Food Research International 74 (2015) 185–198

Contents lists available at ScienceDirect

Food Research International

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

Review

Biologically active peptides: Processes for their generation, purification

and identification and applications as natural additives in the food and
pharmaceutical industries
Ruann Janser Soares de Castro ⁎, Hélia Harumi Sato
Department of Food Science, School of Food Engineering, University of Campinas, 80 Rua Monteiro Lobato, Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Recent technological advances have created great interest in the use of biologically active peptides. Bioactive
Received 19 March 2015 peptides can be defined as specific portions of proteins with 2 to 20 amino acids that have desirable biological
Received in revised form 1 May 2015 activities, including antioxidant, anti-hypertensive, antithrombotic, anti-adipogenic, antimicrobial and anti-
Accepted 8 May 2015
inflammatory effects. Specific characteristics, including low toxicity and high specificity, make these molecules
Available online 12 May 2015
of particular interest to the food and pharmaceutical industries. This review focuses on the production of bioac-
Keywords:
tive peptides, with special emphasis on fermentation and enzymatic hydrolysis. The combination of different
Proteins technologies and the use of auxiliary processes are also addressed. A survey of isolation, purification and peptide
Enzymatic hydrolysis characterization methods was conducted to identify the major techniques used to determine the structures of
Fermentation bioactive peptides. Finally, the antioxidant, antimicrobial, anti-hypertensive, anti-adipogenic activities and
Purification probiotic-bacterial growth-promoting aspects of various peptides are discussed.
Bioactive peptides © 2015 Published by Elsevier Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2. Major processes for obtaining bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2.1. Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2.2. Enzymatic hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
3. Concentration, purification and identification of bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
4. Biological properties of bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
4.1. Peptides with antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4.2. Peptides with antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4.3. Peptides with anti-adipogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
4.4. Peptides with anti-hypertensive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
4.5. Induction of lactic acid bacteria and probiotic growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

1. Introduction physicochemical and sensory properties, such as solubility, viscosity,

Proteins are fundamental food components. Nutritionally, they
are sources of essential amino acids, are indispensible for growth and
maintenance, and are a source of energy. Protein foods are able to affect
⁎ Corresponding author.
E-mail address: ruannjanser@hotmail.com (R.J.S. de Castro).
gelification and emulsion stability. Some proteins in the diet have
specific biological properties, making them potential ingredients for
functional foods (Korhonen, Pihlanto-Leppala, Rantamaki, & Tupasela,
1998). During digestion, proteins are hydrolyzed, generating a large
range of peptides. Some of these peptides have structural characteristics
that allow them to interact with endogenous peptides. Many endoge-
nous peptides have important functions, acting as neurotransmitters,

http://dx.doi.org/10.1016/j.foodres.2015.05.013
0963-9969/© 2015 Published by Elsevier Ltd.
186 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
hormones and regulators (Hernández-Ledesma, García-Nebot,
Fernández-Tomé, Amigo, & Recio, 2014).
Bioactive peptides can be obtained from animal or plant proteins.
Plant sources are generally grains, such as wheat, rice, oats, rye and
corn, and some legumes, such as soy, peas and chickpeas. Of the plant
sources, soy is one of the most widely studied as a source of peptides,
as it is a significant source of dietary protein (Ortiz-Martinez, Winkler,
& García-Lara, 2014). Animal protein sources also have great potential.
One of the most popular and promising lines of research is the produc-
tion of hydrolyzed proteins from meat proteins, which, in addition to
having important biological activities and being excellent sources of nu-
trients, such as minerals and vitamins, can be used as flavor enhancers
and emulsifiers (Lafarga & Hayes, 2014; Mora, Escudero, Fraser,
Aristoy, & Toldrá, 2014). The biological properties of other animal pro-
tein sources, such as egg and fish, have also been studied (Sakanaka,
Tachibana, Ishihara, & Juneja, 2004; Theodore, Raghavan, & Kristinsson,
2008).
Recent studies have linked the prevalence of cardiovascular disease,
obesity, hypertension, diabetes and cancer to nutritional factors. In re-
sponse to increased awareness of the relationship between food and
health, the market for functional foods has expanded. A functional
food is any food that in addition to basic nutritional functions, provides
additional health benefits, regulating one or more functions in the body
(Diplock et al., 1999; Hernández-Ledesma, Contreras, & Recio, 2011).
Processes incorporating protein hydrolysis have been studied to de-
termine whether they produce biologically active peptides. Mellander
(1950) was responsible for the first study relating the ingestion of
bioactive peptides from hydrolyzed casein protein to increased bone
calcification in rachitic newborns. Since then, peptides with countless
bioactivities have been identified. According to the Biopep and BioPD
(Bioactive peptide database) databases, more than 1200 different bioac-
tive peptides have been recorded (Singh, Vij, & Hati, 2014).
Bioactive peptides are defined as specific regions of proteins with
amino acid sequences that have biological activity, including antioxi-
dant, anti-hypertensive, antithrombotic, anti-adipogenic, antimicrobial,
anti-inflammatory and immunomodulatory effects (Ahn, Cho, & Je,
2015; Biziulevicius, Kislukhina, Kazlauskaite, & Zukaite, 2006; Tavares
et al., 2011; Tsou, Kao, Tseng, & Chiang, 2010; Zhang, Li, & Zhou,
2010). These peptides have 2–20 amino acids and molecular masses
of less than 6000 Da. Their bioactivity is mainly determined by their
composition and amino acid sequence (Mora, Reig, & Toldrá, in press;
Sarmadi & Ismail, 2010; Singh et al., 2014; Tsou, Kao, et al., 2010). This
enormous functional diversity places these peptides and proteins at
the forefront of the biotechnology fields (Miranda & Liria, 2008); fur-
thermore, these peptides and proteins have been identified by several
authors as possible substitutes for chemicals used as drugs or food
preservatives (Hong et al., 2008; Uhlig et al., 2014).
According to Uhlig et al. (2014), there is a very good outlook for
using bioactive peptides in the pharmaceutical field. Some peptides in
the clinical trial phase have shown very promising results for treating
cardiovascular, infectious and metabolic diseases. Peptides have an
important competitive advantage over traditional medications for the
following reasons: 1) They have high specificity for their target tissues,
resulting in little or no toxicity, and even low concentrations can be ef-
fective. This characteristic is extremely important for treating chronic
diseases; 2) Synthetic chemical compounds that are typically used as
drugs often have a cumulative effect on the organism. These synthetic
substances may represent an environmental problem due to their ex-
cretion, still in the active form. In contrast, bioactive peptides undergo
little or no accumulation in the organism, and they are easily degraded
in the environment (Uhlig et al., 2014).
Several methods are used to obtain bioactive peptides, including fer-
mentation, enzymatic hydrolysis and a combination of the two process-
es (Fig. 1). In fermentation, the addition of lactic acid bacteria with
proteolytic activity leads to formation of bioactive peptides, especially
during dairy product manufacturing. Enzymatic hydrolysis involves
the use of digestive, plant or microbial proteolytic enzymes in a partial
hydrolysis process, leading to a reduction of allergenic factors, im-
proved digestibility and the formation of biologically active peptides
(Korhonen, 2009). One strategy used in several scientific studies dem-
onstrated that using lactic acid bacteria together with food-grade
enzymes resulted in final products with more interesting characteristics
than either process alone. Combining the techniques, in addition to
increasing the amount of peptides in the fermented products, resulted
in various biological and functional effects (Hafeez et al., 2014). Chen,
Tsai, and Pan (2007) studied a combined process using enzymatic
hydrolysis with the microbial protease Prozyme 6 from Aspergillus and
a commercial starter culture mixture of five lactic acid bacteria as a
strategy to enhance the ACE-inhibitory activity of bioactive peptides
from milk. The results showed that the pre-treatment of the fresh
milk with Prozyme 6 presented a positive impact on the ACE inhibitory
activity of the peptides, in which the IC50 value for the combined
process was 0.24 mg mL−1, in contrast to 0.64 mg mL−1 for the straight
fermentation and 1.18 mg mL−1 for fresh milk.
In addition to the conventional methods mentioned above, combin-
ing several technologies has produced effective results for generating
functional peptides (Korhonen, 2009). Ultrafiltration and nanofiltration
are examples of technologies that have been used to refine and isolate
bioactive peptides, allowing them to be separated by size for use in
specific applications (Picot et al., 2010; Quirós, Chichón, Recio, &
López-Fandiño, 2007).
Understanding the critical parameters for the process is fundamen-
tally important for obtaining hydrolyzed proteins with desirable biolog-
ical and functional characteristics. These parameters include the protein
source and its characteristics, such as chemical composition, pH and
seasonal variations; enzymatic preparation and other aspects related
to purity, substrate specificity, specific activity, pH and temperature
for activity and stability; and processing conditions, including enzyme
and substrate concentrations, pH, temperature and reaction time.
Prior knowledge and identification of these parameters can be used as
tools for obtaining products with distinct functions, for producing mul-
tifunctional peptides or even for producing unique peptides with specif-
ic functions (Li-Chan, 2015; Samaranayaka & Li-Chan, 2011).
This review describes advances in scientific research on the process-
es of obtaining, purifying and identifying the biological activities and
potential applications of bioactive peptides.
2. Major processes for obtaining bioactive peptides
2.1. Fermentation
The use of fermentation processes to make bioactive peptides is
mainly relevant to dairy product manufacturing. Dairy products natu-
rally contain precursor proteins for bioactive molecules (Akalin, 2014;
Schanbacher, Talhouk, & Murray, 1997). Fermenting milk involves a
number of metabolic pathways responsible for generating metabolites,
which significantly contribute to the chemical, biochemical and nutri-
tional properties of the fermented products. The proteolytic system of
lactic acid bacteria (LAB) is complex and consists of three major compo-
nents: proteases bound to the cell wall that promote the initial hydroly-
sis of milk casein into oligopeptides, specific transporters that transfer
the oligopeptides to the cytoplasm and intracellular peptidases that fin-
ish the hydrolysis process to convert oligopeptides into free amino acids
and/or low molecular weight peptides (Chaves-López et al., 2014). The
ability of these microorganisms to produce proteolytic enzymes makes
them potential producers of bioactive peptides, which can be released
during fermented product manufacturing. Several microorganisms
have been extensively reported in the literature as having an effective
proteolytic system for protein hydrolysis and the release of bioactive
peptides, including Lactobacillus helveticus, Lactobacillus delbrueckii ssp.
bulgaricus, Lactococcus lactis ssp. diacetylactis, Lactococcus lactis ssp.
 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198 187

Animal or vegetable protein sources

Proteases: Microbial fermentation

vegetable, animal or
microbial

Proteases produced
during fermentation
High pressure
homogenization

Protein hydrolysates
Bioactive
Peptides
Ultrafiltration or nanofiltration

Biological activities: Isolation, purification

in vitro and in vivo methods and identification

Antihypertensive Antimicrobial Anti-adipogenic

Anti-inflamatory Immunomodulatory Antioxidant

Fig. 1. Major processes for obtaining bioactive peptides and related bioactivities.

cremoris and Streptococcus salivarius ssp. thermophylus (Hernández-
Ledesma et al., 2011). In addition to using live microorganisms, proteo-
lytic enzymes isolated from LAB have also been successfully used in
enzymatic hydrolysis processes and for the production of bioactive
peptides (Choi, Sabikhi, Hassan, & Anand, 2012).
Although dairy products have been highlighted in scientific studies
producing these peptides by fermentation, it has been shown that
fermentation products derived from soy, beans, rice and wheat are
also biologically active (Hati et al., 2014; Inoue et al., 2009; Limón
et al., 2015; Nakahara et al., 2010) (Table 1). Species of filamentous
fungi such as Aspergillus oryzae and Aspergillus sojae have a long tradi-
tion of safe use in the production of fermented foods, in which several
peptides with biological activities were detected, for example antioxi-
dant and antihypertensive activities (Giri, Osako, Okamoto, Okazaki, &
Ohshima, 2011; Inoue et al., 2009; Nakahara et al., 2010).
2.2. Enzymatic hydrolysis
Enzymatic hydrolysis is one of the fastest, safest and most easily con-
trolled techniques for producing bioactive peptides, and it can be used
to improve the functional and biological properties of the proteins
as well as to add value to byproducts with low commercial value
(Luna-Vital, Mojica, de Mejía, Mendoza, & Loarca-Piña, in press; Mora
et al., in press; Singh et al., 2014; Zarei et al., 2014).
Proteases catalyze the hydrolysis of peptide bonds in proteins and
may act on the ester and amide bonds. All proteases have a certain de-
gree of specificity for the substrate, generally based on the sequence of
amino acids directly surrounding the bond that is cleaved (Santos &
Koblitz, 2008). This specificity and the hydrolysis conditions (pH,
temperature, time) affect the size and the amino acid sequences in the
peptide chains as well as the quantity of free amino acids, which can af-
fect the biological activity of the hydrolysates (Luna-Vital et al., in press;
Sarmadi & Ismail, 2010; Su, Ren, Yang, Cui, & Zhao, 2011; Tsou, Kao,
et al., 2010; Zhou, Canning, & Sun, 2013). Proteases with specific activi-
ty, such as trypsin and chymotrypsin, and combinations of different
non-specific proteases, such as Pronase™ E from Streptomyces griseus
and Flavourzyme™ from A. oryzae, have been used to produce more
stable and effective bioactive peptides by reducing the reaction times
needed for hydrolysis and by making it possible to obtain different pro-
files, especially for the composition and molecular mass distribution of
the peptides. These processes are especially useful in the food and phar-
maceutical industries, which rely on animal, plant and microbial prote-
ases (de Castro, Bagagli, & Sato, 2015; Singh et al., 2014; Vanderghem
et al., 2011).
In addition to the commercial enzymes, it is important to note that
several studies reported the use of crude microbial enzymes to hydro-
lyze proteins, suggesting the potential application of novel protease
sources for the production of bioactive peptides.
Table 2 summarizes some studies in which the release of biologically
active peptides after protein hydrolysis using commercial and crude
protease preparations was demonstrated.
3. Concentration, purification and identification of bioactive
peptides
Table 3 summarizes different characteristics for some methods of
purification and identification of bioactive peptides, including their
principle, advantages and limitations. Chromatography techniques are
188 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198

Table 1
Obtaining peptides with different biological activities by fermentation using various protein sources.

