Amino Acids

https://doi.org/10.1007/s00726-022-03152-6

ORIGINAL ARTICLE

Estimation of bioactive peptide content of milk from different species

using an in silico method
Karim Parastouei1 · Masoumeh Jabbari2 · Fardin Javanmardi3 · Meisam Barati1,5 · Yaser Mahmoudi4 ·
Sajad Khalili‑Moghadam5 · Houssein Ahmadi6 · Sayed Hossein Davoodi5,7 · Amin Mousavi Khaneghah8

Received: 13 January 2022 / Accepted: 1 March 2022

© The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2022

Abstract
This study assessed the bioactive peptides content of milk from different species, including humans, camel, bovine, buffalo,
donkey, sheep, goat, and horse. The highest and lowest concentrations of total digestion-resistant peptides were estimated
in sheep and human milk. Donkey milk casein contains a higher angiotensin-converting enzyme (ACE) inhibitory, dipepti-
dyl peptidase III (DPP-III) inhibitory, DPP-IV inhibitory, and antioxidant peptides. On the other hand, camel whey protein
contains the highest ACE-inhibitory peptides. To discover BPs with immunomodulatory and cholesterol-lowering functions,
goat milk casein and sheep milk whey protein can be considered, respectively.

Keywords Milk · Bioactive peptides · In silico · Quantification

Introduction Immunomodulatory effects and antihypertensive, antidia-

Milk is a nutritious food item containing numerous bioac-
tive compounds and nutrients such as minerals, vitamins,
essential fatty acids, conjugated linoleic acid, and bioac-
tive peptides (BPs) (Chalupa-Krebzdak et al. 2018). BPs are
digestion-resistant and absorbable fragments released dur-
ing the digestion process from parent proteins and induce
a couple of beneficial health effects on the body systems.
Handling editor: F. Eisenhaber.
betic, anti-carcinogenic, anti-thrombosis, and cholesterol-
lowering roles are reported beneficial health effects for BPs.
Milk is one of the primary sources of BPs (Barati et al.
2020c, 2020a). Nowadays, a growing body of research has
investigated milk from different animal species to find which
one has more beneficial health effects. Carbohydrates, fats,
proteins, micronutrients, and bioactive compounds in dif-
ferent types of milk differ by animal species. Indeed, these
differences play a considerable role in different biological

5
* Meisam Barati Department of Clinical Nutrition and Dietetics, Faculty
meisam.barati@sbmu.ac.ir of Nutrition Science and Food Technology, National
Nutrition and Food Technology Research Institute, Shahid
* Amin Mousavi Khaneghah
Beheshti University of Medical Sciences, Tehran, Iran
mousavi@unicamp.br; mousavi.amin@gmail.com
6
Department of Biology and Anatomical Sciences, School
1
Health Research Center, Life Style Institute, Baqiyatallah of Medicine, Shahid Beheshti University of Medical
University of Medical Sciences, Tehran, Iran Sciences, Tehran, Iran
2 7
Department of Community Nutrition, Faculty of Nutrition Cancer Research Center, Shahid Beheshti University
Science and Food Technology, National Nutrition and Food of Medical Sciences, Tehran, Iran
Technology Research Institute, Shahid Beheshti University 8
Department of Food Science and Nutrition, Faculty of Food
of Medical Sciences, Tehran, Iran
Engineering, University of Campinas (UNICAMP), Rua
3
Department of Food Science and Technology, Faculty Monteiro Lobato, 80. Caixa Postal: 6121, Campinas,
of Nutrition Science and Food Technology, National São Paulo CEP: 13083‑862, Brazil
Nutrition and Food Technology Research Institute, Shahid
Beheshti University of Medical Sciences, Tehran, Iran
4
Department of Anatomical Sciences, Yasuj University
of Medical Sciences, Yasuj, Iran

