Trends in Food Science & Technology 59 (2017) 30e36

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Trends in Food Science & Technology

journal homepage: http://www.journals.elsevier.com/trends-in-food-science-
and-technology

Review

Prospects of microalgae proteins in producing peptide-based

functional foods for promoting cardiovascular health
Chukwunonso E.C.C. Ejike a, Stephanie A. Collins b, c, Nileeka Balasuriya b, d,
Andrew K. Swanson c, Beth Mason c, Chibuike C. Udenigwe b, e, *
a
Department of Medical Biochemistry, Federal University, Ndufu-Alike, Ikwo, Ebonyi State, Nigeria
b
Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia, B2N 5E3, Canada
c
Verschuren Centre for Sustainability in Energy and the Environment, Cape Breton University, Sydney, Nova Scotia, B1P 6L2, Canada
d
Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, N6A 5C1, Canada
e
School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, Ontario, K1N 6N5, Canada

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

Article history: Background: Bioactive peptides have strong potential for use in functional food formulation for pre-
Received 31 January 2016 vention and management of health conditions, especially cardiovascular disease (CVD). Microalgae can
Received in revised form be used as sustainable protein sources in the production of peptide-based functional foods for preventing
31 August 2016
or treating CVD.
Accepted 10 October 2016
Available online 19 November 2016
Scope and approach: This review discusses the scientic knowledge and current trends in microalgae-
derived peptides, including their chemical composition, production and potential impact in manage-
ment of hypertension and oxidative stress. The prospects for commercial applications as functional food
Keywords:
Microalgae
ingredients are also discussed.
Bioactive peptides Key ndings and conclusions: There is high potential for the production of functional foods containing
Antioxidants microalgae-derived peptides. Peptides that inhibit angiotensin converting enzyme, and those that have
Antihypertensive antihypertensive and antioxidant properties, all of which are important in ameliorating CVD risk factors,
Functional foods have been successfully produced from microalgae. Future research with regards to the microalgae-
Health benets derived peptides will involve the development of large-scale commercial microalgae cultivation,
enhancement of protein extraction and peptide release, understanding of matrix interactions of the
peptides within food products, and in vivo studies in human to validate health benets.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Food components have demonstrated biological activities that
are being applied in the prevention, management and treatment of
health conditions, including cardiovascular disease (CVD). The
global mortality from CVD in 2012 was estimated to account for 37%
of total deaths due to non-communicable diseases (NCD), ahead of
cancer and chronic respiratory diseases which accounted for 27%
and 8% of NCD deaths, respectively (WHO, 2014). About half of the
deaths due to stroke and coronary heart disease are caused by
hypertension (WHO, 2014). Oxidative stress is linked to the path-
ogenesis and progression of CVD, and hypertension has been
* Corresponding author. School of Nutrition Sciences, Faculty of Health Sciences,
University of Ottawa, Ontario, K1N 6N5, Canada
E-mail address: cudenigw@uottawa.ca (C.C. Udenigwe).
identied as the most important controllable risk factor for CVD in
adult humans (Jones et al., 2010; WHO, 2014). The contribution of
hypertension to CVD mortality has increased in some countries
during the last two decades, despite advances in conventional
medicine. For instance, populations in some low-income countries
have the highest prevalence of hypertension (WHO, 2014). Conse-
quently, substantial efforts are focused on the discovery of pre-
vention and management strategies for ameliorating the impact of
CVD on global health. In addition to lifestyle (exercise and diet)
modications, natural remedies are gaining popularity for CVD
management considering that chemotherapeutic agents can have
serious long term side effects. Current research has been devoted to
the mining of food for bioactive molecules for chronic diseases
management. Beyond providing essential nutrients, food can pro-
vide benecial health effects, and consumers tend to prefer this
approach to health promotion (Siro
, K

polna, & Lugasi,
apolna, Ka
2008), possibly due to perceived safety as the functional