Microorganism Protein source Fermentation conditions Peptides Bioactivity Reference

Streptococcus thermophiles Soy milk Submerged fermentation for 5 h Tyr-Pro-Tyr-Tyr Antihypertensive Tsai, Chen, Pan, Gong,
Lactobacillus bulgaricus at 43 °C and Chung (2008)
+
Protease Flavourzyme
Aspergillus oryzae Rice, soy and Solid-state fermentation for 40 h Val-Pro-Pro; Ile-Pro-Pro Antihypertensive Inoue et al. (2009)
casein at 30 °C
Aspergillus oryzae, Okara Sequential submerged Not identified Antioxidant Zhu, Cheng, Wang,
Rhizopus oligosporus, fermentation: Fan, and Li (2008)
Actinomucor elegans, B. subtilis for 48 h at 40 °C
Bacillus subtilis A. oryzae, R. oligosporus and
A. elegans for 60 h at 30 °C
Aspergillus sojae Soy and wheat Solid-state fermentation for 192 h at Gly-Tyr; Ala-Phe; Val-Pro; Antihypertensive Nakahara et al. (2010)
20–45 °C and 95% humidity Ala-Ile; Val-Gly
Enterococcus faecalis Cow's milk Submerged fermentation for 24 h Peptides with molecular Antihypertensive and Regazzo et al. (2010)
TH563 at 37 °C (Enterococcus faecalis) or weights less than 5000 Da immune-regulatory
Lactobacillus delbrueckii subsp. 44 °C (Lactobacillus delbrueckii)
bulgaricus LA2
L. acidophilus ATCC 4356 Sodium Submerged fermentation for 5 h at Peptides with molecular Immunomodulatory Stuknyte, Noni,
Lc. lactis subsp. lactis GR5 Caseinate 30 °C (Lactococcus lactis) or 37 °C weights less than 3000 Da Guglielmetti, Minuzzo,
(L. acidophilus) with agitation at and Mora (2011)
140 rpm
Aspergillus oryzae Squid mantles Solid-state fermentation for 365 Peptides with molecular Antioxidant Giri et al. (2011)
days at 25–30 °C weights less than 1450 Da
B. subtilis 10160 Rapeseed Solid-state fermentation for 6 days Peptides with molecular Antioxidant He et al. (2012)
at 32 °C and 85 ± 5% relative weights between 180 and
humidity 5500 Da
Bifidobacterium longum KACC91563 Casein Submerged fermentation for 24 h Val-Leu-Pro-Val-Gln Antioxidant Chang et al. (2013)
Lactobacillus casei spp. Concentrated Submerged fermentation for 36 h Leu-Ile-Val-Thr-Gln Antihypertensive Vallabha and Tiku (2014)
pseudoplantarum soy protein at 37 °C
B. subtilis ATCC 6051 Bean Solid-state fermentation for 96 h Peptides with molecular Antioxidant Limón et al. (2015)
at 30 °C and 90% relative humidity weights between 6.2 and Antihypertensive
201.2 kDa

amongst the most widely used, such as high performance liquid chro-
matography (HPLC) and ultra high pressure liquid chromatography
(UHPLC) (Singh et al., 2014). UHPLC has shown great potential in the
separation of small bioactive peptides, increasing the throughput of reg-
ular HPLC methods. The main advantages of this method include the in-
crease of throughput, resolution and sensitivity (Everley & Croley, 2008;
Fekete & Guillarme, 2014). Reversed phase HPLC (RP-HPLC) can be used
to separate peptides by hydrophobicity (Pownall, Udenigwe, & Aluko,
2010). Hydrophilic interaction liquid chromatography (HILIC) has
been shown to be a useful method for the separation of hydrophilic sub-
stances. This method is based on increases in retention with increasing
polarity of the stationary phase and of the solutes and the decreasing
polarity of the predominantly organic solvent system used for elution;
the opposite principle of that observed in RP-HPLC (Yoshida, 2004). Le
Maux, Nongonierma, and FitzGerald (2015) reported HLIC method as
a valuable tool to improve the separation of short peptides and differen-
tiation of peptides with homologous sequences by mass spectrometry.
Gel electrophoresis and ultrafiltration techniques have also been used
as auxiliary methods for structural and chemical composition analysis
of peptides (Roblet et al., 2012; Singh et al., 2014).
Mass spectrometry has greatly improved the process of identifying
peptide sequences and studying protein profiles and hydrolysis prod-
ucts. In particular, interfaces have been developed that allow ions to
be generated from analyte molecules that are sensitive to temperature
and/or are not very volatile. Electrospray ionization and matrix assisted
laser desorption/ionization (MALDI-TOF), for example, has recently be-
come important for the identification and characterization of bioactive
peptides and proteins using mass spectrometry. Liquid chromatogra-
phy–mass spectrometry is commonly used to identify peptide se-
quences (Chiaradia, Collins, & Jardim, 2008; Contreras, López-Expósito,
Hernández-Ledesma, Ramos, & Recio, 2008; Singh et al., 2014).
Peptides with anticoagulant activity that were obtained from goby
fish (Awaous guamensis) and a protease from Bacillus licheniformis
were separated by molecular exclusion chromatography and reversed-
phase high-performance liquid chromatography and identified by
mass spectrometry. The hydrolysate solution containing the peptides
was applied to a Sephadex G-25 (5.2 × 56 cm) gel filtration column
pre-equilibrated and eluted with distilled water, and 4.5-mL fractions
were collected using a flow rate of 0.5 mL min− 1. Absorption at
220 nm was measured to determine the peptide elution profile. Frac-
tions with higher anticoagulant activity were recovered and purified
in a reverse-phase Vydac C18 (10 × 250 mm, Grace-Vydac) column
and eluted using a linear acetonitrile gradient (0 to 40% v/v) and a
flow rate of 0.6 mL min− 1. The molecular mass and amino acid
sequence of the peptides were measured using a triple quadrupole
mass spectrometer with an electrospray ionization source (Applied
Biosystems API 3000, PE Sciex, Toronto, Canada). Four peptide
sequences had high anticoagulant activity and were identified as Leu-
Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Leu-Arg and Cys-Arg-Arg (Nasri
et al., 2012).
Tsou, Kao, Lu, Kao, and Chiang (2013) purified and identified bioac-
tive peptides from purified soy protein and the Flavourzyme protease
using sequential fractionation with ultrafiltration membranes of various
sizes, gel chromatography, reversed-phase high-performance liquid
chromatography and mass spectrometry. The hydrolysates were initial-
ly fractionated in ultrafiltration membranes of 30, 10 and 1 kDa. The
fraction retained on the 1-kDa membrane was selected for purification
due to its ability to stimulate lipolysis in 3T3-L1 pre-adipocyte cells.
The 1-kDa retained portion was then applied to a Superdex™ peptide
10/300 GL column (10 × 300 mm; GE Healthcare), equilibrated and
eluted with 30% acetonitrile and a flow rate of 0.5 mL min− 1. One-
milliliter fractions were collected, and elution curves were constructed
based on absorbance measurements at 214 nm. The fractions with the
highest anti-adipogenic activity were collected and purified in a
Develosil ODS-HG-5 reverse-phase column (4.6 × 250 mm, Nomura
Chemical) and eluted using a linear acetonitrile gradient (5.0 to 75.0%)
and a flow rate of 1.0 mL min−1. The fraction with the most anti-
adipogenic activity was purified again using a reverse-phase column
 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198 189

Table 2
Using proteases to generate biologically active peptides from various protein sources.

Protease Hydrolysis Protein Bioactivity of Peptides Identification methods Reference

conditions source the peptides

Alcalase™ pH 8.0; 50 °C; 3 h Soy Antiadipogenesis Peptides with molecular Liquid chromatography mass Mejia et al. (2010)
E:S = 1:20 weights between 754 and spectrometry
[S] = 5.0% 3897 Da
Flavourzyme™ pH 7.0; 50 °C; 2 h Purified soy Antiadipogenesis Peptides with molecular High-performance molecular Tsou, Kao, et al., 2010
E:S = 1:100 protein weights less than 1300 Da exclusion chromatography
[S] = 2.5%
Neutrase™ pH 6.0; 45 °C; 4 h Purified soy Antiadipogenesis Peptides with molecular High-performance molecular Tsou, Lin, et al., 2010
E:S = 1:100 protein weights between 1300 exclusion chromatography
[S] = 2.5% and 2200 Da
Pepsin™ pH 5.5; 23 °C Bovine Antimicrobial Peptides with molecular Electrospray ionization mass Adje et al. (2011)
[S] = 1.0% hemoglobin Antihypertensive weights between 668 and spectrometry
4430 Da (ESI/MS)
Alcalase™ pH 8.0; 50 °C; 3 h Bean Antioxidant Peptides with molecular Matrix-assisted laser Oseguera-Toledo,
[E] = 0.2 mg/mL Anti-inflammatory masses between 445 and desorption/ionization mass Mejia, Dia, and
[S] = 8.0% 2148 Da spectrometry Amaya-Llano (2011)
(MALDI-TOF)
Crude protease from pH 10.0; 50 °C; 5.5 h Goby muscle Anticoagulant Leu-Cys-Arg Liquid chromatography Nasri et al. (2012)
Bacillus licheniformis [S] = 10.0% His-Cys-Phe Electrospray ionization mass
Cys-Leu-Cys-Arg spectrometry
Leu-Cys-Arg-Arg (ESI/MS)
Alcalase™ pH 7.0; 50 °C; 8 h Salmon Antioxidant Peptides with molecular High-performance molecular Ahn, Je, and Cho (2012)
Flavourzyme™ pH 7.0; 50 °C; 8 h Anti-inflammatory masses between 1000 exclusion chromatography
Protamex™ pH 7.0; 50 °C; 8 h and 2000 Da
Neutrase™ pH 7.0; 50 °C; 8 h
Pepsin pH 2.0; 37 °C; 8 h
Trypsin pH 8.0; 37 °C; 8 h
Crude protease from pH 10.0; 50 °C Cuttlefish Antihypertensive Peptides with molecular Liquid chromatography Balti et al. (2015)
Bacillus mojavensis [S] = 5.0% (Sepia masses between 163 and Electrospray ionization mass
officinalis) 1047 Da spectrometry (ESI/MS)
muscle Tandem mass spectrometry
(ESI-MS/MS)