13
Vol.:(0123456789)

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effects of milk from different species. For instance, goat
milk contains a higher amount of vitamin A; while protein
and calcium are more abundant in sheep milk. Many studies
have been done to assess the macronutrient and micronu-
trient content of milk from different species (Derdak et al.
2020; Khan et al. 2019; Potočnik et al. 2011); while the
determination of BPs content in different milk sources are
not quantified yet (Barati et al. 2020b).
The amount and amino-acid sequence of milk pro-
teins, including αs1-casein, αs2-casein, β-casein, k-casein,
β-lactoglobulin, and α-lactalbumin, is varied among mam-
malian species. These differences are the main reason for
the variations in the biological activity of milk proteins from
different species (Shariatikia et al. 2017). Due to the expen-
sive and time-consuming methods of BPs quantifying, no
previous studies have been done to quantify the BPs content
of milk from different species. In our recent work, we devel-
oped and validated an in silico method for quantifying the
BPs content of food items (Barati et al. 2020b). The current
study comprehensively estimated the BPs content of cow,
Protein amount (gram per 100 g milk or milk proteins)
Molar concentration =
molecular weight
buffalo, human, donkey, sheep, goat, and camel milk by this
validated in silico method.
Materials and methods
BPs content of milk from different species was estimated
using an in silico method previously developed and validated
by our research team (Barati et al. 2020b). In brief, BPs con-
tent of cow (Bos taurus), buffalo (Bubalus bubalis), human
(Homo sapiens), donkey (Equus asinus), sheep (Ovis aries),
goat (Capra hircus), horse (Equus caballus), and camel
(Camelus dromedarius) milk were assessed in this study.
The amino-acid sequence of αs1-casein, αs2-casein, β-casein,
k-casein, β-lactoglobulin, and α-lactalbumin (The significant
contributions of milk proteins) from the mentioned species
were obtained from UniProtKB (https://​www.​unipr​ot.​org)
and NCBI (https://​www.​ncbi.​nlm.​nih.​gov) databases (Sup-
plementary Table 1). Using the Expasy peptide mass cal-
culator (https://​web.​expasy.​org/​pepti​de_​mass/), the mass of
the mature proteins (without signal peptide) was estimated
(Supplementary Table 2). The BIOPEP tool was used for the
in silico digestion of milk proteins (Minkiewicz et al. 2008).
To simulate gastrointestinal digestion, the chymotrypsin A
(EC 3.4.21.1), trypsin (EC 3.4.21.4), and pepsin (pH: 1.3;
EC 3.4.23.1) were used simultaneously for proteolysis.
13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
K. Parastouei et al.
Then we recorded the total number of digestion-resistant
peptides, including dipeptides, tripeptides, and peptides with
more than three residues. To find BPs among the obtained
fragments, each peptide was searched on the BIOPEP data-
base, and their reported function(s) were recorded. Then, a
total number of (1) angiotensin-converting enzyme (ACE)
inhibitory peptides, (2) dipeptidyl peptidase III (DPP-III)
inhibitory peptides, (3) immunomodulatory peptides, (4)
anticancer peptides, (5) hypo-cholesterolemic peptides, (6)
antithrombotic peptides and (7) antioxidant peptides for each
protein were recorded.
In the current manuscript, immunomodulatory peptides
referred to the peptides that can modulate lymphocyte pro-
liferation, increase antibody production, phagocytic activity,
or maturation of natural killer cells in vitro and/or in vivo.
Antithrombotic peptides can inhibit fibrinogen, platelet,
fibronectin, or thrombin. Antioxidant peptides included the
peptides scavenge free radicals in vitro and/or in vivo. Anti-
cancer peptides that inhibit cancer cells growth in vitro and/
or in vivo.
The following equations did an estimation of BPs:
.
BPs content (Mol) =[αs1casein (Mol) × peptide number]
+ [αs2casein(Mol) × peptide number]
+ [β casein (Mol) × peptide number]
+ [kcasein (Mol) × peptide number]
+ [βlactoglobulin (Mol) × peptide number]
+ [α lactalbumin (Mol) × peptide number]
The identification and estimation method of digestion-
resistant and bioactive peptides has been illustrated in Fig. 1.
The characteristics of the studied milks in terms of their pro-
tein composition are summarized in Supplementary Table 3.
Results
The current in silico study assessed BPs content of milk
from different species, including human, camel, bovine, buf-
falo, donkey, sheep, goat, and horse after in silico digestion
of αs1-casein, αs2-casein, β-casein, k-casein, β-lactoglobulin,
and α-lactalbumin. Except for human and camel milk, all
the assessed milk contains the aforementioned proteins.
Human milk contains neither αs2-casein nor β-lactoglobulin.
Also, camel milk has no β-lactoglobulin. ҡ-casein in don-
key milk has been reported as non-detectable (Supplemental
Table 3). The fragments obtained from in silico digestion are
Estimation of bioactive peptide content of milk from different species using an in silico method
Fig. 1  Estimation of BP content of milk proteins. (1) digestion of
αs1-casein by simultaneous actions of pepsin, trypsin and chymot-
rypsin; (2) finding ACE inhibitory peptides among the obtained frag-
ments by searching on BIOPEP database; (3) estimation of αs1-casein
derived ACE inhibitory peptides. By sum of ACE inhibitory peptides
from αs1-casein, αs2-casein, β-casein, k-casein, β-lactoglobulin, and
summarized in Table 1. The concentrations were reported
in mmol/100 g milk, mmol/100 g milk protein, mmol/100 g
milk whey protein, and mmol/100 g milk casein.
Estimation of BP content of milk from different
species in mmol/100 g milk
In Table 2, the estimated concentration of digestion-resistant
and bioactive peptides is reported. The highest concentration
of total digestion-resistant peptides was estimated in sheep
milk with 10.73 mmol/100 g milk, while the lowest concen-
tration was obtained from human milk (0.14 mmol/100 g).
Also, among the all studied milk, sheep milk contains the
highest amount of low-molecular-weight (LMW) peptides,
i.e., di and tri-peptides (6.16 mmol/100 g milk), high-molec-
ular-weight (HMW) peptides, i.e., peptides with more than
three residues (4.56 mmol/100 g milk), ACE inhibitory
peptide (2.15 mmol/100 g milk), DPP-III inhibitory peptide
(0.33 mmol/100 g milk), antioxidant peptide (0.36 mmol/100 g
milk), DPP-IV inhibitory peptide (3.14 mmol/100 g milk),
anti-thrombosis peptide (0.08 mmol/100 g milk), immu-
nomodulatory peptide (0.17 mmol/100 g milk) and choles-
terol-lowering peptide (0.03 mmol/100 g milk). Anticancer
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
α-lactalbumin, total ACE inhibitory peptides in a milk is calculated.
BP, Bioactive peptide; ACE, angiotensin-converting enzyme; A, ala-
nine; R, arginine; N, asparagine; D, aspartate; C, cysteine; E, gluta-
mate; 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
peptides only were found in bovine milk. On the other hand,
antithrombotic peptides were only obtained from sheep and
goat milk. Also, cholesterol-lowering peptides only were
observed in sheep, goat, buffalo, and bovine milk. The lowest
levels of all estimated BPs were obtained from human milk.
Estimation of BP concentration per 100 g milk could be
affected by the total content of proteins in milk from dif-
ferent species. For example, sheep milk contains the high-
est amount of BPs per 100 g milk among all other studied
milk because of its highest total protein content. In contrast,
human milk contains the lowest total protein and the low-
est amount of BPs per 100 g milk. Therefore, in the fol-
lowing sub-sections, the BP concentrations were estimated
per 100 g milk protein, 100 g milk casein, and 100 g milk
whey protein. All studied milk was ranked based on their
digestion-resistant and bioactive peptides (Table 3).
Estimation of BP content of milk from different
species in mmol/100 g total protein
Total digestion-resistant (195.05 mmol/100 g protein) and
cholesterol-lowering peptides (0.65 mmol/100 g protein)
13
 Table 1  The peptides retrieved from in silico digestion of different milks proteins by pepsin, trypsin and chymotrypsin
αs1-Casein αs2-Casein k-Casein β-Casein α-Lactalbumin β-Lactoglobulin

13
Cow > sp|P02662|16–214 > sp|P02663|16–222 > sp|P02668|22–190 > sp|P02666|16–224 > sp|P00711|20–142 > sp|P02754|17–178
PK -PIK—QGL— TM—EH—VSSSEESI- QEQN—QEQPIR—CEK— EL—EEL—VPGEIVESL— EQL—TK—CEVF—EL— IVTQTM—GL—DIQK—
PQEVL—EN—VAPF— ISQETY—QEK— DER—SDK—IAK— SSSEESITR—IN— DL—GY—GGVSL— VAGTW—SL—AM—
PEVF—GK—EK— AIN—PSK—EN— IPIQY—VL—SR— IEK—QSEEQQQT- PEW—VCTTF—TSGY— AASDISL—DAQSAPL—
VN—EL—SK—DIG- CSTF—CK—EVVR— PSY—GL—QQK— EDEL—QDK—IH— DTQAIVQN—DSTEY— VY—VEEL—PTPEGDL—
SESTEDQAM—EDIK— AN—EEEY—SIGSS- PVAL—IN—QF—PY— PF—AQTQSL—VY— GL—QIN—IW—CK— EIL—QK—EN—
QM—EAESISSSEE- SEESAEVATEEVK— PY—AK—PAAVR— PF—PGPIPN—SL— DDQN—PH—SSN— GECAQK—IIAEK—
IVPN—SVEQK—IQK— ITVDDK—QK—AL— SPAQIL—QW— PQN—IPPL—TQTPV- ICN—ISCDK—DDDL— TK—IPAVF—IDAL—
EDVPSER—EQL— EIN—QF—QK—PQY— QVL—SN—TVPAK— VVPPF—QPEVM— TDDIM—CVK—IL— EN—VL—VL—DTDY—
VPQL—EIVPN— QY—QGPIVL—PW— SCQAQPTTM—AR— GVSK—VK—EAM— DK—VGIN—AH—AL— CM—EN—SAEPEQSL—
SAEER—SM—EGIH— DQVK—AVPITPTL— PH—PH—SF—AIPPK— APK—EM—PF—PK— CSEK—DQW—CEK ACQCL—VR—TPEVD-
AQQK—EPM—IGVN— EQL—STSEEN—SK— QDK—TEIPTIN—TIAS- PVEPF—TESQSL—TL DEAL—EK—DK—AL—
QEL—AY—PEL—QF— TVDM—ESTEVF—TK— GEPTSTPTTEAVEST- – TDVEN- PL—PL— AL—PM—IR—SF—
QL—DAY—PSGAW— TK—TEEEK—ISQR— VATL—EDSPEVIESP- QSW—QPH—QPL— PTQL—EEQCH
VPL—GTQY— QK—AL—PQY—TVY— PEIN—TVQVTSTAV PPTVM—PPQSVL—
TDAPSF—SDIPN—PIG- QH—QK—AM—PW— SL—SQSK—VL—
SEN—SEK—TTM—PL IQPK—TK—VIPY—VR PVPQK—AVPY—PQR—
DM—PIQAF—QEPVL—
GPVR—GPF—PIIV
Buffalo > sp|O62823|16–214 > NP_001277794.1 > sp|P11840|22–190 > sp|Q9TSI0|16–224 > sp|Q9TSN6|19–142 > sp|P02755|19–180
PK—QPIK—QGL— TM—EH—VSSSEESI- QEQN—QEQPIR— EL—EEL—VPGEIVESL— AEQL—TK—CEVF— IIVTQTM—GL—DIQK—
PQGVL—EN—VAPF— ISQETY—QEK— CEK—EER—DK— SSSEESITH—IN— EL—DL—DY— VAGTW—SL—AM—
PEVF—GK—EK— AIH—PSK—EN— IAK—IPIQY—VL— IEK—QSEEQQQM— GGVSL—PEW— AASDISL—DAQSAPL—
VN—EL—STDIGSEST- CSTF—CK—EVIR— SR—PSY—GL—QQK— EDEL—QDK—IH—PF— VCTAF—TSGY— VY—VEEL—PTPEGDL—
EDQAM—EDIK— AN—EEEY—SIGSS- PVAL—IN—QF—PY— AQTQSL—VY—PF— DTQAIVQN—DSTEY— EIL—QK—EN—
QM—EAESISSSEE- SEESAEVATEEVK— PY—AK—PAAVR— PGPIPK—SL—PQN— GL—QIN—IW—CK— GECAQK—IIAEK—
IVPISVEQK—IQK— ITVDDK—QK—AL— SPAQIL—QW— IPPL—TQTPVVVPPF— DDQN—PH—SSN— TK—IPAVF—IDAL
EDVPSER—GY— EIN—QF—QK—PQY— QVL—PN—TVPAK— QPEIM—GVSK—VK— ICN—ISCDK—DDDL— -EN—VL—VL—
EQL—VPQL—EIVPN— QY—QGPIVL—PW— SCQAQPTTM—TR— EAM—APK—EM—PF— TDDIM—CVK—IL— DTDY—CM—EN—
AEEQL—SM—EGIH— DQVK—AVPITPTL— PH—PH—SF—AIPPK— PK—PVEPF—TESQSL— DK—VGIN—AH—AL— SAEPEQSL—ACQCL—
AQQK—EPM—IGVN— EQL—STSEEN—SK— QDK—TEIPTIN—TIVS- TL—TDVEN—PL— CSEK—DQW—CEK VR—TPEVDDEAL—
QEL—AY- PQL—QF— TVDM—ESTEVF— VEPTSTPTTEAIEN— PL—QSW—QPPQPL— EK—DK—AL—AL—PM
QL—DAY—PSGAW— TK—TK—TEEDK— TVATL—EASSEVIESV- PPTVM—PPQSVL— – IR- SF—PTQL—EEQCH
VPL—GTQY— ISQH—QK—AW— PETN—TAQVTSTVV SL—SQSK—VL—
PDAPSF—SDIPN—PIG- PQY—TVY—QY— PVPQK—AVPY—PQR—
SEN—SGK—TTM—PL QK—AM—PW— DM—PIQAF—QEPVL—
TQPK—TN—VIPY—VR GPVR—GPF—PIIV