http://dx.doi.org/10.1016/j.tifs.2016.10.026
0924-2244/ 2016 Elsevier Ltd. All rights reserved.
 C.E.C.C. Ejike et al. / Trends in Food Science & Technology 59 (2017) 30e36
molecules are derived from natural sources. Moreover, the food
industry is directed towards the present-day consumer demands
with the continuous rise in food products with new functionalities
for health promotion (Bigliardi & Galati, 2013). Therefore, cost-
effective development of functional food products by the food in-
dustry relies heavily on the availability and affordability of food-
grade bioactive ingredients (Udenigwe, 2014).
2. Peptides as bioactive ingredients for functional food
formulation
The use of food protein-derived bioactive peptides as functional
ingredients in foods is a rapidly developing area of food innovation.
Bioactive peptides are short-chain proteinogenic amino acid resi-
dues joined by peptide bonds, and are produced by enzymatic
cleavage of proteins during food digestion, microbial fermentation,
food processing, or exogenous enzymatic hydrolysis (Hernandez-
Ledesma, del Mar Contreras, & Reico, 2011; Udenigwe & Aluko,
2012). The interest in the functional food application of peptides
is due to their demonstrated biological activities relevant to the
management of human health conditions such as hypertension,
oxidative stress, cancer, diabetes, inammation, and immune dis-
orders (Udenigwe & Aluko, 2012). However, a number of factors
may impede the development of peptide-based functional food
products, including sensory, processing, pharmacokinetic, and
regulatory issues, as well as limited clinical evidence to substanti-
ate bioactivity (Udenigwe, 2014). Notably, the use of primary hu-
man food proteins as precursors of bioactive peptides does not
appear sustainable, especially in light of the current and projected
global food security challenges. Therefore, there is a need to iden-
tify sustainable protein sources and optimize technology for their
isolation for niche applications. One area of current focus is the
valorization of protein-rich secondary products of the agri-food
industry, especially dairy whey, oilseed and sh processing meals.
Moreover, marine microalgae have been identied as attractive and
sustainable protein sources for industry-scale production of
bioactive peptides (Udenigwe, 2014).
3. Microalgae as human foods
Algae are a varied polyphyletic group of oxygenic photosyn-
thetic organisms, ranging from the prokaryotic kingdom of the
Monera (blue-green algae) to the eukaryotic kingdom of Protista
(all eukaryotic algae) (Whittaker, 1969). Algae are subdivided into
microalgae and macroalgae. Macrophytic algae, typically
Chlorophyta (green algae), Rhodophyta (red algae) and Phaeophyta
(brown algae), are referred to as macroalgae, while the unicellular
forms are called microalgae (Wang, Xiao, Wang, Zhou, & Tang,
2007). The majority of microalgae exist as small cells (3e20 mm)
representing both photoauto- and hetero-trophic eukaryotes such
as bacilliariophyta (diatoms), dinophyta (dinoagellates), rhaphi-
dophyta, haptophyta, chlorophyta, prasinophyta, prymnesiophyta,
cryptophyta, and chrysophyta (golden, green and yellow-brown
agellates), as well as photoautotrophic prokaryotics such as cya-
nobacteria (El Gamal, 2010; Rasmussen & Morrissey, 2007). It is
estimated that there are more than 50,000 species of microalgae,
although only approximately 30,000 species have been studied and
characterized (Mendes & Vermelho, 2013; Richmond, 2004).
Microalgae are ubiquitous, but are more easily identied in aquatic
systems where they have been isolated in such diverse sites as hot
springs and glacial ice ows (Becker, 2007). Many free-living,
terrestrial microalgae and several symbiotic species are known;
some examples for the symbiotic species are chlorophycean and
cyanobacterial symbionts within lichens, and dinoagellates
within corals (cnidarians). Algae have evolved to survive in highly
31
dynamic and complex competitive environments, producing a wide