and linear acetonitrile gradients of 10 to 40%. Finally, the peptides were
identified by liquid-chromatography coupled with mass spectrometry.
Three peptides with the amino acid sequences Ile-Leu-Leu, Leu-Leu-
Leu and Val-His-Val-Val were identified as being responsible for the
anti-adipogenic activity of the protein hydrolysates isolated from soy.
Peptides with anti-hypertensive activity were isolated and identified
from gelatin hydrolysates extracted from stingray skin (Okamejei
kenojei). The hydrolysates first were subjected to ultrafiltration through
a 1-kDa membrane, and peptides with molecular weights lower than
this cutoff were collected. Purification consisted of sequential steps of
isolation by fast protein liquid chromatography (FPLC) (AKTA,
Amersham Bioscience Co., Uppsala, Sweden) using a HiPrep 16/10
high flow ionic exchange column (16 × 100 mm, Amersham Biosci-
ences, Piscataway, NJ, USA) and a GE Healthcare Superdex™ Peptide
10/300 GL gel filtration column (10 × 300 mm). Purified peptides
were then identified by MALDI-TOF mass spectrometry. Two purified
peptides were found to be very anti-hypertensive and were identified
as Leu-Gly-Pro-Leu-Gly-His-Gln, with an estimated molecular weight
of 720 Da, and Met-Val-Gly-Ser-Ala-Pro-Gly-Val-Leu, with a molecular
weight of 829 Da (Ngo et al., 2015).
Liu et al. (2015) developed a UHPLC-Q-TOF MS/MS method to iden-
tify peptides with antioxidant activities derived from the protein hydro-
lysate of Mactra veneriformis. The hydrolysates were fractionated on a
Sephadex G-25 gel filtration column (2.0 cm × 100 cm; GE Chemicals,
Uppsala, Sweden) using distilled water as the eluting solvent at a flow
rate of 0.4 mL min−1, and separated five fractions. The two most active
fractions were then separated on the basis of their antioxidant activities
and subjected to an analysis using a Waters ACQUITY UHPLC system
with a C18 column (100 mm × 2.1 mm, 1.7 μm) and a linear gradient
of water–acetic acid (eluent A) and methanol (eluent B) at a flow rate
of 0.3 mL min−1. The UHPLC system was coupled to a Synapt Mass
Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF MS/MS) in
which the MS spectra were acquired in the m/z range of 50–2000.
This method allowed for the identification of 21 peptides, and the
most antioxidant peptides were identified as Thr-Asp-Tyr, Leu-Asp-
Tyr, Trp-Asp-Asp-Met-Glu-Lys, Trp-Gly-Asn-Val-Ser-Gly-Ser-Pro, Leu-
Tyr-Glu-Gly-Tyr and Met-Glu-Met-Lys.
It is important to note that each method showed a basic principle for
the separation of the bioactive peptides, as shown in Table 3. However,
in a complex mixture of peptides, common problems are the separation
of small and big peptides or peptides with different physicochemical
properties, which makes their subsequent identification difficult.
These problems can be solved by a combination of different separation
techniques before injection into the mass spectrometer. A practical
example is the separation of peptides containing hydrophobic amino
acids and peptides composed of only hydrophilic amino acids. In this
case, a combination of RP-HPLC with HILIC can be used for an efficient
separation of the peptides with hydrophobic and hydrophilic character-
istics, respectively (Panchaud, Affolter, & Kussmann, 2012).
In addition, an interesting approach was proposed by Le Maux et al.
(2015). These authors affirmed that liquid chromatography coupled to
mass spectrometry (LC–MS/MS) providing the necessary data for pep-
tide sequencing. However, although this strategy has been successfully
used for longer peptides, the identification of short peptides can be
more difficult, due to the presence of peptides with the same amino
acid composition but a different sequence. They showed that the
method HLIC-MS/MS and the parallel determination of the apparent hy-
drophilicity of each peptide for the development of a retention time pre-
diction model could be used as a valuable tool to improve the separation
of short peptides and the differentiation of peptides with homologous
sequences.
4. Biological properties of bioactive peptides
Bioactive peptides from dietary proteins have been extensively
studied over the last decade to determine their potential uses and
their effects on the major systems of the human body, such as the diges-
tive, cardiovascular, nervous and immune systems. Several bioactive
190
Table 3
The main characteristics of the different analytical methods for the purification and identification of bioactive peptides.
Method of
purification/identification
Reversed phase high pressure liquid
chromatography (RP-HPLC)
Affinity chromatography
Ion-exchange chromatography (IEC) Based on the ability of charged
Isoelectric focusing (IEF)
Size exclusion chromatography
(SEC)
Ultra high pressure liquid
chromatography (UHPLC)
Hydrophilic interaction liquid
chromatography (HILIC)
Electrospray ionization mass
spectrometry (ESI/MS)
Matrix-assisted laser
desorption/ionization-time-of-flight analytes when they are mixed with a
mass spectrometry
(MALDI-TOF/MS)
peptides have biological activities that are beneficial for human health,
including antimicrobial (Adje, Balti, Kouach, Guillochon, & Nedjar-
Arroume, 2011), anti-hypertensive (Alemán et al., 2011), antioxidant
R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
Mechanism
Based on the hydrophobicity of
proteins or peptides that can interact
differently to the reversed-phase
material of the chromatography
column.
Based on the affinity of bioactive
peptides to interact specifically and
reversibly with a complementary
molecule bound to a solid support
immobilized on a column.
bioactive peptides to interact with a
solid support bearing the opposite
charge.
Based on the separation of
protein/peptide solutions according to
their isoelectric points (pI). A focusing
cell containing a mixture of
proteins/peptides and a carrier
ampholyte is subjected to an electric
potential, causing the migration of the
proteins/peptides to a position in an
established pH gradient equivalent to
their respective pI.
Based on the fractionation of bioactive
peptides according to the retention
time of the molecules in the stationary
phases particles with a carefully
controlled pore size, in which the
molecules are separated from each
other according to their molecular size.
Based on separation of the molecules
using experimental columns packed
with very small particles of a
non-porous material, carrying out the
analyses at very high pressures.
Based on the polarity and
hydrophilicity of bioactive peptides
separated using polar chromatographic
surfaces (stationary phase) and a
highly organic mobile phase
(N70% solvent) also containing a small
percentage of aqueous solvent/buffer or
other polar solvent.
Based on the transformation of an
aqueous solution with uniform
electrical density to gas-phase ions, by
passing a high voltage through a thin
capillary. The gas-phase is transferred
into a mass analyzer and separated
according to the mass-to-charge
(m/z) ratio.
Based on co-crystallization of the
matrix solution on a target plate. The
co-crystal is subjected to the action of
pulsed laser, causing the accumulation
of high-density energy which results in
vaporization of the analyte and matrix
molecule. MALDI is usually connected
to TOF mass spectrometer which
measures the flight time of ions to the
ion detector, and provides the m/z ratio
mapping results.
Advantage Limitation Reference
Useful method for the Lack of retention of polar Everley and Croley (2008).
isolation of complex molecules. Slow intrapore diffusion Le Maux et al. (2015).
peptide mixtures. times. The presence of unresolved Yang, Boysen, Chowdhury,
structural microheterogeneity and Alam, and Hearn (2015).
conformational isomers. Secondary
interactions with the stationary
phase.
The flexibility of using a Tone must know the Hage et al. (2012).
large number of binding physicochemical properties of the Ortiz-Martinez et al. (2014).
agents, allows for the ligands, which limits its use for a
separation of different complex mixture of unknown
types of peptides. peptides.
Appropriate method for Low selectivity and requires Bouhallab, Henry, and
the separation of highly complementary steps for the Boschetti (1996).
cationic or anionic separation of the fractions. Ortiz-Martinez et al. (2014).
peptides.
The method allows one to Loss of highly hydrophobic Issaq, Conrads, Janini, and
fractionate a complex proteins in the sample Veenstra (2002).
mixture of peptides preparation and precipitation of Guijarro-Díez, García, Crego,
according to their pI. neutral proteins at their pI, which and Marina (2014)
can result in overlapping between
different fractions.
The elution conditions are Long columns are required for Mora et al. (2014).
considered mild, allowing complex peptide mixtures, which Fekete, Beck, Veuthey, and
the characterization of the can be obtained by joining Guillarme (2014).
protein with minimal multiple columns in a series. This
impact on the strategy is necessary to improve
conformational structure the separation resolution.
and the local environment.
Increased throughput, The heat dissipated from the use Everley and Croley (2008).
resolution and sensitivity of small particles at ultra-high Uliyanchenko,
in separation of complex pressures may increase Schoenmakers, and van der
protein mixtures. chromatographic band Wal (2011).
broadening and compromise Fekete and Guillarme (2014).
efficiency of the column.
The method shows great Compared to RP-HPLC, the Gray et al. (2013).
potential for the separation method shows limited flexibility Le Maux et al. (2015).
of short peptide sequences and applicability, problems with
(b5 amino acids) and sample solubility and the
improves the identification retention mechanisms are poorly
using mass spectrometry. understood.
Production of singly and The efficiency of identification is Mano and Goto (2003).
multiply charged ions, directly related to the Contreras et al. (2008).
allowing for an accurate chromatographic method used for Panchaud et al. (2012).
measurement of the the prior separation of the
molecular weight of the bioactive peptides before
peptides. injection into the mass
spectrometer. Therefore, a
combination of different
The method has no separation techniques is
theoretical upper limit to necessary for accurate
the m/z ratio, allowing for identification.
the analysis of complex
samples with a wide range
of molecular weights.
(Zhang et al., 2009) anticancer (Alemán et al., 2011), anti-adipogenic
(Tsou, Kao, et al., 2010), immunomodulatory (Huang, Chen, Chen,
Hong, & Chen, 2010) and anti-inflammatory effects (Ahn et al., 2015).
 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
Therefore, they can potentially be incorporated into functional foods,
nutraceuticals and medications, where this bioactivity can aid in
preventing and controlling diseases (Agyei & Danquah, 2012).
This review describes efforts to identify and characterize peptides
with antimicrobial, antioxidant, anti-adipogenic and anti-hypertensive
activity, and it discusses their use in the growth of lactic acid bacteria
and other probiotic bacteria.
4.1. Peptides with antimicrobial activity
Over the last few decades, a growing number of pathogenic microor-
ganisms have developed resistance to conventional antibiotics, causing
serious problems treating infections, especially in immunocompro-
mised individuals. In addition, the development of new antibiotics has
slowed over this same period. Two major causes underlie the increase
in antibiotic resistance in microorganisms: the indiscriminate use of an-
tibiotics for in small doses or with ineffective treatment times, and the
genetic mutation capacity of the microorganisms, which increases the
difficulty of developing drugs based on specific mechanisms of action
(Harrison, Abdel-Rahman, Miller, & Strong, 2014). Thus, using natural
sources of antimicrobial compounds has enormous potential because
they have characteristics such as low toxicity and high specificity. The
mechanisms of these natural antimicrobial compounds can be better
understood if we compare their modes of action against bacterial
(unicellular) and animal (multicellular) cells. Bacterial cells have a
layer rich in negatively charged phospholipids pointing toward the ex-
ternal environment, facilitating their interactions with peptides, most
of which are positively charged. In contrast, animal cells are mainly
composed of uncharged lipids in the outermost layer, and the negatively
charged regions are pointed toward the cell interior (cytoplasm)
(Matsuzaki, 1999).
Antimicrobial peptides are widely distributed in nature and are es-
sential to the immune system. They are the organism's first line of de-
fense against colonization by exogenous microorganisms, and they
play a fundamental role in regulating bacterial populations on the mu-
cosa and other epithelial surfaces (Bevins & Zasloff, 1990; Boman &
Hultmark, 1987; Zasloff, 2002). More than 800 antimicrobial peptides
have been described in plants and animals (Boman, 2003). Despite
great diversity in their primary structures, most antimicrobial peptides
are similar in that they are short amino acid chains composed primarily
of cationic and hydrophobic amino acids (Dashper, Liu, & Reynolds,
2007; Zasloff, 2002). The low molecular weights of the peptide fractions,
the resulting higher exposure of the amino acids and their charges, and
the formation of small channels in the lipid bilayer are related to their
antimicrobial activity. These features promote interactions between
the peptide and the membrane (Gobbetti, Minervini, & Rizzello, 2004;
Gómez-Guillén et al., 2010; Patrzykat & Douglas, 2005).
The exact mechanisms of action for many antimicrobial peptides
have not been well established. Due to the large number of known pep-
tides, it is likely that there are additional mechanisms of action yet to be
discovered (Dashper et al., 2007).
In addition to the peptides that are naturally present in the defense
systems of plants and animals, peptides with antimicrobial activity