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K. Parastouei et al.
 Table 1  (continued)
αs1-Casein αs2-Casein k-Casein β-Casein α-Lactalbumin β-Lactoglobulin

Human > sp|P47710|16–185 Absent > sp|P07498|21–182 > sp|P05814|16–226 > sp|P00709|20–142 Absent
PK- PL—PER—QN— EVQN—QK—QPACH— ETIESL—SSSEESI- QF—TK—CEL—SQL—
PSESSEPIPL—ESR— EN—DER—PF—QK— TEY—QK—VEK— DIDGY—GGIAL—
EEY—GM—QR—IL— TAPY—VPM—VPN— VK—EDQQQGEDEH— PEL—ICTM—TSGY—
EK—QTDEIK—DTR— SY—PY—GTN—QR— QDK—IY—PSF— DTQAIVEN—ESTEY—
ESTQN—CVVAEPEK— PAIAIN—PY—VPR— QPQPL—IY—PF— GL—QISN—CK—
ESSISSSSEEM—SL— TY—AN—PAVVR— VEPIPY—GF—PQN— SSQVPQSR—ICDIS-
SK—CAEQF—CR— PH—AQIPQR—QY— IL—PL—AQPAVVL— CDK—DDDITDDIM—
EY—QL—QL—QAAH— PN—SH—PPTVVR— PVPQPEIM—EVPK— CAK—IL—DIK—
AQEQIR—EN—SH— PN—PSF—IAIPPK— AK—DTVY—TK—GR— GIDY—AH—AL—
VQVPF—QQL—QL— IQDK—IIIPTIN—TIAT- VM—PVL—SPTIPF— CTEK—EQW—CEK
AAY—PY—AVW— VEPTPAPATEPTVDSV- DPQIPK—TDL—
PQIM—QY—VPF— VTPEAF—SESIITST- EN—PL—PL—QPL—
PPF—SDISN—PTAH— PETTTVAVTPPTA QQVPQPIPQTL—AL—
EN – EK- VM—QW PPQPL—SVPQPK—
VL—PIPQQVVPY—
PQR—AVPVQAL—
QEL—PTH—QIY—
PVTQPL—APVH—PISV
Donkey > sp|P86272|1–202 > sp|B7VGF9|16–236 > CBZ41787.1 > sp|P86273|1–226 > sp|P28546|1–123 > sp|P19647|1–163
PK—PH—PEIIQN— EH- SSSEDSVN – ISQEK EVQN—QEQPTCR— EK—EEL—VSSET- QF—TK—CEL—SQVL— TDIPQTM—QDL—DL—
EQDSR—EK—VL— - QEK- VVIPTSK – DER—DL—TVK— VESL—SSN—EPDSS- SM—DGY—GVTL— QEVAGR—SVAM—
ER—PSF—AL—TPR— ESICSTSCEEATR- IN— IPVY—VL—SSPR— SEESITH—IN—EK— PEW—ICTIF—SSGY— VASDISL—DSESAPL—
EEY—IN—EL—QR— EM—ESAK—PTEVY EPIY—QH—AVL— SQK—EGQQQR— DTQTIVK—- GK— VY—VEEL—PTPEGN—
EL—EK—QK—DEH- – SSSSSSEESAK- IN—QH—PY—QY— EVEH—QDK—ISR— TEY—GL—QIN— EIIL—EGAN—VCVER—
EY—IEDPEQQESSSTSS- PTER—EEK—EVEEK— AR—PAAVR—PH— VQPQPVVY—PY— CR—DN—QIL—PSR— IVAQK—TEDPAVF—
SEEVVPIN—TEQK— QL—IN—QF—EK— VQIPQW—QVL—PN— AEPVPY—AVVPQN— ICGISCN- DDDL— TVN—QGER—ISVL—
Estimation of bioactive peptide content of milk from different species using an in silico method

