914 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO.

4, 2008

SPECIAL GUEST EDITOR SECTION

Bioactive Peptides
FEREIDOON SHAHIDI and YING ZHONG
Memorial University of Newfoundland, Department of Biochemistry, 300 Prince Philip Dr, St. John’s, NL, Canada A1B 3X9

Peptides with biological activities, released during
gastrointestinal digestion or food processing, play
an important role in metabolic regulation and
modulation, suggesting their potential use as
nutraceuticals and functional food ingredients for
health promotion and disease risk reduction. Many
studies have reported that peptides from various
food sources possess bioactivities, including
antihypertensive, antioxidant, anticancer,
antimicrobial, and opioid activities as well as
immunomodulatory and cholesterol-lowering
effects. More studies are being performed
exploring the sources, bioavailabilities, and
possible physiological/functional properties and
the mechanisms of action of bioactive peptides.
Technological approaches in terms of peptide
preparation, purification, and characterization have
also been investigated.
with a molecular weight (MW) of 5400 daltons (Da; 2).
Biologically active peptides, once liberated as independent
entities, act as potential metabolism modulators and
regulatory compounds with hormone-like activities (3).
Possible bioactivities reported for peptides include
antihypertensive, antioxidant, anticancer, antimicrobial, and
opioid activities as well as immunomodulatory and
cholesterol-lowering effects. The activity of these peptides is
dependent on their amino acid composition and sequence. A
database named Biopep, summarizing bioactive peptides and
their regulatory effects, is available, in which more than 1500
different bioactive peptides are included (4). A general
overview of bioactive peptides and their bioactivities, sources,
and technological approaches for preparation/characterization
is presented here.
Physiological/Functional Properties of Bioactive
Peptides

Antihypertensive Activity

T
he word “peptide” comes from the Greek term
Antihypertensive peptides are probably the most
“peptidia” translated as “small digestibles.”
extensively studied bioactive peptides from exogenous
Structurally, peptides are short polymers of amino
sources such as food. There has been a growing interest in
acids linked by peptide bonds. One or more polypeptide
antihypertensive peptides for their effectiveness in lowering
subunits constitute a protein molecule. Proteins are essential
blood pressure, since hypertension has become a serious
components of tissues in biological organisms and participate
health problem, occurring at increasingly high rates,
in a large number of physiological processes within cells. In
especially in developed countries, and has been considered a
foods, proteins are an important macronutrient, serving as a
risk factor for developing cardiovascular diseases.
source of energy and amino acids, which are essential for
Antihypertensive peptides have been found effective in
normal growth and maintenance of the body. Proteins are also
preventing/treating hypertension mainly by inhibiting the
responsible for various physicochemical and sensory
angiotensin-converting enzyme (ACE), which plays a key
properties of foods and may act as functional and
role in the regulation of blood pressure and electrolyte
health-promoting ingredients as well. Many of the
homeostasis (the equilibrium of water and salt in the
physiological and functional properties of proteins are
body; 5, 6). ACE is a nonspecific dipeptidyl carboxypeptidase
believed to attribute to biologically active peptides encrypted
that converts decapeptide angiotensin I into octapeptide
in the protein molecules. These peptides can be released from
angiotensin II, which is known as a potent vasoconstrictor.
their precursor proteins by digestive enzymes during
Angiotensin II also has a regulatory effect on cellular
gastrointestinal digestion or by in vitro proteolytic processes
lipoxygenases which catalyse low-density lipoprotein (LDL)
with exogenous proteases. Bioactive peptides generally
oxidation, a process associated with atherogenesis (7).
contain 3–20 amino acid units (1), but in some cases this range
Furthermore, ACE catalyses the inactivation of the
may be extended. Lunasin, for example, is a food-derived
vasodilator bradykinin, which in turn leads to increased blood
peptide with anticancer activity, composed of 43 amino acids
pressure. ACE inhibitors can decrease the activity of ACE and
indirectly reduce the level of angiotensin II, thereby exerting a
Guest edited as a special report on "Accurate Methodology for Amino vasorelaxing effect on blood vessels. Clinical studies have
Acids and Bioactive Peptides in Functional Foods and Dietary Supplements
for Assessing Protein Adequacy and Health Effects" by G. Sarwar Gilani
revealed that ACE inhibitors significantly reduced the
and Paul J. Moughan. morbidity and mortality of patients with myocardial infarction
Corresponding author's e-mail: fshahidi@mun.ca or heart failure (8, 9).
 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 915
Table 1. ACE-inhibitory peptides from major
food-derived protein sources
Protein source Ref. Protein source Ref.
Milk Meat 33
b-casein, 7, 15, 18, 19, 22–24 Fish 36, 37
as1-Casein,
k-Casein
b-Lactoglobulin, 25, 26 Soy 38, 39
a-Lactalbumin
Egg Sesame 40
Ovalbumin 31, 32 Broccoli 41
Blood Buckwheat 42
Hemoglobin 34 Rice 43
Skin
Gelatin 35
Natural ACE-inhibitory peptides from various sources
have been studied and their antihypertensive effect has been
reported in hypertensive animal models and human
subjects (10–16). Peptides with ACE-inhibitory activity have
been identified from a range of different food proteins from
both animal and plant sources (17; Table 1), among which
milk proteins are the most frequently studied. Peptides
derived from b-casein, the second most abundant protein in
bovine milk, are found to possess inhibitory activity against
ACE (7, 15, 18, 19). The tripeptides IPP and VPP, collectively
known as lactotripeptides (LTPs), are effective ACE
inhibitors that can reduce blood pressure in spontaneously
hypertensive rats (20). These LTPs have been isolated from
hydrolyzed milk casein, and toxicity studies have shown no
related adverse effects, suggesting the safety of
ACE-inhibiting LTP (21). Their use as functional food
ingredients for blood pressure control has been proposed.
Other tripeptides and some longer-chain peptides, including
YQEP, VPKVK, PLPLL, HLPLP, and YQEPVLGP from
b-casein, RPK and RPKHPIKH from as1-casein, as well as
PPEIN from k-casein have also been reported to exhibit
ACE-inhibitory activity (22–24). ACE-inhibiting peptides are
also found in whey proteins, in particular b-lactoglobulin and
a-lactalbumin. Several b-lactoglobulin and a-lactalbumin-
derived peptides have been demonstrated for their
ACE-inhibitory potentials (25, 26).
Bioactive peptides with ACE-inhibitory activity are
produced during enzymatic hydrolysis of milk proteins by
various proteases such as pepsin, pancreatin, Alcalase, and
Flavourzyme (23, 24, 26), during microbial fermentation of
milk (5, 27, 28), or in some cases by combined use of enzymes
and microorganisms (6). The type of enzymes used and
species of bacteria involved during peptide production greatly
influence the cleavage pattern of peptides from the protein,
which determines the inhibitory activity of the resultant
peptides against ACE. For example, milk fermented with
Lactobacillus helveticus bacteria had an increased level of
proline-containing short-chain peptides, such as IPP and VPP,
and therefore showed greater antihypertensive activity than
those fermented with other lactic acid bacteria species (10).
ACE-inhibitory peptides have also been isolated from other
dairy products such as yogurt (5) and cheese (29).
Milk-protein-derived peptides with ACE-inhibitory and
antihypertensive activity and aspects of their physiological
and chemical properties as well as the technological
approaches involved have been reviewed by
Lopez-Fandino et al. (30).
In addition to milk proteins, other sources of
antihypertensive peptides have also been investigated. These
include egg proteins, mainly ovalbumin (31, 32), meat
proteins (33), bovine hemoglobin (34) and skin gelatin (35),
fish proteins (36, 37), and several plant proteins, such as
soybean (38, 39), sesame (40), broccoli (41), buckwheat
proteins (42), and proteins from transgenic rice (43).
Although originated from different sources, these
ACE-inhibitory peptides seem to share one common
structural property, i.e., the presence of hydrophobic
(aromatic or branched side-chains) amino acid residues as the
last 3 amino acids at the C-terminus. ACE appears to prefer
the binding to substrates or competitive inhibitors containing
hydrophobic amino acids in the C-terminal tripeptide (16).
However, C-terminal lysine or arginine with a positive charge
also seems to contribute to the inhibitory activity of peptides,
which suggests a possible interaction between the inhibitor
and an anionic binding site of ACE (16). An inhibitory
mechanism of peptides against ACE has been postulated that
the 2 catalytic sites of ACE have different substrate
specificity (44), and that hydrophobic peptides have the
superiority to bind the occluded N-terminal catalytic site of
ACE, while hydrophilic peptides can only bind to the
C-terminal catalytic site (16). A more detailed action
mechanism of the inhibitory activity of peptides against ACE
is illustrated by FitzGerald and Meisel (45).
Cholesterol-Lowering Effect
Hyperlipidemia, especially hypercholesterolemia, is one of
the most important risk factors contributing to the
development of cardiovascular diseases (46). Numerous
synthetic drugs and natural extracts with cholesterol-lowering
effect have been explored for their potential in prevention and
treatment of hypercholesterolemia. A large body of literature
indicates that proteins from soybean can reduce blood
cholesterol level in experimental animal models as well as in
human subjects (47, 48). An early clinical study clearly
revealed that the substitution of animal proteins with soy
protein resulted in a 22–25% decrease in LDL cholesterol and
a 20–22% decrease in total cholesterol in
hypercholesterolemic patients (49). The hypocholesterolemic
effect of soy protein was later confirmed by more animal and
clinical studies (50–55), and soybean-rich diet has become the
most potent dietary tool for treating hypercholesterolemia,
although the mechanism has not yet been fully established.
The U.S. Food and Drug Administration (FDA)
916 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008