array of functional biomolecules to support growth in extreme
salinity, temperatures, light, and nutrient-deciencies (Plaza,
Cifuentes, & Ibanez, 2008), and defence against micrograzers
(Berry, Gantar, Perez, Berry, & Noriega, 2008).
Generally microalgae contain 40e70% proteins, 12e30% carbo-
hydrates, 4e20% lipids, 8e14% carotene and substantial amounts of
vitamins B1, B2, B3, B6, B12, E, K, and D (Becker, 2007). Not sur-
prisingly, history is replete with records of human reliance on
microalgae for nutrition, with documentation showing Nostoc
(Cyanophyta) species being consumed for human survival in China
as early as 2000 years ago, and from several other different coun-
tries (Ciferri, 1983). Currently, commercially cultivated microalgae
is a mature and sophisticated global industry, and primarily focuses
on the cultivation of Chlorella, Spirulina, Dunaliella, Nannochloris,
Nitzschia, Crypthecodinium, Schizochytrium, Tetraselmis, and Skel-
etonema species (Lee, 1997). Their efcacy in utilizing sunlight,
residual nutrients and seawater, as well as adaptability to non-
arable lands favors the potential global cultivation of microalgae
for both food and non-food based applications and end-products
(Draaisma et al., 2013). These include a wide array of bioactive
compounds (da Silva Vaz et al., 2016; Plaza et al., 2008, Plaza,
Herrero, Cifuentes, & Iba
n
~ ez, 2009), including polyunsaturated
fatty acids (PUFAs) that would compete with conventional vege-
table oil in the development of healthy foods (Draaisma et al.,
2013). Although microalgae PUFAs, as well as sterols, and soluble
bers, possess demonstrated inuence in lowering CVD risk factors
(Plaza et al., 2008), increasingly microalgae proteins are gaining
favour in health outcomes and uses. Despite their positive nutri-
tional composition and attributes, the sourcing of biomass from
traditional large-scale open pond cultivation for microalgae re-
mains challenging (Draaisma et al., 2013). Largely based on
enhanced production control, current microalgae biomass cultiva-
tion has largely shifted away from traditional open pond farm
production models for low-value bio-energy and fertilizer prod-
ucts, towards rigorously controlled large scale closed vessel pro-
duction technologies, adopted from fermentation industries, for the
generation of high margin non-food products such as cosmetics,
tailored industrial oils, and aquaculture feeds. Although open pond
production is viable, albeit at lower production capacity, it
currently is largely limited to artisan sized groups for alternative
food and nutritional supplement markets.
4. Microalgae proteins
Microalgae biomass is rich in proteins that compete favourably,
in terms of quantity and quality, with conventional food proteins
such as soybeans, eggs, and sh, making the unicellular organisms a
promising source of food protein (Batista, Gouveia, Bandarra,
Franco, & Raymundo, 2013; Graziani et al., 2013). For example,
Spirulina sp. is known to contain about 50e70% proteins depending
on the strain (Plaza et al., 2009). The microalgae Dunaliella can
produce 50e100 times more protein per unit area than traditional
plants and animals currently grown for food. Importantly, micro-
algae proteins contain well-balanced amino acid proles, compa-
rable to those of egg, soybean and the FAO/WHO (1973) reference
pattern; however, microalgae proteins may have lower biological
values, digestibility, net protein utilization and protein efciency
ratio than the gold standard, casein, and egg (Becker, 2007).
Microalgae synthesize all 20 proteinogenic amino acids and can be
unconventional sources of essential amino acids for human nutri-
tion (Spolaore, Joannis-Cassan, Duran, & Isambert, 2006). More-
over, the functional properties (nitrogen solubility, water and oil
absorption capacities, emulsication capacity, viscosity) of food
products containing microalgae biomass were found to be
32 C.E.C.C. Ejike et al. / Trends in Food Science & Technology 59 (2017) 30e36