IPR—EDM—QH—TL— QY—QAL—QPR— IY—PSTVVR—PR— IL—VL—AQPPIVPF— TDDVM—CAK—IL— DTDY—AH—CVG-

EQL—SK—QL—QL— IVL—TPW—DQTK— PH—PSF—IAIPPK— QPEIM—EVSQAK— DSEGIDY—AH—PL— PCL—PSAEH—GM—
QAIY—AQEQL—IR— TGASPF—IPIVN— QEK—TVIPK—IN— ETIL—PK—VM—PF— CSEK—EQW—CEEL VCQY—AR—TQK—
EN—SQR—PM—VVN— TEQL—TSEEIPK— TIATVEPTPIPTPEP- SPIVPF—SER—QIL— VDEEVM—EK—SR—
QEQAY—EPF—QPSY— TVDM—ESTEVVTEK— TVN—AVIPDASSEF— PTN—GEN—PVH— AL—QPL—PGH—VQI-
QL—DVY—PY— TEL—TEEEK—IN— IIASTPETTTVPVTSPVV IQPF—QVPQSL—QTL— IQDPSGGQER—CGF
AAW—PAQIM—QH— QY—EK—TL—PQY— PSQPVL—SPPQSK—
VAY—SPF—DTAK— IVH—QH—QTTM— VAPF—PQPVVPY—
IASEN—SEK—TDIIPEW DPQSH—SK—TN— PQR—DTPVQAF—
SY—QIIPVL QDPQL—GL—TGEF—
DPATQPIVPVH—PVIV

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13
 Table 1  (continued)
αs1-Casein αs2-Casein k-Casein β-Casein α-Lactalbumin β-Lactoglobulin

13
Sheep > sp|P04653|16–214 > sp|P04654|16–223 > sp|P02669|22–192 > sp|P11839|16–222 > sp|P09462|20–142 > sp|P67976|19–180
PK—PIK—QGL— EH—VSSSEEPIN— QEQN—QEQR— EQEEL—VVGETVESL— EQL—TK—CEVF— IIVTQTM—GL—DIQK—
SSEVL—EN—VVAPF— ISQEIY—QEK— ICCEK—DER—DDK— SSSEESITH—IN— QEL—DL—DY— VAGTW—SL—AM—
PEVF—EN—IN—EL— AIH—PR—EK—CTTS- IAK—IPIQY—VL— IEK—QSEEQQQT- GGVSL—PEW— AASDISL—DAQSAPL—
SK—DIGSESIEDQAM— CEEVVR—ADEEEY— SR—PSY—GL—QQR— EDEL—QDK—IH— VCTAF—TSGY— VY—VEEL—PTPEGN—
EDAK—QM—AGSSSS- SIR—SSSEESAEVAP- PVAL—IN—QF—PY— PF—AQAQSL—VY— DTQAIVQN—DSTEY— EIL—QK—EN—
SEEIVPN—SAEQK— EEVK—ITVDDK—QK— PY—AK—PVAVR— PF—TGPIPN—SL— GL—QIN—IW—CK— GECAQK—IIAEK—
IQK—EDVPSER—GY— AL—EIN—QF—QK— SPAQTL—QW— PQN—IL—PL—TQT- DDQN—PH—SR— TK—IPAVF—IDAL—
EQL—VPQL—EIVPK— PQY—QY—QGPIVL— QVL—PN—AVPAK— PVVVPPF—QPEIM— ICN—ISCDK—DDDL— EN—VL—VL—DTDY—
SAEEQL—SM—EGN— PW—DQVK—AGPF— SCQDQPTAM—AR— GVPK—VK—ETM— TDDIM—CVK—IL— CM—EN—SAEPEQSL—
PAH—QK—QPM— TPTVN—EQL— PH—PH—SF—AIPPK— VPK—EM—PF—PK— DK—VGIN—AH—AL— ACQCL—VR—TPE-
IAVN—QEL—AY— STSEEN—SK—TIDM— DQDK—TEIPAIN— PVEPF—TESQSL— CSEK—DQW—CEK VDN—EAL—EK—DK—
PQL—QF—QL—DAY— ESTEVF—TK—TK— TIASAEPTVH—STPT- TL—TDVEK—PL— AL—AL—PM—IR—AF—
PSGAW—PL—GTQY— TEEEK—ISQY—QK— TEAVVN—AVDN— PL—VQSW—QPPQPL— PTQL—EGQCH
TDAPSF—SDIPN—PIG- AW—PQY—TVDQH— PEASSESIASAPETN— PPTVM—PPQSVL—
SEN—SGK—ITM—PL QK—AM—PW— TAQVTSTEV SL—SQPK—VL—
TQPK—TN—AIPY—VR PVPQK—AVPQR—
DM—PIQAF—QEPVL—
GPVR—GPF—PIL
Goat > sp|P18626|16–214 > sp|P33049|16–223 > sp|P02670|22–192 > sp|P33048|16–222 > sp|P00712|20–142 > sp|P02756|19–180
PK—PIN—GL—SPE- EH—VSSSEEPIN— QEQN—QEQPICCEK— EQEEL—VVGETVESL— EQL—TK—CEVF—QK— IIVTQTM—GL—DIQK—
VPN—EN—VVAPF— IF—QEIY—QEK— DER—DDK—IAK— SSSEESITH—IN— DL—DY—GGVSL— VAGTW—SL—AM—
PEVF—EN—IN—EL— AIH—PR—EK—CTTS- IPIQY—VL—SR— IEK—QSEEQQQT- PEW—VCTAF— AASDISL—DAQSAPL—
SK—DIGSESTEDQAM— CEEVVR—AN— PSY—GL—QQR— EDEL—QDK—IH— TSGY—DTQAIVQN— VY—VEEL—PTPEGN—
EDAK—QM—AGSSSS- EEEY—SIR—SSSEE- PVAL—IN—QF—PY— PF—AQAQSL—VY— DSTEY—GL—QIN— EIL—QK—EN—
SEEIVPN—SAEQK— SAEVAPEEIK— PY—AK—PVAVR— PF—TGPIPN—SL— IW—CK—DDQN— GECAQK—IIAEK—
IQK—EDVPSER—GY— ITVDDK—QK—AL— SPAQTL—QW— PQN—IL—PL—TQT- PH—SR—ICN—ISCDK TK—IPAVF—IDAL—
EQL—VPQL—EIVPK— EIN—QF—- QK—PQY— QVL—PN—TVPAK— PVVVPPF—QPEIM— -DDDL—TDDIVCAK— EN—VL—VL—DTDY—
SAEEQL—SM—EGN— QY—PY—QGPIVL— SCQDQPTTL—AR— GVPK—VK—ETM— IL—DK – VGIN-AH CM—EN—SAEPEQSL—
PAH—QK—QPM— PW—DQVK—AGPF— PH—PH—SF—AIPPK— VPK—EM—PF—PK— -AL—CSEK -DQW— ACQCL—VR—TPE-
IAVN—QEL—AY— TPTVN—EQL— DQDK—TEVPAIN— PVEPF—TESQSL— CEK VDK—EAL—EK—
PQL—QF—QL—DAY— STSEEN—SK—TIDM— TIASAEPTVH—STPT- TL—TDVEK—PL— DK—AL—AL—PM -IR
PSGAW—PL—GTQY— ESTEVF—TK—TK— TEAIVN—TVDN— PL—VQSW—QPPQPL— -AF—PTQL—EGQCH
TDAPSF—SDIPN—PIG- TEEEK—ISQY—QK— PEASSESIASASETN— SPTVM—PPQSVL—
SEN—SGK—TTM—PL AW—PQY—TVDQH— TAQVTSTEV SL—SQPK—VL—
QK—AM—PW— PVPQK—AVPQR—
TQPK—TN—AIPY—VR DM—PIQAF—QEPVL—
GPVR—GPF—PIL

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K. Parastouei et al.
 Table 1  (continued)
αs1-Casein αs2-Casein k-Casein β-Casein α-Lactalbumin β-Lactoglobulin

