LUNES Biologically Active Peptides PROCESSES For Their Generation, Purification and Isolation

Food Research International 74 (2015) 185198
Contents lists available at ScienceDirect
Food Research International

journal homepage: www.elsevier.com/locate/foodres
Review
Biologically active peptides: Processes for their generation, purication

and identication and applications as natural additives in the food and
pharmaceutical industries
Ruann Janser Soares de Castro , Hlia 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
Article history:
Received 19 March 2015
Received in revised form 1 May 2015
Accepted 8 May 2015
Available online 12 May 2015
Keywords:
Proteins
Enzymatic hydrolysis
Fermentation
Purication
Bioactive peptides
a b s t r a c t
Recent technological advances have created great interest in the use of biologically active peptides. Bioactive
peptides can be dened as specic portions of proteins with 2 to 20 amino acids that have desirable biological
activities, including antioxidant, anti-hypertensive, antithrombotic, anti-adipogenic, antimicrobial and antiinammatory effects. Specic characteristics, including low toxicity and high specicity, make these molecules
of particular interest to the food and pharmaceutical industries. This review focuses on the production of bioactive peptides, with special emphasis on fermentation and enzymatic hydrolysis. The combination of different
technologies and the use of auxiliary processes are also addressed. A survey of isolation, purication and peptide
characterization methods was conducted to identify the major techniques used to determine the structures of
bioactive peptides. Finally, the antioxidant, antimicrobial, anti-hypertensive, anti-adipogenic activities and
probiotic-bacterial growth-promoting aspects of various peptides are discussed.
2015 Elsevier Ltd. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
Major processes for obtaining bioactive peptides . . . . . . . .
2.1.
Fermentation . . . . . . . . . . . . . . . . . . . . .
2.2.
Enzymatic hydrolysis . . . . . . . . . . . . . . . . .
3.
Concentration, purication and identication of bioactive peptides
4.
Biological properties of bioactive peptides . . . . . . . . . . .
4.1.
Peptides with antimicrobial activity . . . . . . . . . . .
4.2.
Peptides with antioxidant activity . . . . . . . . . . . .
4.3.
Peptides with anti-adipogenic activity . . . . . . . . . .
4.4.
Peptides with anti-hypertensive activity . . . . . . . . .
4.5.
Induction of lactic acid bacteria and probiotic growth . . .
5.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
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).
http://dx.doi.org/10.1016/j.foodres.2015.05.013
0963-9969/ 2015 Elsevier Ltd. All rights reserved.
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185
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195
physicochemical and sensory properties, such as solubility, viscosity,

gelication and emulsion stability. Some proteins in the diet have
specic 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 endogenous peptides have important functions, acting as neurotransmitters,
186
R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185198
hormones and regulators (Hernndez-Ledesma, Garca-Nebot,