comparable to those of a similar product containing soybean our

(Guil-Guerrero, Navarro-Jua
rez, Lo

pez-Martnez, Campra-Madrid, Microalgae sample concentrated at ~200 g/L
& Rebolloso-Fuentes, 2004), although these properties cannot be
entirely attributed to the protein components. Fig. 1 shows the
average protein content of selected microalgae species compared
with conventional food proteins. The amount of accumulated pro- Product re-suspended in distilled
teins in microalgae is dependent on several factors, including water at 1:16 (w/v)
species type, growth phase and light quality, and can be modied
through nutrient adjustments and environmental stress (Fontes,
Rivas, Guerrero, & Losada, 1983).
Sample adjusted to pH 11.0 using 2 M NaOH
5. Technology for microalgae protein extraction

Solvent-based extraction techniques, often hexane use on dried

biomass, although effective and scalable for lipid-based energy end
products, can be problematic for food and feed end markets.
Increasing interest in both health qualities of algal proteins, as well
as a strong nancial need to utilize all fractions of the cultivated
biomass has encouraged the development of alternative, safe and
scalable technologies to access quality algal proteins. For example
use of food-safe supercritical-based extractions methods, much like
solvent based methods, initially separates bio-oil fractions, and the
resultant algal protein residues can be qualied for food and feed
inclusion markets. Indeed, some microalgae companies have
bypassed process fractionation altogether, and are directly mar-
keting puried algal biomass as PUFA-rich algal protein powders
(e.g. Solazyme's AlgaVia) for use in speciality niche food and
beverage markets.
Alternatively, pre-extraction of proteins from algal biomass is
also being explored. Recently, Mun ~ oz et al. (2015) developed a
procedure for extraction of soluble proteins under alkaline condi-
tions (Fig. 2), using an adapted method (Kale, 2011), prior to the
removal of lipids for biodiesel production. Such method can be
scaled by the industry and others utilizing microalgae for extracting
useable proteins, augmenting efciencies of follow-on solvent-
based lipid extractions or thermal hydrolysis, and for removal of
components detrimental to process efciencies. Fast pyrolysis, a
promising bio-energy technology proposed for biomass, including
algae, thermally converts carbohydrates and oils to bio-oil, syngas
and biochar. Protein fractions, although convertible, are generally
undesirable as protein-rich biomass creates bio-oils generally rich
Rice
Milk
Soybean
Meat
CE
NI
EG
AC
SO
DS
SP
SM
CV
CP
AF
SS
AM
0 10 20 30 40 50
Sample stirred continuously at 150 rpm for
13 min at room temperature;
Then centrifuge at 4,400 rpm for 15 min
Supernatant adjusted to pH 3.3 with 1 M HCl;
Then centrifuge to recover solubilized proteins
in the supernatant
~ oz et al., 2015).
Fig. 2. Procedure used to extract protein from microalgal biomass (Mun
in nitrogen, creating excessive NOx when fuel is burned (Belottie, de
Caprariis, De Filippis, Scarsella, & Verdone, 2014; Wang, Brown,
Homsy, Martinex, & Sidhu, 2013). These fractions can be recov-
ered for food and non-food applications.
6. Production of bioactive peptides from microalgae proteins
Bioactive peptides derived from microalgae proteins are being
explored as functional ingredients mostly for the management of
hypertension and oxidative stress. These peptides exist as part of
microalgae proteins and remain inactive until they are liberated by
(i) enzymatic hydrolysis via gastrointestinal protease action, (ii)
microbial enzymatic hydrolysis during fermentation, or (iii) pro-
teolytic processing using exogenous enzymes (Samarakoon & Jeon,
2012; Udenigwe & Aluko, 2012). There is limited information on
the detailed proles and primary sequences of microalgae proteins,
making it particularly difcult to use bioinformatic tools in pre-
dicting the pattern of bioactive peptides encrypted within the
proteins prior to experimental analysis and industrial applications.
Nonetheless, efforts are being made to produce protein hydroly-
sates (Kose & Oncel, 2015), and bioactive peptides from microalgae
proteins by hit-and-miss approach. Processing of the peptides has
included several common fractionation and purication tech-
niques, particularly membrane ultraltration and chromatographic
techniques using ion-exchange, afnity, and gel-permeation plat-
forms. The choice of the processing techniques largely depends on
the peptide structural feature of interest, but consideration should
be given to feasibility for industry scale-up of the processing
technology, yield of the resulting peptides, and desired end market.
60 70

Protein (%)
7. Bioactivity of microalgae protein-derived peptides
Fig. 1. Protein contents of selected microalgae and conventional food protein sources;
AM, Arthospira maxima; SS, Synechococcus sp.; AF, Aphanizomenon os-aquae; CP, Microalgae produce a variety of bioactive metabolites that can
Chlorella pyrenoidosa; CV, Chlorella vulgaris; SM, Spirulina maxima; SP, Spirulina pla-
tensis; DS, Dunaliella salina; SO, Scenedesmus obliquus; AC, Anabaena cylindrica; EG,
potentially be used in managing physiological aberrations such as
Euglena gracilis; NI, Navicula incerta; CE, Chlorella ellipsoidea; data derived from hyperlipidemia, cancer, microbial infection, and oxidative stress (da
Spolaore et al., 2006 and Becker, 2007. Silva Vaz et al., 2016; Plaza et al., 2008, Plaza et al., 2009). Several
 C.E.C.C. Ejike et al. / Trends in Food Science & Technology 59 (2017) 30e36 33

