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Food Research International 74 (2015) 185–198

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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-
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.


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,

gelification and emulsion stability. Some proteins in the diet have
Proteins are fundamental food components. Nutritionally, they specific biological properties, making them potential ingredients for
are sources of essential amino acids, are indispensible for growth and functional foods (Korhonen, Pihlanto-Leppala, Rantamaki, & Tupasela,
maintenance, and are a source of energy. Protein foods are able to affect 1998). During digestion, proteins are hydrolyzed, generating a large
range of peptides. Some of these peptides have structural characteristics
⁎ Corresponding author. that allow them to interact with endogenous peptides. Many endoge-
E-mail address: (R.J.S. de Castro). nous peptides have important functions, acting as neurotransmitters,
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, the use of digestive, plant or microbial proteolytic enzymes in a partial
Fernández-Tomé, Amigo, & Recio, 2014). hydrolysis process, leading to a reduction of allergenic factors, im-
Bioactive peptides can be obtained from animal or plant proteins. proved digestibility and the formation of biologically active peptides
Plant sources are generally grains, such as wheat, rice, oats, rye and (Korhonen, 2009). One strategy used in several scientific studies dem-
corn, and some legumes, such as soy, peas and chickpeas. Of the plant onstrated that using lactic acid bacteria together with food-grade
sources, soy is one of the most widely studied as a source of peptides, enzymes resulted in final products with more interesting characteristics
as it is a significant source of dietary protein (Ortiz-Martinez, Winkler, than either process alone. Combining the techniques, in addition to
& García-Lara, 2014). Animal protein sources also have great potential. increasing the amount of peptides in the fermented products, resulted
One of the most popular and promising lines of research is the produc- in various biological and functional effects (Hafeez et al., 2014). Chen,
tion of hydrolyzed proteins from meat proteins, which, in addition to Tsai, and Pan (2007) studied a combined process using enzymatic
having important biological activities and being excellent sources of nu- hydrolysis with the microbial protease Prozyme 6 from Aspergillus and
trients, such as minerals and vitamins, can be used as flavor enhancers a commercial starter culture mixture of five lactic acid bacteria as a
and emulsifiers (Lafarga & Hayes, 2014; Mora, Escudero, Fraser, strategy to enhance the ACE-inhibitory activity of bioactive peptides
Aristoy, & Toldrá, 2014). The biological properties of other animal pro- from milk. The results showed that the pre-treatment of the fresh
tein sources, such as egg and fish, have also been studied (Sakanaka, milk with Prozyme 6 presented a positive impact on the ACE inhibitory
Tachibana, Ishihara, & Juneja, 2004; Theodore, Raghavan, & Kristinsson, activity of the peptides, in which the IC50 value for the combined
2008). process was 0.24 mg mL−1, in contrast to 0.64 mg mL−1 for the straight
Recent studies have linked the prevalence of cardiovascular disease, fermentation and 1.18 mg mL−1 for fresh milk.
obesity, hypertension, diabetes and cancer to nutritional factors. In re- In addition to the conventional methods mentioned above, combin-
sponse to increased awareness of the relationship between food and ing several technologies has produced effective results for generating
health, the market for functional foods has expanded. A functional functional peptides (Korhonen, 2009). Ultrafiltration and nanofiltration
food is any food that in addition to basic nutritional functions, provides are examples of technologies that have been used to refine and isolate
additional health benefits, regulating one or more functions in the body bioactive peptides, allowing them to be separated by size for use in
(Diplock et al., 1999; Hernández-Ledesma, Contreras, & Recio, 2011). specific applications (Picot et al., 2010; Quirós, Chichón, Recio, &
Processes incorporating protein hydrolysis have been studied to de- López-Fandiño, 2007).
termine whether they produce biologically active peptides. Mellander Understanding the critical parameters for the process is fundamen-
(1950) was responsible for the first study relating the ingestion of tally important for obtaining hydrolyzed proteins with desirable biolog-
bioactive peptides from hydrolyzed casein protein to increased bone ical and functional characteristics. These parameters include the protein
calcification in rachitic newborns. Since then, peptides with countless source and its characteristics, such as chemical composition, pH and
bioactivities have been identified. According to the Biopep and BioPD seasonal variations; enzymatic preparation and other aspects related
(Bioactive peptide database) databases, more than 1200 different bioac- to purity, substrate specificity, specific activity, pH and temperature
tive peptides have been recorded (Singh, Vij, & Hati, 2014). for activity and stability; and processing conditions, including enzyme
Bioactive peptides are defined as specific regions of proteins with and substrate concentrations, pH, temperature and reaction time.
amino acid sequences that have biological activity, including antioxi- Prior knowledge and identification of these parameters can be used as
dant, anti-hypertensive, antithrombotic, anti-adipogenic, antimicrobial, tools for obtaining products with distinct functions, for producing mul-
anti-inflammatory and immunomodulatory effects (Ahn, Cho, & Je, tifunctional peptides or even for producing unique peptides with specif-
2015; Biziulevicius, Kislukhina, Kazlauskaite, & Zukaite, 2006; Tavares ic functions (Li-Chan, 2015; Samaranayaka & Li-Chan, 2011).
et al., 2011; Tsou, Kao, Tseng, & Chiang, 2010; Zhang, Li, & Zhou, This review describes advances in scientific research on the process-
2010). These peptides have 2–20 amino acids and molecular masses es of obtaining, purifying and identifying the biological activities and
of less than 6000 Da. Their bioactivity is mainly determined by their potential applications of bioactive peptides.
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 2. Major processes for obtaining bioactive peptides
the forefront of the biotechnology fields (Miranda & Liria, 2008); fur-
thermore, these peptides and proteins have been identified by several 2.1. Fermentation
authors as possible substitutes for chemicals used as drugs or food
preservatives (Hong et al., 2008; Uhlig et al., 2014). The use of fermentation processes to make bioactive peptides is
According to Uhlig et al. (2014), there is a very good outlook for mainly relevant to dairy product manufacturing. Dairy products natu-
using bioactive peptides in the pharmaceutical field. Some peptides in rally contain precursor proteins for bioactive molecules (Akalin, 2014;
the clinical trial phase have shown very promising results for treating Schanbacher, Talhouk, & Murray, 1997). Fermenting milk involves a
cardiovascular, infectious and metabolic diseases. Peptides have an number of metabolic pathways responsible for generating metabolites,
important competitive advantage over traditional medications for the which significantly contribute to the chemical, biochemical and nutri-
following reasons: 1) They have high specificity for their target tissues, tional properties of the fermented products. The proteolytic system of
resulting in little or no toxicity, and even low concentrations can be ef- lactic acid bacteria (LAB) is complex and consists of three major compo-
fective. This characteristic is extremely important for treating chronic nents: proteases bound to the cell wall that promote the initial hydroly-
diseases; 2) Synthetic chemical compounds that are typically used as sis of milk casein into oligopeptides, specific transporters that transfer
drugs often have a cumulative effect on the organism. These synthetic the oligopeptides to the cytoplasm and intracellular peptidases that fin-
substances may represent an environmental problem due to their ex- ish the hydrolysis process to convert oligopeptides into free amino acids
cretion, still in the active form. In contrast, bioactive peptides undergo and/or low molecular weight peptides (Chaves-López et al., 2014). The
little or no accumulation in the organism, and they are easily degraded ability of these microorganisms to produce proteolytic enzymes makes
in the environment (Uhlig et al., 2014). them potential producers of bioactive peptides, which can be released
Several methods are used to obtain bioactive peptides, including fer- during fermented product manufacturing. Several microorganisms
mentation, enzymatic hydrolysis and a combination of the two process- have been extensively reported in the literature as having an effective
es (Fig. 1). In fermentation, the addition of lactic acid bacteria with proteolytic system for protein hydrolysis and the release of bioactive
proteolytic activity leads to formation of bioactive peptides, especially peptides, including Lactobacillus helveticus, Lactobacillus delbrueckii ssp.
during dairy product manufacturing. Enzymatic hydrolysis involves 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