Table 2. Selected hypocholesterolemic proteins and peptides and their cholesterol-lowering effects in different
model systems

Protein/peptide Model Effect Reference

Soy protein Human Decrease in triacylglycerol and cholesterol circulation 52

Soy 7S globulin Rats Decrease in plasma cholesterol level 69
Soy protein hydrolysate Rats Decrease in serum cholesterol level 62
Soy protein hydrolysate Mice Decrease in total serum cholesterol and LDL level 70
Soy protein hydrolysate In vitro Suppression of cholesterol uptake by Caco-2 cells 63
Enterostatin Mice Decrease in serum cholesterol level 72, 73
Soy glycinin fragment In vitro Bile acid-binding ability 74
Milk b-lactoglobulin hydrolysate In vitro Inhibition of micellar cholesterol absorption in Caco-2 cells 65
Rats Decrease in cholesterol level 65
Pork protein hydrolysate Rats Decrease in cholesterol level 76, 77

recommended a daily intake of 25 g of soybean protein for
lower level of serum cholesterol and reduction in the risk of
cardiovascular disease (56).
The hypocholesterolemic effect of soybean was first
attributed to the presence of isoflavones, according to Adams
et al. (57), Ali et al. (58), and Zhan and Ho (59), since
ethanol-washed isoflavone-free soy protein showed less
cholesterol-lowering capacity than that containing
isoflavones. However, another experiment by Fukui et al. (60)
indicated that isoflavones alone did not exhibit a
cholesterol-lowering effect. Hence, it was speculated that the
isoflavone-protein interaction may contribute to the
hypocholesterolemic effect of soy proteins (61). More
recently, it has been demonstrated that soy peptides may be
responsible, at least in part, for the hypocholesterolemic
property of soy proteins, based on the observation that soy
protein hydrolysate showed a stronger serum cholesterol-
lowering effect than intact soy protein (50, 62, 63). Soy
proteins given to animals or human subjects by oral
administration are subjected to protease digestion in the
gastrointestinal tract, releasing the bioactive peptides which
inhibit cholesterol absorption, possibly due to the suppression
of the micellar solubility of cholesterol. Cholesterol is a
water-insoluble molecule and, similar to triacylglycerols,
requires a micellar solubilization step prior to intestinal
absorption (64). Cholesterol is rendered soluble in bile
salt-mixed micelles, and the presence of certain peptides
derived from digestion of food proteins has been found to
suppress its solubility by binding to bile acid and competing
for the micellar composition (65). It is hypothesized that a
peptide with high bile acid-binding capacity can inhibit the
reabsorption of bile acid in the ileum or decrease the micellar
solubility of cholesterol in small intestinal epithelial cells and
therefore decrease the blood cholesterol concentration. This
hypothesis is supported by many studies on the
hypocholesterolemic effects of soy proteins (62, 63, 66) and
the suppression of cholesterol micelle solubility in artificially
prepared or naturally derived micelles has been used as an in
vitro test for hypocholesterolemic efficiency of proteins and
peptides. Another hypothesis on cholesterol-lowering
mechanism is that some peptides can upregulate LDL
receptors, which are chronically suppressed by hyper-
cholesterolemia or dietary cholesterol administration (67).
The stimulatory effect of peptides on LDL receptors has been
observed for b-conglycinin and glycinin from soybean, which
significantly increased the binding of labeled LDL to
high-affinity receptors in liver cells (68).
Many proteins and their peptides are known to exert a
cholesterol-lowering effect (Table 2), among which soybean
is the most well recognized source of hypocholesterolemic
proteins and peptides. It has been shown that soy protein
reduces circulation of triacylglycerols and cholesterol in
hypercholesterol individuals (52). Sirtori et al. (69) have
reported that 7S globulin, a major storage protein in soybean,
decreased plasma cholesterol concentration by 35% in rats.
Soy protein hydrolysates showed a stronger serum
cholesterol-lowering effect than natural soy protein in
rats (62). Compared to casein hydrolysate, cholesterol
micelles containing soy protein peptic hydrolysate more
significantly suppressed the cholesterol uptake by Caco-2
cells (63). Zhong et al. (70) investigated soy protein
hydrolysates prepared with different enzymes at various
degrees of hydrolysis (DH) and found that soy protein
hydrolysate produced with Alcalase at DH 18% had the
highest hypocholesterolemic activity in mice, decreasing the
total serum cholesterol and LDL level by 24 and 34%,
respectively. Kwon et al. (71) isolated a tetrapeptide LPYP
from soy glycinin hydrolysate possessing a
hypocholesterolemic effect. Also, from soy glycinin subunits,
2 pentapeptides with hypocholesterolemic activity were
identified. Enterostatin (VPDPR) and its homolog LPYPR
were released during chymotrypsin and trypsin hydrolysis of
soy glycinin, and were indicated to reduce serum cholesterol
concentration in mice (72, 73). Choi et al. (74) reported the
bile acid-binding ability of a peptide fragment from soy
glycinin. The peptide region (VAWWMY) with high
 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 917
hydrophobicity was believed to be responsible for the bile
acid-binding ability of soy glycinin subunit A1ab1b,
suggesting its hypocholesterolemic potential. Kwon et al. (71)
synthesized a tetrapeptide LPYP and its 2 derivatives LPYPR
and SPYPR; the first 2 are hypocholesterolemic peptides
present in glycinin hydrolysates, and the last one had a serine
instead of a leucine residue at the N-terminal. Administration
of the 3 synthetic peptides LPYP, LPYPR, and SPYPR
resulted in 30, 31, and 14% reduction in total serum
cholesterol concentration in hypercholesterolemic mice,
respectively, which suggests that hypocholesterolemic
activity of LPYP is strongly influenced by the presence of the
leucine residue at the N terminus.
Milk is another important source of bioactive peptides with
cholesterol-lowering effect. Nagaoka et al. (65) isolated from
milk b-lactoglobulin tryptic hydrolysate a center responsible for the antioxidant activity of soy protein
hypocholesterolemic peptide, which was identified to be
IIAEK. Those authors demonstrated that this peptide inhibited
the absorption of micellar cholesterol in Caco-2 cells in vitro
and exhibited greater hypocholesterolemic activity than the
medicine b-sitosterol in rats. Kirana et al. (75) evaluated the
suppression of cholesterol absorption in Caco-2 cells by
IIAEK using both artificial micelles and pig bile-derived
natural micelle. Pork protein hydrolysate prepared with
papain has also been shown to exert a hypocholesterolemic
effect in cholesterol-fed rats (76, 77). Plant protein
hydrolysates with hypocholesterolemic activity, such as
soybean (63) and Brassica carinata protein hydrolysate have
also been reported (78).
Antioxidant Activity
Proteins, protein hydrolysates, individual peptides, and
amino acids have been shown to have significant antioxidant
activities. Some amino acids were found to possess strong
antioxidant activity in linoleic acid and methyl linoleate model
systems (79). A mixture of tryptophan and lysine was able to
inhibit oxidation of butter fat (80). Moreover, antioxidant
properties of proline in sardine oil (81), methionine in
vegetable oils (82), and histidine, threonine, lysine, and
methionine in a sunflower oil emulsion (83) have been
reported. Taurine, hypotaurine, carnosine, and anserine have
been demonstrated to exert antioxidant effect in vivo (84, 85).
Proteins from a variety of plant, animal, and microbial
sources, such as gluten, egg albumin, casein, soy protein, and
yeast protein, also exhibit antioxidant activity. However, in
many cases, peptide fractions or protein hydrolysates showed
greater antioxidant activity than intact proteins or amino acid
mixtures, suggesting that peptides play a major role in
antioxidant action of proteins. Matoba (86) demonstrated that
forming peptides was more critical than maintaining protein
structure, since heating did not affect the antioxidant
efficiency of proteins. More recently, individual peptides
responsible for antioxidant activity of protein or protein
hydrolysates have been separated and identified.
Protein digests have varied antioxidant activities
depending on the peptide structure, i.e., size of the peptides
and their amino acid sequences, which are influenced by the
source of protein and conditions of the hydrolysis process
involved. Soy peptides, for example, obtained from native or
heated soy protein by different enzymes, such as pepsin,
papain, chymotrypsin, Alcalase, Protamex, and Flavourzyme,
resulted in different degrees of hydrolysis, ranging from 1.7 to
20.6% and antioxidant activity ranging from 28 to 65%,
measured as inhibition against formation of thiobarbituric
acid-reactive substances (TBARS) in a liposome-oxidizing
system (87). Chen et al. (88) identified 6 antioxidant peptides
from the proteolytic digest of soybean protein, and found that
the peptides were composed of 5–16 amino acid residues with
hydrophobic amino acids V or L at the N-terminal position
and P, H, or Y in the sequence. Upon comparing the
antioxidant capacity of 28 small peptides structurally related
to LLPHH, the tripeptide unit PHH was found to be an active
hydrolysate (89). It was later believed that
histidine-containing peptides can act as metal chelator, active
oxygen quencher, and hydroxyl radical scavenger, thus
contributing to the antioxidant activity of the protein
hydrolysate (90). Saito et al. (91) constructed 2 tripeptide
libraries and investigated their antioxidant capacity by
different means. One was a library of 108 tripeptides
containing either histidine or tyrosine residues, and the other
was composed of 114 tripeptides structurally related to PHH.
The results indicated that tripeptides containing 2 tyrosine
units had higher capacity than those containing 2 histidine
units in inhibiting linoleic acid oxidation, and that
cysteine-containing tripeptides were strong peroxynitrite
scavengers. The authors also demonstrated that the presence
of W or Y residues at the C-terminus played an important role
in the radical scavenging activities but not in peroxynitrite
scavenging activity of the tested tripeptides. These findings
shed light on explaining the variation between protein digests
in antioxidant properties. Another study on whey protein
hydrolysates revealed a relationship between the antioxidant
properties of the whey peptides and the presence of
aromatic/imino amino acids (92). It was found that the peptide
fractions with high DPPH scavenging capacity had high levels
of histidine and hydrophobic amino acids. The radical
scavenging of peptides is likely due to the hydrogen donor
activity of the hydroxyl groups of aromatic amino acids, as
observed in most phenolic antioxidants.
Proteins from many natural sources and industrial
by-products have been explored for production of protein
hydrolysates with antioxidant potential (Table 3). Whey
protein hydrolysates have received much attention for
their radical scavenging and lipid oxidation-inhibiting
activities (93, 94). Cheison et al. (95) prepared protein
hydrolysates from whey using both a single- and a 2-stage
enzymatic membrane reactor and found better DPPH
scavenging activity for hydrolysates so produced than that for
the native whey protein isolate. Hernandez-Ledesma et
al. (26) evaluated the radical scavenging activity of several
b-lactoglobulin-derived peptides and suggested that the
presence and position of amino acids W, Y, and M in the
peptides are responsible for their antioxidant activity. Their
918 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008