studies have demonstrated that peptides derived from enzymatic
hydrolysis of microalgae proteins possess antioxidative and anti-
hypertensive properties that can be applied in human health
promotion.
7.1. Antioxidative microalgae peptides
Oxidative stress has been identied as one of the key etiological
factors in the development of hypertension (Briones & Touyz,
2010). Elevated levels of reactive oxygen species (ROS) can cause
oxidation of biological macromolecules, ultimately leading to
pathological conditions that include endothelial dysfunction
(Lahera et al., 2007). Oxidative stress is not only a causative factor of
hypertension, but also an important mediator in the imbalance
between vasoconstrictor and vasodilator mechanisms. Oxidative
stress-mediated events can therefore be considered important
factors in the development of hypertension (de Champlain et al.,
2004; Dhalla, Temsah, & Netticadan, 2000). Bioactive compounds
in food, including microalgae-derived peptides, are widely inves-
tigated for their ability to mitigate oxidative stress and associated
disease complications.
The antioxidant capacity of bioactive peptides depends on
several factors such as the peptide molecular weight, amino acid
composition, molecular and surface hydrophobicity, and the type of
ROS/free radical (Garcia et al., 2013; Samaranayaka & Li-Chan, 2011;
Udenigwe & Aluko, 2012). Most bioactive peptides have been
investigated for antioxidant properties using in vitro and cell-based
assays. For instance, the ability of the peptides to scavenge free
radicals (e.g. a,a-diphenyl-b-picrylhydrazyl [DPPH] and oxygen
radical absorbance capacity [ORAC] assays), reduce ferric ion (e.g.
ferric reducing ability of plasma [FRAP] assay) and inhibit lipid
peroxidation (e.g. thiobarbituric acid reactive substances [TBARS]
assay) are being utilized for assessing antioxidant capacity (Garcia
et al., 2013). As shown in Table 1, most known antioxidant pep-
tides from microalgae proteins were derived from Navicula incerta
and Chlorella sp.
The high availability of microalgae species has made them a
promising protein source for bioactive peptide production. Peptides
derived from Chlorella sp. have demonstrated potent antioxidant
capacity in vitro and in cell-based assays. Peptic hydrolysis of
Chlorella ellipsiodea yielded a pentapeptide (Leu-Asn-Gly-Asp-Val-
Trp) with potent free radical scavenging ability. The peptide was
shown to have peroxyl radical and DPPH radical scavenging anti-
oxidant activities in vitro, and intracellular radical scavenging ac-
tivity in monkey kidney cells (Ko, Kim, & Jeon, 2012a). Moreover,
Chlorella pyrenoidosa-derived peptide mixture also showed anti-
oxidant properties in skin broblasts. In the study with UV irradi-
ation, skin cells treated with the Chlorella-derived peptides (5 and
10 mg/L) showed 100% cytoprotection over a period of 72 h (Shih &
Cherng, 2012). It is not apparent however if the microalgal peptides
were transported into cells to exhibit their antioxidant activity, or
only acted extracellularly in cytoprotection. Other protein hydro-
lysates produced from Chlorella ellipsiodea and Tetraselmis suecica
using different microbial proteases were found to yield digests with
moderate free radical scavenging capacity (Lee, Chang, Lee, & Jeon,
2009). In the same study, the Neutrase and kojizyme hydrolysates
of Tetrasel missuecica were found to scavenge hydrogen peroxide in
African green monkey kidney cell line (Lee et al., 2009). Further-
more, the use of papain, pepsin, a-chymotrypsin, pronase-E and
Neutrase to hydrolyze Navicula incerta also yielded antioxidant
hydrolysates, and the resulting peptides (Pro-Gly-Trp-Asn-Gln-Trp-
Phe-Leu and Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu) from the
papain reaction demonstrated antioxidant effects in HepG2/
CYP2E1 cells (Kang, Qian, Ryu, & Kim, 2011, Kang et al., 2012a, Kang,
Qian, Ryu, Kim & Kim, 2012b). The abundance of aromatic amino
acids (Tyr, Trp, Met, Lys, Cys, His), hydrophobic amino acids (Leu,
Val), and particular functional group (sulfhydryl group of Cys) is
thought to facilitate higher antioxidant capacity of peptides (Elias,
Kellerby, & Decker, 2008; Udenigwe & Aluko, 2011). Most micro-
algal peptide mixtures investigated for antioxidant activity possess
these amino acid properties. However, only a few studies have
identied the amino acid sequence of the microalgal peptides. To
date, enzymatic hydrolysis is the most common method of pro-
ducing antioxidant peptides from microalgae. The lack of evidence
to demonstrate physiological antioxidant capacity of the
microalgae-derived peptides makes it challenging to draw con-
clusions on their use in combating oxidative stress.
7.2. Antihypertensive properties of algae peptides
Hypertension is a complex disorder, resulting from an interac-
tion of genetic and environmental factors, and is dened as a

Table 1
Antioxidant peptides derived from microalgae.