have been identified in several protein hydrolysates.
Hydrolysates of casein from cow's milk obtained by enzymatic hy-
drolysis using chymosin were analyzed for their antimicrobial power.
Five different antibacterial peptides were isolated from the carboxylic
end of αs2-casein. Peptide fractions f (181–207), f (175–207) and f
(164–207) had a wide spectrum of activity and were able to inhibit sev-
eral Gram+ and Gram− bacteria; the minimum inhibitory concentra-
tion (MIC) of each fraction ranged from 21.0 to 168.0 mg mL−1, 10.7
to 171.2 mg mL−1 and 4.8 to 76.2 mg mL−1, respectively. The inhibitory
power of these peptides against Gram+ bacteria was as strong as the
known antimicrobial peptides nisin and lactoferricin B (Mccann et al.,
2005).
191
Peptides with antimicrobial activity were prepared from gelatin hy-
drolysate with Alcalase™ 2.4 L (Sigma-Aldrich, United States). Fractions
obtained from ultrafiltration through 1- and 10-kDa membranes were
used for antimicrobial tests against 18 bacteria. The most sensitive bacte-
ria in the presence of the tested fractions were Lactobacillus acidophilus,
Bifidobacterium lactis, Shewanella putrafaciens and Photobacterium
phosphoreum (Gómez-Guillén et al., 2010). Hydrolysates of bovine hemo-
globin treated with pepsin were purified by HPLC and tested for their
antimicrobial power against two Gram− (Escherichia coli, Salmonella
enteritidis) and three Gram+ strains (Kocuria luteus A270, Staphylococcus
aureus and Listeria innocua). The results showed that the purified peptide
fractions had a wide spectrum of action, affecting 4 of the 5 tested bacteria
(Kocuria luteus A270, L. innocua, E. coli and S. aureus), with a MIC between
35.2 and 187.1 μM (Adje et al., 2011).
Tellez, Corredig, Turner, Morales, and Griffiths (2011) demonstrated
the efficiency of a peptide fraction isolated from milk fermented with
L. helveticus against an experimental infection of S. enteritidis in rats.
The survival rate of the group fed with the peptide fraction (0.02 μg
per day) was higher than the group fed with half of the dose (0.01 μg
per day) and higher than the control group.
The antimicrobial powers of protein isolated from whey hydrolyzed
with various gastrointestinal enzymes were demonstrated by Théolier,
Hammami, Labelle, Fliss, and Jean (2013). These authors showed that
hydrolyzed proteins from trypsin and chymotrypsin digests did not
have antibacterial activity against Listeria ivanovii HPB28 and E. coli
MC4100, but they found that hydrolysates of pepsin had significant
activity. Hydrolysates were fractionated by reverse-phase high-
performance liquid chromatography, resulting in five fractions with
high antibacterial activity and MIC values between 20.0 and
35.0 μg mL− 1. A peptide fraction obtained from wastewater from
cooking anchovies (Engraulis japonicus) was digested by the Protamex
enzyme and had high antimicrobial activity against S. aureus. The iden-
tified fraction had the peptide sequence Gly-Leu-Ser-Arg-Leu-Phe-Thr-
Ala-Leu-Lys and an estimated molecular weight of 1.1 kDa (Tang, Zhang,
Wang, Qian, & Qi, 2015). Due to their hydrophobicity, bioactive peptides
containing sequences rich in the amino acids Gly and Leu were reported
as potent antimicrobial molecules. The presence of the Arg residue in
the peptide sequence also plays an important role in antimicrobial
activity, increasing interactions with bacterial cell walls, due its cationic
characteristic (Amadou, Le, Amza, Sun, & Shi, 2013; Sousa et al., 2009;
Tang et al., 2015).
4.2. Peptides with antioxidant activity
The creation of free radicals, such as superoxide (O− 2 ) and hydroxyl
(OH), is one of the inevitable consequences of respiration in aerobic
organisms. These radicals are very unstable and react quickly with
other groups or substances in the organism, causing cellular and tissue
damage (Zhang et al., 2009). An excessive amount of these radicals in
the organism has been linked to the development of several diseases,
such as atherosclerosis, arthritis, diabetes and cancer (Gu et al., 2015).
Because they are highly reactive species, free radicals can damage
proteins, mutate DNA, oxidize membrane phospholipids and modify
low-density lipoproteins (LDL) (Pihlanto, 2006). In food, oxidation
also directly affects quality, negatively affecting characteristics such as
taste, aroma and color. Thus, substances that inhibit oxidation reactions
are useful for maintaining food quality.
An antioxidant's ability to remove free radicals is determined by var-
ious factors, including chemical reactivity, the rate of removal of the
compound, the fate of the product of the antioxidant–radical reaction,
interactions with other antioxidants, concentration and mobility in the
environment and the compound's absorption, distribution, retention
and metabolism (Niki, 2010).
Antioxidants are thought to be important nutraceuticals with vari-
ous health benefits. They are defined as substances that significantly
slow or inhibit the oxidation of a substrate (Bougatef et al., 2009).
192 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
Currently, some artificially synthesized antioxidants, such as butylated
hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert-
butylhydroquinone (TBHQ), are used to prevent oxidative damage in
foods and biosystems. However, these products are being used less
due to their potential risks to human health, such as DNA damage and
toxicity (Chi, Wang, Wang, Zhang, & Deng, 2015; Wang et al., 2014).
Consequently, there is growing interest in finding safer antioxidants
from natural sources, such as peptides from hydrolyzed proteins (Chi,
Wang, et al., 2015; Senphan & Benjakul, 2014).
Some peptides with antioxidant activity occur naturally in food.
Glutathione (γ-Glu-Cys-Gly) and carnosine (β-alanyl-L-histidine) are
antioxidants that are naturally present in muscle tissues. They can
donate electrons, chelate metals and ions and inhibit lipid peroxidation
(Samaranayaka & Li-Chan, 2011). In addition to the antioxidants that
are naturally present, peptides from hydrolyzed dietary proteins have
been reported to have antioxidant capabilities similar to or better than
synthetic antioxidants, such as BHT, making them safe for food applica-
tions (Chi, Hu, Wang, Li, & Ding, 2015; Mora et al., 2014; Rao et al., 2012;
Yasufumi, Shigeki, Keiji, & Tomoyuki, 2001).
The mechanisms of action for the antioxidant activities of peptides
are not fully understood, but several studies have demonstrated the
ability of peptides to inhibit lipid peroxidation (Sakanaka et al., 2004),
remove free radicals (Gómez-Guillén et al., 2010), chelate metal ions
(Alemán et al., 2011) and eliminate reactive oxygen species (Zhuang
& Sun, 2011). Similar to other biological activities, the antioxidant prop-
erties of peptides are related to their composition, structure and hydro-
phobicity (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998).
The presence of the amino acids Tyr, Trp, Met, Lys and Cys was reported
to be an important factor in the antioxidant activities of the peptides, es-
pecially due to their ability to reduce Fe3+ to Fe2+ and to chelate Fe2+
and Cu2+ ions (Carrasco-Castilla et al., 2012; Huang et al., 2010; Wang
& De Mejia, 2005). Aromatic amino acids, such as Trp, Tyr, Phe have phe-
nolic, indole and imidazole groups, respectively, which can act as proton
donors to electron deficient radicals and efficiently scavenge them
(Duan et al., 2014; Sarmadi & Ismail, 2010). The basic amino acid His
has shown great potential in radical scavenging as a result of the chelat-
ing, lipid trapping and decomposition of the imidazole ring (Saidi,
Deratani, Belleville, & Amar, 2014). Not only the presence but also the
sequence of the peptide chain plays an important role in determining
antioxidant power (Rajapakse, Mendis, Jung, Je, & Kim, 2005).
The antioxidant abilities of peptides can be analyzed by several
in vitro methods, which test for various mechanisms of action and
thus measure distinct activities (Table 4).
In addition to in vitro analysis, antioxidant activity can be analyzed
in vivo using animal models. Antioxidant ability is measured in vivo
using several factors, as these substances must be absorbed, transported,
distributed and retained sufficiently in biological fluids, cells and tissues.
The bioavailability of these compounds, the effects of dose and the
duration of the treatments have been studied by analyzing human
and animal biological fluids and tissues after ingestion (Alam, Bristi, &
Rafiquzzaman, 2013; Niki, 2010). Table 5 shows some of these methods
and the principles underlying the analyses.
In vitro methods are more commonly used for analyzing antioxidant
activity in hydrolyzed proteins. Nazeer and Kulandai (2012) analyzed
the antioxidant properties of fish protein hydrolysates obtained from
enzymatic treatment with various proteases (papain, pepsin, trypsin
and chymotrypsin). Antioxidant activity was measured in terms of
DPPH radical reduction, iron reduction and the ability to chelate metals.
All of the hydrolysates had antioxidant activity, especially those made
with pepsin and trypsin. Li, Luo, Shen, and You (2012) showed that pro-
tein hydrolysates from carp prepared with Alcalase™ 2.4 L and papain
had antioxidant activity according to tests using ABTS, DPPH, Fe3+ re-
duction and Fe2+ chelation. Najafian and Babji (2015) studied the anti-
oxidant activity of myofibril protein from fish (Pangasius sutchi) that
was hydrolyzed using enzymatic preparations of the proteases papain,
Alcalase and Flavourzyme. The degree of hydrolysis varied depending
on the enzyme used, with values ranging from 36.53 to 89.17%. The hy-
drolysates obtained from 60 min of hydrolysis using papain had the
highest antioxidant activity by TBARS testing, Fe2+ chelation and Fe3+
reduction. The hydrolysates obtained with Alcalase had higher activity
against the DPPH radical, whereas those obtained with Flavourzyme
had greater activity against the ABTS radical.
Measuring antioxidant activity by in vivo methods is mainly based
on measurements of enzymatic activity. Decreases in the activities of
antioxidant enzymes, such as catalase (CAT), glutathione peroxidase
(GPx), superoxide dismutase (SOD) and glutathione-S-transferase
(GST), can dramatically affect the susceptibility of many tissues to oxi-
dative stress, and impairments of these enzymes are associated with
several diseases (Ktari et al., 2014). Liu et al. (in press) studied the anti-
oxidant properties of protein hydrolysates from corn gluten prepared
using the proteases Alcalase, Flavourzyme and Protamex. The antioxi-
dant activities of the hydrolysates were analyzed by in vivo methods.
The experiments were performed in 40 4–5 week old Kunming line
mice (25.0 ± 2.0 g). Mice were randomly divided into five experimental
groups, with eight animals and an equal number of males and females in
each group. The groups were then treated as follows: group I was used
as a control and received the base diet common to all groups; group II
was treated daily with vitamin E (83 mg/kg/day) for 10 days and groups
III, IV and V were treated with 300, 700 and 1000 mg/kg/day of hydro-
lysates, respectively, for 10 days. The results showed that the hydroly-
sates created with a combination of Alcalase and Protamex enzymes
produced peptides with the highest levels of antioxidant activity.
The distribution of the molecular weights in the hydrolysates showed
that the peptides were between 250 and 1200 Da. The ingestion of
300 mg peptides/kg resulted in increased superoxide dismutase and
glutathione-peroxidase activity and reduced the malonaldehyde levels
in the liver and blood of the mice compared to the control group, indi-
cating great antioxidant potential.
4.3. Peptides with anti-adipogenic activity
Obesity results from disequilibrium between energy intake and
energy expenditure, leading to pathological growth of adipocytes
(Aoyama, Fukui, Takamatsu, Hashimoto, & Yamamoto, 2000). The quan-
tity of adipose tissue can be controlled by inhibiting adipogenesis in
precursor or pre-adipocyte cells, such as pre-adipocyte 3T3-L1 cells,
which are the best-characterized models for studying adipogenesis.
Many transcription factors are involved in the differentiation of pre-
adipocyte cells into adipocytes, and their inhibition or regulation may
lead to decreased fat accumulation in the organism (Tsou, Kao, et al.,
2010). Glycerol-3-phosphate dehydrogenase (GPDH) is a key enzyme
for glucose metabolism and is linked to biosynthesis of phospholipids
and triglycerides (Harding, Pyeritz, Copeland, & White, 1975; Tsou,
Lin, Lu, Tsui, & Chiang, 2010). Suppressing GPDH activity may inhibit
differentiation and reduce lipid accumulation in 3T3-L1 cells. Thus, the
activity of this enzyme can be measured to evaluate anti-adipogenic
effects (Hirai, Yamanaka, Kawachi, Matsui, & Yano, 2005). Another
enzyme involved in adipogenesis is fatty-acid synthase (FAS), which
participates in the endogenous synthesis of saturated long-chain
fatty-acids from the precursors acetyl-CoA and malonyl-CoA (Maier,
Leibundgut, & Ban, 2008; Rahman et al., 2008). It has been reported
that certain fractions of hydrolyzed proteins are able to inhibit the activ-
ity of these enzymes, thus regulating the process of cell differentiation
and relative accumulation of lipids. According to Kim, Bae, Ahn, Lee,
and Lee (2007), these hydrolysates have great potential in obesity treat-
ments because they decrease fat accumulation in the organism.
Martinez-Villaluenga, Bringe, Berhow, and Mejia (2008) studied the
production of soy protein hydrolysates using a commercial preparation
of Alcalase proteases. The hydrolysates were analyzed for their effects
on relative lipid accumulation in 3T3-L1 pre-adipocyte cells, and they
showed a 29.0 to 46.0% decrease in lipid accumulation. The authors
also analyzed the anti-adipogenic activities of various soy protein
 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198 193