Proteases produced
during fermentation
High pressure

Protein hydrolysates
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- temperature, time) affect the size and the amino acid sequences in the
Ledesma et al., 2011). In addition to using live microorganisms, proteo- peptide chains as well as the quantity of free amino acids, which can af-
lytic enzymes isolated from LAB have also been successfully used in fect the biological activity of the hydrolysates (Luna-Vital et al., in press;
enzymatic hydrolysis processes and for the production of bioactive Sarmadi & Ismail, 2010; Su, Ren, Yang, Cui, & Zhao, 2011; Tsou, Kao,
peptides (Choi, Sabikhi, Hassan, & Anand, 2012). et al., 2010; Zhou, Canning, & Sun, 2013). Proteases with specific activi-
Although dairy products have been highlighted in scientific studies ty, such as trypsin and chymotrypsin, and combinations of different
producing these peptides by fermentation, it has been shown that non-specific proteases, such as Pronase™ E from Streptomyces griseus
fermentation products derived from soy, beans, rice and wheat are and Flavourzyme™ from A. oryzae, have been used to produce more
also biologically active (Hati et al., 2014; Inoue et al., 2009; Limón stable and effective bioactive peptides by reducing the reaction times
et al., 2015; Nakahara et al., 2010) (Table 1). Species of filamentous needed for hydrolysis and by making it possible to obtain different pro-
fungi such as Aspergillus oryzae and Aspergillus sojae have a long tradi- files, especially for the composition and molecular mass distribution of
tion of safe use in the production of fermented foods, in which several the peptides. These processes are especially useful in the food and phar-
peptides with biological activities were detected, for example antioxi- maceutical industries, which rely on animal, plant and microbial prote-
dant and antihypertensive activities (Giri, Osako, Okamoto, Okazaki, & ases (de Castro, Bagagli, & Sato, 2015; Singh et al., 2014; Vanderghem
Ohshima, 2011; Inoue et al., 2009; Nakahara et al., 2010). et al., 2011).
In addition to the commercial enzymes, it is important to note that
2.2. Enzymatic hydrolysis several studies reported the use of crude microbial enzymes to hydro-
lyze proteins, suggesting the potential application of novel protease
Enzymatic hydrolysis is one of the fastest, safest and most easily con- sources for the production of bioactive peptides.
trolled techniques for producing bioactive peptides, and it can be used Table 2 summarizes some studies in which the release of biologically
to improve the functional and biological properties of the proteins active peptides after protein hydrolysis using commercial and crude
as well as to add value to byproducts with low commercial value protease preparations was demonstrated.
(Luna-Vital, Mojica, de Mejía, Mendoza, & Loarca-Piña, in press; Mora
et al., in press; Singh et al., 2014; Zarei et al., 2014). 3. Concentration, purification and identification of bioactive
Proteases catalyze the hydrolysis of peptide bonds in proteins and peptides
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 Table 3 summarizes different characteristics for some methods of
amino acids directly surrounding the bond that is cleaved (Santos & purification and identification of bioactive peptides, including their
Koblitz, 2008). This specificity and the hydrolysis conditions (pH, 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- phase high-performance liquid chromatography and identified by
matography (HPLC) and ultra high pressure liquid chromatography mass spectrometry. The hydrolysate solution containing the peptides
(UHPLC) (Singh et al., 2014). UHPLC has shown great potential in the was applied to a Sephadex G-25 (5.2 × 56 cm) gel filtration column
separation of small bioactive peptides, increasing the throughput of reg- pre-equilibrated and eluted with distilled water, and 4.5-mL fractions
ular HPLC methods. The main advantages of this method include the in- were collected using a flow rate of 0.5 mL min− 1. Absorption at
crease of throughput, resolution and sensitivity (Everley & Croley, 2008; 220 nm was measured to determine the peptide elution profile. Frac-
Fekete & Guillarme, 2014). Reversed phase HPLC (RP-HPLC) can be used tions with higher anticoagulant activity were recovered and purified
to separate peptides by hydrophobicity (Pownall, Udenigwe, & Aluko, in a reverse-phase Vydac C18 (10 × 250 mm, Grace-Vydac) column
2010). Hydrophilic interaction liquid chromatography (HILIC) has and eluted using a linear acetonitrile gradient (0 to 40% v/v) and a
been shown to be a useful method for the separation of hydrophilic sub- flow rate of 0.6 mL min− 1. The molecular mass and amino acid
stances. This method is based on increases in retention with increasing sequence of the peptides were measured using a triple quadrupole
polarity of the stationary phase and of the solutes and the decreasing mass spectrometer with an electrospray ionization source (Applied
polarity of the predominantly organic solvent system used for elution; Biosystems API 3000, PE Sciex, Toronto, Canada). Four peptide
the opposite principle of that observed in RP-HPLC (Yoshida, 2004). Le sequences had high anticoagulant activity and were identified as Leu-
Maux, Nongonierma, and FitzGerald (2015) reported HLIC method as Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Leu-Arg and Cys-Arg-Arg (Nasri
a valuable tool to improve the separation of short peptides and differen- et al., 2012).
tiation of peptides with homologous sequences by mass spectrometry. Tsou, Kao, Lu, Kao, and Chiang (2013) purified and identified bioac-
Gel electrophoresis and ultrafiltration techniques have also been used tive peptides from purified soy protein and the Flavourzyme protease
as auxiliary methods for structural and chemical composition analysis using sequential fractionation with ultrafiltration membranes of various
of peptides (Roblet et al., 2012; Singh et al., 2014). sizes, gel chromatography, reversed-phase high-performance liquid
Mass spectrometry has greatly improved the process of identifying chromatography and mass spectrometry. The hydrolysates were initial-
peptide sequences and studying protein profiles and hydrolysis prod- ly fractionated in ultrafiltration membranes of 30, 10 and 1 kDa. The
ucts. In particular, interfaces have been developed that allow ions to fraction retained on the 1-kDa membrane was selected for purification
be generated from analyte molecules that are sensitive to temperature due to its ability to stimulate lipolysis in 3T3-L1 pre-adipocyte cells.
and/or are not very volatile. Electrospray ionization and matrix assisted The 1-kDa retained portion was then applied to a Superdex™ peptide
laser desorption/ionization (MALDI-TOF), for example, has recently be- 10/300 GL column (10 × 300 mm; GE Healthcare), equilibrated and
come important for the identification and characterization of bioactive eluted with 30% acetonitrile and a flow rate of 0.5 mL min− 1. One-
peptides and proteins using mass spectrometry. Liquid chromatogra- milliliter fractions were collected, and elution curves were constructed
phy–mass spectrometry is commonly used to identify peptide se- based on absorbance measurements at 214 nm. The fractions with the
quences (Chiaradia, Collins, & Jardim, 2008; Contreras, López-Expósito, highest anti-adipogenic activity were collected and purified in a
Hernández-Ledesma, Ramos, & Recio, 2008; Singh et al., 2014). Develosil ODS-HG-5 reverse-phase column (4.6 × 250 mm, Nomura
Peptides with anticoagulant activity that were obtained from goby Chemical) and eluted using a linear acetonitrile gradient (5.0 to 75.0%)
fish (Awaous guamensis) and a protease from Bacillus licheniformis and a flow rate of 1.0 mL min−1. The fraction with the most anti-
were separated by molecular exclusion chromatography and reversed- 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)
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