Fernndez-Tom, 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 signicant source of dietary protein (Ortiz-Martinez, Winkler,
& Garca-Lara, 2014). Animal protein sources also have great potential.
One of the most popular and promising lines of research is the production of hydrolyzed proteins from meat proteins, which, in addition to
having important biological activities and being excellent sources of nutrients, such as minerals and vitamins, can be used as avor enhancers
and emulsiers (Lafarga & Hayes, 2014; Mora, Escudero, Fraser,
Aristoy, & Toldr, 2014). The biological properties of other animal protein sources, such as egg and sh, 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 response 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 benets, regulating one or more functions in the body
(Diplock et al., 1999; Hernndez-Ledesma, Contreras, & Recio, 2011).
Processes incorporating protein hydrolysis have been studied to determine whether they produce biologically active peptides. Mellander
(1950) was responsible for the rst study relating the ingestion of
bioactive peptides from hydrolyzed casein protein to increased bone
calcication in rachitic newborns. Since then, peptides with countless
bioactivities have been identied. According to the Biopep and BioPD
(Bioactive peptide database) databases, more than 1200 different bioactive peptides have been recorded (Singh, Vij, & Hati, 2014).
Bioactive peptides are dened as specic regions of proteins with
amino acid sequences that have biological activity, including antioxidant, anti-hypertensive, antithrombotic, anti-adipogenic, antimicrobial,
anti-inammatory 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 220 amino acids and molecular masses
of less than 6000 Da. Their bioactivity is mainly determined by their
composition and amino acid sequence (Mora, Reig, & Toldr, 2014;
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 elds (Miranda & Liria, 2008); furthermore, these peptides and proteins have been identied 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 eld. 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 specicity for their target tissues,
resulting in little or no toxicity, and even low concentrations can be effective. 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 excretion, 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 fermentation, enzymatic hydrolysis and a combination of the two processes (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, improved digestibility and the formation of biologically active peptides
(Korhonen, 2009). One strategy used in several scientic studies demonstrated that using lactic acid bacteria together with food-grade
enzymes resulted in nal 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 ve 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 mL1, in contrast to 0.64 mg mL1 for the straight
fermentation and 1.18 mg mL1 for fresh milk.
In addition to the conventional methods mentioned above, combining several technologies has produced effective results for generating
functional peptides (Korhonen, 2009). Ultraltration and nanoltration
are examples of technologies that have been used to rene and isolate
bioactive peptides, allowing them to be separated by size for use in
specic applications (Picot et al., 2010; Quirs, Chichn, Recio, &
Lpez-Fandio, 2007).
Understanding the critical parameters for the process is fundamentally important for obtaining hydrolyzed proteins with desirable biological 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 specicity, specic activity, pH and temperature
for activity and stability; and processing conditions, including enzyme
and substrate concentrations, pH, temperature and reaction time.
Prior knowledge and identication of these parameters can be used as
tools for obtaining products with distinct functions, for producing multifunctional peptides or even for producing unique peptides with specific functions (Li-Chan, 2015; Samaranayaka & Li-Chan, 2011).
This review describes advances in scientic research on the processes 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 naturally 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 signicantly contribute to the chemical, biochemical and nutritional properties of the fermented products. The proteolytic system of
lactic acid bacteria (LAB) is complex and consists of three major components: proteases bound to the cell wall that promote the initial hydrolysis of milk casein into oligopeptides, specic transporters that transfer
the oligopeptides to the cytoplasm and intracellular peptidases that nish the hydrolysis process to convert oligopeptides into free amino acids
and/or low molecular weight peptides (Chaves-Lpez 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.
187
Animal or vegetable protein sources
Proteases:
vegetable, animal or
microbial
Microbial fermentation
Proteases produced
during fermentation
High pressure
homogenization
Protein hydrolysates
Bioactive
Peptides
Ultrafiltration or nanofiltration
Isolation, purification
and identification
Biological activities:
in vitro and in vivo methods
Antihypertensive
Anti-inflamatory
Antimicrobial
Immunomodulatory
Anti-adipogenic
Antioxidant
Fig. 1. Major processes for obtaining bioactive peptides and related bioactivities.
cremoris and Streptococcus salivarius ssp. thermophylus (HernndezLedesma et al., 2011). In addition to using live microorganisms, proteolytic 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 scientic 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; Limn
et al., 2015; Nakahara et al., 2010) (Table 1). Species of lamentous
fungi such as Aspergillus oryzae and Aspergillus sojae have a long tradition of safe use in the production of fermented foods, in which several
peptides with biological activities were detected, for example antioxidant 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 controlled 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 Meja, Mendoza, & Loarca-Pia, in press; Mora
et al., 2014; 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 degree of specicity for the substrate, generally based on the sequence of
amino acids directly surrounding the bond that is cleaved (Santos &
Koblitz, 2008). This specicity 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 affect 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 specic activity, such as trypsin and chymotrypsin, and combinations of different
non-specic 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 proles, especially for the composition and molecular mass distribution of
the peptides. These processes are especially useful in the food and pharmaceutical industries, which rely on animal, plant and microbial proteases (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 hydrolyze 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, purication and identication of bioactive
peptides
Table 3 summarizes different characteristics for some methods of
purication and identication of bioactive peptides, including their
principle, advantages and limitations. Chromatography techniques are
188
Table 1
Obtaining peptides with different biological activities by fermentation using various protein sources.
Microorganism
Protein source
Fermentation conditions
Peptides
Bioactivity
Reference
Streptococcus thermophiles
Lactobacillus bulgaricus
+
Protease Flavourzyme
Aspergillus oryzae
Soy milk
Submerged fermentation for 5 h

at 43 C
Tyr-Pro-Tyr-Tyr
Antihypertensive
Tsai, Chen, Pan, Gong,

and Chung (2008)
Rice, soy and

casein
Okara
Solid-state fermentation for 40 h

at 30 C
Sequential submerged
fermentation:
B. subtilis for 48 h at 40 C
A. oryzae, R. oligosporus and
A. elegans for 60 h at 30 C
Solid-state fermentation for 192 h at
2045 C and 95% humidity
at 37 C (Enterococcus faecalis) or
44 C (Lactobacillus delbrueckii)
Val-Pro-Pro; Ile-Pro-Pro
Antihypertensive
Inoue et al. (2009)
Not identied
Antioxidant
Zhu, Cheng, Wang,

Fan, and Li (2008)
Gly-Tyr; Ala-Phe; Val-Pro;

