International Journal of Peptide Research and Therapeutics

https://doi.org/10.1007/s10989-019-09823-5

A Review on Bioactive Peptides: Physiological Functions,

Bioavailability and Safety
Divya Bhandari1 · Shafiya Rafiq1 · Yogesh Gat1   · Punam Gat1 · Roji Waghmare2 · Vikas Kumar1

Accepted: 7 February 2019

© Springer Nature B.V. 2019

Abstract
Bioactive peptides can be defined as isolated small fragments of proteins which provide some physiological health benefits.
They act as potential modifiers reducing the risk of many chronic diseases. To the best of our knowledge, limited literature
is available for the methods of isolation of these peptides from different protein sources with their in vitro and vivo physi-
ological effects. Also, there is a need to adopt healthy lifestyle choices for prevention of diseases, to counter increase in con-
sumption of functional foods and nutraceuticals day-by-day. Thus, these peptides play a major role in the development of
various functional foods. In the present study, attempts are made to review different physiological effects from peptides. Also,
effects of processing on the peptides are discussed with special emphasis on its bioavailability, safety and future application
for further development.

Keywords  Bioactive peptides · Physiological benefits · Effect of processing · Bioavailability

Introduction a pharmaceutical approach which can lead to drug toxicity

Bioactive peptides are the protein derived fragments
which when consumed by humans provide positive influ-
ence on health by acting as source of nutrition and dem-
onstrates numerous potential physiological functions in the
body (Kaur et al. 2019). These peptides are released as such
by enzymatic proteolysis during gastrointestinal digestion.
In vitro, peptides from foods can be released by hydrolysis
of proteins with the use of food grade proteolytic enzymes
and also during the processing of foods (cooking, ripening
and fermentation), small fragments of bioactive peptides
can be obtained in hydrolysates (Abdel-Hamid et al. 2017).
The bioavailability of isolated peptides majorly depends on
the degree of hydrolysis during the isolation which is to be
determined in vitro and in vivo for their commercialization.
There is a global prevalence of diseases like cardiovas-
cular diseases, blood pressure, diabetes, cancer, etc. and
direct treatment for these diseases has always been through
* Yogesh Gat
yogeshcft10@gmail.com
1
Department of Food Technology and Nutrition, Lovely
Professional University, Jalandhar 144411, India
2
School of Biotechnology and Bioinformatics, DY Patil
University, Navi Mumbai 400614, India
(Girgih et al. 2014). Also, prolonged usage of any medi-
cation has its own negative side effects which can lead to
deterioration in health, thus, increase in health care costs.
Therefore, change in the era demands the prevention of
diseases through healthy lifestyle choices and intriguing in
early treatments of individuals. Hence, natural food sources
containing these bioactive peptides can significantly help in
preventing the diseases and also, reducing the health care
costs globally, decreasing the dependence on drug therapy
(Girgih et al. 2014).
Emerging new researches are focusing on the develop-
ment of functional foods and nutraceuticals, which involves
the application of bioactive peptides endeavouring their
respective physiological/health benefits (Cheung et  al.
2015). Hence, there is need to increase the commercial usage
of peptides in the market by incorporating in various food
products and making them bioavailable to claim the proper
health functions. In this review, the isolation methods, their
physiological functionalities, and bioavailability and safety
concern are taken into considerations.
Different Sources of Bioactive Peptides
A number of plant and animal sources have been used for the
isolation or production of bioactive peptides. Food proteins

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are selected as a source of bioactive peptides on the basis
of two criteria, i.e. (i) a pursuit of value-added use of abun-
dant underutilized proteins or protein-rich food industry
by-products, (ii) utilization of proteins containing specific
peptide sequences or amino acid residues of particular phar-
macological interest (Udenigwe and Aluko 2011). Studies
on peptide extraction from animal and plant based sources
are shown in Tables 1, 2 and 3.
Means of Production of Bioactive Peptides
Bioactive peptides include the sequence of amino acids,
which are present in an inactive form and can be produced
from precursor proteins through two different methods:
enzymatic hydrolysis and fermentation. Combination of
the above mentioned methods results in producing short
chain functional peptides (Korhonen and Pihlanto 2006).
The importance of each of the method is specific, as in,
hydrolysis of peptides is important to liberate the poten-
tial peptides from the parent proteins because of their
more bioavailability in target tissues. Also, the bioactiv-
ity of peptides majorly depends upon the enzymes used
for hydrolysis, conditions of processing and size of the
isolated peptides (Udenigwe and Aluko 2011). The size
of the active peptide sequence varies in the range of 2–20