and linear acetonitrile gradients of 10 to 40%. Finally, the peptides were most antioxidant peptides were identified as Thr-Asp-Tyr, Leu-Asp-
identified by liquid-chromatography coupled with mass spectrometry. Tyr, Trp-Asp-Asp-Met-Glu-Lys, Trp-Gly-Asn-Val-Ser-Gly-Ser-Pro, Leu-
Three peptides with the amino acid sequences Ile-Leu-Leu, Leu-Leu- Tyr-Glu-Gly-Tyr and Met-Glu-Met-Lys.
Leu and Val-His-Val-Val were identified as being responsible for the It is important to note that each method showed a basic principle for
anti-adipogenic activity of the protein hydrolysates isolated from soy. the separation of the bioactive peptides, as shown in Table 3. However,
Peptides with anti-hypertensive activity were isolated and identified in a complex mixture of peptides, common problems are the separation
from gelatin hydrolysates extracted from stingray skin (Okamejei of small and big peptides or peptides with different physicochemical
kenojei). The hydrolysates first were subjected to ultrafiltration through properties, which makes their subsequent identification difficult.
a 1-kDa membrane, and peptides with molecular weights lower than These problems can be solved by a combination of different separation
this cutoff were collected. Purification consisted of sequential steps of techniques before injection into the mass spectrometer. A practical
isolation by fast protein liquid chromatography (FPLC) (AKTA, example is the separation of peptides containing hydrophobic amino
Amersham Bioscience Co., Uppsala, Sweden) using a HiPrep 16/10 acids and peptides composed of only hydrophilic amino acids. In this
high flow ionic exchange column (16 × 100 mm, Amersham Biosci- case, a combination of RP-HPLC with HILIC can be used for an efficient
ences, Piscataway, NJ, USA) and a GE Healthcare Superdex™ Peptide separation of the peptides with hydrophobic and hydrophilic character-
10/300 GL gel filtration column (10 × 300 mm). Purified peptides istics, respectively (Panchaud, Affolter, & Kussmann, 2012).
were then identified by MALDI-TOF mass spectrometry. Two purified In addition, an interesting approach was proposed by Le Maux et al.
peptides were found to be very anti-hypertensive and were identified (2015). These authors affirmed that liquid chromatography coupled to
as Leu-Gly-Pro-Leu-Gly-His-Gln, with an estimated molecular weight mass spectrometry (LC–MS/MS) providing the necessary data for pep-
of 720 Da, and Met-Val-Gly-Ser-Ala-Pro-Gly-Val-Leu, with a molecular tide sequencing. However, although this strategy has been successfully
weight of 829 Da (Ngo et al., 2015). used for longer peptides, the identification of short peptides can be
Liu et al. (2015) developed a UHPLC-Q-TOF MS/MS method to iden- more difficult, due to the presence of peptides with the same amino
tify peptides with antioxidant activities derived from the protein hydro- acid composition but a different sequence. They showed that the
lysate of Mactra veneriformis. The hydrolysates were fractionated on a method HLIC-MS/MS and the parallel determination of the apparent hy-
Sephadex G-25 gel filtration column (2.0 cm × 100 cm; GE Chemicals, drophilicity of each peptide for the development of a retention time pre-
Uppsala, Sweden) using distilled water as the eluting solvent at a flow diction model could be used as a valuable tool to improve the separation
rate of 0.4 mL min−1, and separated five fractions. The two most active of short peptides and the differentiation of peptides with homologous
fractions were then separated on the basis of their antioxidant activities sequences.
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 4. Biological properties of bioactive peptides
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 Bioactive peptides from dietary proteins have been extensively
Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF MS/MS) in studied over the last decade to determine their potential uses and
which the MS spectra were acquired in the m/z range of 50–2000. their effects on the major systems of the human body, such as the diges-
This method allowed for the identification of 21 peptides, and the tive, cardiovascular, nervous and immune systems. Several bioactive
190 R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198

Table 3
The main characteristics of the different analytical methods for the purification and identification of bioactive peptides.