Table 3. Food-derived bioactive peptides and their antioxidant activities

Peptides Antioxidant activity Reference

Soy peptides Inhibition of thiobarbituric acid reactive substances (TBARS) 87

Inhibition of liposome oxidation 87
Whey protein hydrolysate Radical scavenging activity 95
b-Lactoglobulin-derived peptides Radical scavenging activity 26
Inhibition against lipid oxidation in oil-in-water emulsions 96
Casein hydrolysate Radical scavenging activity 97
Caseinophosphopeptides Radical scavenging activity 98
Inhibition of liposome oxidation 99
Egg yolk protein hydrolysate Radical scavenging activity 100
Inhibition of TBARS 100
Porcine skin collagen hydrolyaste Radical scavenging activity 102
Capelin protein hydrolysate Inhibition of carotene-linoleate oxidation 104
Synergistic effects with BHA, BHT, and TBHQ 105
Seal protein hydrolysates Inhibition of carotene-linoleate oxidation 111
Canola protein hydrolysates Radical scavenging activity 120

results also support the hypothesis that the peptidic bond or
structural peptidic conformation improves the hydrogen
donor capacity of the amino acid residues, enhancing their
antioxidant activity. A synergetic effect of these
b-lactoglobulin-derived peptides with ascorbic acid was also
reported. Chymotryptic hydrolysates of b-lactoglobulin have
been shown to inhibit lipid oxidation in oil-in-water
emulsions, possibly due to the presence of oxidatively labile
amino acids M and Y (96). Also derived from milk protein,
casein hydrolysates produced with pepsin and pancreatin
exhibited antioxidant activity with the peptides HLPLP and
WSVPQPK being identified responsible for the antioxidant
activity (22). Another peptide from peptic digest of casein
YFYPEL with radical scavenging activity was identified by
Suetsuna et al. (97) and its antioxidant activity was attributed
to the sequence of G-L. Caseinophosphopeptides have been
reported to have radical scavenging activity (98) and an
inhibitory effect against liposome oxidation (99).
Hydrolysates prepared by enzymatic hydrolysis of egg yolk
protein exhibited superoxide, hydroxyl, and DPPH radical
scavenging activities and inhibited formation of TBARS in
food model systems such as in ground beef and tuna
homogenates (100). Fat-free egg yolk proteins hydrolyzed
sequentially with Orientase (EC 3.4.21.62) and a proteinase
(EC 3.4.11.12) yielded peptides with low MW (maximum
mass <1000 Da and average chain length of 2.6), which were
more effective in inhibiting linoleic acid oxidation than the
original egg yolk or a mixture of amino acids with the same
composition (101). By-products from meat production
provide a source of antioxidative peptides. The hydrolysate of
porcine skin collagen by cocktail mixture of proteases showed
substantial scavenging activity against DPPH radical (102).
Chicken cracklings and feathers hydrolyzed enzymatically or
chemically with acid also possessed antioxidant activity,
according to Flaczyk et al. (103).
Aquatic species and by-products from aquaculture
industry are probably the most extensively investigated as an
important source of antioxidative peptides. Amarowicz and
Shahidi (104) separated 4 peptide fractions from protein
hydrolysates of capelin with one fraction possessing notable
antioxidant activity, another 2 a weak antioxidant efficacy,
and the fourth exerting a pro-oxidant effect. In another study,
synergistic effects of capelin protein hydrolysates with
synthetic antioxidants butylated hydroxylanisole (BHA),
butylated hydroxyltoluene (BHT), and tert-butylhydroquinone
(TBHQ) were observed (105). Synergistic effects have also
been reported for sardine protein hydrolysates with BHA,
BHT, sodium ascorbate, and a-tocopherol both in vitro and in
vivo (106, 107). Li et al. (108) demonstrated the radical
activity of carp muscle hydrolysates and the optimal
hydrolysis conditions for production of peptides with strong
antioxidant activity. Protein hydrolysates prepared from
mussel and fermented mussel sauce have shown antioxidant
activity (109, 110). Marine mammal protein hydrolysates
have been explored for potential antioxidant activity. Shahidi
and Amarowicz (111) evaluated the antioxidant effectiveness
of harp seal protein hydrolysates and found that the
hydrolysates displayed antioxidant property at certain
concentrations while a pro-oxidant property was observed at
higher concentrations. A variety of fish processing waste has
recently been considered a potential source of peptides with
antioxidant potential. These include protein hydrolysates from
tuna backbones (112), Alaska Pollack frame and
skin (113, 114), hoki frame (115), and other marine
wastes (116).
 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 919