Source of peptide Treatment Amino acid sequence Activity Reference

Navicula incerta Hydrolysis with papain Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu Cytotoxicity in Kang et al. (2012a)
Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu HepG2/CYP2E1
cells
Navicula incerta Hydrolysis with pepsin, Not available DPPH and Kang et al. (2011)
a-chymotrypsin and superoxide radical
Neutrase quenching
Navicula incerta Hydrolysis with papain Not available Antioxidant in Kang et al. (2012b)
Pronase-E HepG2/CYP2E1
a-chymotrypsin cells
Chlorella pyrenoidosa Aqueous extraction; Not available Cytotoxicity in skin Shih and Cherng (2012)
centrifugation and ltration broblasts
Chlorella vulgaris Hydrolysis with pepsin Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Glu-Phe Superoxide radical Sheih, Wu, and Fang (2009)
quenching
Chlorella ellipsiodea Hydrolysis with pepsin Leu-Asn-Gly-Asp-Val-Trp Free radical Ko et al. (2012a)
scavenging
Chlorella ellipsiodea Aqueous extraction; hydrolysis with Not available Moderate free Lee et al. (2009)
protamex, alcalase, avourzyme, radical scavenging
neutrase, and kojizyme
Tetraselmis suecica Aqueous extraction; hydrolysis Not available Free radical Lee et al. (2009)
with neutrase, and kojizyme scavenging; H2O2
scavenging in a
monkey kidney cell
line
34 C.E.C.C. Ejike et al. / Trends in Food Science & Technology 59 (2017) 30e36
sustained increase in systemic arterial pressure above 140/90 Hg
mm (Giles, Materson, Cohn, & Kostis, 2009). Hypertension is the
most prevalent NCD globally. Progression of hypertension results in
cardiac and vascular abnormalities such as endothelial dysfunction,
peripheral resistance, altered contractility, and vascular remodel-
ing. Maintaining homeostasis in blood pressure regulation is
dependent on the balance between the vasoconstrictive mecha-
nisms (the renin-angiotensin-aldosterone system, RAAS), the
sympathetic nervous system, the vasopressin system, the endo-
thelin system, ROS-induced vasoconstriction, and vasodilatory
mechanisms (function of nitric oxide, the atrial natriuretic peptide
system, the bradykinin system, and the prostaglandin systems).
Many antihypertensive drugs interfere with one or more of these
mechanisms (de Champlain et al., 2004).
Food-derived bioactive peptides are best known for their anti-
hypertensive properties. Their effect is mainly due to the inhibition
of angiotensin I-converting enzyme (ACE) in the renin-angiotensin-
aldosterone system (RAAS) (Hernandez-Ledesma et al., 2011;
Udenigwe & Aluko, 2012). RAAS plays an important role in the
regulation of blood volume and is responsible for blood pressure
and uid balance regulation in humans (Fitzgerald, Gallagher,
Tasdemir, & Hayes, 2011). ACE is a monomeric, membrane-bound
zinc metalloprotease that catalyzes the proteolytic cleavage of a
carboxyl-terminal dipeptide in angiotensin-I to form angiotensin-II
(Sagardia, Roa-Ureta, & Bald, 2013). Inhibiting ACE can lead to the
lowering of blood pressure and has been widely exploited in the
management of hypertension. The structure and the composition of
the peptide are key factors in determining the ACE inhibition. The
most effective antihypertensive peptides are those that contain
hydrophobic amino acid residues such as Pro (Garcia et al., 2013;
Hernandez-Ledesma et al., 2011; Udenigwe & Aluko, 2012). Most
of the ACE inhibitory peptides are short-chain peptides with 2e20
amino acid residues.
Similar to antioxidant activity, most antihypertensive peptides
from microalgal sources are derived from Chlorella sp. Some
microalgae species (Chlorella vulgaris, Chlorella ellipsiodea, Spirulina
platensis and Nannochloropsis oculata) have been used to produce
peptides with possible antihypertensive property, particularly ACE
inhibition in vitro (Table 2) and blood pressure reduction in hy-
pertensive rats (Table 3). The hydrolysis of Chlorella vulgaris with
pepsin was found to yield peptides with both in vitro and in vivo
antihypertensive properties (Sheih, Fang, & Wu, 2009; Suetsuna &
Chen, 2001). Chlorella vulgaris processing by-product was used to