Method of Mechanism Advantage Limitation Reference


Reversed phase high pressure liquid Based on the hydrophobicity of Useful method for the Lack of retention of polar Everley and Croley (2008).
chromatography (RP-HPLC) proteins or peptides that can interact isolation of complex molecules. Slow intrapore diffusion Le Maux et al. (2015).
differently to the reversed-phase peptide mixtures. times. The presence of unresolved Yang, Boysen, Chowdhury,
material of the chromatography structural microheterogeneity and Alam, and Hearn (2015).
column. conformational isomers. Secondary
interactions with the stationary
Affinity chromatography Based on the affinity of bioactive The flexibility of using a Tone must know the Hage et al. (2012).
peptides to interact specifically and large number of binding physicochemical properties of the Ortiz-Martinez et al. (2014).
reversibly with a complementary agents, allows for the ligands, which limits its use for a
molecule bound to a solid support separation of different complex mixture of unknown
immobilized on a column. types of peptides. peptides.
Ion-exchange chromatography (IEC) Based on the ability of charged Appropriate method for Low selectivity and requires Bouhallab, Henry, and
bioactive peptides to interact with a the separation of highly complementary steps for the Boschetti (1996).
solid support bearing the opposite cationic or anionic separation of the fractions. Ortiz-Martinez et al. (2014).
charge. peptides.
Isoelectric focusing (IEF) Based on the separation of The method allows one to Loss of highly hydrophobic Issaq, Conrads, Janini, and
protein/peptide solutions according to fractionate a complex proteins in the sample Veenstra (2002).
their isoelectric points (pI). A focusing mixture of peptides preparation and precipitation of Guijarro-Díez, García, Crego,
cell containing a mixture of according to their pI. neutral proteins at their pI, which and Marina (2014)
proteins/peptides and a carrier can result in overlapping between
ampholyte is subjected to an electric different fractions.
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 The elution conditions are Long columns are required for Mora et al. (2014).
(SEC) peptides according to the retention considered mild, allowing complex peptide mixtures, which Fekete, Beck, Veuthey, and
time of the molecules in the stationary the characterization of the can be obtained by joining Guillarme (2014).
phases particles with a carefully protein with minimal multiple columns in a series. This
controlled pore size, in which the impact on the strategy is necessary to improve
molecules are separated from each conformational structure the separation resolution.
other according to their molecular size. and the local environment.
Ultra high pressure liquid Based on separation of the molecules Increased throughput, The heat dissipated from the use Everley and Croley (2008).
chromatography (UHPLC) using experimental columns packed resolution and sensitivity of small particles at ultra-high Uliyanchenko,
with very small particles of a in separation of complex pressures may increase Schoenmakers, and van der
non-porous material, carrying out the protein mixtures. chromatographic band Wal (2011).
analyses at very high pressures. broadening and compromise Fekete and Guillarme (2014).
efficiency of the column.
Hydrophilic interaction liquid Based on the polarity and The method shows great Compared to RP-HPLC, the Gray et al. (2013).
chromatography (HILIC) hydrophilicity of bioactive peptides potential for the separation method shows limited flexibility Le Maux et al. (2015).
separated using polar chromatographic of short peptide sequences and applicability, problems with
surfaces (stationary phase) and a (b5 amino acids) and sample solubility and the
highly organic mobile phase improves the identification retention mechanisms are poorly
(N70% solvent) also containing a small using mass spectrometry. understood.
percentage of aqueous solvent/buffer or
other polar solvent.
Electrospray ionization mass Based on the transformation of an Production of singly and The efficiency of identification is Mano and Goto (2003).
spectrometry (ESI/MS) aqueous solution with uniform multiply charged ions, directly related to the Contreras et al. (2008).
electrical density to gas-phase ions, by allowing for an accurate chromatographic method used for Panchaud et al. (2012).
passing a high voltage through a thin measurement of the the prior separation of the
capillary. The gas-phase is transferred molecular weight of the bioactive peptides before
into a mass analyzer and separated peptides. injection into the mass
according to the mass-to-charge spectrometer. Therefore, a
(m/z) ratio. combination of different
Matrix-assisted laser Based on co-crystallization of the The method has no separation techniques is
desorption/ionization-time-of-flight analytes when they are mixed with a theoretical upper limit to necessary for accurate
mass spectrometry matrix solution on a target plate. The the m/z ratio, allowing for identification.
(MALDI-TOF/MS) co-crystal is subjected to the action of the analysis of complex
pulsed laser, causing the accumulation samples with a wide range
of high-density energy which results in of molecular weights.
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.

peptides have biological activities that are beneficial for human health, (Zhang et al., 2009) anticancer (Alemán et al., 2011), anti-adipogenic
including antimicrobial (Adje, Balti, Kouach, Guillochon, & Nedjar- (Tsou, Kao, et al., 2010), immunomodulatory (Huang, Chen, Chen,
Arroume, 2011), anti-hypertensive (Alemán et al., 2011), antioxidant Hong, & Chen, 2010) and anti-inflammatory effects (Ahn et al., 2015).
R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198 191