Table 4
Major methods for measuring antioxidant activities of peptides in vitro and their respective mechanisms.

Method Mechanism Reaction Measurement

DPPH DPPH capture DPPH radical (2,2-diphenyl-1-picryl-hydrazyl) reacts with Reduction in the Sharma and Bhat (2009)
hydrogen-donating antioxidants, changing the color from absorbance at 517 nm
violet to yellow.
ORAC Peroxyl radical capture The peroxyl radical, generated from the breakdown of Reduction in fluorescence Dávalos, Gómez-Cordovés, and
AAPH [2,2′-Azobis(2-amidinopropane) dihydrochloride] (excitation at 485 nm and Bartolomé (2004)
in the presence of atmospheric oxygen, reacts with a emission at 520 nm)
fluorescent indicator to produce a non-fluorescent
product. In the presence of antioxidants, the fluorescence
is maintained.
FRAP Iron reducing power In the presence of electron-donating antioxidants, the Increase in the absorbance Ou, Huang, Hampsch-Woodill,
Fe3+-TPTZ [2,4,6-Tripyridyl-S-Triazine] complex is at 593 nm Flanagan, and Deemer (2002)
reduced to Fe2+-TPTZ, changing the color from light blue
to dark blue.
ABTS ABTS capture The radical ABTS Reduction in the Gómez-Guillén et al. (2010)
(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) absorbance at 734 nm
is stabilized in the presence of hydrogen-donating free
radicals, changing the color from dark green to light green.
Ability to chelate Chelation of Cu2+ Complexation reaction of Cu2+ with pyrocatechol violet to Reduction in the Theodore et al. (2008)
transition metals generate a colored product. The presence of antioxidants absorbance at 620 nm
(Cu2+) decreases the formation of the Cu2+-pyrocatechol
complex, reducing the intensity of the color.
Ability to chelate Chelation of Fe2+ Complexation reaction of Fe2+ with ferrozine, generating Reduction in the Nazeer and Kulandai (2012)
transition metals a colored product. The presence of antioxidants decreases absorbance at 562 nm
(Fe2+) the formation if the Fe2+-ferrozine complex, reducing
color intensity.
TBARS Quantification of lipid Reaction of thiobarbituric acid with hydroperoxide Increase in the absorbance Raghavan and Kristinsson
peroxidation products decomposition products. Malonaldehyde is the main at 532 nm (2008)
compound quantified. Absorbance and antioxidant activity
are inversely proportional.

fractions and found that subunits from the β-conglicinin fraction had a
larger number of peptides responsible for the inhibition of lipid accu-
mulation in 3T3-L1 cells than the glycinin subunits.
Tsou, Kao, et al., 2010 studied the use of commercial preparations
of Flavourzyme™ proteases on protein hydrolysates isolated from soy
by analyzing the anti-adipogenic capacity of the hydrolysate fractions
obtained by ultrafiltration. The results revealed that the partial hydroly-
sis of proteins isolated from soy provided hydrolysates with strong
anti-adipogenic capacity, and fractions obtained by ultrafiltration
more efficiently inhibited GPDH activity. Specifically, the 1-kDa mem-
brane fraction was the most effective (59.0% inhibition). The anti-
adipogenic activity of the hydrolysates from soy protein after enzymatic

Table 5
Major methods for measuring antioxidant activities of peptides in vivo and their respective mechanisms.

Method Sample analyzed Animals Tissue/organ Mechanism of action and principle Reference
analyzed underlying measurement