Ala-Ile; Val-Gly
Peptides with molecular
weights less than 5000 Da
Antihypertensive
Nakahara et al. (2010)
Antihypertensive and
immune-regulatory
Regazzo et al. (2010)
Submerged fermentation for 5 h at

30 C (Lactococcus lactis) or 37 C
(L. acidophilus) with agitation at
140 rpm
Solid-state fermentation for 365

Immunomodulatory
Stuknyte, Noni,
Guglielmetti, Minuzzo,
and Mora (2011)

Antioxidant
Giri et al. (2011)

weights between 180 and
5500 Da
Val-Leu-Pro-Val-Gln
Leu-Ile-Val-Thr-Gln
Antioxidant
He et al. (2012)
Antioxidant
Antihypertensive
Chang et al. (2013)

Vallabha and Tiku (2014)

weights between 6.2 and
201.2 kDa
Antioxidant
Antihypertensive
Limn et al. (2015)
Aspergillus oryzae,
Rhizopus oligosporus,
Actinomucor elegans,
Bacillus subtilis
Aspergillus sojae
Soy and wheat
Enterococcus faecalis
TH563
Lactobacillus delbrueckii subsp.
bulgaricus LA2
L. acidophilus ATCC 4356
Lc. lactis subsp. lactis GR5
Cow's milk
Sodium
Caseinate
Aspergillus oryzae
Squid mantles
B. subtilis 10160
Rapeseed
Bidobacterium longum KACC91563

Lactobacillus casei spp.
pseudoplantarum
B. subtilis ATCC 6051
Casein
Concentrated
soy protein
Bean
days at 2530 C
Solid-state fermentation for 6 days
at 32 C and 85 5% relative
humidity
at 37 C
Solid-state fermentation for 96 h
at 30 C and 90% relative humidity
amongst the most widely used, such as high performance liquid chromatography (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 regular HPLC methods. The main advantages of this method include the increase 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 substances. 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 differentiation of peptides with homologous sequences by mass spectrometry.
Gel electrophoresis and ultraltration 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 proles and hydrolysis products. 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 become important for the identication and characterization of bioactive
peptides and proteins using mass spectrometry. Liquid chromatographymass spectrometry is commonly used to identify peptide sequences (Chiaradia, Collins, & Jardim, 2008; Contreras, Lpez-Expsito,
Hernndez-Ledesma, Ramos, & Recio, 2008; Singh et al., 2014).
Peptides with anticoagulant activity that were obtained from goby
sh (Awaous guamensis) and a protease from Bacillus licheniformis
were separated by molecular exclusion chromatography and reversed-
phase high-performance liquid chromatography and identied by

mass spectrometry. The hydrolysate solution containing the peptides
was applied to a Sephadex G-25 (5.2 56 cm) gel ltration column
pre-equilibrated and eluted with distilled water, and 4.5-mL fractions
were collected using a ow rate of 0.5 mL min 1. Absorption at
220 nm was measured to determine the peptide elution prole. Fractions with higher anticoagulant activity were recovered and puried
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
ow 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 identied as LeuCys-Arg, His-Cys-Phe, Cys-Leu-Cys-Leu-Arg and Cys-Arg-Arg (Nasri
et al., 2012).
Tsou, Kao, Lu, Kao, and Chiang (2013) puried and identied bioactive peptides from puried soy protein and the Flavourzyme protease
using sequential fractionation with ultraltration membranes of various
sizes, gel chromatography, reversed-phase high-performance liquid
chromatography and mass spectrometry. The hydrolysates were initially fractionated in ultraltration membranes of 30, 10 and 1 kDa. The
fraction retained on the 1-kDa membrane was selected for purication
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 ow rate of 0.5 mL min 1. Onemilliliter 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 puried 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 ow rate of 1.0 mL min1. The fraction with the most antiadipogenic activity was puried again using a reverse-phase column
189
Table 2
Using proteases to generate biologically active peptides from various protein sources.
Protease
Hydrolysis
conditions
Protein
source
Bioactivity of
the peptides
Peptides
Identication methods
Reference
Alcalase
pH 8.0; 50 C; 3 h
E:S = 1:20
[S] = 5.0%
pH 7.0; 50 C; 2 h
E:S = 1:100
[S] = 2.5%
pH 6.0; 45 C; 4 h
E:S = 1:100
[S] = 2.5%
pH 5.5; 23 C
[S] = 1.0%
Soy
Antiadipogenesis
Liquid chromatography mass

spectrometry
Mejia et al. (2010)
Puried soy
protein
Antiadipogenesis

3897 Da
High-performance molecular
exclusion chromatography
Tsou, Kao, et al., 2010
Puried soy
protein
Antiadipogenesis
Tsou, Lin, et al., 2010
Bovine
hemoglobin
Antimicrobial
Antihypertensive
pH 8.0; 50 C; 3 h
[E] = 0.2 mg/mL
[S] = 8.0%
Bean
Antioxidant
Anti-inammatory
Crude protease from