Therefore, they can potentially be incorporated into functional foods, Peptides with antimicrobial activity were prepared from gelatin hy-
nutraceuticals and medications, where this bioactivity can aid in drolysate with Alcalase™ 2.4 L (Sigma-Aldrich, United States). Fractions
preventing and controlling diseases (Agyei & Danquah, 2012). obtained from ultrafiltration through 1- and 10-kDa membranes were
This review describes efforts to identify and characterize peptides used for antimicrobial tests against 18 bacteria. The most sensitive bacte-
with antimicrobial, antioxidant, anti-adipogenic and anti-hypertensive ria in the presence of the tested fractions were Lactobacillus acidophilus,
activity, and it discusses their use in the growth of lactic acid bacteria Bifidobacterium lactis, Shewanella putrafaciens and Photobacterium
and other probiotic bacteria. 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
4.1. Peptides with antimicrobial activity enteritidis) and three Gram+ strains (Kocuria luteus A270, Staphylococcus
aureus and Listeria innocua). The results showed that the purified peptide
Over the last few decades, a growing number of pathogenic microor- fractions had a wide spectrum of action, affecting 4 of the 5 tested bacteria
ganisms have developed resistance to conventional antibiotics, causing (Kocuria luteus A270, L. innocua, E. coli and S. aureus), with a MIC between
serious problems treating infections, especially in immunocompro- 35.2 and 187.1 μM (Adje et al., 2011).
mised individuals. In addition, the development of new antibiotics has Tellez, Corredig, Turner, Morales, and Griffiths (2011) demonstrated
slowed over this same period. Two major causes underlie the increase the efficiency of a peptide fraction isolated from milk fermented with
in antibiotic resistance in microorganisms: the indiscriminate use of an- L. helveticus against an experimental infection of S. enteritidis in rats.
tibiotics for in small doses or with ineffective treatment times, and the The survival rate of the group fed with the peptide fraction (0.02 μg
genetic mutation capacity of the microorganisms, which increases the per day) was higher than the group fed with half of the dose (0.01 μg
difficulty of developing drugs based on specific mechanisms of action per day) and higher than the control group.
(Harrison, Abdel-Rahman, Miller, & Strong, 2014). Thus, using natural The antimicrobial powers of protein isolated from whey hydrolyzed
sources of antimicrobial compounds has enormous potential because with various gastrointestinal enzymes were demonstrated by Théolier,
they have characteristics such as low toxicity and high specificity. The Hammami, Labelle, Fliss, and Jean (2013). These authors showed that
mechanisms of these natural antimicrobial compounds can be better hydrolyzed proteins from trypsin and chymotrypsin digests did not
understood if we compare their modes of action against bacterial have antibacterial activity against Listeria ivanovii HPB28 and E. coli
(unicellular) and animal (multicellular) cells. Bacterial cells have a MC4100, but they found that hydrolysates of pepsin had significant
layer rich in negatively charged phospholipids pointing toward the ex- activity. Hydrolysates were fractionated by reverse-phase high-
ternal environment, facilitating their interactions with peptides, most performance liquid chromatography, resulting in five fractions with
of which are positively charged. In contrast, animal cells are mainly high antibacterial activity and MIC values between 20.0 and
composed of uncharged lipids in the outermost layer, and the negatively 35.0 μg mL− 1. A peptide fraction obtained from wastewater from
charged regions are pointed toward the cell interior (cytoplasm) cooking anchovies (Engraulis japonicus) was digested by the Protamex
(Matsuzaki, 1999). enzyme and had high antimicrobial activity against S. aureus. The iden-
Antimicrobial peptides are widely distributed in nature and are es- tified fraction had the peptide sequence Gly-Leu-Ser-Arg-Leu-Phe-Thr-
sential to the immune system. They are the organism's first line of de- Ala-Leu-Lys and an estimated molecular weight of 1.1 kDa (Tang, Zhang,
fense against colonization by exogenous microorganisms, and they Wang, Qian, & Qi, 2015). Due to their hydrophobicity, bioactive peptides
play a fundamental role in regulating bacterial populations on the mu- containing sequences rich in the amino acids Gly and Leu were reported
cosa and other epithelial surfaces (Bevins & Zasloff, 1990; Boman & as potent antimicrobial molecules. The presence of the Arg residue in
Hultmark, 1987; Zasloff, 2002). More than 800 antimicrobial peptides the peptide sequence also plays an important role in antimicrobial
have been described in plants and animals (Boman, 2003). Despite activity, increasing interactions with bacterial cell walls, due its cationic
great diversity in their primary structures, most antimicrobial peptides characteristic (Amadou, Le, Amza, Sun, & Shi, 2013; Sousa et al., 2009;
are similar in that they are short amino acid chains composed primarily Tang et al., 2015).
of cationic and hydrophobic amino acids (Dashper, Liu, & Reynolds,
2007; Zasloff, 2002). The low molecular weights of the peptide fractions, 4.2. Peptides with antioxidant activity
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 The creation of free radicals, such as superoxide (O− 2 ) and hydroxyl
antimicrobial activity. These features promote interactions between (OH), is one of the inevitable consequences of respiration in aerobic
the peptide and the membrane (Gobbetti, Minervini, & Rizzello, 2004; organisms. These radicals are very unstable and react quickly with
Gómez-Guillén et al., 2010; Patrzykat & Douglas, 2005). other groups or substances in the organism, causing cellular and tissue
The exact mechanisms of action for many antimicrobial peptides damage (Zhang et al., 2009). An excessive amount of these radicals in
have not been well established. Due to the large number of known pep- the organism has been linked to the development of several diseases,
tides, it is likely that there are additional mechanisms of action yet to be such as atherosclerosis, arthritis, diabetes and cancer (Gu et al., 2015).
discovered (Dashper et al., 2007). Because they are highly reactive species, free radicals can damage
In addition to the peptides that are naturally present in the defense proteins, mutate DNA, oxidize membrane phospholipids and modify
systems of plants and animals, peptides with antimicrobial activity low-density lipoproteins (LDL) (Pihlanto, 2006). In food, oxidation
have been identified in several protein hydrolysates. also directly affects quality, negatively affecting characteristics such as
Hydrolysates of casein from cow's milk obtained by enzymatic hy- taste, aroma and color. Thus, substances that inhibit oxidation reactions
drolysis using chymosin were analyzed for their antimicrobial power. are useful for maintaining food quality.
Five different antibacterial peptides were isolated from the carboxylic An antioxidant's ability to remove free radicals is determined by var-
end of αs2-casein. Peptide fractions f (181–207), f (175–207) and f ious factors, including chemical reactivity, the rate of removal of the
(164–207) had a wide spectrum of activity and were able to inhibit sev- compound, the fate of the product of the antioxidant–radical reaction,
eral Gram+ and Gram− bacteria; the minimum inhibitory concentra- interactions with other antioxidants, concentration and mobility in the
tion (MIC) of each fraction ranged from 21.0 to 168.0 mg mL−1, 10.7 environment and the compound's absorption, distribution, retention
to 171.2 mg mL−1 and 4.8 to 76.2 mg mL−1, respectively. The inhibitory and metabolism (Niki, 2010).
power of these peptides against Gram+ bacteria was as strong as the Antioxidants are thought to be important nutraceuticals with vari-
known antimicrobial peptides nisin and lactoferricin B (Mccann et al., ous health benefits. They are defined as substances that significantly
2005). 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 on the enzyme used, with values ranging from 36.53 to 89.17%. The hy-
hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert- drolysates obtained from 60 min of hydrolysis using papain had the