Horse > NP_001075352.1 > NP_001164238.1 > sp|P82187|21–185 > sp|Q9GKK3|16–241 > sp|P08334|1–123 > sp|P02758|19–180
PK—PH—QPEIIQN— EH—SSSEDSVN— EVQN—QEQPTCH— EK—EEL—VSSET- QF—TK—CEL— TN—IPQTM—QDL—DL—
EQDSR—EK—ER— ISQEK—QEK—VVI- DER—DL—TVK— VESL—SSN—EPDSS- SQVL—SM—DGY— QEVAGK—SVAM—AAS-
PSF—AL—EY—IN— PTSK—ESICSTS- IPIY—VL—SSPR— SEESITH—IN—EK— GVTL—PEW— DISL—DSESAPL—VY—
EL—QR—EL—EK— CEEATR—IN—EM— EPIY—QH—AL— QK—EGQQQR— ICTIF—SSGY—DTQ- IEK—PTPEDN—EIIL—
QK—DEH—EY— ESAK—PTER—EEK— IN—QH—PY—QY— EVER—QDK—ISR— TIVK—GK—TEY—GL EGEN—GCAEK—IF—
IEDPEQQESSSTSS- EVEEK—QL—IN— AR—PAAVR—PH— VQPQPVVY—PY— -QIN—CR—DN—QIL— AEK—TESPAEF—IN—
SEEVVPIN—TEQK— QF—EK—QY—QAL— VQIPQW—QVL—PN— AEPVPY—AVVPQSIL— PSR—ICGISCDK— DEDTVF—AL—DTDY—
IPR—EDM—QH—TL— QPR—IVL—TPW— IY—PSTVVR—PCPH— PL—AQPPIL—PF— DDDL—TDDVM— CM—AATPGQSL—
EQL—SK—QL—QL— DQTK—TGDSPF— PSF—IAIPPK—QEIT- QPEIM—EVSQAK— CAK—IL—DSEGIDY— VCQY—AR—TQM—
QAIH—AQEQL—IR— IPIVN—TEQL—TSEE- VIPK—IN—TIATEPT- ETIL—PK—VM—PF— AH—PL—CSEK— VDEEIM—EK—AL—
EN—SQR—PM—VVN— IPK—TVDM—ESTEV- PIPTPEPTVN—AVIP- SPIVPF—SER—QIL— EQW—CEEL QPL—PGR—VQIVPDL—
QEQAY—EPF—QPSY— VTEK—TEL—TEEEK— DASSEF—IIASTPETTT- PTN—GEN—PVH— TR—AER—CR
QL—DVY—PY— EK—TL—PQY—IVR— VPVTSPVVQK IQPF—QVPQSL—
AAW—PAQIM—QH— QH—QTTM—DPR— QTL—PSQPVL—
VAY—SPF—DTAK— SH—TN—SY—QIIPVL SPPQSK—VAPF—
IASEN—SEK—TDIIPEW PQPVVPY—PQR
– DTPVQAF-QDPR—
GPTGEL—DPATQPIV-
AVH—PVIV
Camel > sp|O97943|16–230 > sp|O97944|16–193 > sp|P79139|21–182 > sp|Q9TVD0|16–232 > sp|P00710|1–123 Absent
PK—PL—PEVF—QN— EM—DQGSSSEESIN— EVQN—QEQPTCF— EK—EEF—TAGEAL— QF—TK—CK—SDEL—
EPDSIEEVL—IL—EL— VSQQK—QVK— EK—VER—EK—TVK— ESISSSEESITH—IN— DM—GH—GGITL—
AVVSPIQF—QEN— VAIH—PSK—EDIC- PIQF—VQSR—PSY— QK—IEK—IEEQQQT- AEW—ICIIF—SGY—
IDEL—DTR—EPT- STF—CEEAVR—IK— GIN—QH—AVPIN— EDEQQDK—IY— DTETVVSN—GN—
EDH—IM—EDTER— EVESAEVPTEN— QF—IPY—PN—AK— TF—PQPQSL—VY— EY—GL—QIN—IW—
ESGSSSSEEVVS- ISQF—QK—QY PVAIR—AQIPQCQAL— SH—TEPIPY—PIL— CR—DN—EN—QSR—
Estimation of bioactive peptide content of milk from different species using an in silico method

STTEQK—DIL— -QAL—QGQIVM— PN—IDPPTVER—PR— PQN—PPL—QPAVM— ICDISCDK—DDDL—

EDM—PSQR—EEL— PW—DQGK—TR— PR—PSF—IAIPPK— VPF—QPK—VM— TDDK—CAK—IL—
QL—EAIR—DQK AY—PF—IPTVN— TQDK—TVN—PAIN— DVPK—TK—ETI- DK—EGIDY—AH—
-IPR—VK—SSH— TEQL—SISEESTE- TVATVEPPVIP- IPK—EM—PL—QSPV- PL—CSEK—EQW—
PY—EQL—IN— VPTEESTEVF—TK— TAEPAVN—TVVIAE- VPF—TESQSL—TL— QCEK
EDN—PQL—GEPVK— TEL—TEEEK—DH— ASSEF—ITTSTPETTTV- TDL—EN—PL—PL—
VVTQEQAY—EPF— QK—IY—QY—QTF— QITSTEI QSL—QIPQPVPQTPM—
PQF—QL—GASPY— PEY—TVY—QY—QK— IPPQSL—SL—SQF—
VAW—PPQVM—QY— TM—TPW—IK VL—PVPQQM—VPY—
IAH—PSSY—DTPE- PQR—AM—PVQAVL—
GIASEDGGK—TDVM— PF—QEPVPDPVR—

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PQW GL—PVPQPL—VPVIA

A, Alanine; R, Arginine; N, Asparagine; D, Aspartate; C, Cysteine; E, Glutamate; 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

13
 Table 2  Number of peptides with different functionality obtained from in silico digestion and the concentration of them in milk and milk proteins isolate
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic

13
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Bovine αs1-Casein 43 24 19 6 1 4 9 0 0 0 0
αs2-Casein 46 30 16 7 0 2 14 0 0 0 0
k-Casein 35 22 13 12 0 0 7 2 0 0 0
β-Casein 49 28 21 6 5 2 14 1 1 0 0
α-Lactalbumin 32 20 12 6 0 2 8 0 0 0 0
β-Lactoglobulin 38 21 17 9 1 2 14 0 0 1 0
Total 243 145 98 46 7 12 66 3 1 1 0
Concentration 6.61 3.90 2.70 1.38 0.14 0.33 1.61 0.12 0.01 0.01 0
(mmol per 100 g
milk)
Concentration 192.19 113.55 78.63 40.13 4.31 9.69 47.01 3.49 0.43 0.58 0
(mmol per 100 g
protein)
Concentration 188.86 112.25 76.61 38.92 4.35 9.35 42.96 4.07 0.50 0 0
(mmol per 100 g
casein)
Concentration 212.36 121.52 90.84 47.45 4.07 11.74 71.42 0 0 4.07 0
(mmol per 100 g
whey)
Buffalo αs1-Casein 42 23 19 9 1 3 9 0 0 0 0
αs2-Casein 46 30 16 8 0 3 14 0 0 0 0
k-Casein 36 22 14 7 0 0 13 2 0 0 0
β-Casein 49 26 23 6 4 2 14 0 0 0 0
α-Lactalbumin 32 19 13 6 0 2 7 0 0 0 0
β-Lactoglobulin 38 21 17 9 1 2 14 0 0 1 0
Total 243 141 102 45 6 12 71 2 0 1 0
Concentration 7.48 4.35 3.13 1.47 0.13 0.32 2.20 0.12 0 0.02 0
(mmol per 100 g
milk)
Concentration 189.49 110.24 79.24 37.26 3.33 8.27 55.74 3.25 0 0.52 0
(mmol per g