Bacillus licheniformis
pH 10.0; 50 C; 5.5 h
[S] = 10.0%
Goby muscle
Anticoagulant
Alcalase
Flavourzyme
Protamex
Neutrase
Pepsin
Trypsin
Crude protease from
Bacillus mojavensis
pH 7.0; 50 C; 8 h
pH 7.0; 50 C; 8 h
pH 7.0; 50 C; 8 h
pH 7.0; 50 C; 8 h
pH 2.0; 37 C; 8 h
pH 8.0; 37 C; 8 h
pH 10.0; 50 C
[S] = 5.0%
Salmon
Antioxidant
Anti-inammatory
Electrospray ionization mass

spectrometry
(ESI/MS)
Matrix-assisted laser
desorption/ionization mass
spectrometry
(MALDI-TOF)
Liquid chromatography
spectrometry
(ESI/MS)
Adje et al. (2011)
Alcalase

weights between 1300
and 2200 Da
4430 Da
masses between 445 and
2148 Da
Cuttlesh
(Sepia
ofcinalis)
muscle
Antihypertensive
Flavourzyme
Neutrase
Pepsin
and linear acetonitrile gradients of 10 to 40%. Finally, the peptides were

identied by liquid-chromatography coupled with mass spectrometry.
Three peptides with the amino acid sequences Ile-Leu-Leu, Leu-LeuLeu and Val-His-Val-Val were identied as being responsible for the
anti-adipogenic activity of the protein hydrolysates isolated from soy.
Peptides with anti-hypertensive activity were isolated and identied
from gelatin hydrolysates extracted from stingray skin (Okamejei
kenojei). The hydrolysates rst were subjected to ultraltration through
a 1-kDa membrane, and peptides with molecular weights lower than
this cutoff were collected. Purication consisted of sequential steps of
isolation by fast protein liquid chromatography (FPLC) (AKTA,
Amersham Bioscience Co., Uppsala, Sweden) using a HiPrep 16/10
high ow ionic exchange column (16 100 mm, Amersham Biosciences, Piscataway, NJ, USA) and a GE Healthcare Superdex Peptide
10/300 GL gel ltration column (10 300 mm). Puried peptides
were then identied by MALDI-TOF mass spectrometry. Two puried
peptides were found to be very anti-hypertensive and were identied
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 identify peptides with antioxidant activities derived from the protein hydrolysate of Mactra veneriformis. The hydrolysates were fractionated on a
Sephadex G-25 gel ltration column (2.0 cm 100 cm; GE Chemicals,
Uppsala, Sweden) using distilled water as the eluting solvent at a ow
rate of 0.4 mL min1, and separated ve 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 m) and a linear gradient
of wateracetic acid (eluent A) and methanol (eluent B) at a ow rate
of 0.3 mL min1. 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 502000.
This method allowed for the identication of 21 peptides, and the
Leu-Cys-Arg
His-Cys-Phe
Cys-Leu-Cys-Arg
Leu-Cys-Arg-Arg
masses between 1000
and 2000 Da

masses between 163 and
1047 Da
Liquid chromatography
spectrometry (ESI/MS)
Tandem mass spectrometry
(ESI-MS/MS)
Oseguera-Toledo,
Mejia, Dia, and
Amaya-Llano (2011)
Nasri et al. (2012)
Ahn, Je, and Cho (2012)
Balti et al. (2015)
most antioxidant peptides were identied as Thr-Asp-Tyr, Leu-AspTyr, Trp-Asp-Asp-Met-Glu-Lys, Trp-Gly-Asn-Val-Ser-Gly-Ser-Pro, LeuTyr-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 identication difcult.
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 efcient
separation of the peptides with hydrophobic and hydrophilic characteristics, respectively (Panchaud, Affolter, & Kussmann, 2012).
In addition, an interesting approach was proposed by Le Maux et al.
(2015). These authors afrmed that liquid chromatography coupled to
mass spectrometry (LCMS/MS) providing the necessary data for peptide sequencing. However, although this strategy has been successfully
used for longer peptides, the identication of short peptides can be
more difcult, 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 hydrophilicity of each peptide for the development of a retention time prediction 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 digestive, cardiovascular, nervous and immune systems. Several bioactive
190
Table 3
The main characteristics of the different analytical methods for the purication and identication of bioactive peptides.
Method of
purication/identication
Mechanism
Advantage
Limitation
Reference
Reversed phase high pressure liquid

chromatography (RP-HPLC)
Based on the hydrophobicity of

proteins or peptides that can interact
differently to the reversed-phase
material of the chromatography
column.
Useful method for the

isolation of complex
peptide mixtures.
Lack of retention of polar

molecules. Slow intrapore diffusion
times. The presence of unresolved
structural microheterogeneity and
conformational isomers. Secondary
interactions with the stationary
phase.
Tone must know the
physicochemical properties of the
ligands, which limits its use for a
complex mixture of unknown
peptides.
Low selectivity and requires
complementary steps for the
separation of the fractions.
Everley and Croley (2008).