butylhydroquinone (TBHQ), are used to prevent oxidative damage in highest antioxidant activity by TBARS testing, Fe2+ chelation and Fe3+
foods and biosystems. However, these products are being used less reduction. The hydrolysates obtained with Alcalase had higher activity
due to their potential risks to human health, such as DNA damage and against the DPPH radical, whereas those obtained with Flavourzyme
toxicity (Chi, Wang, Wang, Zhang, & Deng, 2015; Wang et al., 2014). had greater activity against the ABTS radical.
Consequently, there is growing interest in finding safer antioxidants Measuring antioxidant activity by in vivo methods is mainly based
from natural sources, such as peptides from hydrolyzed proteins (Chi, on measurements of enzymatic activity. Decreases in the activities of
Wang, et al., 2015; Senphan & Benjakul, 2014). antioxidant enzymes, such as catalase (CAT), glutathione peroxidase
Some peptides with antioxidant activity occur naturally in food. (GPx), superoxide dismutase (SOD) and glutathione-S-transferase
Glutathione (γ-Glu-Cys-Gly) and carnosine (β-alanyl-L-histidine) are (GST), can dramatically affect the susceptibility of many tissues to oxi-
antioxidants that are naturally present in muscle tissues. They can dative stress, and impairments of these enzymes are associated with
donate electrons, chelate metals and ions and inhibit lipid peroxidation several diseases (Ktari et al., 2014). Liu et al. (in press) studied the anti-
(Samaranayaka & Li-Chan, 2011). In addition to the antioxidants that oxidant properties of protein hydrolysates from corn gluten prepared
are naturally present, peptides from hydrolyzed dietary proteins have using the proteases Alcalase, Flavourzyme and Protamex. The antioxi-
been reported to have antioxidant capabilities similar to or better than dant activities of the hydrolysates were analyzed by in vivo methods.
synthetic antioxidants, such as BHT, making them safe for food applica- The experiments were performed in 40 4–5 week old Kunming line
tions (Chi, Hu, Wang, Li, & Ding, 2015; Mora et al., 2014; Rao et al., 2012; mice (25.0 ± 2.0 g). Mice were randomly divided into five experimental
Yasufumi, Shigeki, Keiji, & Tomoyuki, 2001). groups, with eight animals and an equal number of males and females in
The mechanisms of action for the antioxidant activities of peptides each group. The groups were then treated as follows: group I was used
are not fully understood, but several studies have demonstrated the as a control and received the base diet common to all groups; group II
ability of peptides to inhibit lipid peroxidation (Sakanaka et al., 2004), was treated daily with vitamin E (83 mg/kg/day) for 10 days and groups
remove free radicals (Gómez-Guillén et al., 2010), chelate metal ions III, IV and V were treated with 300, 700 and 1000 mg/kg/day of hydro-
(Alemán et al., 2011) and eliminate reactive oxygen species (Zhuang lysates, respectively, for 10 days. The results showed that the hydroly-
& Sun, 2011). Similar to other biological activities, the antioxidant prop- sates created with a combination of Alcalase and Protamex enzymes
erties of peptides are related to their composition, structure and hydro- produced peptides with the highest levels of antioxidant activity.
phobicity (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998). The distribution of the molecular weights in the hydrolysates showed
The presence of the amino acids Tyr, Trp, Met, Lys and Cys was reported that the peptides were between 250 and 1200 Da. The ingestion of
to be an important factor in the antioxidant activities of the peptides, es- 300 mg peptides/kg resulted in increased superoxide dismutase and
pecially due to their ability to reduce Fe3+ to Fe2+ and to chelate Fe2+ glutathione-peroxidase activity and reduced the malonaldehyde levels
and Cu2+ ions (Carrasco-Castilla et al., 2012; Huang et al., 2010; Wang in the liver and blood of the mice compared to the control group, indi-
& De Mejia, 2005). Aromatic amino acids, such as Trp, Tyr, Phe have phe- cating great antioxidant potential.
nolic, indole and imidazole groups, respectively, which can act as proton
donors to electron deficient radicals and efficiently scavenge them 4.3. Peptides with anti-adipogenic activity
(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- Obesity results from disequilibrium between energy intake and
ing, lipid trapping and decomposition of the imidazole ring (Saidi, energy expenditure, leading to pathological growth of adipocytes
Deratani, Belleville, & Amar, 2014). Not only the presence but also the (Aoyama, Fukui, Takamatsu, Hashimoto, & Yamamoto, 2000). The quan-
sequence of the peptide chain plays an important role in determining tity of adipose tissue can be controlled by inhibiting adipogenesis in
antioxidant power (Rajapakse, Mendis, Jung, Je, & Kim, 2005). precursor or pre-adipocyte cells, such as pre-adipocyte 3T3-L1 cells,
The antioxidant abilities of peptides can be analyzed by several which are the best-characterized models for studying adipogenesis.
in vitro methods, which test for various mechanisms of action and Many transcription factors are involved in the differentiation of pre-
thus measure distinct activities (Table 4). adipocyte cells into adipocytes, and their inhibition or regulation may
In addition to in vitro analysis, antioxidant activity can be analyzed lead to decreased fat accumulation in the organism (Tsou, Kao, et al.,
in vivo using animal models. Antioxidant ability is measured in vivo 2010). Glycerol-3-phosphate dehydrogenase (GPDH) is a key enzyme
using several factors, as these substances must be absorbed, transported, for glucose metabolism and is linked to biosynthesis of phospholipids
distributed and retained sufficiently in biological fluids, cells and tissues. and triglycerides (Harding, Pyeritz, Copeland, & White, 1975; Tsou,
The bioavailability of these compounds, the effects of dose and the Lin, Lu, Tsui, & Chiang, 2010). Suppressing GPDH activity may inhibit
duration of the treatments have been studied by analyzing human differentiation and reduce lipid accumulation in 3T3-L1 cells. Thus, the
and animal biological fluids and tissues after ingestion (Alam, Bristi, & activity of this enzyme can be measured to evaluate anti-adipogenic
Rafiquzzaman, 2013; Niki, 2010). Table 5 shows some of these methods effects (Hirai, Yamanaka, Kawachi, Matsui, & Yano, 2005). Another
and the principles underlying the analyses. enzyme involved in adipogenesis is fatty-acid synthase (FAS), which
In vitro methods are more commonly used for analyzing antioxidant participates in the endogenous synthesis of saturated long-chain
activity in hydrolyzed proteins. Nazeer and Kulandai (2012) analyzed fatty-acids from the precursors acetyl-CoA and malonyl-CoA (Maier,
the antioxidant properties of fish protein hydrolysates obtained from Leibundgut, & Ban, 2008; Rahman et al., 2008). It has been reported
enzymatic treatment with various proteases (papain, pepsin, trypsin that certain fractions of hydrolyzed proteins are able to inhibit the activ-
and chymotrypsin). Antioxidant activity was measured in terms of ity of these enzymes, thus regulating the process of cell differentiation
DPPH radical reduction, iron reduction and the ability to chelate metals. and relative accumulation of lipids. According to Kim, Bae, Ahn, Lee,
All of the hydrolysates had antioxidant activity, especially those made and Lee (2007), these hydrolysates have great potential in obesity treat-
with pepsin and trypsin. Li, Luo, Shen, and You (2012) showed that pro- ments because they decrease fat accumulation in the organism.
tein hydrolysates from carp prepared with Alcalase™ 2.4 L and papain Martinez-Villaluenga, Bringe, Berhow, and Mejia (2008) studied the
had antioxidant activity according to tests using ABTS, DPPH, Fe3+ re- production of soy protein hydrolysates using a commercial preparation
duction and Fe2+ chelation. Najafian and Babji (2015) studied the anti- of Alcalase proteases. The hydrolysates were analyzed for their effects
oxidant activity of myofibril protein from fish (Pangasius sutchi) that on relative lipid accumulation in 3T3-L1 pre-adipocyte cells, and they
was hydrolyzed using enzymatic preparations of the proteases papain, showed a 29.0 to 46.0% decrease in lipid accumulation. The authors