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protein)
Concentration 188.39 110.37 78.01 36.13 3.18 7.91 54.31 3.73 0 0 0
(mmol per 100 g
Casein)
Concentration 211.53 118.83 92.70 47.78 4.33 11.59 70.90 0 0 4.33 0
(mmol per 100 g
whey)
K. Parastouei et al.
 Table 2  (continued)
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Human αs1-Casein 43 31 12 7 0 0 17 1 0 0 0
k-Casein 34 21 13 4 1 1 9 0 0 0 0
β-Casein 47 27 20 12 2 2 11 0 0 0 0
α-Lactalbumin 26 14 12 4 0 2 6 0 0 0 0
Total 150 93 57 27 3 5 43 1 0 0 0
Concentration 0.14 0.08 0.05 0.02 0.002 0.006 0.03 0.0002 0 0 0
(mmol per 100 g
milk)
Concentration 189.08 111.16 77.92 28.74 3.02 9.17 48.74 0.36 0 0 0
(mmol per g
protein)
Concentration 192.50 120.15 72.34 29.00 5.33 5.33 53.43 0.64 0 0 0
(mmol per 100 g
casein)
Concentration 184.68 99.44 85.23 28.41 0 14.20 42.61 0 0 0 0
(mmol per 100 g
whey)
Estimation of bioactive peptide content of milk from different species using an in silico method

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

13
 Table 2  (continued)
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic

13
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Sheep αs1-Casein 44 27 17 7 2 2 9 0 0 0 0
αs2-Casein 44 26 18 9 1 3 13 0 0 0 0
k-Casein 37 20 17 9 0 0 13 2 0 0 1
β-Casein 47 25 22 8 5 1 16 0 0 0 0
α-Lactalbumin 32 20 12 6 0 2 7 0 0 0 0
β-Lactoglobulin 39 22 17 9 1 2 14 0 0 1 0
Total 243 140 103 48 7 10 72 2 0 1 1
Concentration 10.73 6.16 4.56 2.15 0.33 0.36 3.14 0.17 0 0.03 0.08
(mmol per 100 g
milk)
Concentration 195.05 112.15 82.90 39.18 6.11 6.56 57.19 3.13 0 0.65 1.56
(mmol per g
protein)
Concentration 190.16 109.38 80.77 37.46 6.22 5.73 54.27 3.63 0 0 1.81
(mmol per 100 g
casein)
Concentration 214.94 123.08 91.86 48.30 4.74 11.36 72.94 0 0 4.74 0
(mmol per 100 g
whey)

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K. Parastouei et al.
 Table 2  (continued)
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Gout αs1-Casein 44 27 17 8 1 3 10 0 0 0 0
αs2-Casein 47 29 18 10 1 3 14 1 0 0 0
k-Casein 36 20 16 8 0 0 12 2 0 0 1
β-Casein 47 25 22 8 5 1 16 0 0 0 0
α-Lactalbumin 31 19 12 7 0 1 7 0 0 0 0
β-Lactoglobulin 39 22 17 9 1 2 14 0 0 1 0
Total 244 142 102 50 8 10 73 3 0 1 1
Concentration 5.78 3.29 2.48 1.20 0.13 0.14 1.81 0.16 0 0.01 0.07
(mmol per 100 g
milk)
Concentration 193.89 110.63 83.26 40.56 4.56 4.93 60.92 5.65 0 0.38 2.54
(mmol per g
protein)
Concentration 191.98 109.24 82.74 39.67 4.66 4.36 60.45 6.41 0 0 2.88
(mmol per 100 g
casein)
Concentration 215.20 125.27 89.92 49.26 3.48 9.52 66.67 0 0 3.48 0
(mmol per 100 g
whey)
Estimation of bioactive peptide content of milk from different species using an in silico method

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

13
 Table 2  (continued)
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic

13
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Camel αs1-Casein 44 28 16 3 0 1 11 1 0 0 0
αs2-Casein 38 25 13 6 1 3 9 0 0 0 0
k-Casein 30 16 14 7 2 0 6 0 0 0 0
β-Casein 49 32 17 12 3 2 16 0 0 0 0
α-Lactalbumin 32 22 10 10 0 1 9 0 0 0 0
Total 193 123 70 38 6 6 51 1 0 0 0
Concentration 4.30 2.57 1.73 0.89 0.16 0.08 1.00 0.01 0 0 0
(mmol per 100 g
milk)
Concentration 176.97 105.79 71.18 36.77 6.59 3.35 41.20 0.81 0 0 0
(mmol per g
protein)
Concentration 169.42 97.93 71.49 31.29 7.70 2.75 37.63 0.94 0 0 0
(mmol per 100 g
casein)
Concentration 221.76 152.46 69.30 69.30 0 6.93 62.73 0 0 0 0
(mmol per 100 g
whey)

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K. Parastouei et al.
 Table 2  (continued)
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Donkey αs1-Casein 51 39 12 7 0 3 18 1 0 0 0
αs2-Casein 45 25 20 4 0 0 15 0 0 0 0
k-Casein 33 19 14 9 2 2 12 1 0 0 0
β-Casein 45 22 23 6 1 0 10 1 0 0 0
α-Lactalbumin 30 19 11 6 1 1 7 0 0 0 0
β-Lactoglobulin 34 14 20 5 1 2 4 0 0 0 0
Total 238 138 100 37 5 8 62 3 0 0 0
Concentration 1.92 1.05 0.86 0.38 0.07 0.09 0.47 0.02 0 0 0
(mmol per 100 g
milk)
Concentration 183.02 100.45 82.56 36.61 6.97 9.44 45.21 2.03 0 0 0
(mmol per g
protein)
Concentration 180.48 109.10 71.37 43.94 8.74 10.41 65.08 4.92 0 0 0
(mmol per 100 g
casein)
Concentration 196.51 100.30 96.21 33.53 6.12 9.32 33.24 0 0 0 0
(mmol per 100 g
whey)
Estimation of bioactive peptide content of milk from different species using an in silico method

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

13
 Table 2  (continued)
Total LMW peptides HMW peptides ACE DPP-III Anti- DPP-IV Immu- Anti- hypocholes- antithrom-
Peptide inhibitory inhibitory oxidant inhibitory nomodulatory cancer terolemia botic

13
fragment peptides peptides peptides peptides peptides peptides peptides peptides

Horse αs1-Casein 49 37 12 8 0 3 18 1 0 0 0
αs2-Casein 41 24 17 5 0 0 13 0 0 0 0
k-Casein 31 17 14 3 0 1 10 1 0 0 0
β-Casein 44 21 23 6 2 0 9 1 0 0 0
α-Lactalbumin 30 19 11 6 1 1 7 0 0 0 0
β-Lactoglobulin 35 19 16 4 1 1 7 0 0 0 0
Total 230 137 93 32 4 6 64 3 0 0 0
Concentration 2.75 1.65 1.00 0.37 0.03 0.08 0.79 0.03 0 0 0
(mmol per 100 g
milk)
Concentration 180.25 108.32 71.93 24.56 2.36 5.51 51.76 2.50 0 0 0
(mmol per g
protein)
Concentration 171.07 103.60 67.46 21.02 0.59 5.19 55.45 3.64 0 0 0
(mmol per 100 g
casein)
Concentration 200.49 118.77 81.71 32.32 6.25 6.25 43.75 0 0 0 0
(mmol per 100 g
whey)