Table 4. Peptides with anticancer activities

Peptides Anticancer activity Reference

Lunasin in soybean Inhibition of carcinogens in mammalian cells 2, 131–133

Soy protein hydrolysates
Soy glycopeptide
Buckwheat peptide
Ginseng peptides
Egg protein hydrolysates
Fish protein hydrolysates
Suppression of colon and liver tumorogenesis 138, 139
Cytotoxicity to lymphoma cells 140
Antiproliferation to hepatoma, leukemia, and breast cancer cells 144
Antitumor activity to lymphoma cells 145
Antiproliferation to lymphoma cells 148
Inhibition of breast cancer cells 150

In addition to animal proteins, proteins from plant sources
are also of great interest for providing potential antioxidative
peptides. Hydrolysates from native or preheated soybean
proteins were effective antioxidants (87, 117). Wheat gluten
and germ protein hydrolysates have shown good antioxidant
activity comparable to that of a-tocopherol (118, 119). Other
known plant-derived protein hydrolysates with antioxidant
properties include canola (120), corn (121), potato (122),
peanut (123), sunflower (124), and B. carinata (78).
Anticancer Activity
Proteins, peptides, and amino acids have been implicated
in preventing the development of different types of cancer.
Bowman Birk protease inhibitor (BBI), a water-soluble
protein isolated from legumes and many monocotyledonous
seeds, has shown anticarcinogenic activity in in vitro and
animal models and is now intensively studied as a cancer
chemopreventive agent in clinical trials (125, 126). Another
protease inhibitor, soybean Kunitz trypsin inhibitor, was
reported to suppress ovarian cancer cell invasion by blocking
urokinase upregulation (127). Bovine lactoferrin and
lactoferricin from bovine milk were able to inhibit lung
metastasis and angiogenesis in mice transplanted with murine
melanoma, lymphoma, or colon carcinoma 26 cells (128, 129).
Lectins from mistletoe extract induced powerful anticancer
effects in mice inoculated with tumor cells (130). The
anticancer activities of these proteins may, at least partially, be
attributed to encrypted bioactive peptides.
Numerous peptides in different sizes from various sources
have been indicated to render an anticancer effect in in vivo
studies (Table 4). Lunasin, a novel chemopreventive peptide
from soybean, has been found to suppress chemical
carcinogen and viral oncogene-induced transformation of
mammalian cells and inhibit skin carcinogens in
mice (2, 131–133). Lunasin is a 43-amino-acid peptide
containing 9 aspartic acid residues at the C-terminus, a
tripeptide arginine-glycine-aspartic acid cell adhesion motif,
and a predicted helix whose structure is similar to a conserved
region of chromatin-binding proteins (131). Lunasin exhibits
an inhibition effect against core histone acetylation in
mammalian cells (134, 135), suggesting its involvement in
chromatin modification, a process implicated in cell-cycle
control and suppression of carcinogenesis. It is possible that
lunasin selectively kills cells being transformed or newly
transformed by binding to exposed deacetylated core histones,
disrupting the histone acetylation-deacetylation dynamics and
leading to cell death (133). The affinity of lunasin for
hypoacetylated chromatin is attributed to its polyaspartyl
structure at the carboxyl end (136). It has recently been found
that orally administered lunasin can be protected from
digestion by BBI, another bioactive component in
soybean (137). Originally isolated from soybean, lunasin has
also been found in barley and wheat (135).
Other bioactive peptides from soybean with
cancer-preventive effects have also been reported. Azuma et
al. (138) and Kanamoto et al. (139) demonstrated that a high
MW fraction (HMF) of proteinase-treated soybean protein
isolate suppressed colon and liver tumorgenesis in
experimental animals. Those authors suggested that the HMF
inhibited intestinal resorption of bile acids through
hydrophobic binding, and therefore lowered the
tumor-promoting activity of bile acids on the colon and liver.
A glycopeptide isolated from soybean hydrolysate containing
mainly D, E, P, G, and L has been shown to be cytotoxic
against P388D1 mouse lymphoma cells (140). Kim et
al. (141) purified an anticancer peptide from the hydrophobic
peptide fraction of thermoase-treated soy protein hydrolysate.
The peptide was identified as a nonapeptide with a MW of
1157 Da and a sequence of X-MLPSYSPY. In addition to
soybean, bioactive peptides with anticancer activity have been
found in other leguminous plants such as definsin-like
peptides in Yunnan bean (142) and pinto bean (143). Other
plant sources of anticancer peptides reported include those of
buckwheat and ginseng. A peptide isolated from buckwheat
seeds with an MW of 4 kDa showed antiproliferative activity
against hepatoma, leukemia, and breast cancer cells (144). A
hydrophobic fraction of ginseng peptide isolate demonstrated
high antitumor activity against mouse lymphoma cell line
P388D1 (145). The peptides present in the fraction were
identified to be either dipeptides or tripeptides composed
entirely of tryptophan.
Dairy milk proteins and their peptide derivatives play a
role in cancer prevention (25, 146). Kim et al. (147) studied
the anticarcinogenicity of hydrophobic peptide fractions
920 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
isolated from cheese slurries and found that the purified
peptide fractions exhibited high cytotoxic activity against
tumor cell lines SNU-C2A, SNU-1, and P388D1. Egg protein
hydrolysates were found to exert antiproliferative effect on
mouse lymphoma cells (148). Glycopeptides from
pronase-treated ovomucin showed antitumor effects in a
double-grafted tumor system in mice, which has been
associated with their antiangiogenic activity (149). In
addition, anticancer peptides are present in marine animals
and microorganisms. Picot et al. (150) assessed the
antiproliferative activity of 18 fish protein hydrolysates in
vitro and identified 3 blue whiting, 3 cod, 3 plaice, and
1 salmon hydrolysates as significant growth inhibitors against
2 human breast cancer cell lines. Shark-cartilage-derived
peptides have shown antiangiogenic activity, as evaluated in a
variety of in vitro and in vivo models (151). Vitilevuamide, a
bicyclic 13 amino acid peptide isolated from marine ascidians,
was found to be cytotoxic in several human tumor cell
lines (152). An antitumor peptide symplostatin 3 has been
identified from marine cyanobacteria which is analogous in
structure to dolastatin 10, a compound in human clinical trials
for cancer treatment (153).
Immunomodulatory Effects
The association between nutrition and immunity has long
been recognized. It has been demonstrated that bioactive
peptides derived from various protein sources exert
immunomodulatory effects in in vitro and in vivo studies.
However, most studies focused on evaluation of the effect of
peptides and protein hydrolysates on specific immune systems
and only a limited number of investigations examined their
impact on nonspecific (innate) immune systems. Moreover,
characterization of these bioactive peptides and illustration of
their mechanism of action as well as clinical data to support
their physiological properties are needed.
Immunomodulatory peptides can be released naturally
from proteins during the digestion process in the
gastrointestinal tract, thus affecting downstream
immunological responses and cellular functions, or obtained
by enzymatic hydrolysis in vitro prior to the administration to
achieve a therapeutic effect as nutraceuticals. Dairy milk,
while providing a number of essential nutrients, is also found
to play an important role in the immune system of newborns
and have been investigated for peptides with possible
immunomodulating properties (154). Mercier et al. (155)
reported the stimulation of in vitro proliferation of murine
spleen lymphocytes by whey protein enzymatic hydrolsates
(prepared with a trypsin/chymotrypsin mixture), although to a
lesser extent than that by the intact whey protein isolate. A
similar trend was observed by Wong et al. (156), who
demonstrated that b-lactoglobulin promoted proliferation of
murine spleen cells in vitro and that the stimulating effect was
reduced by tryptic hydrolysis of the protein. However, another
study by Mahmud et al. (157) showed that mitogenic activity
of b-lactoglobulin on proliferation of mouse resting spleen
cells was retained following reduction of the protein and was
enhanced after digestion with pepsin, trypsin, chymotrypsin,
or pancreatin, suggesting that the activity depends on the
primary structure of the protein and that its peptides may be
responsible for this activity. The authors also indicated that
most mitogenic pancreatic hydrolysates significantly induced
the growth of a human B cell line (U266) without influencing
the growth of other cell lines (Ball human B cells, MOLT-4,
and Jurkat T cells). Contradictory results for native and
hydrolyzed proteins may be due to different methodologies
used, including preparation of proteins, conditions for
enzymatic hydrolysis process, origins of the lymphocytes, and
conditions used for proliferation assays, among others. In
addition, presence of mitogens such as lipopolysaccharides
(LPS), phytohemagglutinin, and concanavalin A may affect
the proliferation of targeted cells, thus interfering with the
results. Brix et al. (158) showed that endotoxin contamination,
especially LPS contamination, in commercial b-lactoglobulin
preparations may account for the marked in vitro proliferative
effect of this protein on murine splenocytes and mesenteric
lymph node cells and other immunostimulatory effects. A
peptic hydrolysate of bovine lactoferrin was found to promote
proliferation of murine splenocyte, while the intact lactoferrin
inhibited cell proliferation (159). Further digestion of the
hydrolysates with pepsin led to the loss of mitogenic activity
and the authors concluded that pepsic hydrolysates of bovine
lactoferrin may contain both immunostimulatory and
immunoinhibitory peptides. Milk-derived peptides have also
shown a suppressive effect on mitogen-induced proliferative
responses, as observed for glycomacropeptides
(GMP; 160–162) and pancreatin and trypsin digests of
as1-casein and b-casein (163). In addition, the IL-6 (a
cytokine) response in an LPS-stimulated monocytic cell line
was suppressed by bovine lactoferrin or its peptide
lactoferricin (164). Certain peptides derived from as1-casein
and b-casein have shown more sophisticated
immunomodulatory effects, i.e., suppressing
mitogen-induced proliferation of human lymphocytes and
peripheral blood mononuclear cells while stimulating
lymphocyte proliferation in the absence of
mitogens (165, 166). The down-regulation property of
bioactive peptide on immune response depends on the nature
of the peptide and has been associated with their potential
anti-inflammatory activity (167).
Dairy peptides have also been demonstrated to modulate
antibody production. A pepsic hydrolysate of bovine
lactoferrin containing lactoferricin significantly enhanced
immunoglobulin (Ig) production in cultured murine
splenocytes and Peyer’s patch cells (159). Whey protein
pancreatic hydrolysate exerted an impact on the humoral
immune system by producing larger amounts of antibody to
T-cell-dependent antigens in mouse spleen (168). Bovine
lactoferrin hydrolysate in the diet resulted in greater
anticholera toxin IgA levels in the intestines of mice, which
suggests its potential benefit in enhancing mucosal
immunity (159). However, an opposite trend was observed for
GMP, which inhibited in vitro antibody production to sheep
red blood cells in murine splenic cell cultures (161) and
 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 921