Alcalase and Flavourzyme. The degree of hydrolysis varied depending 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 obtained by ultrafiltration. The results revealed that the partial hydroly-
larger number of peptides responsible for the inhibition of lipid accu- sis of proteins isolated from soy provided hydrolysates with strong
mulation in 3T3-L1 cells than the glycinin subunits. anti-adipogenic capacity, and fractions obtained by ultrafiltration
Tsou, Kao, et al., 2010 studied the use of commercial preparations more efficiently inhibited GPDH activity. Specifically, the 1-kDa mem-
of Flavourzyme™ proteases on protein hydrolysates isolated from soy brane fraction was the most effective (59.0% inhibition). The anti-
by analyzing the anti-adipogenic capacity of the hydrolysate fractions 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) Peptide isolate from Male adult Liver SOD is an enzyme that catalyzes the Liu, Kong, Li, Liu, and
hydrolyzed pig plasma Wistar rats dismutation of superoxide radicals into Xia (2011)
hydrogen and oxygen, thus playing an
important role in protecting cells against
reactive oxygen species
Catalase (CAT) Peptide isolated from Albino male Erythrocyte CAT is an enzyme that converts hydrogen Nazeer, Kumar, and
hydrolyzed fish protein 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
Level of reduced glutathione Protein isolates from Albino male rats Liver and blood GSH is an intracellular reducer that plays Soliman, Abu-El-Zahab,
(GSH) seeds from Syrian rue plasma an important role for protecting cells and Alswiai (2013)
(Peganum harmala) from free radicals, peroxides and other
toxic compounds
Glutathione-S-transferase Peptide isolated from Male adult rats Liver GST is an enzymatic complex in the Kim et al. (2013)
(GST) hydrolyzed mussel cytosol that catalyzes the binding of
protein reactive electrophilic molecules with
glutathione, facilitating the metabolism
and excretion of toxins and consequently
reducing cell damage and DNA damage
Glutathione peroxidase Hydrolyzed corn gluten Male and female Liver and blood GPx is an enzyme that catalyzes the Liu et al. (in press)
(GPx) Kunming mice plasma reaction between hydroperoxide and
reduced glutathione leading to the
formation of glutathione disulfite and the
product of hydroperoxide reduction
Measurement of the level of Hydrolyzed fish protein Male adult Liver and blood Malanodialdehyde is an intermediate Ktari et al. (2014)
malonaldehydes (Salaria basilisca) 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 and obtained an interesting result in which no relationship was found
on activity were studied by Tsou, Lin, et al., 2010. Similar to the previous between hydrophobicity/size and the IC50 values of the ACE-inhibitory
study, the results showed that low molecular weight peptides (between activity, indicating that it was the sequence of peptides from the
1300 and 2200 Da) most effectively inhibited GPDH activity. wheat gluten hydrolysate that was mainly responsible for the ACE-
Mejia, Martinez-Villaluenga, Roman, and Bringe (2010) analyzed the inhibitory activity, although several studies have shown that the size
effects of soy protein hydrolysates enriched with β-conglycinin (a and hydrophobic character of the peptides exert a strong influence on
protein naturally found in soy) on FAS activity and adipogenesis in this bioactivity (Aluko et al., in press; Li, Le, Shi, & Shrestha, 2004;
human adipocytes in vitro. The results showed that genotypic alterations Wijesekara et al., 2011). According to Li et al. (2004), the hydrophilic–
in the subunits of the soy protein (enriched with β-conglycinin) pro- hydrophobic partitioning in the peptide sequence was a critical factor
duced peptide profiles that inhibited FAS and decreased lipid accumula- in ACE-inhibitory activity, because hydrophilic amino acid residues
tion in vitro. The quantity of soy protein hydrolysates necessary to could disrupt the access of the peptide to the active site of ACE. This
inhibit 50% of FAS activity (IC50) ranged from 50–175 μM. A peptide concept was reinforced by Yuan, Wu, and Aluko (2007) and Aluko
with anti-adipogenic abilities was isolated by ultrafiltration, gel filtra- et al. (in press), who showed an important role of branched-chain
tion and reversed-phase HPLC from soy protein hydrolysates, and its amino acids such as Val, Leu and Ile, in enhancing the hydrophobic char-
anti-adipogenic ability was confirmed by the inhibition of 3T3-L1 pre- acter of peptides, which is an important structural feature that enables
adipocyte cell differentiation. The inhibitor was identified as a tripeptide strong peptide interactions with non-polar amino acid residues within
(Ile-Gln-Asn) with an IC50 value of 0.014 mg mL−1 (Kim et al., 2007). the enzyme active site.
Peptide fractions from soy protein hydrolyzed with pepsin were
4.4. Peptides with anti-hypertensive activity separated by ion exchange chromatography, gel filtration and HPLC,
and they were found to have ACE-inhibiting activity. Four amino
Arterial hypertension affects approximately 25% of the adult popula- acid sequences were identified as potential ACE inhibitors: Ile-Ala
tion worldwide and is predicted to reach 29% of the population by 2025, (IC50 153 μM), Tyr-Leu-Ala-Gly-Asn-Gln (IC50 14 μM), Phe-Phe-Leu
representing a total of 1.56 billion people (Ngo et al., 2015). Although it (IC50 37 μM) and Ile-Tir-Leu-Leu (IC50 42 μM). When administered at
is a controllable disease, hypertension is associated with several cardio- a dose of 2.0 g/kg body weight in hypertensive rats over 15 weeks, the
vascular diseases, such as atherosclerosis, myocardial infarction and peptide fractions considerably reduced arterial pressure (Chen, Okada,
stroke (Sheih, Fang, & Wu, 2009). Angiotensin converting enzyme Muramoto, Suetsuna, & Yang, 2003).
(ACE) plays an important role in the regulation of arterial pressure be- Peptides with anti-hypertensive activity were isolated from protein
cause it catalyzes the conversion of angiotensin-I (the inactive form) hydrolysates of milk after fermentation with lactic bacteria and enzy-
to angiotensin-II (a vasoconstrictor) and inactivates bradykinin (a vaso- matic hydrolysis with the commercial protease Prozyme 6. The peptides
dilator). Consequently, synthetic ACE inhibitors, such as captopril and were identified as Gly-Thr-Trp and Gly-Val-Trp and had ACE inhibitor
enalapril, are often used to treat hypertension and other related heart activity with IC50 values of 464.4 and 240.0 μM, respectively (Chen
diseases. However, synthetic inhibitors can cause various side effects, et al., 2007). Hernández-Ledesma, Quirós, Amigo, and Recio (2007)
such as cough, altered taste, rash and angioedema (Alemán et al., 2011). hydrolyzed human milk proteins with pepsin and pancreatin to study
It is well known that dietary proteins have primary sequences of the anti-hypertensive properties of the peptides and showed that
peptides able to modulate specific physiological functions (Hong et al., hydrolysates derived from β-casein were strong inhibitors of ACE,
2008). Many types of bioactive peptides that inhibit ACE were isolated with an IC50 of 21 μM.
from protein hydrolysates and fermented products. The dipeptide Chaves-López et al. (2014) studied the effects of combined microbial
Ala-Pro and the tripeptide Phe-Ala-Pro, for example, have structures cultures previously identified as proteolytic and their ability to release
analogous to the drugs captopril and enalapril, respectively (Fig. 2). ACE-inhibiting peptides during the production of fermented milk. The
The presence of aromatic and aliphatic amino acids, such as Pro, Phe yeast strains Torulaspora delbruekii KL66A, Galactomyces geotrichum
or Tyr at the C-terminal and of Val and Ile at the N-terminal position of KL20B, Pichia kudriavzevii KL84A and Kluyveromyces marxianus KL26A
the bioactive peptides has been associated with ACE-inhibitory activity and the lactic acid bacteria Lactobacillus plantarum LAT03, Lb. plantarum
(Kapel, Rahhou, Lecouturier, Guillochon, & Dhulster, 2006; Wijesekara, KLAT01 and Enterococcus faecalis KE06 (non-virulent) were used. The
Qian, Ryu, Ngo, & Kim, 2011). Cian, Vioque, and Drago (2015) studied results indicated that the combination of different cultures can signifi-
the ACE-inhibitory activity of peptides from wheat gluten hydrolysate cantly increase the levels of anti-hypertensive peptides. The most