Table 1  Marine sources with their respective isolated peptides and physiological function
Marine source Species Bioactive peptide Physiological function References

Sponges Jaspis spp. Jaspamide Anticancer Odaka et al. (2000)

Heniastrella spp.
Auletta hemiasterlins Hemiasterlins Cytotoxic Gamble et al. (1999)
Siphonochalina hemiasterlins Antitubulin
Geodia corticostylifera Geodiamolide H Antiproliferative Freitas et al. (2008)
Dysidia arenaria Arenastatin A cytotoxic Kobayashi (1994)
Homophymia sp Homophymines Cytotoxic Zampella (2008)
Haliclona nigra Haligramides A and B Cytotoxic Rashid et al. (2000)
Mollusks Conus magus Ziconolide Analgesic Olivera (2000)
Dolabella auricularia Dolastatins Cytotoxic Bai et al. (1990)
Pleurobranchus forskalii Keenamide A Cytotoxic Wesson et al. (1996)
Elysia rufescens Kahalalides Antitumor Garcia-Rocha et al. (1996)
Ascidians Tridemnum solidum Didemnin B Anticancer Palanisamy et al. (2015)
Styela sp Styelin D Cytotoxic Vervoort et al. (2000)
Antimicrobial
Seaweeds Eucheuma serra Lectins Anticancer Hori et al. (2007)
Cytotoxic
Porphyridium Phycobiliproteins hepatoprotective, Neuroprotective Harnedy and Fitzgerald (2011)

Table 2  Peptides isolated from milk products or its components with their bioactive action
Milk product/component Description/peptide Bioactive action References

Yoghurt Bovine and ovine milk Antihypertensive and anti- López-Expósito et al. (2006)
microbial
Fortified with whey proteins Antioxidative Unal and Akalin (2012)
κ - casein : Met-Ala-Ile Antithrombotic Manso and Lopez (2003)
Fermented milk Val-Pro-Pro, Ile-Pro-Pro Antihypertensive Nakamura et al. (1995)
Casein hydrosylates Arg-Tyr-Lue-Gly-Tyr Antihypertensive Contreras et al. (2009)
Ala-Tyr-Phe-Tyr-Pro-Glu Antihypertensive Contreras et al. (2009)
Casokinins Antihypertensive Fitzgerald et al. 2004
β- lactoglobulin β - lactosin B Antihypertensive Murakami et al. (2004)
Bovine milk Bovine milk fragment f(106–116) Antithrombotic Chabance et al. (1995)
Bovine milk fragment f(17–111) Antimicrobial Wakabayashi et al. (2003)
Whey protein : Lactoferrin Lactoferricin Antimicrobial Bellamy et al. (1992)

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International Journal of Peptide Research and Therapeutics

Table 3  Plant source based bioactive peptides with their physiological action

Physiological function Source Peptide isolated References

Antihypertensive Maize LPP (zein) Garcia et al. (2016)

VHLPPP (γ-zein) Maruyama et al. (1989)
FNQ, FSQ, LAY, LNPA Yano, Suzuki and Funatsu (1996)
LSPA, AF, LPN, FYQQ
ASY, LVP, FLP (α-zein)
LQP, LSP, LRP Puchalska et al. (2013)
Common beans KKSSG, KTYGL, CPGNK Bitocchi et al. (2012)
(Phaseolus vulgaris) LSFNT, KMARPV, GHVPP
Amaranth YL, RW, RR, KL, LF, EG Montoya- Rodriguez et al. (2014)
GT, HK, RP, HP, PG, GG
Antioxidant Maize YA, LMCH (zein) Tang et al. (2015)
Phaseolus vulgaris TACKD, GGGLHK, KTYGL Bitocchi et al. (2012)
MPHLK
Amaranth PYY, RWY, WY, RW Montoya- Rodriguez et al. ((2014)
PWW, PWR, PW, PWY
WYS
Antimicrobial Maize RSGRGECRRQCLRRHEGQ Duvick et al. (1992)
PWETQECMRRC​RRR​
Amaranth VGECVRGRCPSGMCCSQF Broekaert et al. (1992)
GYCGKGPKYCG​
Antidiabetic Phaseolus vulgaris EGLELLLLLLAG Mojica and Mejía (2016)
FEELN Mojica and Mejía (2016
TTGGKGGK, KKSSG Mojica and Mejía (2016
KTYGL, GGGLHK
CPGNK, VKFMT
Amaranth GGV, IVG, VGVL Soares et al. (2015)
Anticancer Phaseolus vulgaris GLTSK, LSGNK Luna-Vital et al. (2015)
GEGSGA, MPACGSS
MTEEY
Amaranth VW, GQ Montoya- Rodriguez et al. (2014)