Table 5. Immunomodulatory effects of food-derived peptides

Peptides Immunomodulatory effect Reference

Whey protein hydrolysate Stimulating in vitro proliferation of murine spleen lymphocytes 155
Stimulating antibody production in mouse spleen 168
b-Lactoglobulin hydrolysates Stimulating in vitro proliferation of murine spleen cells 156
Stimulating proliferation of mouse resting spleen cells 157
Promoting growth of a human B cell line 157
Lactoferrin hydrolysate Stimulating proliferation of murine splenocyte 159
Stimulating antibody production in mice intestine 159
Increasing phagocytic activity of human neutrophils 170
Suppressing IL-6 response in mitogen-induced monocytic cells 164
Glycomacropeptides Stimulating proliferation and phagocytic activity of human macrophage-like cells 171
Inhibiting in vitro antibody production in murine splenic cell cultures 161
Suppressing cytokine secretion in mitogen-induced murine splenic lymphocyte culture 169
as1-Casein and b-casein hydrolysates Stimulating lymphocyte proliferation in the absence of mitogens 165
Suppressing mitogen-induced proliferation of human lymphocytes 166
Ovalbumin peptides Enhancing phagocytic activity of macrophages 176
Egg yolk peptides Enhancing mucosal immune responses 181
Soy protein hydrolysate Stimulating phagocytosis 182