Le Maux et al. (2015).
Yang, Boysen, Chowdhury,
Alam, and Hearn (2015).
Loss of highly hydrophobic

proteins in the sample
preparation and precipitation of
neutral proteins at their pI, which
can result in overlapping between
different fractions.
Issaq, Conrads, Janini, and

Veenstra (2002).
Guijarro-Dez, Garca, Crego,
and Marina (2014)
Long columns are required for

complex peptide mixtures, which
can be obtained by joining
multiple columns in a series. This
strategy is necessary to improve
the separation resolution.
Mora et al. (2014).

Fekete, Beck, Veuthey, and
Guillarme (2014).
Afnity chromatography
Based on the afnity of bioactive

peptides to interact specically and
reversibly with a complementary
molecule bound to a solid support
immobilized on a column.
Ion-exchange chromatography (IEC) Based on the ability of charged
bioactive peptides to interact with a
solid support bearing the opposite
charge.
Isoelectric focusing (IEF)
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.
Size exclusion chromatography
Based on the fractionation of bioactive
(SEC)
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.
Ultra high pressure liquid
Based on separation of the molecules
chromatography (UHPLC)
using experimental columns packed
with very small particles of a
non-porous material, carrying out the
analyses at very high pressures.
Hydrophilic interaction liquid
chromatography (HILIC)
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
spectrometry (ESI/MS)
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-assisted laser
desorption/ionization-time-of-ight analytes when they are mixed with a
matrix solution on a target plate. The
mass spectrometry
co-crystal is subjected to the action of
(MALDI-TOF/MS)
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 ight time of ions to the
ion detector, and provides the m/z ratio
mapping results.
The exibility of using a

large number of binding
agents, allows for the
separation of different
types of peptides.
Appropriate method for
the separation of highly
cationic or anionic
peptides.
The method allows one to
fractionate a complex
mixture of peptides
according to their pI.
The elution conditions are

considered mild, allowing
the characterization of the
protein with minimal
impact on the
conformational structure
and the local environment.
Increased throughput,
resolution and sensitivity
in separation of complex
protein mixtures.
The heat dissipated from the use

of small particles at ultra-high
pressures may increase
chromatographic band
broadening and compromise
efciency of the column.
Compared to RP-HPLC, the
The method shows great
potential for the separation method shows limited exibility
of short peptide sequences and applicability, problems with
sample solubility and the
(b5 amino acids) and
improves the identication retention mechanisms are poorly
understood.
using mass spectrometry.
Production of singly and

multiply charged ions,
allowing for an accurate
measurement of the
molecular weight of the
peptides.
The method has no

theoretical upper limit to
the m/z ratio, allowing for
the analysis of complex
samples with a wide range
of molecular weights.
peptides have biological activities that are benecial for human health,
including antimicrobial (Adje, Balti, Kouach, Guillochon, & NedjarArroume, 2011), anti-hypertensive (Alemn et al., 2011), antioxidant
Hage et al. (2012).

Ortiz-Martinez et al. (2014).
Bouhallab, Henry, and

Boschetti (1996).
Ortiz-Martinez et al. (2014).
Everley and Croley (2008).

Uliyanchenko,
Schoenmakers, and van der
Wal (2011).
Fekete and Guillarme (2014).
Gray et al. (2013).
Le Maux et al. (2015).
Mano and Goto (2003).

The efciency of identication is
Contreras et al. (2008).
directly related to the
chromatographic method used for Panchaud et al. (2012).
the prior separation of the
bioactive peptides before
injection into the mass
spectrometer. Therefore, a
combination of different
separation techniques is
necessary for accurate
identication.
(Zhang et al., 2009) anticancer (Alemn et al., 2011), anti-adipogenic