or even more amino acid sequences and they may possess
multifunctional properties (Girgih et al. 2014).
Enzymatic Hydrolysis
Different proteolytic enzymes are used for enzymatic
hydrolysis such as papain, pepsin, protease, pancreatin,
trypsin, chymotrypsin, alkalies, thermolysin, along with
the enzymes from bacterial and fungal sources, depending
upon the parent protein to produce protein hydrolysates
(Korhonen and Pihlanto 2006). Specific conditions are
provided to carry out the enzyme hydrolysis including pH,
enzyme to substrate ratio, hydrolysis time and temperature.
This method is considered to be more suitable as compared
to microbial fermentation as it is easy to adapt, short reac-
tion time, and predictability. Subtilisin enzyme produces
smaller, low molecular weight peptides, out of which,
some are bioactive (Morato et  al. 2000). Also, during
the hydrolysis of snail foot muscle protein, i.e. Achatina
Fulica (Huang et al. 2017), and rice bran proteins (Zhang
et al. 2012), subtilisin produced an excessive number of
smaller bioactive peptides when compared to papain,
trypsin, cysteine endopeptidase and pepsin in their respec-
tive studies (Inouye et al. 2009). Depending upon the type
of enzyme used, the biological activity and the sequence
of peptides varies (Mojica and Mejía (2016). Proteases
producing low molecular weight peptides (< 10 kDa) are
preferred as these are more effective antioxidants and anti-
hypertensive peptides (Wattanasiritham et al. 2016).
It is recommended to remove other compounds from the
protein source to avoid their interference in the bioavail-
ability assay at a later stage. For example, phenolic com-
pounds are known for their physiological functionalities like
antihypertensive, antidiabetic, antimicrobial activities, etc.,
hence, need to be separated preceding enzymatic hydrolysis
(Daliri et al. 2017). Thermal treatment of proteins enhances