LMW Low Molecular Weight, i.e. di and tri-peptides; HMW, High Molecular Weight, i.e. peptides with more than three residues. ACEI, Angiotensin-converting-enzyme inhibitors; DPP, Dipep-
tidyl-peptidase. The concentrations have been identified in bold format

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K. Parastouei et al.
Estimation of bioactive peptide content of milk from different species using an in silico method
in sheep milk protein are higher than the other types of
milk protein isolates. Among the studied milk, goat milk
protein contains the highest amounts of HMW-peptides
(83.26 mmol/100 g protein), ACE inhibitor peptides
(40.56 mmol/100 g protein), DPP-IV inhibitor peptides
(60.92 mmol/100 g protein), immunomodulatory peptides
(5.65 mmol/100 g protein) and antithrombotic peptides
(2.54 mmol/100 g protein). On the other hand, LMW-
peptides (113.55 mmol/100 g protein), antioxidant pep-
tides (9.69 mmol/100 g protein), and anticancer peptides
(0.43 mmol/100 g protein) are higher in bovine milk protein
compared to the other species. Also, the total protein of don-
key milk contains a higher amount of DPP-III inhibitory
peptides (6.97 mmol/100 g protein) (Table 2).
Our in silico work showed that among the all assessed
milk, total digestion-resistant peptides (176.97 mmol/100 g
protein), HMW-peptides (71.18 mmol/100 g protein), anti-
oxidant peptides (3.35 mmol/100 g protein), and DPP-IV
peptides (41.20 mmol/100 g protein) have the lowest amount
in camel milk protein. Also, the lowest concentration of
LMW-peptides (100.45 mmol/100 g protein) was obtained
from donkey milk. Compared to the other protein isolates,
horse milk protein contains the lowest amount of ACE
inhibitor peptides (24.56 mmol/100 g protein) and DPP-III
inhibitory peptides (2.36 mmol/100 g protein).
Estimation of casein and whey protein‑derived BPs
in milk from different species
The estimation and ranking of digestion-resistant and bio-
active peptides content of casein and whey proteins in the
assessed milk are reported in Tables 2 and 3, respectively.
The results showed that casein from donkey milk contains
the highest amount of ACE inhibitory (43.94 mmol/100 g
casein), DPP-III inhibitory (8.74 mmol/100 g casein),
antioxidant (10.41 mmol/100 g casein), and DPP-IV
inhibitory (65.08 mmol/100 g casein) peptides. Also,
among the whey proteins, the highest amount of pep-
tides with ACE inhibitory, DPP-III inhibitory, antioxi-
dant, and DPP-IV inhibitory functions was found in camel
(69.30 mmol/100 g whey protein), horse (6.25 mmol/100 g
whey protein), human (14.20 mmol/100 g whey protein)
and sheep (72.94 mmol/100 g whey protein) whey proteins,
respectively.
Discussion
The current in silico study was designed to evaluate BPs
content of milk from different species, including humans,
camel, bovine, buffalo, donkey, sheep, goat, and horse. Only
a few studies have been done to compare the biological func-
tions of milk protein hydrolysates among different species.
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Their long-term effects should be considered to evaluate
the clinical effects of milk-derived BPs from different spe-
cies. On the other hand, long-term effects of milk-derived BP
consumption only can be assessed in observational studies
(case–control and cohort studies). Without quantifying BP
content, the long-term effect assessment cannot be done. Our
previous work quantified BPs content of bovine dairy prod-
ucts (Barati et al. 2020b). Then, the association of bovine
dairy-derived BPs with breast cancer (Jabbari et al. 2022)
and hypertension (Barati et al. 2021) risk has been assessed.
It is interesting to know that the long-term consumption of
dairy-derived BPs from bovine milk had an inverse asso-
ciation with breast cancer risk (Jabbari et al. 2022) while
increasing the risk of hypertension (Barati et al. 2021). The
current work quantified the BPs content of milk from differ-
ent species. Therefore, according to available data/reports,
researchers can conduct the risk assessment analysis for
consumption of different milk (including humans, camel,
bovine, buffalo, donkey, sheep, goat, and horse milk) with
chronic diseases to observe the long-term effects of them on
health condition and disease.
There are many studies, including experimental (Naka-
mura et al. 2013), observational (Barati et al. 2021; Jabbari
et al. 2022) as well as randomized controlled trials (Boelsma
and Kloek 2010) studies to evaluate the effects of bovine
milk-derived BPs on different pathological conditions. How-
ever, there is limited evidence about comparing the biologi-
cal effects of milk from different species to the best of our
knowledge.
Akan et al., in an in vitro study, examined the antidia-
betic (DPP-IV inhibitory) and antioxidant (DPPH inhibi-
tory) function of camel and donkey milk proteins (casein
and whey proteins) after in vitro digestion by porcine pep-
sin and pancreatic enzymes. Their findings showed that
the casein hydrolysate from donkey milk compared to the
camel milk casein has more antioxidant potential (DPPH
inhibitory function) and considerably inhibits the DPP-IV
enzyme (Akan 2020). Following Akan et al.’s findings, the
current in silico results showed that the peptides released
from donkey milk caseins have a higher antioxidant potential
than camel milk. Furthermore, an in vitro study showed that
casein hydrolysate from caprine milk has superiority over
bovine milk to inhibit DPP-IV enzyme (Zhang et al. 2016).
In an experimental study, Koresh et al. examined the
effects of whole camel’s and bovine’s milk on serum level of
fibrinogen in diabetic rats (Korish et al. 2020). Their findings
showed that bovine milk could improve serum fibrinogen
levels more effectively. Fibrinogen is an indicator of malnu-
trition status. Improving the quantity and quality of dietary
protein increases fibrinogen serum and plasma levels. The
current in silico work also revealed that bovine milk con-
tains higher levels of LMW-peptides. LMW-peptides more
efficiently supply required amino acids for the human body
13