(Tsou, Kao, et al., 2010), immunomodulatory (Huang, Chen, Chen,
Hong, & Chen, 2010) and anti-inammatory effects (Ahn et al., 2015).
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 microorganisms have developed resistance to conventional antibiotics, causing
serious problems treating infections, especially in immunocompromised 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 antibiotics for in small doses or with ineffective treatment times, and the
genetic mutation capacity of the microorganisms, which increases the
difculty of developing drugs based on specic 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 specicity. 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 external 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 essential to the immune system. They are the organism's rst line of defense against colonization by exogenous microorganisms, and they
play a fundamental role in regulating bacterial populations on the mucosa 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;
Gmez-Guilln 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 peptides, 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 identied in several protein hydrolysates.
Hydrolysates of casein from cow's milk obtained by enzymatic hydrolysis using chymosin were analyzed for their antimicrobial power.
Five different antibacterial peptides were isolated from the carboxylic
end of s2-casein. Peptide fractions f (181207), f (175207) and f
(164207) had a wide spectrum of activity and were able to inhibit several Gram+ and Gram bacteria; the minimum inhibitory concentration (MIC) of each fraction ranged from 21.0 to 168.0 mg mL1, 10.7
to 171.2 mg mL1 and 4.8 to 76.2 mg mL1, 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 hydrolysate with Alcalase 2.4 L (Sigma-Aldrich, United States). Fractions
obtained from ultraltration through 1- and 10-kDa membranes were
used for antimicrobial tests against 18 bacteria. The most sensitive bacteria in the presence of the tested fractions were Lactobacillus acidophilus,
Bidobacterium lactis, Shewanella putrafaciens and Photobacterium
phosphoreum (Gmez-Guilln et al., 2010). Hydrolysates of bovine hemoglobin treated with pepsin were puried 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 puried 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 M (Adje et al., 2011).
Tellez, Corredig, Turner, Morales, and Grifths (2011) demonstrated
the efciency 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 g
per day) was higher than the group fed with half of the dose (0.01 g
per day) and higher than the control group.
The antimicrobial powers of protein isolated from whey hydrolyzed
with various gastrointestinal enzymes were demonstrated by Tholier,
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 signicant
activity. Hydrolysates were fractionated by reverse-phase highperformance liquid chromatography, resulting in ve fractions with
high antibacterial activity and MIC values between 20.0 and
35.0 g 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 identied fraction had the peptide sequence Gly-Leu-Ser-Arg-Leu-Phe-ThrAla-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 various factors, including chemical reactivity, the rate of removal of the
compound, the fate of the product of the antioxidantradical 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 various health benets. They are dened as substances that signicantly
slow or inhibit the oxidation of a substrate (Bougatef et al., 2009).
192
Currently, some articially synthesized antioxidants, such as butylated

hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertbutylhydroquinone (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 nding 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 (-Glu-Cys-Gly) and carnosine (-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 applications (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 (Gmez-Guilln et al., 2010), chelate metal ions
(Alemn et al., 2011) and eliminate reactive oxygen species (Zhuang
& Sun, 2011). Similar to other biological activities, the antioxidant properties of peptides are related to their composition, structure and hydrophobicity (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, especially 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 phenolic, indole and imidazole groups, respectively, which can act as proton
donors to electron decient radicals and efciently 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 chelating, 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 sufciently in biological uids, 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 uids and tissues after ingestion (Alam, Bristi, &
Raquzzaman, 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 sh 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 protein hydrolysates from carp prepared with Alcalase 2.4 L and papain
had antioxidant activity according to tests using ABTS, DPPH, Fe3+ reduction and Fe2+ chelation. Najaan and Babji (2015) studied the antioxidant activity of myobril protein from sh (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 hydrolysates 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 oxidative stress, and impairments of these enzymes are associated with
several diseases (Ktari et al., 2014). Liu et al. (in press) studied the antioxidant properties of protein hydrolysates from corn gluten prepared
using the proteases Alcalase, Flavourzyme and Protamex. The antioxidant activities of the hydrolysates were analyzed by in vivo methods.
The experiments were performed in 40 45 week old Kunming line
mice (25.0 2.0 g). Mice were randomly divided into ve 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 hydrolysates, respectively, for 10 days. The results showed that the hydrolysates 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, indicating 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 quantity 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 preadipocyte 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 activity 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 treatments 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
193
Table 4
Major methods for measuring antioxidant activities of peptides in vitro and their respective mechanisms.
Method
Mechanism
Reaction
Measurement
DPPH
DPPH capture
Reduction in the
absorbance at 517 nm
Sharma and Bhat (2009)
ORAC
Peroxyl radical capture
Reduction in uorescence
(excitation at 485 nm and
emission at 520 nm)
Dvalos, Gmez-Cordovs, and