suppressed cytokine secretion in a mitogen-induced murine
splenic lymphocyte culture (169).
The effect of milk-derived peptides on a nonspecific
immune response such as enhancement of phagocytosis has
been reported. Lactoferricin was found to increase the
phagocytic activity of human neutrophils, possibly due to the
presence of specific binding sites on human blood phagocytic
cells that can bind to the peptide (170). GMP and its pepsic
digest were able to promote proliferation and phagocytic
activities of human macrophage-like cells (U937; 171). A
synthetic peptide GLL, corresponding to sequence f51-53 of
a-lactoalbumin, markedly increased phagocytosis of sheep
red blood cells by murine peritoneal macrophages (172).
The mechanism by which milk-derived bioactive peptides
exert their modulatory effects on the immune system is not yet
fully uncovered. However, the stimulatory role of these
peptides in maturation and proliferation of T cells and natural
killer cells has been suggested (173). The hypothesized
mechanism, which is based on the relationship between
immune system and opioid peptides, suggests that the opioid
peptides in milk affect the immunoreactivity of lymphocytes
via the opiate receptor, since opioid m receptors for endorphins
are present in T lymphocytes and human phagocytic
leukocytes (174). Moreover, receptors for many biologically
active mediators are expressed by lymphocytes and
macrophages, and it is believed that the arginine residue at the
N- or C-terminal region may be the dominant entity
recognized by specific surface membrane receptors (174).
Some ACE-inhibitory peptides with arginine in the
C-terminus exhibit immunomodulatory effects. For example,
b-casein-derived ACE-inhibitory peptides were found to
stimulate phagocytosis and protect mice against Klebsiella
pneumoniae infection (175).
In addition to milk, immunomodulatory peptides are also
present in other protein sources, such as egg and soybean
proteins. Egg white protein-derived peptides have shown
immunomodulatory activity. Ovalbumin peptides have been
found to enhance the phagocytic activity of
macrophages (176) and have been used for promoting
immune responses for cancer immunotherapy (177, 178).
Ovomucoid peptides have also been shown to possess
immunomodulatory effect by stimulating macrophage
activity (179) and inducing cytokine secretion (180). Egg yolk
peptic digests have been demonstrated to enhance mucosal
immune responses (181). Immunomodulatory peptides have
been identified from soybean protein hydrolysates. A
bioactive peptide with sequence MITLAIPVNKPGR isolated
from trypsin digests of soy protein exhibited
phagocytosis-stimulating activity, which was attributed to the
methionine residue at the N-terminus and influenced by the
3rd amino acid residue from the N-terminus (182). Major
immunomodulatory peptides are summarized in Table 5.
It is noteworthy that bioactive peptides with
immunomodulatory properties may be of benefit in treating
acquired immune deficiency syndrome. Preliminary trials
have revealed a decreased progression of the
disease (183, 184), a reduced number of symptoms (185), and
more recently the restoration of cytokine receptor expression
in patients with human immunodeficiency virus (186).
922 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
Antimicrobial Activity
Peptides with antimicrobial property have been identified
in a broad variety of natural sources from microorganisms to
animals and plants. Antimicrobial peptides display inhibitory
effects against microbe-caused food deterioration and
invasion of a wide range of pathogens in vivo, including
bacteria, fungi, and virus as well as eukaryotic parasites. The
action mode and effectiveness of these biologically active
peptides as antimicrobial agents vary depending on their
structural characteristics, i.e., peptide size, amino acid
composition, charge, hydrophobicity, and secondary
structure (187). Moreover, they show varied selectivity and
sensitivity on target microorganisms. In general,
animal-derived antimicrobial peptides exhibit inhibitory
activity against a much larger spectrum of microorganisms
than those produced by bacteria (188), while the latter show
higher efficiency at extremely low concentrations of even
nanomolar level (189). However, antimicrobial peptides
possess certain common features. Most antimicrobial peptides
are composed of less than 50 amino acids with approximately
50% being hydrophobic amino acids, and often fold into
amphipathic 3D structures (188). Having an excess of basic
amino acids (lysine and arginine), antimicrobial peptides
usually bear a net positive charge. Being cationic and
amphipathic are 2 important structural features that account
for the antimicrobial activity of these bioactive peptides.
Antimicrobial peptides are of great interest for their
potential application in food preservation and for therapeutic
purpose in health care. The appearance of low pH and high
salt-tolerant microorganisms has been a big challenge for
foodborne pathogen control, since traditional food
preservation has only limited efficacy in inhibiting the growth
of these microorganisms. Thus, emergence of novel
antimicrobial agents, especially those of natural origin is
needed in order to fill this gap. Bioactive peptides with
antimicrobial property such as those produced by lactic acid
bacteria (LAB) in fermented foods and many food component
peptides are good candidates as food additives. Advantages of
antimicrobial peptides over chemical preservatives include
fewer adverse effects introduced, lower intensity of heat
treatment required (minimal processing), and retaining of
organoleptic and nutritional properties of food, such as less
acidic and/or lower salt content (190). In therapeutic
applications, antimicrobial peptides are superior to
conventional bactericidal antibiotics because they kill bacteria
faster and are not affected by antibiotic-resistance
mechanisms that are often encountered by other
antibiotics (188).
A large group of antimicrobial peptides belong to
bacteriocins, which are ribosomally synthesized and
post-translationally modified peptides produced by different
groups of bacteria against other closely related bacteria (191).
Bacteriocins are categorized into several classes and
subclasses according to their biochemical and genetic
properties: class I (lantibiotics), class IIa (Listeria active or
pediocin-like bacteriocins), class IIb (2-peptide bacteriocins),
class IIc (sec-dependent bacteriocins), class IId (other class II
bacteriocins), class III (thermosensitive proteins), and
class IV (complex bacteriocins that require lipid or
carbohydrate moiety for activity; 192–194). Class I
bacteriocins, also known as lantibiotics, are a group of small
and post-translationally modified peptides produced by a
large number of Gram-positive bacteria that contain unusual
amino acids such as thioether-cross-linked amino acids,
lanthionine and 3-methyllanthionine, and dehydrated amino
acids (195, 196). These unusual amino acids are believed to
contribute solely to the structural stability and antimicrobial
activity of lantibiotics (189). Nisin, the first identified and
most predominant lantibiotic produced by Lactococcus lactis,
has obtained FDA approval and has been used commercially
as a food preservative in many food products (197). A
comprehensive review on lantibiotics and their biosynthesis
and antimicrobial role is presented by Nagao et al. (189). Most
of the LAB-produced bacteriocins belong to class II, in
particular class IIa bacteriocins, which are active in inhibiting
the growth of Gram-positive bacteria such as Listeria
monocytogenes, Bacillus cereus, and Clostridium
perfringens. These peptides are considered one of the most
interesting bacteriocins owing to their large number,
significant bioactivity, and potential application in the food
and medicinal industries as antibiotics and antiviral
agents (193, 198). Bacteriocins have been identified in a wide
array of bacteria, and novel bacteriocins from new bacterial
sources are being added. Furthermore, various approaches
such as gene cloning and heterologous peptide production for
bacteriocin overexpression have been proposed to promote
production of high-value bacteriocins (199).
Antimicrobial activity of bacteriocins against pathogenic
microorganisms has been elucidated at cellular and molecular
levels. The inhibition has been associated with the interaction
of peptides with membrane and other cellular components.
Many bacteriocins elicit their lethal effect on target
microorganisms by disintegrating the cell wall, increasing the
permeability of the cell membrane, and causing efflux of small
molecules such as amino acids and adenosine
triphosphate (189). The positively charged antimicrobial
peptides traverse the negatively charged LPS-containing outer
wall of Gram- negative bacteria or the acidic
polysaccharides-containing outer wall of Gram-positive
bacteria by competitively displacing the divalent cations that
bridge and neutralize the polysaccharides (192). At low
peptide concentrations, this leads to outer cell wall disruption,
while at higher concentrations bacteria are partially lysed and
disintegrated (192). In some cases, the bacteriocins bind to
certain target molecules (docking molecules) which are
essential intermediates for cell wall biosynthesis and therefore
inhibit cell wall formation, as observed for some
lantibiotics (189) and class IIa bacteriocins (193). Upon
reaching the phospholipid’s cytoplasmic membrane, the
amphipathic peptides perpendicularly insert themselves into
the interface of phospholipid bilayers, causing formation of
transmembrane pores, leakage of cytoplasmic contents, and
eventually cell death (192). In addition to the interaction with
 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 923
cell membrane, bacteriocins and other antimicrobial peptides
exert an inhibitory effect on target microorganisms also
through interaction with intracellular anionic components
such as DNA and RNA, thereby inhibiting protein synthesis
and cell division (200). Moreover, some peptides are involved
in activation of autolysin in target microorganisms (200).
Eukaryotic organisms such as fungi, animals, and plants
also produce bioactive peptides with antimicrobial activity.
Eukaryotic antimicrobial peptides such as animal-derived
antimicrobial peptides are synthesized as primary translation
products, which are later converted into active forms by
post-translational modification and proteolytic processing,
including glycosylation, carboxy-terminal amidation,