13

enzymatic hydrolysis as they become more susceptible to
disintegrate into smaller peptides (Inouye et al. 2008).
Fermentation of Precursor Proteins by Proteolytic Starter
Cultures
Microbial fermentation is a next feasible way for isolation
of bioactive peptides. Many starter and non-starter cultures
used in industries are proteolytic in nature, hence, can be
used for the isolation and release of these peptides from food
protein sources (Girgih et al. 2014). Bacteria or yeast cul-
tures are grown over protein substrates which, when release
enzymes result in hydrolysis of proteins and release of pep-
tides. The extent of hydrolysis mainly depends upon the
type of starter culture used, protein source and fermentation
time (Fitzgerald and Murray 2006). Pihlanto et al. (2010)
reported that Lactobacillus brevis fermented whey exert
a stronger ACE inhibitory effect than those incorporated
with other Lactobacillus spp. Also, different ACE inhibi-
tory and antioxidant activities were observed when milk
was fermented using 14 different starter cultures (El- Fattah
et al. 2016). Hence, it was concluded that the functional-
ity of hydrolysed peptides depends upon the strain used for
hydrolysis as each micro-organism has an unique proteolytic
system. However, this method came out to be less useful
for isolation of peptides from meat proteins (Arihara 2006).
Also, no bioactive peptides have been produced by fermenta-
tion of muscle proteins (Ryan et al. 2011).
Following production of peptides through above men-
tioned methods, mixture obtained is centrifuged and low
molecular weight peptides need to be separated using vari-
ous techniques like freeze drying desalting, gel filtration,
cross-flow membrane filtration, hydrostatic pressure treat-
ments, membrane ultrafiltration, size exclusion chromatog-
raphy, electro dialysis ultrafiltration (EDUF) and column
chromatography based on their sizes. At last, a synthetic
version of a peptide is synthesized and assay is repeated to
verify bioactivity (Sharma et al. 2011).
Physiological Functionalities of Bioactive Peptides
Proteins are required for the growth and maintenance of
various body functions are also responsible for the improved
physicochemical and sensory characteristics of food. The
knowledge about physiologically active peptides is increas-
ing rapidly nowadays, acting as potential modifiers for many
regulatory processes in the body. The studies are being con-
ducted and a considerable amount of scientific data has been
collected to check for the bioactivities of peptides. These
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International Journal of Peptide Research and Therapeutics
functions relate to general health conditions or a reduced
risk of certain chronic diseases.
Antihypertensive
Antihypertensive peptides are the peptides which once
released from the parent protein have the ability to modu-
late the renin—angiotensin systems and regulate blood
pressure in the body. This hypertensive effect has been
already put to use with the application of commercial
drugs and now, this effect is also known to be shown by
ACE inhibitory peptides which have been isolated from
barley, garlic, sunflower, gelatin, etc. with the use of
β-lacto globulin, α-lactalbumin, γ-Zein, like enzymes.
Blood pressure is physiologically controlled by renin-
angiotensin system (RAS) and kinin- nitric oxide system
(NO). Angiotensin-I converting enzyme (ACE) is the most
important enzyme in the regulation of blood pressure as
it catalysis the conversion of Angiotensin-I, a peptide
hormone, to Angiotensin-II which results in Vasco-con-
striction. ACE inhibitory peptides inhibit the conversion
resulting in dilation of blood vessels and hence, control-
ling the hypersensitivity. Also, ACE degrades bradykinin
(produced by kinin—NO system) which has vasco dila-
tory properties. Therefore, ACE inhibitors control blood
pressure with the prevention of cardiovascular diseases
(Fig. 1).
A fraction of peptides of sequence SDNRNQGY,
IQVPL and KGLWE isolated from egg yolk proteins
exhibited 69.2% of ACE inhibitory activity due to the pres-
ence of leucine, positively charged lysine and arginine and
also, hydrophobic amino acid tryptophan, that can bind to
­Zn2+ at the enzyme active site controlling the blood pres-
sure (Yousr and Howell 2015).The ACE inhibitory peptide
was found to be derived from tuna muscle having a novel