Bartolom (2004)
FRAP
Iron reducing power
Increase in the absorbance

at 593 nm
Ou, Huang, Hampsch-Woodill,

Flanagan, and Deemer (2002)
ABTS
ABTS capture
Reduction in the
Gmez-Guilln et al. (2010)
Ability to chelate
transition metals
(Cu2+)
Chelation of Cu2+
Reduction in the
Theodore et al. (2008)
Ability to chelate
transition metals
(Fe2+)
Chelation of Fe2+
Reduction in the
Nazeer and Kulandai (2012)
TBARS
Quantication of lipid
peroxidation products
DPPH radical (2,2-diphenyl-1-picryl-hydrazyl) reacts with

hydrogen-donating antioxidants, changing the color from
violet to yellow.
The peroxyl radical, generated from the breakdown of
AAPH [2,2-Azobis(2-amidinopropane) dihydrochloride]
in the presence of atmospheric oxygen, reacts with a
uorescent indicator to produce a non-uorescent
product. In the presence of antioxidants, the uorescence
is maintained.
In the presence of electron-donating antioxidants, the
Fe3+-TPTZ [2,4,6-Tripyridyl-S-Triazine] complex is
reduced to Fe2+-TPTZ, changing the color from light blue
to dark blue.
The radical ABTS
(2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))
is stabilized in the presence of hydrogen-donating free
radicals, changing the color from dark green to light green.
Complexation reaction of Cu2+ with pyrocatechol violet to
generate a colored product. The presence of antioxidants
decreases the formation of the Cu2+-pyrocatechol
complex, reducing the intensity of the color.
Complexation reaction of Fe2+ with ferrozine, generating
a colored product. The presence of antioxidants decreases
the formation if the Fe2+-ferrozine complex, reducing
color intensity.
Reaction of thiobarbituric acid with hydroperoxide
decomposition products. Malonaldehyde is the main
compound quantied. Absorbance and antioxidant activity
are inversely proportional.
Increase in the absorbance

at 532 nm
Raghavan and Kristinsson

(2008)
fractions and found that subunits from the -conglicinin fraction had a
larger number of peptides responsible for the inhibition of lipid accumulation 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 ultraltration. The results revealed that the partial hydrolysis of proteins isolated from soy provided hydrolysates with strong
anti-adipogenic capacity, and fractions obtained by ultraltration
more efciently inhibited GPDH activity. Specically, the 1-kDa membrane fraction was the most effective (59.0% inhibition). The antiadipogenic 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
analyzed
Mechanism of action and principle

underlying measurement
Reference
Superoxide dismutase (SOD)
Peptide isolate from

hydrolyzed pig plasma
Male adult
Wistar rats
Liver
Liu, Kong, Li, Liu, and

Xia (2011)
Catalase (CAT)
Peptide isolated from

hydrolyzed sh protein
Albino male
adult Wistar rats
Erythrocyte
lysate (blood)
Level of reduced glutathione

(GSH)
Protein isolates from

seeds from Syrian rue
(Peganum harmala)
Albino male rats
Liver and blood

plasma
Glutathione-S-transferase
(GST)
Peptide isolated from

hydrolyzed mussel
protein
Male adult rats
Liver
Glutathione peroxidase
(GPx)
Hydrolyzed corn gluten
Male and female

Kunming mice
Liver and blood

plasma
Measurement of the level of

malonaldehydes
Hydrolyzed sh protein
(Salaria basilisca)
Male adult
Wistar rats
Liver and blood

plasma
SOD is an enzyme that catalyzes the

dismutation of superoxide radicals into
hydrogen and oxygen, thus playing an
important role in protecting cells against
reactive oxygen species
CAT is an enzyme that converts hydrogen
peroxide into water and oxygen, thus
having one of the major mechanisms for
removing free radicals in the organism
GSH is an intracellular reducer that plays
an important role for protecting cells
from free radicals, peroxides and other
toxic compounds
GST is an enzymatic complex in the
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
GPx is an enzyme that catalyzes the
reaction between hydroperoxide and
reduced glutathione leading to the
formation of glutathione disulte and the
product of hydroperoxide reduction
Malanodialdehyde is an intermediate
product for lipid peroxidation and thus
can be used as an indicator for the
presence of free radicals
Nazeer, Kumar, and

Ganesh (2012)
Soliman, Abu-El-Zahab,
and Alswiai (2013)
Kim et al. (2013)
Liu et al. (in press)
Ktari et al. (2014)
194
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 ACEinhibitory activity, although several studies have shown that the size
and hydrophobic character of the peptides exert a strong inuence 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 character 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 ltration and HPLC,
and they were found to have ACE-inhibiting activity. Four amino
acid sequences were identied as potential ACE inhibitors: Ile-Ala
(IC50 153 M), Tyr-Leu-Ala-Gly-Asn-Gln (IC50 14 M), Phe-Phe-Leu
(IC50 37 M) and Ile-Tir-Leu-Leu (IC50 42 M). 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 enzymatic hydrolysis with the commercial protease Prozyme 6. The peptides
were identied as Gly-Thr-Trp and Gly-Val-Trp and had ACE inhibitor
activity with IC50 values of 464.4 and 240.0 M, respectively (Chen
et al., 2007). Hernndez-Ledesma, Quirs, 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 -casein were strong inhibitors of ACE,
with an IC50 of 21 M.
Chaves-Lpez et al. (2014) studied the effects of combined microbial
cultures previously identied 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 signicantly increase the levels of anti-hypertensive peptides. The most
treatment with Neutrase and the effect of fractionation by ultraltration