Superoxide dismutase (SOD)
Catalase (CAT)
Level of reduced glutathione
(GSH)
Glutathione-S-transferase
(GST)
Glutathione peroxidase
(GPx)
Measurement of the level of
malonaldehydes
Peptide isolate from
hydrolyzed pig plasma
Peptide isolated from
hydrolyzed fish protein
Protein isolates from
seeds from Syrian rue
(Peganum harmala)
Peptide isolated from
hydrolyzed mussel
protein
Hydrolyzed corn gluten
Hydrolyzed fish protein
(Salaria basilisca)
Male adult Liver SOD is an enzyme that catalyzes the Liu, Kong, Li, Liu, and
Wistar rats dismutation of superoxide radicals into Xia (2011)
hydrogen and oxygen, thus playing an
important role in protecting cells against
reactive oxygen species
Albino male Erythrocyte CAT is an enzyme that converts hydrogen Nazeer, Kumar, and
adult Wistar rats lysate (blood) peroxide into water and oxygen, thus Ganesh (2012)
having one of the major mechanisms for
removing free radicals in the organism
Albino male rats Liver and blood GSH is an intracellular reducer that plays Soliman, Abu-El-Zahab,
plasma an important role for protecting cells and Alswiai (2013)
from free radicals, peroxides and other
toxic compounds
Male adult rats Liver GST is an enzymatic complex in the Kim et al. (2013)
cytosol that catalyzes the binding of
reactive electrophilic molecules with
glutathione, facilitating the metabolism
and excretion of toxins and consequently
reducing cell damage and DNA damage
Male and female Liver and blood GPx is an enzyme that catalyzes the Liu et al. (in press)
Kunming mice plasma reaction between hydroperoxide and
reduced glutathione leading to the
formation of glutathione disulfite and the
product of hydroperoxide reduction
Male adult Liver and blood Malanodialdehyde is an intermediate Ktari et al. (2014)
Wistar rats plasma product for lipid peroxidation and thus
can be used as an indicator for the
presence of free radicals
194 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
treatment with Neutrase and the effect of fractionation by ultrafiltration
on activity were studied by Tsou, Lin, et al., 2010. Similar to the previous
study, the results showed that low molecular weight peptides (between
1300 and 2200 Da) most effectively inhibited GPDH activity.
Mejia, Martinez-Villaluenga, Roman, and Bringe (2010) analyzed the
effects of soy protein hydrolysates enriched with β-conglycinin (a
protein naturally found in soy) on FAS activity and adipogenesis in
human adipocytes in vitro. The results showed that genotypic alterations
in the subunits of the soy protein (enriched with β-conglycinin) pro-
duced peptide profiles that inhibited FAS and decreased lipid accumula-
tion in vitro. The quantity of soy protein hydrolysates necessary to
inhibit 50% of FAS activity (IC50) ranged from 50–175 μM. A peptide
with anti-adipogenic abilities was isolated by ultrafiltration, gel filtra-
tion and reversed-phase HPLC from soy protein hydrolysates, and its
anti-adipogenic ability was confirmed by the inhibition of 3T3-L1 pre-
adipocyte cell differentiation. The inhibitor was identified as a tripeptide
(Ile-Gln-Asn) with an IC50 value of 0.014 mg mL−1 (Kim et al., 2007).
4.4. Peptides with anti-hypertensive activity
Arterial hypertension affects approximately 25% of the adult popula-
tion worldwide and is predicted to reach 29% of the population by 2025,
representing a total of 1.56 billion people (Ngo et al., 2015). Although it
is a controllable disease, hypertension is associated with several cardio-
vascular diseases, such as atherosclerosis, myocardial infarction and
stroke (Sheih, Fang, & Wu, 2009). Angiotensin converting enzyme
(ACE) plays an important role in the regulation of arterial pressure be-
cause it catalyzes the conversion of angiotensin-I (the inactive form)
to angiotensin-II (a vasoconstrictor) and inactivates bradykinin (a vaso-
dilator). Consequently, synthetic ACE inhibitors, such as captopril and
enalapril, are often used to treat hypertension and other related heart
diseases. However, synthetic inhibitors can cause various side effects,
such as cough, altered taste, rash and angioedema (Alemán et al., 2011).
It is well known that dietary proteins have primary sequences of
peptides able to modulate specific physiological functions (Hong et al.,
2008). Many types of bioactive peptides that inhibit ACE were isolated
from protein hydrolysates and fermented products. The dipeptide
Ala-Pro and the tripeptide Phe-Ala-Pro, for example, have structures
analogous to the drugs captopril and enalapril, respectively (Fig. 2).
The presence of aromatic and aliphatic amino acids, such as Pro, Phe
or Tyr at the C-terminal and of Val and Ile at the N-terminal position of
the bioactive peptides has been associated with ACE-inhibitory activity
(Kapel, Rahhou, Lecouturier, Guillochon, & Dhulster, 2006; Wijesekara,
Qian, Ryu, Ngo, & Kim, 2011). Cian, Vioque, and Drago (2015) studied
the ACE-inhibitory activity of peptides from wheat gluten hydrolysate
Captopril
H CH
3
HS C C C N
COOH
H H O H
Ala-Pro
CH 3
H N C C N H N
2 COOH
H O
Fig. 2. Structures of ACE inhibitor drugs and their analogous peptides (Matsui & Matsumoto, 2006).
and obtained an interesting result in which no relationship was found
between hydrophobicity/size and the IC50 values of the ACE-inhibitory
activity, indicating that it was the sequence of peptides from the
wheat gluten hydrolysate that was mainly responsible for the ACE-
inhibitory activity, although several studies have shown that the size
and hydrophobic character of the peptides exert a strong influence on
this bioactivity (Aluko et al., in press; Li, Le, Shi, & Shrestha, 2004;
Wijesekara et al., 2011). According to Li et al. (2004), the hydrophilic–
hydrophobic partitioning in the peptide sequence was a critical factor
in ACE-inhibitory activity, because hydrophilic amino acid residues
could disrupt the access of the peptide to the active site of ACE. This
concept was reinforced by Yuan, Wu, and Aluko (2007) and Aluko
et al. (in press), who showed an important role of branched-chain
amino acids such as Val, Leu and Ile, in enhancing the hydrophobic char-
acter of peptides, which is an important structural feature that enables
strong peptide interactions with non-polar amino acid residues within
the enzyme active site.
Peptide fractions from soy protein hydrolyzed with pepsin were
separated by ion exchange chromatography, gel filtration and HPLC,
and they were found to have ACE-inhibiting activity. Four amino
acid sequences were identified as potential ACE inhibitors: Ile-Ala
(IC50 153 μM), Tyr-Leu-Ala-Gly-Asn-Gln (IC50 14 μM), Phe-Phe-Leu
(IC50 37 μM) and Ile-Tir-Leu-Leu (IC50 42 μM). When administered at
a dose of 2.0 g/kg body weight in hypertensive rats over 15 weeks, the
peptide fractions considerably reduced arterial pressure (Chen, Okada,
Muramoto, Suetsuna, & Yang, 2003).
Peptides with anti-hypertensive activity were isolated from protein
hydrolysates of milk after fermentation with lactic bacteria and enzy-
matic hydrolysis with the commercial protease Prozyme 6. The peptides
were identified as Gly-Thr-Trp and Gly-Val-Trp and had ACE inhibitor
activity with IC50 values of 464.4 and 240.0 μM, respectively (Chen
et al., 2007). Hernández-Ledesma, Quirós, Amigo, and Recio (2007)
hydrolyzed human milk proteins with pepsin and pancreatin to study
the anti-hypertensive properties of the peptides and showed that
hydrolysates derived from β-casein were strong inhibitors of ACE,
with an IC50 of 21 μM.
Chaves-López et al. (2014) studied the effects of combined microbial
cultures previously identified as proteolytic and their ability to release
ACE-inhibiting peptides during the production of fermented milk. The
yeast strains Torulaspora delbruekii KL66A, Galactomyces geotrichum
KL20B, Pichia kudriavzevii KL84A and Kluyveromyces marxianus KL26A
and the lactic acid bacteria Lactobacillus plantarum LAT03, Lb. plantarum
KLAT01 and Enterococcus faecalis KE06 (non-virulent) were used. The
results indicated that the combination of different cultures can signifi-
cantly increase the levels of anti-hypertensive peptides. The most
Enalapril
CH 2 CO2 CH 3
H C C N C C N
2 COOH
H H O
Phe-Ala-Pro
CH 2 CH 3
C C N C C N
2 COOH
H O H H O
 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
effective combination for producing these peptides was a mixture of
P. kudriavzevii KL84A, Lb. plantarum LAT3 and E. faecalis KL06, which
had an IC50 for ACE inhibition of 30.63 μg mL−1.
The anti-hypertensive effect of a bovine casein peptide previously
identified as Met-Lys-Pro was analyzed in vitro and in vivo. The in vitro
analyses were based on the ability to inhibit ACE, and the in vivo studies
were conducted using groups of naturally hypertensive rats. Animals
were treated with peptide solutions (10 mg/kg) two times per day
and a single daily dose of enalapril (10 mg/kg) for 28 consecutive
days. The in vitro assay showed that the peptide had ACE inhibition ac-
tivity with an IC50 of 0.43 μM. For the in vivo assays, the arterial pressure
of the animals was 171.7, 163.3 and 139.7 for the control, peptide solu-
tion and enalapril groups, respectively, indicating significant differences
and reductions in arterial pressure between the control group and the
peptide (p b 0.05) and enalapril (p b 0.01) treatment groups (Yamada
et al., 2015).
4.5. Induction of lactic acid bacteria and probiotic growth
Lactic acid bacteria are not able to synthesize all of the amino acids
necessary for their growth. Thus, these microorganisms must hydrolyze
proteins while fermenting dairy products to obtain free amino acids and
small peptides as nutritional sources. The proteolytic system of lactic
acid bacteria has three basic mechanisms: 1) one or more proteolytic
enzymes located in the cell wall, also known as cell-envelope proteases,
which can hydrolyze milk proteins into peptides containing 4 to 30
amino acid residues; 2) a peptide transport system, composed of bind-
ing proteins, two permeases to create the transport channels and two
ATPases to provide energy to the system, and 3) a group of intracellular
peptidases that catalyzes the hydrolysis of the peptides transported into
the cell interior into amino acids (Hafeez et al., 2014).
Despite the existence of this proteolytic apparatus, several studies
have shown that supplementing milk with sources of hydrolyzed pro-
teins positively affects the growth of lactic acid and probiotic bacteria.
These studies are mainly driven by two main characteristics of this
group of microorganisms: 1) lactic acid and probiotic bacteria are re-
quired nutritionally, especially for amino acid uptake and 2) the free
amino acids and peptides in the milk are not sufficient to ensure ideal
bacterial growth on this substrate, possibly impeding fermentation by
these microorganisms (Zhang et al., 2011). Thus, various protein
sources have been analyzed as supplements to be used in the culture
media to study the induction of lactic acid and probiotic bacteria growth
(Mccomas & Gilliland, 2003; Prasanna, Grandison, & Charalampopoulos,
2012; Zhang et al., 2011).
Mccomas and Gilliland (2003) studied the growth of lactic acid and
probiotic bacteria in cow's milk samples supplemented with hydrolyzed
whey protein. The results showed that the hydrolysates did not affect
L. delbrueckii ssp. bulgaricus and Streptococcus thermophiles growth but
significant increases in Bifidobacterium longum and L. acidophilus growth
were observed.
Prasanna et al. (2012) studied the supplementation of skim milk
with various hydrolyzed proteins and their effects of probiotic bacteria
growth and found that the type or fraction of the protein used directly
affected microorganism growth. The final concentration of B. longum
subsp. infantis CCUG 52486 and B. infantis NCIMB 702.205 cells was
higher when these strains were cultured in milk supplemented with hy-
drolyzed casein than with the other hydrolyzed protein fractions from
lacto-albumin, concentrated whey protein or purified whey protein.
5. Conclusion
Biologically active peptides can be defined as specific amino-acid se-
quences that promote beneficial physiological effects. Technologies for
obtaining bioactive peptides include protein hydrolysis by exogenous
microbial, plant or animal enzymes and fermentation using fungi or
bacteria. The wide biochemical diversity of the proteins and the
195
existence of protein sources with various amino-acid compositions
make it possible to obtain peptides with distinct biological functions
and/or multiple functions. Important techniques for purification and
identification include chromatography and mass spectrometry. Studies
on production processes, studies on multifunctionality and analyses
using in vitro and in vivo methods all contribute to our understanding
of bioactive peptides as powerful natural biological agents that can be
used together with or in place of synthetic substances in food preserva-
tion, functional foods and pharmaceutical production.
References
Adje, E.Y., Balti, R., Kouach, M., Guillochon, D., & Nedjar-Arroume, N. (2011). α 67–106 of
bovine hemoglobin: A new family of antimicrobial angiotensin I-converting enzyme
inhibitory peptides. European Food Research and Technology, 232, 637–646.
Agyei, D., & Danquah, M.K. (2012). Rethinking food-derived bioactive peptides for antimi-
crobial and immunomodulatory activities. Trends in Food Science & Technology, 23,
62–69.
Ahn, C. -B., Cho, Y. -S., & Je, J. -Y. (2015). Purification and anti-inflammatory action of
tripeptide from salmon pectoral fin byproduct protein hydrolysate. Food Chemistry,
168, 151–156.
Ahn, C. -B., Je, J. -Y., & Cho, Y. -S. (2012). Antioxidant and anti-inflammatory peptide frac-
tion from salmon byproduct protein hydrolysates by peptic hydrolysis. Food Research
International, 49, 92–98.
Akalin, A.S. (2014). Dairy-derived antimicrobial peptides: Action mechanisms, pharma-
ceutical uses and production proposals. Trends in Food Science & Technology, 36,
79–95.
Alam, M.N., Bristi, N.J., & Rafiquzzaman, M. (2013). Review on in vivo and in vitro
methods evaluation of antioxidant activity. Saudi Pharmaceutical Journal, 21,
143–152.
Alemán, A., Pérez-Santín, E., Bordenave-Juchereau, S., Arnaudin, I., Gómez-Guillén, M.C., &
Montero, P. (2011). Squid gelatin hydrolysates with antihypertensive, anticancer and
antioxidant activity. Food Research International, 44, 1044–1051.
Aluko, R.E., Girgih, A.T., He, R., Malomo, S., Li, H., Offengenden, M., et al. (2015). Structural
and functional characterization of yellow field pea seed (Pisum sativum L.) protein-
derived antihypertensive peptides. Food Research International. http://dx.doi.org/10.
1016/j.foodres.2015.03.029 (in press).
Amadou, I., Le, G. -W., Amza, T., Sun, J., & Shi, Y. -H. (2013). Purification and characteriza-
tion of foxtail millet-derived peptides with antioxidant and antimicrobial activities.
Food Research International, 51, 422–428.
Aoyama, T., Fukui, K., Takamatsu, K., Hashimoto, Y., & Yamamoto, T. (2000). Soy protein
isolate and its hydrolysate reduce body fat of dietary obese rats and genetically
obese mice (yellow KK). Nutrition, 16, 349–354.
Balti, R., Bougatef, A., Sila, A., Guillochon, D., Dhulster, P., & Nedjar-Arroume, N. (2015).
Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttle-