amino acid sequence of Pro-Thr-His-Ile-Lys-Trp-Gly-Asp
in which antepenultimate aromatic amino acid residue
increases its binding capacity to ACE, further preventing
its action with biological substrate (Kohama et al. 1991).
In vitro and in vivo studies of ACE inhibitory peptides
have also been done to check for the absorption and activ-
ity of these peptides in our body. It was studied that for
ACE inhibitory peptides to function in vivo, it must enter
the circulatory system unimpaired. But there can be pos-
sible changes in the peptides while passing through our
body by action of different enzymes, which lead to the
variation in the activity of these peptides. A study dem-
onstrated the bioavailability of the β – casein inhibitory
peptide (LHLPLP) which was noted to be hydrolysed to
HLPLP and was further stable to different proteases, suc-
cessful in maintaining the blood pressure (Quiros et al.
2008). Hence, the method of isolation with its bioavail-
ability needs to be taken into consideration.
International Journal of Peptide Research and Therapeutics
Fig. 1  Mechanism of action of antihypertensive activity of bioactive peptides
Antimicrobial Peptides
Antimicrobial peptides are the peptides which act directly
to protect animals against a wide range of bacteria, fungi,
viruses and protozoans i.e. has antimicrobial activity. Anti-
microbial peptides have selective toxicity towards bacterial
pathogens without impairing the host tissue. The microbial
cell wall is made up of lipoproteins which act as the princi-
ple target for disintegration to take into account antimicro-
bial activity of peptides. Antimicrobial peptides mainly and
majorly contain α- helical structure which are cationic and
amphipathic in nature, while less number are hydrophobic α-
helical peptides. This cationic part of peptides is responsible
for the electrostatic interaction with anionic phospholipids
leading to formation of pores in the cytoplasmic membrane.
Due to formation of pores, there occurs ionic imbalance
across the membrane and hence, cell lysis (Fig. 2). Also,
these AMP’s assemble themselves over the surface of cell
membrane and incorporate into it (Shai 1999).
Comprehensive studies are done to demonstrate the anti-
microbial activity of milk. Lactoferrin when hydrolysed
into lactoferricin in the gastrointestinal tract from milk act
as an important precursor for the formation of many other
bioactive peptides and itself shows potential antimicrobial
activity. Lactoferrin in combination with milk Ig has syner-
gistic action on micro-organisms. AMP’s are isolated from
casein, α- lacto globulin and β-lactalbumin. It shows that
the growth of Listeria innocua, Micrococcus luteus, Sal-
monella enteritidis and E. coli was inhibited by the isolated
peptide (SSSEESII) from αS2–casein. IKHQGLPQE, a nano
peptide, withdrawn from casein hydrolysates was effective
in decreasing the microbial load in infant formula (Kamali
and Ehsani 2017). Fish and fish products have also found
to be a rich source of AMPs. GLSRLFTALK, isolated
from anchovy cooked waste water inhibited the growth of
many microbes like S. aureus, B. subtilis, S. pneumoniae,
E. coli, S. dysenteriae, P. aeruginosa and S. typhimurium
(Tang et al. 2015). Misgurin, a protein from fish source has
also reported to show antimicrobial activity against a broad
range of microbes without significant haemolytic activity
(Park et al. 1997). Human’s blood, i.e. haemoglobin when
hydrolysed with pepsin under controlled conditions yielded
a number of antimicrobial peptides which were effective
against Salmonella enteritidis, Escherichia coli, Shigella
sonnei, Micrococcus luteus, Enterococcus faecalis, Listeria
innocua, Staphylococcus saprophyticus, Bacillus cereus and
Staphylococcus stimulants (Adje et al. 2011).
Mineral Binding
There are several peptides with specific sequences which
form complexes by binding with minerals like Ca, P and
any other in a solution, at intestinal pH. The greater anionic
character of these peptides results in the formation of soluble
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Fig. 2  Antimicrobial action of bioactive peptides
complexes as they become resistant to further proteolytic
attack, preventing the formation of insoluble complexes with
minerals.
Peptides having the sequence Ser-P:Ser-P:Ser-P:Glu-Ile-
Val-Pro-Asn is isolated from αS1-casein. Milk caseins are