isomerization, halogenation, and cyclization as well as
proteolysis (188). Similar to most bacteriocins, eukaryotic
peptides inhibit the growth of target microorganisms by
nonspecific mechanisms involving interaction with multiple
membrane and intracellular targets that results in membrane
perturbation, cytoplasmic translocation, and interaction with
intracellular elements (188). Eukaryotic antimicrobial
peptides are widely distributed in many living organisms,
including mammals, amphibians, insects, fish, fungi, and
plants. Antimicrobial peptides from food sources have been of
special interest because of consumers’ preference for
food-derived components over other food additives. Protein
hydrolysates from various food materials have been evaluated
for their antimicrobial activity, including hydrolysates of dairy
protein (201–206), egg protein (207–209), marine shellfish
protein (210, 211), and plant proteins such as proteins from
buckwheat seeds (144) and chili pepper seeds (212).
Other Bioactivities: Opioid, Antiobesity, and
Mineral-Binding Activity
Endogenous and many exogenous opioid agonists and
antagonists have been characterized as peptides (213). Their
binding to opioid receptors in the central nervous system as
well as in many peripheral tissues has been related to a number
of physiological and pathophysiological functions, including
immunological functions, gastrointestinal function control,
reproductive mechanism control, and regulation of many
central nervous functions such as stress handling, depression,
and other emotional behaviors (213). Although endogenous
opioids are identified as peptides with the presence of a
tyrosine residue at the N-terminus (except a-casein opioid)
and another aromatic amino acid in the third or fourth
position, exogenous opioid peptides are classified into typical
opioid peptides, which have the same N-terminal sequence
YGGF, and atypical opioid peptides, which only have a
tyrosine as obligatory residue in the N-terminal sequence but
various amino acids from parent proteins (214). The negative
potential, localized in the vicinity of the phenolic hydroxyl
group of tyrosine seems to be essential for opioid activity, and
lack of the tyrosine results in a total absence of
bioactivity (215). Most food-derived opioid peptides are
atypical opiods, resulting from in vivo or in vitro digestion of
their precursor proteins. Exogenous opioid agonists and
antagonists have been of important significance in nutritional
and pharmacological areas.
Peptides with opioid activity originating from food
proteins have been identified and characterized, including
milk (214, 216), wheat (217), barley, maize, and
soybean (218, 219) opioid-like peptides, with milk
protein-derived opioid peptides being the most intensively
studied. Opioid peptides derived from milk protein have been
effective in prolonging gastrointestinal transit time, inhibiting
diarrhea, modulating intestinal transport of amino acids, and
stimulating insulin and somatostatin secretion as well as in
producing analgesia and modulating social behavior (174).
b-Casomorphins from b-casein were the first opioid peptides
identified from food protein sources and have so far been most
frequently studied. Although exhibiting limited efficacy in
adult humans, b-casomorphins can be transported across
intestinal mucosa in neonates and provide an analgesic effect
on the nervous system, which results in calmness and sleep in
infants (214). In a recent study, the presence of
b-casomorphin-5 has been reported in a peptide extract from
an infant formula and its pepsin-trypsin hydrolysate (220).
b-Casomorphins have also been demonstrated to play a role in
appetite regulation and social behavior in experimental
animals (221, 222). In addition to b-casomorphins, a- and
b-lactorphin from bovine and human a-lactalbumin and
b-lactoglubulin (223), exorphins from bovine
as1-casein (224), and serorphin from whey protein (225) have
been reported to have opioid agonistic activity. Milk proteins
are also a source of antagonistic peptides, such as
casoxins (226), lactoferroxin (227), and hemorphins (228).
Opioid peptides derived from milk proteins have been
reviewed by Kostyra et al. (229).
Opioid peptides have been demonstrated to play an
important role in the control of food intake, which is
implicated to its potential antiobesity activity. A large body of
evidence has shown that opioid antagonists reduce feeding in
most species including humans (230). Yeomans and
Gray (231) reported that naltrexone reduced food intake and
eating rate and abolished the stimulation of appetite through
palatability in human male subjects. The opioid receptor, in
particular the k-receptor, is believed to be responsible for food
intake regulation and initiates several satiety signals when
activated by agonist peptides. Moreover, opioid peptides exert
appetite regulation effect by modifying the endocrine activity
of the pancreas to increase insulin output (221). In addition to
the opioid activity, other mechanisms also apply to the
antiobesity activity of peptides, such as inhibiting the
absorption of dietary lipids, increasing postprandial energy
expenditure, accelerating lipid metabolism, and decreasing
body fat, all of which lead to reduced weight gain and
modulated lipid profile as well. Several food-derived and
synthesized peptides have been reported to have antiobesity
activity. Anorectic peptides from soybean (e.g., LPYPR and
PGP) have been shown to exert antiobesity activity through
decreasing food intake and body weight (232). Soybean
protein hydrolysates were found to decrease serum or hepatic
triacylglycerol level and body weight in rats (232). A synthetic
924 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
peptide e-polylysine has been demonstrated to have an
antiobesity function in mice by inhibiting intestinal absorption
of dietary fat (233).
Peptides derived from in vivo and/or in vitro enzymatic
proteolysis of whole protein exhibit mineral-binding activity,
which contributes both to their antioxidant activity and
mineral absorption-enhancing activity. Bioactive peptides
such as caseinophosphopeptides from b-casein are able to
chelate pro-oxidant metals, in particular iron and copper, and
therefore protect from lipid oxidation-caused food
deterioration and oxidative stress-mediated cellular damage.
Copper-chelating peptides have been purified from sunflower
protein hydrolysates, and their metal-chelating activity was
found to be dependent on histidine content and peptide
size (124). The metal-chelation property of the peptides was
attributed to the imidazole ring in histidine, which is directly
implicated in peptide binding to copper. The results suggested
the potential antioxidant role of food-derived peptides in
preventing copper-induced in vivo oxidative damage,
including DNA and LDL oxidation, which are involved in
atherogenesis and other chronic diseases (124). More
importantly, mineral-binding peptides are effective in
enhancing in vivo absorption of metals, including copper,
calcium, iron, zinc, and other trace elements and therefore
improving their bioavailability. Binding of copper to certain
amino acids such as histidine, methionine, and cysteine in
small peptides mediates absorption of copper through these
amino acid transporters (234). Caseinophosphopeptides,
which in most cases contain a serine phosphate cluster and
glutamyl residues in the sequence of 3 phosphoseryl groups
followed by 2 glutamic acid residues, can bind to calcium at
the negatively charged side chains and form a soluble
complex (174). This leads to enhancement of calcium
absorption across enterocytes in the distal intestine (173). Use
of caseinophosphopeptides in preventing dental caries has
also been proposed because of their role in recalcification of
the dental enamel (235).
Technological Approaches to Bioactive Peptide
Production
Preparation of Bioactive Peptides
Because bioactive peptides are inactive within the parent
protein molecules, certain processing approaches releasing
the peptide entities from the protein precursors are necessary
for their utilization as food additives and/or nutraceuticals.
Bioactive peptides are generally produced by means of
digestive proteolysis in the gastrointestinal tract, chemical or
enzymatic hydrolysis in vitro, and bacterial fermentation as
well as gene expression approaches and chemical synthesis.
Variations in peptide yield, composition, and bioactivity exist
among different methodologies and should be considered
when selecting one or combined methods for a certain
purpose. Orally ingested proteins undergo proteolytic
reactions in the digestive system with the aid of digestive
enzymes and/or resident microflora in the gut, and release
peptides of different size and amino acid composition. These
peptides are then absorbed from the gastrointestinal tract and
transported to different sites such as the peripheral blood and
cellular receptor sites, and exert their physiological functions.
Experimental evidence has demonstrated that bioactive casein
peptides are naturally generated during the normal digestive
process of milk (173). Hence, milk is currently considered as a
source of high quality proteins with good bioavailability.
However, due to different proteolysis patterns, digestion of
some other proteins may result in different peptide
composition, which leads to altered bioactivity or loss of
efficacy. In vitro hydrolysis of the source proteins provides an
effective approach for preparation and better utilization of
bioactive peptides.
Peptides can be produced in vitro through chemical or
enzymatic hydrolysis of proteins. Chemical hydrolysis, which
involves use of acids or alkali, is relatively simple and less
expensive; however, it lacks selectivity and specificity, and is
more difficult to control and more prone to amino acid
damage. Enzymatic methods use mild conditions and are
more predictable with respect to the end products. They are
the commonly used methods for peptide production in
laboratories and in industry. In enzymatic protein hydrolysis,
the type of enzyme is very important as it dictates the cleavage
pattern of the peptide bonds. Proteinases, especially
endopeptidases such as trypsin, subtilisin, chymotrypsin,
thermolysin, pepsin, proteinase K, papain, and plasmin as
well as commercial proteases such as Alcalase and
Flavourzyme are major enzymes used for peptide preparation.
These enzymes are obtained from different sources, including
plants, animals, and microorganisms, and each requires
optimal conditions (temperature, pH, time course,
