSKVL (Bouzerzour et al., 2012), SQSKVLPVPQK (Barbé et al.,
(Wada et al., 2017), HQPHQPLPPTVM (Barbé 2014)
et al., 2014)
GPVR LLYQEPVLGPVRGPF (Barbé et al., 2014), GVSK SLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEA (Wada et al., 2017),
GPVRGPFP (Wada et al., 2017) MGVSKVKE (Barbé et al., 2014)
PPQSVL HQPHQPLPPTVMFPPQSVLS (Wada et al., QDK DELQDKIHP (Wada et al., 2017), LQDKIHP (Barbé et al., 2014)
2017), MHQPHQPLPPTVMFPPQSVL (Barbé
et al., 2014)
(continued on next page)

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M. Barati, et al. LWT - Food Science and Technology 130 (2020) 109616

Table 6 (continued)

QPH QPHQPLPPT (Wada et al., 2017), TESQSL PVEPFTESQSLT (Barbé et al., 2014)
HQPHQPLPPT (Barbé et al., 2014),
QPHQPLPPT (Nongonierma et al., 2017)
AVPY AVPYPQR (Tu et al., 2019), KAVPYPQR (Barbé QSEEQQQTEDEL FQSEEQQQTEDEL (Barbé et al., 2014)
et al., 2014)
APK VKEAMAPK (Barbé et al., 2014) IEK
PQN LPQNIPP (Nongonierma et al., 2017), EEL
SLPQNIPPLT (Wada et al., 2017), PQNIPPLTQT
(Barbé et al., 2014)
QPEVM QPEVM (Bouzerzour et al., 2012), TDVEN TDVENLHLPLP (Barbé et al., 2014)
TQTPVVVPPFLQPEVMGV (Wada et al., 2017),
PVVVPPFLQPEVM (Barbé et al., 2014)
PIIV GPFPIIV (Tu et al., 2019) TQTPVVVPPF TQTPVVVPPFL (Bouzerzour et al., 2012), VVVPPFLQP (Barbé et al.,
2014)
PVPQK SKVLPVPQK (Barbé et al., 2014)
k-casein
SPAQIL VRSPAQIL (Barbé et al., 2014), SPAQILQWQ PAAVR YAKPAAVR (Barbé et al., 2014)
(Tu et al., 2019)
AIPPK MAIPPK (Wada et al., 2017), MAIPPKKNQDK EDSPEVIESPPEIN EDSPEVIESPPEINT (Barbé et al., 2014), EDSPEVIESPPEINT (Wada
(Barbé et al., 2014) et al., 2017)
SCQAQPTTM SNTVPAKSCQAQPT (Wada et al., 2017) CEK EQPIRCEKDE (Wada et al., 2017)
QEQPIR EQNQEQPI (Wada et al., 2017), SN SNTVPAKSC (Wada et al., 2017)
SR QVL SPAQILQWQVL (Wada et al., 2017)
PVAL YQQKPVAL (Barbé et al., 2014) AK
TEIPTIN TEIPTINT (Wada et al., 2017), DKTEIPTINT TVPAK SNTVPAKSC (Wada et al., 2017)
(Barbé et al., 2014)
DER – SDK FFSDKIAK (Tu et al., 2019), FSDKIAK (Barbé et al., 2014)
QQK YQQKPVAL (Barbé et al., 2014) QDK NQDKTEIP (Barbé et al., 2014), MAIPPKKNQDK (Barbé et al., 2014)
QEQN QEQNQEQPI (Wada et al., 2017) TVQVTSTAV TVQVTSTAV (Skrzypczak et al., 2017)
TIASGEPTSTPTTEAVESTVATL PTTEAVES (Bouzerzour et al., 2012),
TIASGEPTSTPTTEAVEST (Wada et al., 2017)
α-Lactalbumin
PEW LPEWV (Wada et al., 2017) CEVF EQLTKCEVFR (Wada et al., 2017), TKCEVFRE (Nongonierma et al.,
2017)
DQW DQWLCE (Wada et al., 2017) CK
ICN SSNICNI (Wada et al., 2017) VCTTF CTTFHTSGYDTQA (Wada et al., 2017)