known to stabilize calcium and phosphate ions, a reason for
which, several phosphopeptides have been identified from
enzymatic digest of milk proteins, including αS1-casein
comprising fragments (f43–f58, f59–f79 & f43–f49), αS2-
casein possessing two fragments (f1–f24 and f46–f70) and
β-casein with four fragments (f1–f28, f2–f28, f1–f25 &
f33–f48) (Reynolds 2004). When casein proteins are sub-
jected to tryptic digestion, caseinophosphopeptides (CPPs)
are released from the N-terminus polar domain containing
cluster of phosphorylated seryl residues and are responsi-
ble for interaction between calcium phosphate and casein,
resulting in the formation of casein micelle. Also, these
phosphorylated peptides bind to calcium and phosphate ions
increasing their bioavailability (Meisel and Fitzgerald 2003).
However, in vivo studies showed that CPPs were unable to
enhance the absorption of calcium ion in the intestine.
Antithrombotic
Antithrombotic means the prevention of formation or
enlargement of blood clots in the body. Increased level of
fibrinogen, hyper reactive platelets, irregular fibrinolysis
lead to more chances of thrombotic activities. Antithrom-
botic drugs are given to increase fibrinolysis and reduc-
ing platelet aggregation, but drugs are always associated
with their side effects. The mechanism involved in milk
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International Journal of Peptide Research and Therapeutics
coagulation, includes the interaction of k-casein with chy-
mosin is remarkably identical to the mechanism involved in
clotting of blood, characterized by the interaction of fibrino-
gen with thrombin. The k-casein fragment, casoplatelins,
isolated from tryptic hydrolysates, inhibits the activity of
fibrinogen binding platelet, showing antithrombotic action.
These peptides are liberated during gastrointestinal diges-
tion and are easily assimilated into the blood, which aids the
concept that they show an antithrombotic activity in vivo.
A study showed that bovine k-casein f106–f116, with
amino acid sequence MAIPPKKNQDK, inhibited platelet
aggregation and bind with the receptor site, further, pre-
venting fibrinogen binding with blood platelets (Erdmann
et al. 2008). The two smaller tryptic peptides, when isolated,
(k-casein f106–f112 and f113–f116) did not exert much
effect on platelet aggregation and were unable to inhibit
fibrinogen binding. The behaviour of k-casein f106–f116 is
found to be identical to that of the C-terminal peptide of the
human fibrinogen g-chain (Clare and Swaisgood 2000). The
potential physiological effects of these antithrombotic pep-
tides have not been yet established, but such peptides have
been observed in the plasma of infants after breastfeeding or
on ingestion of cow milk-based infant formula.
Antioxidant
Various antioxidant peptides have been isolated from pea,
soy, flaxseed, fish, milk casein, whey, egg, etc. Peptides
involved in antioxidant activity must be able to induce or
take part in single electron transfer reactions (Huang et al.
2005). Therefore, amino acids, which can transfer the
International Journal of Peptide Research and Therapeutics
electrons at a physiological pH are considered to be able to
participate in antioxidant activities. There are some other
mechanisms followed by peptides to enhance the antioxida-
tive action, such as metal chelation, ferric reducing power,
etc (Poljsak and Milisav 2013).
Amino acids, which can contribute to antioxidant activ-
ity are histidine, cysteine, proline, methionine and other
aromatic amino acids. Aromatic amino acids, sometimes
chelate the pro-oxidant metal ions or scavenge OH radical
to show their activity. Hence, each amino acid, according to
its type, contributes as antioxidant in its own way (Zhuang
et al. 2013).
Besides the direct effect of scavenging by amino acid,
they are also used to synthesize other peptides which can
show antioxidant property, such as, cysteine residues (sulf-
hydryl groups) act as a precursor for synthesis of glutathione
which is a tripeptide also a part of antioxidant defense sys-
tem (Meisel 2005). Endothelial cells, a dipeptide of methio-
nine and tyrosine isolated from sardine muscle protein acti-
vated the gene expression of heme oxygenase-1 and ferritin
resulting in protection from any kind of oxidative stress
(Erdmann et al. 2006). Casein hydrolysates isolated with