produce an 11-amino acid residue peptide (Val-Glu-Cys-Tyr-Gly-
Pro-Asn-Arg-Pro-Gln-Phe), which dose-dependently inhibited ACE
activity (Sheih, Fang, & Wu, 2009). Enzyme kinetics demonstrated
that the peptide inhibited ACE activity non-competitively, which
can be due to its large size that would be expected to hinder active
Table 2
ACE (angiotensin converting enzyme) inhibitory peptides derived from microalgae.
Source of peptide Treatment
Chlorella vulgaris Hydrolysis with pepsin; chromatography
Chlorella vulgaris Hydrolysis with pepsin
Chlorella ellipsoidea Hydrolysis with alcalase
Spirulina platensis Hydrolysis with pepsin; chromatography
Nannochloropsis oculata Hydrolysis with pepsin; chromatography
site binding. On the other hand, pepsin hydrolysates of Chlorella
vulgaris and Spirulina platensis yielded several short chain peptides
that were found to be effective in reducing the high blood pressure
of spontaneously hypertensive rats (SHR) within a time period of
6 h after administration (Suetsuna & Chen, 2001). However, the
single-dose administration effect would not indicate the sustained,
long-term benecial effects of the peptides. Another peptide (Val-
Glu-Gly-Tyr) derived from the Chlorella ellipsiodea hydrolysate was
also reported to have both in vitro ACE inhibitory activity and in vivo
blood pressure lowering effect (Ko et al., 2012b). The short-chain
peptide was found to be a competitive ACE inhibitor with an IC50
value of 128.4 mM, demonstrating its accessibility and binding to
the enzyme active site. Furthermore, Nannochloropsis oculata hy-
drolysis with pepsin yielded two ACE inhibitory peptides, which
also increased nitric oxide production in human umbilical vein
endothelial cells (HUVEC) (Samarakoon et al., 2013). If bioavailable,
especially in the vascular endothelium, the latter activity of the
microalgae peptides is expected to promote vasodilation and sub-
sequent blood pressure reduction during hypertension.
Unlike antioxidant capacity, the antihypertensive properties of
food peptides have been widely investigated. Although short chain,
low-molecular weight peptides are clearly more effective
compared to larger peptides, due in part to higher bioavailability
and active site accessibility, the IC50 values reported for the
different microalgae-derived peptides do not follow any particular
molecular size-activity pattern (Table 2). Similar to antioxidant
activity, further evaluation is needed to establish the physiological
relevance of the microalgal peptides, relative to peptides from
other food sources, in reducing blood pressure and other clinical
signs of CVD in hypertensive human subjects. Of particular
importance is the need to obtain more in vivo evidence of the
mechanisms of peptides that demonstrate in vitro ACE inhibitory
activity, especially as the latter does not necessarily translate into
physiological antihypertensive effects.
8. Prospective use of algae protein-derived peptides in
functional food formulation
The potential for producing functional foods using microalgae-
derived peptides is enormous. High biomass, easy production,
high levels of proteins and other bioactive compounds, and less
competitiveness with other food sources as a raw material are key
factors that make microalgae an effective peptide-based functional
food source. The current market for microalgae is mainly focused
on biofuel production, niche nutritional supplements, alginates in
food, and as sh feed. Cultivating microalgae as functional in-
gredients for the food, cosmetic, and pharmaceutical industries is
expected to expand as a result of emerging evidence of their
Amino acid sequence IC50 (mM) Reference
Ile-Val-Val-Glu 315.3 Suetsuna and Chen (2001)
Ala-Phe-Leu 63.8
Phe-Ala-Leu 26.3
Ala-Glu-Leu 57.1
Val-Val-Pro-Pro-Ala 79.5
Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe 29.6 Sheih, Fang, and Wu (2009)
ValeGlueGlyeTyr 128.4 Ko et al. (2012b)
Ile-Ala-Glu 34.7 Suetsuna and Chen (2001)
Phe-Ala-Leu 11.4
Ala-Glu-Leu 11.4
Ile-Ala-Pro-Gly 11.4
Val-Ala-Phe 35.8
Gly-Met-Asn-Asn-Leu-Thr-Pro 123 Samarakoon et al. (2013)
Leu-Glu-Gln 173