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 -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 -conglycinin) produced peptide proles that inhibited FAS and decreased lipid accumulation in vitro. The quantity of soy protein hydrolysates necessary to
inhibit 50% of FAS activity (IC50) ranged from 50175 M. A peptide
with anti-adipogenic abilities was isolated by ultraltration, gel ltration and reversed-phase HPLC from soy protein hydrolysates, and its
anti-adipogenic ability was conrmed by the inhibition of 3T3-L1 preadipocyte cell differentiation. The inhibitor was identied as a tripeptide
(Ile-Gln-Asn) with an IC50 value of 0.014 mg mL1 (Kim et al., 2007).
4.4. Peptides with anti-hypertensive activity
Arterial hypertension affects approximately 25% of the adult population 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 cardiovascular 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 because it catalyzes the conversion of angiotensin-I (the inactive form)
to angiotensin-II (a vasoconstrictor) and inactivates bradykinin (a vasodilator). 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 (Alemn et al., 2011).
It is well known that dietary proteins have primary sequences of
peptides able to modulate specic 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
HS
CH
Enalapril
H C
COOH
Ala-Pro
COOH
CH 3
CH 2
Phe-Ala-Pro
CH 3
H N
CH 3
CH 2 CO2
COOH
H N
2
COOH
Fig. 2. Structures of ACE inhibitor drugs and their analogous peptides (Matsui & Matsumoto, 2006).
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 g mL1.
The anti-hypertensive effect of a bovine casein peptide previously
identied 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 activity with an IC50 of 0.43 M. For the in vivo assays, the arterial pressure
of the animals was 171.7, 163.3 and 139.7 for the control, peptide solution and enalapril groups, respectively, indicating signicant 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 binding 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 proteins 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 required nutritionally, especially for amino acid uptake and 2) the free
amino acids and peptides in the milk are not sufcient 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
signicant increases in Bidobacterium 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 nal 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 hydrolyzed casein than with the other hydrolyzed protein fractions from
lacto-albumin, concentrated whey protein or puried whey protein.
5. Conclusion
Biologically active peptides can be dened as specic amino-acid sequences that promote benecial 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 purication and
identication 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 preservation, functional foods and pharmaceutical production.
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Food Research International 74 (2015) 185198

Contents lists available at ScienceDirect

Food Research International

Biologically active peptides: Processes for their generation, purication

physicochemical and sensory properties, such as solubility, viscosity,

R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185198

hormones and regulators (Hernndez-Ledesma, Garca-Nebot,

the use of digestive, plant or microbial proteolytic enzymes in a partial

2. Major processes for obtaining bioactive peptides

R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185198

Animal or vegetable protein sources

R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185198

Submerged fermentation for 5 h

Tsai, Chen, Pan, Gong,

Rice, soy and

Solid-state fermentation for 40 h

Inoue et al. (2009)

Zhu, Cheng, Wang,

Gly-Tyr; Ala-Phe; Val-Pro;

Nakahara et al. (2010)

Regazzo et al. (2010)

Submerged fermentation for 5 h at

Peptides with molecular

Peptides with molecular

Giri et al. (2011)

Peptides with molecular

Chang et al. (2013)

Peptides with molecular

Limn et al. (2015)

Soy and wheat

Bidobacterium longum KACC91563

phase high-performance liquid chromatography and identied by

R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185198

Liquid chromatography mass

Mejia et al. (2010)

Peptides with molecular

Tsou, Kao, et al., 2010

Tsou, Lin, et al., 2010

Crude protease from

Electrospray ionization mass

Adje et al. (2011)

Peptides with molecular

and linear acetonitrile gradients of 10 to 40%. Finally, the peptides were

Peptides with molecular

Ahn, Je, and Cho (2012)

Balti et al. (2015)

R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185198

Reversed phase high pressure liquid

Based on the hydrophobicity of

Useful method for the

Lack of retention of polar

Everley and Croley (2008).

Loss of highly hydrophobic

Issaq, Conrads, Janini, and

Long columns are required for

Mora et al. (2014).

Based on the afnity of bioactive

Based on the polarity and

The exibility of using a

The elution conditions are

The heat dissipated from the use

Production of singly and

The method has no