the use of different proteolytic enzyme showed different
antioxidant activities, independent of degree of hydrolysis,
in human T cells by increasing cellular catalase activity and
amount of reduced glutathione but without any effect on
superoxide dismutase (SOD) activity (Phelan et al. 2009;
Lahart et al. 2011). Alcalase hydrolysed hemp seed were
found to possess protective effect against cell death due to
oxidation (Lu et al. 2010). Also, rapeseed hydrolysate, when
hydrolysed with alcalase and proteinase k, demonstrated
more antioxidant activity, as compared to when hydrolysed
with combination of pepsin and pancreatin (He et al. 2013).
Anticancer
Anticancer implies the treatment or use of peptides in
prevention of cancer or malignant tumors (uncontrolled
multiplication of cells). The maximum number of stud-
ies for anticancer property on peptides is done on lunasin
i.e. peptide isolated from soy or cereal grains (Wang et al.
2008). It is mainly effective against chemical and viral onco-
gene induced cancers and works by inhibiting histone acetyl
transferase which further inhibits the acetylation of H3 and
H4, resulting in repression of cell cycle multiplication, and
apoptosis of cancerous cells (Hernández–Ledesma et al.
2009). Jeong et al. (2009) reported that with the ingestion of
rye into the liver, kidney and blood, there is efficient absorp-
tion of lunasin and the tissue-derived extracts retained the
anticancer HAT-inhibitory property of the parent molecule.
Also, there is need to take care that the food in which lunasin
is incorporated, must not contain any protease inhibitor, oth-
erwise, will slow down its activity against gastro-intestinal
ingestion. Further studies are still needed on lunasin activity,
its absorption kinetics, cancer cell targets, etc.
Except lunasin, other soy protein derived peptides also
show anticancer property but not as effective as lunasin
(IC50 3.5 to 6.2 mg/mL) against leukaemia cells (Wang et al.
2008). HVLSRAPR, peptide isolated from Spirulina platen-
sis hydrolysates demonstrates a little effect against normal
liver cells, but did not allow the multiplication of HT-29
cancer cell (Wei et al. 2017; Wang and Zhang 2016). PC-3,
DU-145, H-1299 and HeLa cell lines were inhibited by the
tripeptide WPP hydrolysed from blood clam muscle. Tuna is
cooked juice isolated peptides (KPEGMDPPLSEPEDRRD-
GAAGPK and KLPPLLLAKLLMSGKLLAEPCTGR) were
found to be effective against breast cancer cell line MCF–7
(Hung et al. 2014). Human Hep-G2 liver cancer and breast
cancer MCF-7 were inhibited by rapeseed protein fer-
ments. Until now, these anticancer peptides have only been
researched in vitro, in vivo studies are still required to know
its bioavailability.
Osteoprotective
Osteoprotective literally means bone protecting, i.e. the pep-
tides helping in protection of bones in our body. To cure
osteoporosis in our body, we need a higher intake of calcium
in our diet which is bioavailable. It is found that in milk
components, whey derived antioxidant peptides (YVEEL)
showed more osteoprotective activity than angiotensin con-
verting enzyme inhibitory peptide (YLLF) (Pandey et al.
2018). Also, milk casein derived peptide (NAVPITPTL)
showed osteoprotective activity (Reddi et al. 2018).
Whey components in the milk are found to increase the
bone metabolism. These components are termed as milk
basic proteins which promote bone formation, bone mineral
density, bone break force resistance, content of osteocalcin,
but suppress bone resorption, pit formation and deoxypyri-
dinoline levels in urine in animal and human model (Vel-
liyagounder et al. 2014; Toba et al. 2000). Lactoferrin, on
the other hand is found to be promoting factor for bones and
anabolic factor in osteoporosis (Naot et al. 2005). Hence,
both casein and whey proteins are effective for osteoporosis
provided the diet of the person is adequate.
Hypolipidemic and Hypocholestrolenic
Any agent that reduces the level of lipids and lipoproteins
in the blood is said to be hypolipedemic and they can be
used in the prevention of accumulation of high levels of fats
(cholesterol) in blood vessels. The studies have shown that
high postprandial triglyceride levels in the blood can result
in insulin resistance, atherosclerosis, obesity, etc which can
be reduced by decreased activity of the lipase and its cofac-
tor colipase which are required for the cleavage of dietary
13