Table 3  The bioactive peptide content of different milks in order from high to low
Total Peptide fragments
LMW-peptides
HMW-peptides
ACE inhibitory peptides
DPP-III inhibitory peptides
Antioxidant peptides
DPP-IV inhibitory peptides
Immunomodulatory peptides
Anti-cancer peptides
Cholesterol lowering peptides
Anti-thrombosis peptides
LMW-peptides, low molecular peptides, i.e. di and tri-peptides; HMW-peptides, high molecular weight peptides, i.e. peptides with more than
three residues; ACE, angiotensin converting enzyme; DPP, Dipeptidyl-peptidase
*Human and camel whey protein do not contain DPP-III inhibitory peptides
13
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Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
Per 100 g milk
Per 100 g milk protein
Per 100 g casein
Per 100 g whey protein
K. Parastouei et al.
Sheep > Buffalo > Cow > Goat > Camel > Horse > Donkey > Human
Sheep > Goat > Cow > Buffalo > Human > Donkey > Horse > Camel
Human > Goat > Sheep > Cow > Buffalo > Donkey > Horse > Camel
Camel > Goat > Sheep > Cow > Buffalo > Horse > Donkey > Human
Sheep > Buffalo > Cow > Goat > Camel > Horse > Donkey > Human
Cow > Sheep > Human > Goat > Buffalo > Horse > Camel > Donkey
Human > Cow > Buffalo > Sheep > Goat > Donkey > Horse > Camel
Camel > Goat > Sheep > Cow > Buffalo > Horse > Donkey > Human
Sheep > Buffalo > Cow > Goat > Camel > Horse > Donkey > Human
Goat > Sheep > Donkey > Buffalo > Cow > Human > Horse > Camel
Goat > Sheep > Buffalo > Cow > Human > Camel > Donkey > Horse
Donkey > Buffalo > Sheep > Cow > Goat > Human > Horse > Camel
Sheep > Buffalo > Cow > Goat > Camel > Donkey > Horse > Human
Goat > Cow > Sheep > Buffalo > Camel > Donkey > Human > Horse
Donkey > Goat > Cow > Sheep > Buffalo > Camel > Human > Horse
Camel > Goat > Sheep > Buffalo > Cow > Donkey > Horse > Human
Sheep > Camel > Cow > Buffalo > Goat > Donkey > Horse > Human
Donkey > Camel > Sheep > Goat > Cow > Buffalo > Human > Horse
Donkey > Camel > Sheep > Human > Goat > Cow > Buffalo > Horse
Horse > Donkey > Sheep > Buffalo > Cow > Goat
Sheep > Cow > Buffalo > Goat > Donkey > Camel > Horse > Human
Cow > Donkey > Human > Buffalo > Sheep > Horse > Goat > Camel
Donkey > Cow > Buffalo > Sheep > Human > Horse > Goat > Camel
Human > Cow > Buffalo > Sheep > Goat > Donkey > Camel > Horse
Sheep > Buffalo > Goat > Cow > Camel > Horse > Donkey > Human
Goat > Sheep > Buffalo > Horse > Human > Cow > Donkey > Camel
Donkey > Goat > Horse > Human > Buffalo > Sheep > Cow > Camel
Sheep > Buffalo > Cow > Goat > Camel > Horse > Human > Donkey
Sheep > Goat > Buffalo > Cow > Horse > Donkey > Camel > Human
Goat > Cow > Buffalo > Sheep > Horse > Donkey > Camel > Human
Goat > Donkey > Cow > Buffalo > Horse > Sheep > Camel > Human
Not found
Cow
Cow
Cow
Not found
Sheep > Buffalo > Cow > Goat
Sheep > Cow > Buffalo > Goat
Not found
Sheep > Goat > Buffalo > Cow
Sheep > Goat
Goat > Sheep
Goat > Sheep
Not found
Estimation of bioactive peptide content of milk from different species using an in silico method
(Soop et al. 2012; Amirani et al. 2020). Koresh et al. also
declared that camel milk exerts more efficient antidiabetic
effects (Korish et al. 2020), while the findings of our in silico
study showed that bovine milk contains higher levels of DPP-
IV inhibitor peptides compared to camel milk. This contra-
diction suggests that camel milk's antidiabetic effects do not
necessarily rely only on their proteins. Other components of
camel milk, including lactose, fats, micronutrients, bioactive
compounds, and probiotics, also play an important role in this
regard (Ayoub et al. 2018; Ayyash et al. 2018; Parodi 2016).
Our in silico study showed that the hypo-cholesterolemic
function of the milk protein hydrolysates has limited to whey
protein. An interventional study evaluated the effects of
whey, casein, cod protein, and gluten on postprandial lipemia
in type 2 diabetes patients. The results showed that serum tri-
glyceride (TG) increment in patients receiving whey protein
was significantly lower than patients who received the other
proteins (Mortensen et al. 2009). Also, an experimental work
tried to evaluate the effects of different doses of dietary whey
and casein proteins on lipid profile in hypercholesterolemic
rats (Zhang and Beynen 1993). The results showed that whey
protein improves the serum level of very-low-density lipo-
protein (VLDL) dose-dependent. On the other hand, no con-
siderable differences were found between different doses of
dietary casein protein on lipid profile components, except
for low-density lipoprotein (LDL), which had increased by
casein (Zhang and Beynen 1993).
As the other considerable findings of the current in silico
work, results showed that the immunomodulatory function of
all assessed milk was limited to their casein proteins. Based
on the previous evidence, the hydrolysates of both whey and
casein proteins have immunomodulatory effects. However, it
has been suggested that this role is predominantly assigned to
the casein-derived peptides (Reyes-Díaz et al. 2018).
There is limited evidence comparing the biological effects
of milk from different species. To the best of our knowledge,
the current in silico work is the first comprehensive work
that has been conducted to assess the BPs profile of milk
from different species and has compared these profiles with
each other.
It should be noted that the current in silico method that
has been used for BPs quantification has its limitation. Many
variables such as time, pH, and temperature can affect the
efficacy of the digestion process. The current simulation was
performed under the optimum conditions. Due to the cost
and time-consuming nature of BPs' content quantification,
there are no in vitro, in vivo, and/or in silico methods for
BPs quantification, and the currently validated method is the
only proposed method in this regard. Although this method
is thoroughly in silico, the validation part of this work was
performed using the retrieved data from an in vivo study
(Barati et al. 2020b). The current method could predict about
80% of the released peptides after in vivo digestion. This
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
proposed model has its limitations, but it is the only available
model for quantifying BPs' content in food items. As one of
its limitations, the effect of fermentation on the BPs produc-
tion cannot be predicted by this model. Fermentation makes
new BPs and increases the bioactivity of food items. The
type of microbes are used for fermentation is the most crucial
influential factor on BPs content (Chaudhary et al. 2021).
The proposed model cannot predict and quantify BPs content
after microbial fermentation because only human intestinal
proteases have been used for digestion in this model. It is
necessary to develop and validate another in silico model for
predicting and quantifying BPs content after fermentation.
Conclusion and future perspective
Many studies have explored the novel BPs in different
protein isolates. The results of our simulation showed
that casein of donkey milk is expected to be a valuable
source of antihypertensive (ACE inhibitors), anticancer
(DPP-III inhibitors), antioxidant, and antidiabetic (DPP-
IV inhibitors) BPs. Also, the whey proteins of camel,
horse, human, and sheep milk are the precious sources of
the antihypertensive, anticancer, antioxidant, and antidia-
betic BPs, respectively. In the current study, our research
team has proposed a validated in silico simulation method
to quantify BPs content in milk from different species.
Regarding the valuable content and diverse components
of milk-derived BPs, assessing immunomodulatory and
cholesterol-lowering BPs from the casein of goat milk and
whey protein of sheep milk are critical issues worth explor-
ing in future research.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00726-0​ 22-0​ 3152-6.
Author contributions MB and KP developed the main idea. MB, KP,
MJ, and SHD developed the study protocol. AMK, YM, SKM, HA,
MB, and MJ performed the study protocol and manuscript writing.
KP and SHD contributed to the critical revision of the manuscript and
approved the final version.
Funding This study was not supported.
Availability of data and materials Not applicable.
Declarations
Conflict of interest The authors declare no conflict of interest relevant
to this article.
Research involving human participants and/or animals This study did
not involve human participants or animals.
Ethical approval. Not applicable.
13
 K. Parastouei et al.

Consent to participate The authors declare their consent to participate Derdak R, Sakoui S, Pop OL, Muresan CI, Vodnar DC, Addoum B,
in this article. Vulturar R, Chis A, Suharoschi R, Soukri A, El Khalfi B (2020)
Insights on health and food applications of Equus asinus (Donkey)
Consent to publish The authors declare their consent to publish this milk bioactive proteins and peptides—an overview. Foods 9(9):1302
article. Jabbari M, Barati M, Shabani M, Kazemian E, Khalili-Moghadam S,
Javanmardi F, Hatami E, Zeinalian R, Davoodi SH, Rashidkhani B,
Jafarzadeh S, Huseyn E, Mousavi Khaneghah A (2022) The Asso-
ciation between consumption of dairy-originated digestion resistant
and bioactive peptides and breast cancer risk: a case-control study.
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13

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