DL GGVSL GYGGVSLP (Wada et al., 2017)
DDDL FLDDDLTDD (Wada et al., 2017) ISCDK NISCDK (Wada et al., 2017)
TSGY TTFHTSGYDT TDDIM DLTDDIMC (Wada et al., 2017)
CVK – CEK
VGIN ILDKVGINY (Nongonierma et al., 2017) CSEK CSEKLDQ (Wada et al., 2017), LCSEKLDQ (Nongonierma et al.,
2017)
QIN GLFQINNKIWCKDDQNPHSSNIC (Wada et al., SSN SSNICNI (Wada et al., 2017)
2017)
DDQN DDQNPH (Barbé et al., 2014) DK NISCDK (Wada et al., 2017)
DSTEY IVQNNDSTEYGLF (Nongonierma et al., 2017) DTQAIVQN TTFHTSGYDTQA (Wada et al., 2017)
EQL EQLTKCEVFR (Wada et al., 2017)
β-lactoglobulin
CM CMENSAEP (Wada et al., 2017) PTQL NPTQLE (Wada et al., 2017), LSFNPTQLEEQCHI (Barbé et al., 2014)
ACQCL MENSAEPEQSLACQC (Wada et al., 2017) SAEPEQSL NSAEPEQSL (Wada et al., 2017), MENSAEPEQSL (Barbé et al.,
2014)
AM GECAQK WENGECAQ (Wada et al., 2017)
DAQSAPL DAQSAPLR (Wada et al., 2017), LDAQSAPLR DTDY VLDTDY (Nongonierma et al., 2017), VLDTDYKK (Wada et al.,
(Barbé et al., 2014) 2017)
PTPEGDL ELKPTPEGD (Bouzerzour et al., 2012), EIL LKPTPEGDLEIL (Nongonierma et al., 2017), VEELKPTPEGDLEIL
LKPTPEGDL (Barbé et al., 2014) (Barbé et al., 2014) VEELKPTPEGDLEILLQKWEN (Wada et al.,
KPTPEGDLE (Wada et al., 2017) 2017)
IDAL KIDALNE (Barbé et al., 2014) EEQCH EEQCHI (Wada et al., 2017), LSFNPTQLEEQCH (Barbé et al., 2014)
AASDISL SDISLLDAQSAPLR (Wada et al., 2017), TPEVDDEAL TPEVDDEALEK ( Nongonierma et al., 2017), VRTPEVDDEAL (Barbé
SDISLLDAQSAPL (Barbé et al., 2014) et al., 2014)
DIQK LIVTQTMKGLDIQKVAGT (Wada et al., 2017), EN
LDIQKVAGTW (Barbé et al., 2014)
IVTQTM IVTQTMKGLDIQ (Wada et al., 2017), VEEL RVYVEEL (Bouzerzour et al., 2012), YVEELKPT (Wada et al., 2017),
IVTQTMK (Barbé et al., 2014) VEELKPTPEGD (Barbé et al., 2014)
DK ALEKFDK (Barbé et al., 2014)

Declaration of competing interest Committee, Shahid Beheshti University of Medical Sciences, Tehran,
Iran. We also appreciate the “Student Research Committee” and
The authors declare that they have no conflict of interest. “Research & Technology Chancellor” at Shahid Beheshti University of
Medical Sciences for their financial support of this study.

Acknowledgements Appendix A. Supplementary data

This study is related to the project NO 20803 with a code of ethics Supplementary data to this article can be found online at https://
“IR.SBMU.RETECH.REC.1398.774” from the Student Research doi.org/10.1016/j.lwt.2020.109616.

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M. Barati, et al.
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