triglycerides and hence its absorption (Moller et al. 2008)
(Fig. 3). Therefore, lipase inhibition results in preventing
type 2 diabetes, accelerated weight loss and improving the
metabolism. Peptides isolated from soy protein, milk pro-
tein, buckwheat protein, egg white protein and fish protein
show hypolipedemic and hypocholestrolenic activity.
A study showed that prolamine (isolated from fish
source), was able to reduce the triglyceride levels when
incorporated in animals, but did not show any effect in
human trial (Yang et al. 2007). Rats supplemented with soy
or fish protein hydrolysate had more high density lipopro-
tein to total cholesterol ratio than casein hydrolysates. The
α subunit of soy 7S globulin was found to be responsible for
a rise in low density lipoprotein degradation by effectively
increasing the response in cultured hepatocytes for LDL
receptor (Lovati et al. 2000). CSP1, CSP2, CSP3 peptides
isolated from cumin seeds repress cholesterol micelle for-
mation and adhere to bile acids inhibiting the lipase activity
results in hypocholestrolenic effect (Siow and Gan 2016).
Lupin (soy proteins) when hydrolyzed suppress the activity
of HMG CoA reductase in HepG2 cells, accounting for the
lowering of cholesterol in blood (Cicero and Colletti 2016).
There is a need to conduct more in vivo studies for com-
mercial application of these peptides.
Effect of Processing on Bioactive Proteins
and Peptides
Different processing treatments are applied to raw materials
according to the product requirement, depending on which,
proteins present are subjected to alterations in relation to
their functional and biological properties and these altera-
tions are mainly caused due to pH changes i.e. acidic or
alkaline treatments, and application of chemical treatments
(Korhonen and Pihlanto 2003). The changes occurred may
have some positive impact, such as, developing better tex-
tural or organoleptic characteristics, improving stability, but
Fig. 3  Activity of lipase
enzyme for hypolipidemic effect
of peptides
13
International Journal of Peptide Research and Therapeutics
also can have some negative effect, like changes in struc-
ture of amino acids and production of allergenic compounds
(Toldra et al. 2018).
Heat treatments make food proteins more nourished and
digestible. Cooking, in aged beef meat, doesn’t affect bio-
activity of proteins (Mora et al. 2017). While studying the
in vitro digestibility and functional analysis of peptides in
egg white proteins, effect of temperature was also taken into
consideration which states that digestibility of enzymes pep-
sin and pancreatin increased with increase in temperature.
Therefore, a positive correlation was found between the pep-
tides produced and digestibility of same enzymes, when used
(Wang et al. 2018). Also, fermentation with Bacillus subtilis
to obtain peptides from tomato waste, resulted in increase
in number of aromatic and positively charged amino acids
(Moayedi et al. 2017).
Ultra high pressure processing (UHP) is the non-thermal
method of processing in which foods are subjected to high
isostatic pressures of ~ 100–1000 MPa at room temperature.
It is found that at lower pressures, proteins retain their struc-
tures and are stable (Garcia-Mora et al. 2015), while when
high pressures are applied, there is breakage of non-covalent
and disulphide bonds, resulting in increase in production of
low molecular weight protein fragments (Zhou et al. 2016).
Extrusion is the process of converting raw material to pro-
cessed food on exposure to high pressure, high temperature
and high shear forces and is used for preparation of instant
and snack foods. It is observed that if extrusion is carried
out at high barrel moisture i.e. 36%, low concentration of
protein is extracted from canola meal because protein aggre-
gate themselves in high moisture and vice versa (Zhang et al.
2017).
Bioavailability of Bioactive Peptides
Bioactivity of different peptides is different while in obser-
vation in vitro than the real life applications as there are
International Journal of Peptide Research and Therapeutics
concerns for its bioavailability, absorption and suscepti-
bility to breakdown into inactive fragments on action of
physiological enzymes. For effective health promoting
activity from these peptides, resistance against gastro-
intestinal proteases and easy absorption into the blood
serum without any degradation is necessary (Fitzgerald
et al. 2004). This stability makes certain that the ingested
peptide sequences which showed in vitro bioactivity have
safely reached to the desired cellular sites of action. It
is reported that MAP1 and MAP2, milk proteins derived
peptides, showed in  vitro ACE inhibitory activity for
hypertensivity but only MAP1 could show potent inhibi-
tory effect and reduction in blood pressure (Boelsma and
Klooek 2010). Intestinal-expressed peptide transporters
help in carrying the smaller peptides across the interests,
whereas oligopeptides are transported passively through
hydrophobic regions of epithelial membrane (Darewicz
et al. 2011). Also, small sizes peptides have demonstrated
more in vivo bioactivity and can be easily absorbed into
blood circulation.
Safety of Bioactive Peptides
Till now, there is a bit of concern related to bio peptides
derived from food source. Intestinal wall disruption, eryth-
rocytes and lymphocytes toxicity, free radical production,
enzymopathic and immunopathic tissue damage and cyto-
toxicity due to the consumption of peptides are the main
problems in the biological system that lead to various com-
plicated disorders. Therefore, before considering biologi-
cally active peptides for food production and for therapeutic
purpose, it is first necessary to evaluate the immunogenic-
ity and toxicities of peptides (Niaz et al. 2018). There have
been various studies which have demonstrated the absence
of toxicity of these isolated bioactive peptides when incor-
porated in cell cultures. It has been studied that single doses
of 2000 mg/kg and daily doses of 1000 mg/kg for 4 weeks
of casein hydrolysates comprising antihypertensive peptides,
when subjected to rats, showed no adverse effects on dif-
ferent clinical parameters such as blood biochemistry, his-
topathological, haematology, etc. or on its mortality (Ana-
don et al. 2010). The sample doses were selected as such
that peptide amounts were greater than those required to
observe pharmacological activities. Additionally, no adverse
effects were found on serum biochemistry and different urine
parameters, when pre hypertensive and hypertensive humans
were subjected to lacto-tripeptide (Ile-Pro-Pro) -rich milk
protein hydrolysates as compared to those who received pla-
cebo treatment (Boelsma and Kloek 2010). Hence, bioactive
peptides are generally regarded as safe, but there is need to
avoid processing treatments that would degrade the quality
of peptides and its safety.
Future Applications
Bioactive peptides have proven to be remarkably applicable
in developing various health promoting functional foods.
Also, with the emergence of new technologies, the appli-
cation of bioactive peptides has significantly increased.
Membrane separation techniques are used for enrichment
of peptides of specific molecular weight range. Nano and
ultrafiltration techniques give peptides specifically on the
basis of casein or whey hydrolysates (Girgih et al. 2014).
Only a few of the peptides have been proven to be effective
in vitro and in vivo conditions as well. Concern needs to be
given in producing bioactive peptides for the treatment of
chronic diseases such as Alzheimer’s disease, kidney, liver
and cancer related diseases. Also, research area should be
extended in case of human trials for incorporation of bioac-
tive peptides to retrieve the approval of disease reduction
claims. This will lead to increase in development and utili-
zation of value added bioactive peptides with health claim
benefits (Kaspar and Reichert 2013).
Conclusion
Bioactive peptides have a limited usage in the market or at
the global level. Though, new technologies are emerging
nowadays, to get the most active form of these peptides and
incorporated to get the novel food product. The main focus
needs to be given on bioavailability of these peptides after
its intake. Further studies, in vivo and in vitro need to be
performed to demonstrate its use on a social level for the
prevention of various chronic diseases. Also, to increase the
market of value- added products or functional foods globally,
bioactive peptides act as a potential candidate.
Compliance with Ethical Standards 
Conflict of interest  Authors declare no any conflict of interest.
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