fish (Sepia officinalis) muscle protein hydrolysates and antihypertensive effect of
the potent active peptide in spontaneously hypertensive rats. Food Chemistry, 170,
519–525.
Bevins, C.L., & Zasloff, M. (1990). Peptides from frog skin. Annual Review of Biochemistry,
59, 395–414.
Biziulevicius, G.A., Kislukhina, O.V., Kazlauskaite, J., & Zukaite, V. (2006). Food-protein en-
zymatic hydrolysates possess both antimicrobial and immunostimulatory activities:
A ‘cause and effect’ theory of bifunctionality. FEMS Immunology and Medical
Microbiology, 46, 131–138.
Boman, H.G. (2003). Antibacterial peptides: Basic facts and emerging concepts. Journal of
Internal Medicine, 254, 197–215.
Boman, H.G., & Hultmark, D. (1987). Cell-free immunity in insect. Annual Review of
Microbiology, 41, 103–126.
Bougatef, A., Hajji, M., Balti, R., Lassoued, I., Triki-Ellouz, Y., & Nasril, M. (2009). Antioxi-
dant and free radical-scavenging activities of smooth hound (Mustelus mustelus)
muscle protein hydrolysates obtained by gastrointestinal proteases. Food Chemistry,
114, 1198–1205.
Bouhallab, S., Henry, G., & Boschetti, E. (1996). Separation of small cationic bioactive pep-
tides by strong ion-exchange chromatography. Journal of Chromatography A, 724(1),
137–145.
Carrasco-Castilla, J., Hernández-Álvarez, A.J., Jiménez-Martínez, C., Jacinto-Hernández, C.,
Alaiz, M., Girón-Calle, J., et al. (2012). Antioxidant and metal chelating activities of
Phaseolus vulgaris L. var. Jamapa protein isolates, phaseolin and lectin hydrolysates.
Food Chemistry, 131, 1157–1164.
Chang, O.K., Seol, K.H., Jeong, S.G., Oh, M.H., Park, B.Y., Perrin, C., et al. (2013). Casein hy-
drolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides
derived therefrom. Journal of Dairy Science, 96(9), 5544–5555.
Chaves-López, C., Serio, A., Paparella, A., Martuscelli, M., Corsetti, A., Tofalo, R., et al.
(2014). Impact of microbial cultures on proteolysis and release of bioactive peptides
in fermented milk. Food Microbiology, 42, 117–121.
Chen, H.M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara, K. (1998). Antioxidative
properties of histidine-containing peptides designed from peptide fragments found
in the digests of a soybean protein. Journal of Agricultural and Food Chemistry, 46,
49–53.
Chen, J., Okada, T., Muramoto, K., Suetsuna, K., & Yang, S. (2003). Identification of angio-
tensin I-converting enzyme inhibitory peptides derived from the peptic digest of
soybean protein. Journal of Food Biochemistry, 26, 543–554.
196 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
Chen, G., Tsai, J., & Pan, B. (2007). Purification of angiotensin I-converting enzyme inhib-
itory peptides and antihypertensive effect of milk produced by protease-facilitated
lactic fermentation. International Dairy Journal, 17, 641–647.
Chi, C. -F., Hu, F. -Y., Wang, B., Li, T., & Ding, G. -F. (2015a). Antioxidant and anticancer
peptides from the protein hydrolysate of blood clam (Tegillarca granosa) muscle.
Journal of Functional Foods, 15, 301–313.
Chi, C. -F., Wang, B., Wang, Y. -M., Zhang, B., & Deng, S. -G. (2015b). Isolation and charac-
terization of three antioxidant peptides from protein hydrolysate of bluefin
leatherjacket (Navodon septentrionalis) heads. Journal of Functional Foods, 12, 1–10.
Chiaradia, M.C., Collins, C.H., & Jardim, I.C.S.F. (2008). O estado da arte da cromatografia
associada à espectrometria de massas acoplada à espectrometria de massas na
análise de compostos tóxicos em alimentos. Quimica Nova, 31(3), 623–636.
Choi, J., Sabikhi, L., Hassan, A., & Anand, S. (2012). Bioactive peptides in dairy products.
International Journal of Dairy Technology, 65(1), 1–12.
Cian, R. E., Vioque, J., & Drago, S. R. (2015). Structure–mechanism relationship of antiox-
idant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by
pH. Food Research International, 69, 216–223.
Contreras, M.M., López-Expósito, I., Hernández-Ledesma, B., Ramos, M., & Recio, I. (2008).
Application of mass spectrometry to the characterization and quantification of food-
derived bioactive peptides. Journal of AOAC International, 91(4), 981–994.
Dashper, S.G., Liu, S.W., & Reynolds, E.C. (2007). Antimicrobial peptides and their
potential as oral therapeutic agents. International Journal of Peptide Research and
Therapeutics, 13(4), 505–516.
Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending applicability of the
oxygen radical absorbance capacity (ORAC-fluorescein) assay. Journal of Agricultural
and Food Chemistry, 52, 48–54.
de Castro, R.J.S., Bagagli, M.P., & Sato, H.H. (2015). Improving the functional properties of
milk proteins: Focus on the specificities of proteolytic enzymes. Current Opinion in
Food Science, 1, 64–69.
Diplock, A.T., Aggett, P.J., Ashwell, M., Bornet, F., Fern, E.B., & Roberfroid, M.B. (1999). Sci-
entific concepts of functional foods in Europe: Consensus document. British Journal of
Nutrition, 81, 1–27.
Duan, X., Ocen, D., Wu, F., Li, M., Yang, N., Xu, J., et al. (2014). Purification and character-
ization of a natural antioxidant peptide from fertilized eggs. Food Research
International, 56, 18–24.
Everley, R.A., & Croley, T.R. (2008). Ultra-performance liquid chromatography/mass spec-
trometry of intact proteins. Journal of Chromatography A, 1192, 239–247.
Fekete, S., Beck, A., Veuthey, J. -L., & Guillarme, D. (2014). Theory and practice of size
exclusion chromatography for the analysis of protein aggregates. Journal of
Pharmaceutical and Biomedical Analysis, 101, 161–173.
Fekete, S., & Guillarme, D. (2014). Ultra-high-performance liquid chromatography for the
characterization of therapeutic proteins. Trends in Analytical Chemistry, 63, 76–84.
Giri, A., Osako, K., Okamoto, A., Okazaki, E., & Ohshima, T. (2011). Antioxidative properties
of aqueous and aroma extracts of squid miso prepared with Aspergillus oryzae-
inoculated koji. Food Research International, 44(1), 317–325.
Gobbetti, M., Minervini, F., & Rizzello, C.G. (2004). Angiotensin I-converting enzyme-
inhibitory and antimicrobial bioactive peptides. International Journal of Dairy
Technology, 57, 173–188.
Gómez-Guillén, M.C., López-Caballero, M.E., López De Lacey, A., Alemán, A., Giménez, B., &
Montero, P. (2010). Antioxidant and antimicrobial peptide fractions from squid and
tuna skin gelatin. In E.L. Bihan, & N. Koueta (Eds.), Sea by-products as a real material:
New ways of application (pp. 89–115). Kerala: Transworld Research Network Signpost.
Gray, N., Heaton, J., Musenga, A., Cowan, D.A., Plumb, R.S., & Smith, N.W. (2013). Compar-
ison of reversed-phase and hydrophilic interaction liquid chromatography for the
quantification of ephedrines using medium-resolution accurate mass spectrometry.
Journal of Chromatography A, 1289, 37–46.
Gu, M., Chen, H. -P., Zhao, M. -M., Wang, X., Yang, B., Ren, J. -Y., et al. (2015). Identification
of antioxidant peptides released from defatted walnut (Juglans Sigillata Dode) meal
proteins with pancreatin. LWT — Food Science and Technology, 60, 213–220.
Guijarro-Díez, M., García, M.C., Crego, A.L., & Marina, M.L. (2014). Off-line two dimension-
al isoelectrofocusing-liquidchromatography/mass spectrometry (time of flight) for
the determination of the bioactive peptide lunasin. Journal of Chromatography A,
1371, 117–124.
Hafeez, Z., Cakir-Kiefer, C., Roux, E., Perrin, C., Miclo, L., & Dary-Mourot, A. (2014). Strate-
gies of producing bioactive peptides from milk proteins to functionalize fermented
milk products. Food Research International, 63, 71–80.
Hage, D.S., Anguizola, J.A., Bi, C., Li, R., Matsuda, R., Papastavros, E., et al. (2012). Pharma-
ceutical and biomedical applications of affinity chromatography: Recent trends and
developments. Journal of Pharmaceutical and Biomedical Analysis, 69, 93–105.
Harding, J.W., Pyeritz, E.A., Copeland, E.S., & White, H.B. (1975). Role of glycerol
3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme
activities in chicken liver. Biochemistry Journal, 146, 223–229.
Harrison, P.L., Abdel-Rahman, M.A., Miller, K., & Strong, P.N. (2014). Antimicrobial
peptides from scorpion venoms. Toxicon, 88, 115–137.
Hati, S., Vij, S., Mandal, S., Malik, R.K., Kumari, V., & Khetra, Y. (2014). α-Galactosidase
activity and oligosaccharides utilization by lactobacilli during fermentation of soy
milk. Journal of Food Processing and Preservation, 38, 1065–1071.
He, R., Ju, X., Yuan, J., Wang, L., Girgih, A.T., & Aluko, R.E. (2012). Antioxidant activities of
rapeseed peptides produced by solid state fermentation. Food Research International,
49, 432–438.
Hernández-Ledesma, B., Contreras, M.M., & Recio, I. (2011). Antihypertensive peptides:
Production, bioavailability and incorporation into foods. Advances in Colloid and
Interface Science, 165, 23–35.
Hernández-Ledesma, B., García-Nebot, M.J., Fernández-Tomé, S., Amigo, L., & Recio, I.
(2014). Dairy protein hydrolysates: Peptides for health benefits. International Dairy
Journal, 38, 82–100.
Hernández-Ledesma, B., Quirós, A., Amigo, L., & Recio, I. (2007). Identification of bioactive
peptides after digestion of human milk and infant formula with pepsin and pancrea-
tin. International Dairy Journal, 17, 42–49.
Hirai, S., Yamanaka, M., Kawachi, H., Matsui, T., & Yano, H. (2005). Activin A inhibits dif-
ferentiation of 3T3-L1 preadipocyte. Molecular and Cellular Endocrinology, 232, 21–26.
Hong, F., Ming, L., Yi, S., Zhanxia, L., Yongquan, W., & Chi, L. (2008). The antihypertensive
effect of peptides: A novel alternative to drugs? Peptides, 29, 1062–1071.
Huang, S., Chen, K. -N., Chen, Y. -P., Hong, W. -S., & Chen, M. -J. (2010). Immunomodula-
tory properties of the milk whey products obtained by enzymatic and microbial
hydrolysis. International Journal of Food Science and Technology, 45, 1061–1067.
Inoue, K., Gotou, T., Kitajima, Hirokuni, Mizuno, S., Nakazawa, T., & Yamamoto, N. (2009).
Release of antihypertensive peptides in miso paste during its fermentation, by the ad-
dition of casein. Journal of Bioscience and Bioengineering, 108(2), 111–115.
Issaq, H.J., Conrads, T.P., Janini, G.M., & Veenstra, T.D. (2002). Methods for fractionation,
separation and profiling of proteins and peptides. Electrophoresis, 23, 3048–3061.
Kapel, R., Rahhou, E., Lecouturier, D., Guillochon, D., & Dhulster, P. (2006). Characteriza-
tion of an antihypertensive peptide from an alfalfa white protein hydrolysate
produced by continuous enzymatic membrane reactor. Process Biochemistry, 41,
1961–1966.
Kim, H.J., Bae, I.Y., Ahn, C., Lee, S., & Lee, H.G. (2007). Purification and identification of ad-
ipogenesis inhibitory peptide from black soybean protein hydrolysate. Peptides, 28,
2098–2103.
Kim, E. -K., Oh, H. -J., Kim, Y. -S., Hwang, J. -W., Ahn, C. -B., Lee, J.S., et al. (2013). Purifica-
tion of a novel peptide derived from Mytilus coruscus and in vitro/in vivo evaluation of
its bioactive properties. Fish & Shellfish Immunology, 34, 1078–1084.
Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications.
Journal of Functional Foods, 1, 177–187.
Korhonen, H., Pihlanto-Leppala, A., Rantamaki, P., & Tupasela, T. (1998). Impact of
processing on bioactive proteins and peptides. Trends in Food Science & Technology,
9, 307–319.
Ktari, N., Nasri, R., Mnafgui, K., Hamden, K., Belguith, O., Boudaouara, T., et al. (2014).
Antioxidative and ACE inhibitory activities of protein hydrolysates from zebra
blenny (Salaria basilisca) in alloxan-induced diabetic rats. Process Biochemistry,
49, 890–897.
Lafarga, T., & Hayes, M. (2014). Bioactive peptides from meat muscle and by-products:
Generation, functionality and application as functional ingredients. Meat Science, 98,
227–239.
Le Maux, S., Nongonierma, A.B., & FitzGerald, R.J. (2015). Improved short peptide identi-
fication using HILIC–MS/MS: Retention time prediction model based on the impact of
amino acid position in the peptide sequence. Food Chemistry, 173, 847–854.
Li, G.H., Le, G.W., Shi, Y.H., & Shrestha, S. (2004). Angiotensin I-converting enzyme inhib-
itory peptides derived from food proteins and their physiological and pharmacologi-
cal effects. Nutrition Research, 24, 469–486.
Li, X., Luo, Y., Shen, H., & You, J. (2012). Antioxidant activities and functional properties of
grass carp (Ctenopharyngodon idellus) protein hydrolysates. Journal of the Science of
Food and Agriculture, 92, 292–298.
Li-Chan, E.C. (2015). Bioactive peptides and protein hydrolysates: Research trends and
challenges for application as nutraceuticals and functional food ingredients. Current
Opinion in Food Science, 1, 28–37.
Limón, R.I., Peñas, E., Torino, M.I., Martínez-Villaluenga, C., Dueñas, M., & Frias, J. (2015).
Fermentation enhances the content of bioactive compounds in kidney bean extracts.
Food Chemistry, 172, 343–352.
Liu, Q., Kong, B., Li, G., Liu, N., & Xia, X. (2011). Hepatoprotective and antioxidant effects of
porcine plasma protein hydrolysates on carbon tetrachloride-induced liver damage
in rats. Food and Chemical Toxicology, 49, 1316–1321.
Liu, R., Zheng, W., Li, J., Wang, L., Wu, H., Wang, X., et al. (2015). Rapid identification of
bioactive peptides with antioxidant activity from the enzymatic hydrolysate of
Mactra veneriformis by UHPLC–Q-TOF mass spectrometry. Food Chemistry, 167,
484–489.
Liu, X., Zheng, X., Song, Z., Liu, X., Kopparapu, N.K., Wang, X., et al. (2014). Preparation of
enzymatic pretreated corn gluten meal hydrolysate and in vivo evaluation of its an-
tioxidant activity. Journal of Functional Foods. http://dx.doi.org/10.1016/j.jff.2014.10.
013 (in press).
Luna-Vital, D.A., Mojica, L., de Mejía, E.G., Mendoza, S., & Loarca-Piña, G. (2014). Biological
potential of protein hydrolysates and peptides from common bean (Phaseolus
vulgaris L.): A review. Food Research International. http://dx.doi.org/10.1016/j.
foodres.2014.11.024 (in press).
Maier, T., Leibundgut, M., & Ban, N. (2008). The crystal structure of mammalian fatty acid
synthase. Science, 321, 1315–1322.
Mano, N., & Goto, J. (2003). Biomedical and biological mass spectrometry. Analytical
Sciences, 19, 3–14.
Martinez-Villaluenga, C., Bringe, N.A., Berhow, M.A., & Mejia, E.G. (2008). β-Conglycinin
embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes
in vitro. Journal of Agricultural and Food Chemistry, 56, 10533–10543.
Matsui, T., & Matsumoto, K. (2006). Antihypertensive peptides from natural resources. In
M.T.H. Khan, & A. Ather (Eds.), Lead molecules from natural products (pp. 255–271).
Amsterdam: Elsevier.
Matsuzaki, K. (1999). Why and how are peptide-lipid interactions utilized for self-
defense? Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta,
1462, 1–10.
Mccann, K.B., Shiell, B.J., Michalski, W.P., Lee, A., Wan, J., Roginski, H., et al. (2005). Isola-
tion and characterisation of antibacterial peptides derived from the f (164–207)
region of bovine αS2-casein. International Dairy Journal, 15, 133–143.
Mccomas, K.A., Jr., & Gilliland, S.E. (2003). Growth of probiotic and traditional yogurt cul-
tures in milk supplemented with whey protein hydrolysate. Journal of Food Science,
68, 2090–2095.
 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
Mejia, E.G., Martinez-Villaluenga, C., Roman, M., & Bringe, N.A. (2010). Fatty acid synthase
and in vitro adipogenic response of human adipocytes inhibited by α and α′ subunits