Captopril Enalapril

H CH CH 2 CO2 CH 3

Ala-Pro Phe-Ala-Pro

CH 3 CH 2 CH 3


Fig. 2. Structures of ACE inhibitor drugs and their analogous peptides (Matsui & Matsumoto, 2006).
R.J.S. de Castro, H.H. Sato / Food Research International 74 (2015) 185–198 195

effective combination for producing these peptides was a mixture of existence of protein sources with various amino-acid compositions
P. kudriavzevii KL84A, Lb. plantarum LAT3 and E. faecalis KL06, which make it possible to obtain peptides with distinct biological functions
had an IC50 for ACE inhibition of 30.63 μg mL−1. and/or multiple functions. Important techniques for purification and
The anti-hypertensive effect of a bovine casein peptide previously identification include chromatography and mass spectrometry. Studies
identified as Met-Lys-Pro was analyzed in vitro and in vivo. The in vitro on production processes, studies on multifunctionality and analyses
analyses were based on the ability to inhibit ACE, and the in vivo studies using in vitro and in vivo methods all contribute to our understanding
were conducted using groups of naturally hypertensive rats. Animals of bioactive peptides as powerful natural biological agents that can be
were treated with peptide solutions (10 mg/kg) two times per day used together with or in place of synthetic substances in food preserva-
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days. The in vitro assay showed that the peptide had ACE inhibition ac-
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