enzyme/substrate ratio, etc.) for best performance and results
in unique peptide profiles. Optimization of hydrolysis
conditions for bioactive peptide production has been
conducted by many researchers. Jiang et al. (109) investigated
protein hydrolysates of mussel meat prepared by papain,
trysin, and pepsin under different conditions for their
antioxidant potential. The highest antioxidant activity for
papain hydrolysates was achieved when hydrolysis was
carried out at 60°C and pH 7.0 for 45 min at an enzyme
content of 1%, whereas optimal conditions for trypsin were
temperature, 50°C; pH, 8.5; time, 60 min; and enzyme
content, 0.76%. Li et al. (108) reported the optimum
conditions for preparing carp muscle Alcalase hydrolysate
with radical-scavenging effectiveness to be 4.8 h and 4.8
AU/kg, 6 h and 3.84 AU/kg, and 4.3 h and 4.8 AU/kg all at pH
7.5. Another study compared 2 proteases, Alcalase and
Flavourzyme, in canola protein hydrolysis in terms of
antioxidant activity and water-binding capacity of the
resultant hydrolysates (120). Flavourzyme hydrolysates,
although having a lower degree of hydrolysis, exhibited
higher antioxidant efficiency and water-binding capacity than
hydrolysate produced using Alcalase, possibly due to smaller
size of peptides produced. In addition to the type of enzyme,
temperature, pH, time, and enzyme concentration, other
factors may also contribute to peptide composition in the
hydrolysate and its bioactivity. Release of bioactive peptides
 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 925
Table 6. Major techniques for purification and identification of bioactive peptides
Purification
Membrane-based techniques
Microfiltration (MF)
Ultrafiltration (UF)
Nanofiltration (NF)
Reverse osmosis (RO)
Chromatographic techniques
High-performance liquid chromatography (HPLC)
Ion-exchange chromatography (IEC)
Capillary electrophoresis (CE)
Capillary isoelectric focusing separation (CIEF) Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF)
Size-exclusion chromatography (SEC)
Counter-current chromatography (CCC)
Hydrophobic interaction chromatography (HIC)
Affinity chromatography (AC)
from ovalbumin was promoted by use of high hydrostatic
pressure (32). Hydrolysis of ovalbumin with chymotrypsin,
trypsin, and pepsin under hydrostatic pressure of
200–400 Mpa showed enhanced degree of hydrolysis and
accelerated release of anti-ACE peptides, as a result of altered
proteolytic pattern. The authors suggested that this could be
due to conformational changes in the protein which makes it
more vulnerable to proteolysis, because protein unfolding can
expose new cleavage sites to enzymatic hydrolysis. Moreover,
high pressure may affect enzyme structure/function and/or
substrate-enzyme interaction, which may partially account for
the improved yield of bioactive peptides. High pressure
during hydrolysis was also found to induce and, when used in
combination with high temperature (600–800 Mpa/60–80°C),
to accelerate cyclization of a dipeptide LG at the C-terminus,
and the product cyclo LG was identified as a bioactive
peptide (236).
New technologies have been developed for improving the
processing of enzymatic hydrolysis such as introduction of
immobilized enzymes. Compared to traditional soluble
enzymes, immobilized enzymes have the following
advantages: allowing milder and more easily controlled
conditions; preventing generation of secondary metabolites
from autolysis of enzymes; avoiding product damage caused
by heat or acidification which is required for process
termination in traditional methods; and allowing recovery and
reuse of enzymes (78). Furthermore, methods combining
bacterial fermentation and enzymatic hydrolysis have been
proposed. Tsai et al. (6) developed a combination method
using lactic acid fermentation by 2 LAB and hydrolysis by
Flavourzyme to produce a milk product which is more
Identification/detection
Spectrophotometric techniques
Ultraviolet (UV) detection
Fluorescence (FL) detection
Evaporative light scattering detection (ELSD)
Mass spectrometric techniques
Atmospheric pressure ionization triple quadrupole (API-III)
Electrospray ionization (ESI-MS/MS)
Other mass spectrometries
abundant in bioactive peptides than the traditional
fermentation or hydrolytic products of milk. It is noteworthy
that, during the enzymatic hydrolytic processing of proteins,
each bioactive peptide shows different release kinetics. In
general, peptides of larger size appear in quantity at the early
stage of hydrolysis. As enzymatic hydrolysis proceeds, larger
bioactive peptides are cleaved into small peptides, sometimes
with different bioactivities, as observed for
hemorphins (237, 238). Biotransformation has also been
reported for peptic hydrolysis of bovine hemoglobin, which
turned the antimicrobial peptide a107-136 to the
bradykinin-potentiating peptide a110-125 (34).
In addition to in vivo and in vitro proteolysis of protein
precursors, production of bioactive peptides can also be
achieved via molecular genetic engineering, in which
bioactive peptides with specific amino acid sequences can be
synthesized through gene cloning, overexpression, and
site-directed mutagenesis (73, 199).
Characterization of Bioactive Peptides
The isolation and purification of bioactive peptides are
very important for exploration of their physicochemical
properties and evaluation of their in vitro and in vivo
bioactivities. Bioactive peptides can be separated from a
protein hydrolysate mixture by a number of approaches,
mainly, different kinds of chromatography and
membrane-based separation techniques. Prior to the
separation process, a peptide mixture can be subjected to
ammonium sulfate precipitation, salting out, and solvent
extraction in order to remove proteins, enzymes, and other
components in the source material. Most chromatographic
926 SHAHIDI & ZHONG: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
techniques for protein purification are also applicable to
peptide purification, given special consideration on size
difference, such as size-exclusion chromatography (SEC; 232).
Different kinds of chromatography have been adopted in
peptide separation, including high-performance liquid
chromatography (HPLC), especially reversed-phase HPLC,
ion-exchange chromatography (IEC), capillary
electrophoresis (CE), capillary isoelectric focusing (CIEF)
separation, gel filtration chromatography (GFC) for aqueous
systems, gel-permeation chromatography (GPC) for
nonaqueous systems, counter-current chromatography
(CCC), centrifugal partition chromatography (CPC), and
low-pressure hydrophobic interaction chromatography (HIC).
One or more of these techniques in combination can be
selected based on the properties of the peptides.
HPLC is the most commonly used method for peptide
separation. Commercially available reversed-phase columns
allow rapid separation and detection of peptides from a
mixture and meanwhile indicate their hydrophilicity and
hydrophobicity. Use of reversed-phase columns with a
polystyrene-divinylbenzene copolymer-based packing is
recommended for separation of peptides with different surface
hydrophobicity (239). Less common than reversed-phase,
normal-phase HPLC is used preferentially for separation of
more hydrophilic peptides (232). HPLC is usually coupled
with quantitative/qualitative analyzing equipment such as a
UV detector or mass spectrometer. Similar to HPLC,
techniques such as CCC, CPC, and HIC are
hydrophilicity/hydrophobicity-based chromatographic
methods with the absence of solid adsorbent, except for HIC,
in which peptides are adsorbed to a phenyl agarose matrix.
Instead of using the hydrophilic/hydrophobic properties,
another large group of chromatographic methods separate
peptides based on their charge properties; these include IEC,
CE, and CIEF. In addition, GFC and GPC, collectively known
as SEC, are also widely used separation methods that are
solely based on molecular size of the peptides.
More recently, affinity chromatography appears as an
effective purification method for bioactive peptides that relies
on the formation of specific reversible complexes between the
peptide to be purified and an immobilized ligand (124). This
docking and virtual screening method makes it possible to
“dock” small molecules (ligands) towards bioactive peptides
and “score” their potential binding (240). The mixture
containing bioactive peptides is first incubated with affinity
absorbent, followed by washing to remove unbound
molecules; the retained peptides are then recovered by
specific or nonspecific elution agents. Affinity
chromatography has been successfully used for purification of
ACE-inhibitory and copper-chelating peptides using
immobilized ACE on a glyoxyl-agarose support and
immobilized copper, respectively (124, 241, 242).
Membrane-based separation processes are techniques in
which a component is separated from a mixture as the latter is
forced through a porous membrane under applied pressure;
hence the name pressure-driven membrane-based
separation (243). They are further divided into microfiltration
(MF), ultrafiltration (UF), nanofiltration (NF), and reverse
osmosis (RO), depending on the properties of the membrane
used. Peptide size and/or charge are important features for the
retention mechanism of these methods. A wide variety of
membranes are available to serve different fractionation
purposes. The pore size of membrane can range from <0.1 nm
in RO to >0.1 mm in MF, and separation domain varies
accordingly from monovalent salts to particles. Operating
pressure usually varies inversely with membrane pore size,
i.e., the highest pressure for RO and lowest for MF (243).
Other than selecting one separation method, different
techniques are normally used in combination to achieve best
separation of bioactive peptides. For example, peptide
fractions obtained from membrane filtration can be further
purified using HPLC.
Bioactive peptides, once isolated and purified, can be
subjected to qualitative analysis. Amino acid analyzer and
protein sequencer are common tools for determining amino
acid composition and sequence of unknown peptides. Other
methods such as UV detection, fluorescence detection,
evaporative light scattering detection, mass spectrometry,
atmospheric pressure ionization triple quadrupole,
electrospray ionization-tandem mass spectrometry, and
matrix-assisted laser desorption ionization-time of flight
techniques are also becoming popular for peptide
identification. Table 6 presents a summary of major
techniques for characterization of bioactive peptides.
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