of soybean β-conglycinin hydrolysates. Food Chemistry, 119, 1571–1577.
Mellander, O. (1950). The physiological importance of the casein phosphopeptides calci-
um salts. II. Per oral calcium dosage of infants. Acta Society Medicine Uppsala, 55,
247–255.
Miranda, M.T.M., & Liria, C.W. (2008). Técnicas de análise e caracterização de peptídeos e
proteínas. In A. PessoaJr., & B.V. Kilikian (Eds.), Purificação de Produtos Biotecnológicos
(pp. 411–427). Barueri: Manole.
Mora, L., Escudero, E., Fraser, P.D., Aristoy, M. -C., & Toldrá, F. (2014). Proteomic identifi-
cation of antioxidant peptides from 400 to 2500 Da generated in Spanish dry-cured
ham contained in a size-exclusion chromatography fraction. Food Research
International, 56, 68–76.
Mora, L., Reig, M., & Toldrá, F. (2014). Bioactive peptides generated from meat industry by
products. Food Research International. http://dx.doi.org/10.1016/j.foodres.2014.09.
014 (in press).
Najafian, L., & Babji, A.S. (2015). Isolation, purification and identification of three novel
antioxidative peptides from patin (Pangasius sutchi) myofibrillar protein hydroly-
sates. LWT — Food Science and Technology, 60, 452–461.
Nakahara, T., Sano, A., Yamaguchi, H., Sugimoto, K., Chikata, H., Kinoshita, E., et al. (2010).
Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identifica-
tion of its angiotensin I-converting enzyme inhibitory substances. Journal of
Agricultural and Food Chemistry, 58, 821–827.
Nasri, R., Amor, I.B., Bougatef, A., Nedjar-Arroume, N., Dhulster, P., Gargouri, J., et al.
(2012). Anticoagulant activities of goby muscle protein hydrolysates. Food
Chemistry, 133, 835–841.
Nazeer, R.A., & Kulandai, K.A. (2012). Evaluation of antioxidant activity of muscle and skin
protein hydrolysates from giant kingfish, Caranx ignobilis (Forsskal, 1775).
International Journal of Food Science and Technology, 47, 274–281.
Nazeer, R.A., Kumar, N.S.S., & Ganesh, R.J. (2012). In vitro and in vivo studies on the anti-
oxidant activity of fish peptide isolated from the croaker (Otolithes ruber) muscle pro-
tein hydrolysate. Peptides, 35, 261–268.
Ngo, D. -H., Kang, K. -H., Ryu, B., Vo, T. -S., Jung, W. -K., Byun, H. -G., et al. (2015).
Angiotensin-I converting enzyme inhibitory peptides from antihypertensive skate
(Okamejei kenojei) skin gelatin hydrolysate in spontaneously hypertensive rats.
Food Chemistry, 174, 37–43.
Niki, E. (2010). Assessment of antioxidant capacity in vitro and in vivo. Free Radical Biology
& Medicine, 49, 503–515.
Ortiz-Martinez, M., Winkler, R., & García-Lara, S. (2014). Preventive and therapeutic po-
tential of peptides from cereals against cancer. Journal of Proteomics, 111, 165–183.
Oseguera-Toledo, M.E., Mejia, E.G., Dia, V.P., & Amaya-Llano, S.L. (2011). Common bean
(Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macro-
phages through suppression of NF-κB pathways. Food Chemistry, 127, 1175–1185.
Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., & Deemer, E.K. (2002). Analysis of
antioxidant activities of common vegetables employing oxygen radical absorbance
capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: A comparative
study. Journal of Agricultural and Food Chemistry, 50, 3122–3128.
Panchaud, A., Affolter, M., & Kussmann, M. (2012). Mass spectrometry for nutritional
peptidomics: How to analyze food bioactives and their health effects. Journal of
Proteomics, 75, 3546–3559.
Patrzykat, A., & Douglas, S.E. (2005). Antimicrobial peptides: Cooperative approaches to
protection. Protein and Peptide Letters, 12, 19–25.
Picot, L., Ravallec, R., Fouchereau-Péron, M., Vandanjon, L., Jaouen, P., Chaplain-
Derouiniot, M., et al. (2010). Impact of ultrafiltration and nanofiltration of an indus-
trial fish protein hydrolysate on its bioactive properties. Journal of the Science of
Food and Agriculture, 90, 1819–1826.
Pihlanto, A. (2006). Antioxidative peptides derived from milk proteins. International Dairy
Journal, 16, 1306–1314.
Pownall, T.L., Udenigwe, C.C., & Aluko, R.E. (2010). Amino acid composition and antioxi-
dant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate
fractions. Journal of Agricultural and Food Chemistry, 58, 4712–4718.
Prasanna, P.H.P., Grandison, A.S., & Charalampopoulos, D. (2012). Effect of dairy-based
protein sources and temperature on growth, acidification and exopolysaccharide pro-
duction of Bifidobacterium strains in skim milk. Food Research International, 47, 6–12.
Quirós, A., Chichón, R., Recio, I., & López-Fandiño, R. (2007). The use of high hydrostatic
pressure to promote the proteolysis and release of bioactive peptides from ovalbu-
min. Food Chemistry, 104, 1734–1739.
Raghavan, S., & Kristinsson, H.G. (2008). Antioxidative efficacy of alkali-treated tilapia
protein hydrolysates: A comparative study of five enzymes. Journal of Agricultural
and Food Chemistry, 56, 1434–1441.
Rahman, M.A., Kumar, S.G., Kim, S.W., Hwang, H.J., Baek, Y.M., Lee, S.H., et al. (2008). Pro-
teomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte
differentiation. Proteomics, 8, 569–581.
Rajapakse, N., Mendis, E., Jung, W., Je, J., & Kim, S. (2005). Purification of a radical scaveng-
ing peptide from fermented mussel sauce and its antioxidant properties. Food
Research International, 38, 175–182.
Rao, S., Sun, J., Liu, Y., Zeng, H., Su, Y., & Yang, Y. (2012). ACE inhibitory peptides and
antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white
lysozyme. Food Chemistry, 135(3), 1245–1252.
Regazzo, D., Da Dalt, L., Lombardi, A., Andrighetto, C., Negro, A., & Gabai, G. (2010).
Fermented milks from Enterococcus faecalis TH563 and Lactobacillus delbrueckii
subsp. bulgaricus LA2 manifest different degrees of ACE-inhibitory and immunomod-
ulatory activities. Dairy Science & Technology, 90, 469–476.
Roblet, C., Amiot, J., Lavigne, C., Marette, A., Lessard, M., Jean, J., et al. (2012). Screening of
in vitro bioactivities of a soy protein hydrolysate separated by hollow fiber and spiral-
wound ultrafiltration membranes. Food Research International, 46, 237–249.
197
Saidi, S., Deratani, A., Belleville, M. -P., & Amar, R.B. (2014). Antioxidant properties of
peptide fractions from tuna dark muscle protein by-product hydrolysate produced
by membrane fractionation process. Food Research International, 65, 329–336.
Sakanaka, S., Tachibana, Y., Ishihara, N., & Juneja, L.R. (2004). Antioxidant activity of
egg-yolk protein hydrolysates in linoleic acid oxidation system. Food Chemistry,
86(1), 99–103.
Samaranayaka, A.G.P., & Li-Chan, E.C.Y. (2011). Food-derived peptidic antioxidants: A re-
view of their production, assessment, and potential applications. Journal of Functional
Foods, 3, 229–254.
Santos, L.F., & Koblitz, M.G.B. (2008). Proteases. In M.G.B. Koblitz (Ed.), Bioquímica de
Alimentos (pp. 78–103). Rio de Janeiro: Guanabara Koogan.
Sarmadi, B.H., & Ismail, A. (2010). Antioxidative peptides from food proteins: A review.
Peptides, 31, 1949–1956.
Schanbacher, F.L., Talhouk, R.S., & Murray, F.A. (1997). Biology and origin of bioactive pep-
tides in milk. Livestock Production Science, 50, 105–123.
Senphan, T., & Benjakul, S. (2014). Antioxidative activities of hydrolysates from seabass
skin prepared using protease from hepatopancreas of Pacific white shrimp. Journal
of Functional Foods, 6, 147–156.
Sharma, O.P., & Bhat, T.K. (2009). DPPH antioxidant assay revisited. Food Chemistry, 113,
1202–1205.
Sheih, I.C., Fang, T.J., & Wu, T.K. (2009). Isolation and characterisation of a novel angioten-
sin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste. Food
Chemistry, 115, 279–284.
Singh, B.P., Vij, S., & Hati, S. (2014). Functional significance of bioactive peptides derived
from soybean. Peptides, 54, 171–179.
Soliman, A.M., Abu-El-Zahab, H.S., & Alswiai, G.A. (2013). Efficacy evaluation of the
protein isolated from Peganum harmala seeds as an antioxidant in liver of rats.
Asian Pacific Journal of Tropical Medicine, 6(4), 285–295.
Sousa, J.C., Berto, R.F., Gois, E.A., Fontenele-Cardi, N.C., Honório-Júnior, J.E.R., Konno, K.,
et al. (2009). Leptoglycin: A new glycine/leucine-rich antimicrobial peptide isolated
from the skin secretion of the South American frog Leptodactylus pentadactylus
(Leptodactylidae). Toxicon, 54, 23–32.
Stuknyte, M., Noni, I.D., Guglielmetti, S., Minuzzo, M., & Mora, D. (2011). Potential immu-
nomodulatory activity of bovine casein hydrolysates produced after digestion with
proteinases of lactic acid bacteria. International Dairy Journal, 21, 763–769.
Su, G., Ren, J., Yang, B., Cui, C., & Zhao, M. (2011). Comparison of hydrolysis characteristics
on defatted peanut meal proteins between a protease extract from Aspergillus oryzae
and commercial proteases. Food Chemistry, 126, 1306–1311.
Tang, W., Zhang, H., Wang, L., Qian, H., & Qi, X. (2015). Targeted separation of antibacterial
peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium
dialysis. Food Chemistry, 168, 115–123.
Tavares, T.G., Contreras, M.M., Amorim, M., Martín-Álvarez, P.J., Pintado, M.E., Recio, I.,
et al. (2011). Optimization, by response surface methodology, of degree of hydrolysis
and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained
with cardoon extract. International Dairy Journal, 21, 926–933.
Tellez, A., Corredig, M., Turner, P.V., Morales, R., & Griffiths, M. (2011). A peptidic fraction
from milk fermented with Lactobacillus helveticus protects mice against Salmonella
infection. International Dairy Journal, 21, 607–614.
Theodore, A.E., Raghavan, S., & Kristinsson, H.G. (2008). Antioxidative activity of protein
hydrolysates prepared from alkaline-aided channel catfish protein isolates. Journal
of Agricultural and Food Chemistry, 56, 7459–7466.
Théolier, J., Hammami, R., Labelle, P., Fliss, I., & Jean, J. (2013). Isolation and identification
of antimicrobial peptides derived by peptic cleavage of whey protein isolate. Journal
of Functional Foods, 5, 706–714.
Tsai, J. -S., Chen, T. -J., Pan, B.S., Gong, S. -D., & Chung, M. -Y. (2008). Antihypertensive
effect of bioactive peptides produced by protease-facilitated lactic acid fermentation
of milk. Food Chemistry, 106, 552–558.
Tsou, M. -J., Kao, F. -J., Lu, H. -C., Kao, H. -C., & Chiang, W. -D. (2013). Purification and iden-
tification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy
protein. Food Chemistry, 138, 1454–1460.
Tsou, M. -J., Kao, F.J., Tseng, C.K., & Chiang, W.D. (2010a). Enhancing the anti-adipogenic
activity of soy protein by limited hydrolysis with flavourzyme and ultrafiltration.
Food Chemistry, 122, 243–248.
Tsou, M. -J., Lin, W., Lu, H., Tsui, Y., & Chiang, W. (2010b). The effect of limited hydrolysis
with neutrase and ultrafiltration on the anti-adipogenic activity of soy protein.
Process Biochemistry, 45, 217–222.
Uhlig, T., Kyprianou, T., Martinelli, F.G., Oppici, C.A., Heiligers, D., Hills, D., et al. (2014). The
emergence of peptides in the pharmaceutical business: From exploration to
exploitation. EuPA Open Proteomics, 4, 58–69.
Uliyanchenko, E., Schoenmakers, P.J., & van der Wal, S. (2011). Fast and efficient size-
based separations of polymers using ultra-high-pressure liquid chromatography.
Journal of Chromatography A, 1218, 1509–1518.
Vallabha, V.S., & Tiku, P.K. (2014). Antihypertensive peptides derived from soy
protein by fermentation. International Journal of Peptide Research and Therapeutics,
20, 161–168.
Vanderghem, C., Francis, F., Danthine, S., Deroanne, C., Paquot, M., Pauw, E.D., et al.
(2011). Study on the susceptibility of the bovine milk fat globule membrane proteins
to enzymatic hydrolysis and organization of some of the proteins. International Dairy
Journal, 21, 312–318.
Wang, W.Y., & De Mejia, E.G. (2005). A new frontier in soy bioactive peptides that may
prevent age-related chronic diseases. Comprehensive Reviews in Food Science and
Food Safety, 4, 63–78.
Wang, B., Gong, Y.D., Li, Z.R., Yu, D., Chi, C.F., & Ma, J.Y. (2014). Isolation and characterisa-
tion of five novel antioxidant peptides from ethanol-soluble proteins hydrolysate of
spotless smoothhound (Mustelus griseus) muscle. Journal of Functional Foods, 6,
176–185.
198 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198
Wijesekara, I., Qian, Z., Ryu, B., Ngo, D., & Kim, S. (2011). Purification and identification
of antihypertensive peptides from seaweed pipefish (Syngnathus schlegeli) muscle
protein hydrolysate. Food Research International, 44, 703–707.
Yamada, A., Sakurai, T., Ochi, D., Mitsuyama, E., Yamauchi, K., & Abe, F. (2015). Antihyper-
tensive effect of the bovine casein-derived peptide Met-Lys-Pro. Food Chemistry, 172,
441–446.
Yang, Y., Boysen, R.I., Chowdhury, J., Alam, A., & Hearn, M.T.W. (2015). Analysis of pep-
tides and protein digests by reversed phase high performance liquid chromatogra-
phy–electrospray ionisation mass spectrometry using neutral pH elution conditions.
Analytica Chimica Acta, 872, 84–94.
Yasufumi, K., Shigeki, M., Keiji, I., & Tomoyuki, O. (2001). Antioxidative peptides from
milk fermented with Lactobacillus delbrueckii subsp. bulgaricus IFO 13953. Journal of
the Japanese Society for Food Science and Technology, 48, 44–50.
Yoshida, T. (2004). Peptide separation by hydrophilic-interaction chromatography: A
review. Journal of Biochemical and Biophysical Methods, 60, 265–280.
Yuan, L., Wu, J., & Aluko, R.E. (2007). Size of the aliphatic chain of sodium houttuyfonate
analogs determines their affinity for renin and angiotensin I converting enzyme.
International Journal of Biological Macromolecules, 41, 274–280.
Zarei, M., Ebrahimpour, A., Abdul-Hamid, A., Anwar, F., Bakar, F.A., Philip, R., et al. (2014).
Identification and characterization of papain-generated antioxidant peptides from
palm kernel cake proteins. Food Research International, 62, 726–734.
Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature, 145,
389–395.
Zhang, L., Li, J., & Zhou, K. (2010). Chelating and radical scavenging activities of soy pro-
tein hydrolysates prepared from microbial proteases and their effect on meat lipid
peroxidation. Bioresource Technology, 101, 2084–2089.
Zhang, Q., Ren, J., Zhao, H., Zhao, M., Xu, J., & Zhao, Q. (2011). Influence of casein hydroly-
sates on the growth and lactic acid production of Lactobacillus delbrueckii subsp.
bulgaricus and Streptococcus thermophilus. International Journal of Food Science and
Technology, 46, 1014–1020.
Zhang, J., Zhang, H., Wang, L., Guo, X., Wang, X., & Yao, H. (2009). Antioxidant activities of
the rice endosperm protein hydrolysate: Identification of the active peptide.
European Food Research and Technology, 229, 709–719.
Zhou, K., Canning, C., & Sun, S. (2013). Effects of rice protein hydrolysates prepared by
microbial proteases and ultrafiltration on free radicals and meat lipid oxidation.
LWT — Food Science and Technology, 50, 331–335.
Zhu, Y.P., Cheng, Y.Q., Wang, L.J., Fan, J.F., & Li, L.T. (2008). Enhanced antioxidative activity
of chinese traditionally fermented Okara (Meitauza) prepared with various microor-
ganism. International Journal of Food Properties, 11, 519–529.
Zhuang, Y., & Sun, L. (2011). Preparation of reactive oxygen scavenging peptides from
tilapia (Oreochromis niloticus) skin gelatin: Optimization using response surface
methodology. Journal of Food Science, 76(3), 483–489.