 C.E.C.C. Ejike et al. / Trends in Food Science & Technology 59 (2017) 30e36
Table 3
Antihypertensive effects of microalgae derived peptides in vivo.
Source of peptide Animal model Treatment
Chlorella vulgaris Spontaneously Oral dose of peptidic fractions e 200 mg/kg body
hypertensive rats weight
Chlorella ellipsoidea Spontaneously Oral dose of peptide (ValeGlueGlyeTyr)
hypertensive rats body weight
Spirulina platensis Spontaneously Oral dose of peptidic fractions e 200 mg/kg body
hypertensive rats weight
benecial effects. The production of microalgae-derived, bioactive-
rich functional foods with antioxidant and antihypertensive prop-
erties can contribute to ongoing efforts to reduce the global CVD
burden. There are a few commercial functional food products
containing bioactive peptides currently available on the market.
However, there are limitations on the incorporation of peptides
into foods under most food regulatory systems (EFSA, FDA, etc.).
Fitzgerald et al. (2011) estimated that there are approximately
seven functional products sold on the market that are fortied with
milk-derived blood pressure modulatory peptides. Two widely
known examples are Calpis Ameal-S drink and Evolus, both of
which contain the bioactive peptides Ile-Pro-Pro and Val-Pro-Pro,
which are known to possess ACE inhibitory properties (Arihara,
2006). Solazyme, a U.S.-based microalgae rm, has successfully
penetrated several speciality food markets with its AlgaVia algal
protein our substitute. To our knowledge, microalgae-derived
bioactive peptide products are yet to be commercialized and mar-
keted as functional foods for cardiovascular or related health
benets.
There are many challenges to overcome during processing of
bioactive peptide-rich foods. Certain conventional food processing
techniques result in detrimental effects on the bioactive peptide
structure (Hernandez-Ledesma et al., 2011; Udenigwe, 2014). For
example, heating, which ordinarily improves texture and avor of
foods, can modify the molecular structure of amino acid residues in
a bioactive peptide, thereby resulting in the loss of activity or
bioavailability of the peptide (Korhonen, Pihlanto-Leppala, Ranta-
maki, & Tupasela, 1998; Udenigwe, 2014). Bioactive peptides from
microalgae can also interact with different matrices in the medium
during processing or storage, and this can result in accentuation or
attenuation of their functional and nutritional properties (Moller,
Scholz-Ahrens, Roos, & Schrezenmeir, 2008). Digestion may be
another challenge, as enzymatic hydrolysis of the peptides in the
gastrointestinal tract may render them inactive (Hernandez-
Ledesma et al., 2011; Udenigwe, 2014). The bitter taste of most
bioactive peptides containing hydrophobic amino acids is another
challenge that can limit their use in functional foods (Hernandez-
Ledesma et al., 2011; Kristinsson & Rasco, 2000; Udenigwe,
2014). However, treatment of such peptides with activated car-
bon, extraction with alcohol, and concealing the taste with addi-
tives have successfully removed or reduced peptide bitterness
(Saha & Hayashi, 2001). Furthermore, many of these challenges can
be overcome by encapsulation, which can mask undesirable avors,
reduce heat sensitivity and reactivity, improve stability, and ensure
a controlled release of the functional peptides of the product
(Mohan, Rajendran, He, Bazinet, & Udenigwe, 2015). There is also a
need to investigate other important factors such as allergenicity (of
the peptides or their possible interaction products) that may hinder
the use of peptides in the production of functional foods and
nutraceuticals. Although this review is focused to peptides that
35
Activity Reference
Signicant reduction of systolic blood Suetsuna and Chen (2001)
pressure from 200 mmHg by 49.9 mm
Hg at 1 h post-administration; activity
continued over a 4-h period
10 mg/kg Signicant reduction of systolic blood Ko et al. (2012b)
pressure from 189.5 mmHg by 22.8 mm
Hg at 4 h post-administration
Signicant reduction of systolic blood Suetsuna and Chen (2001)
pressure from 200 mmHg by 39.5 mm
Hg at 2 h post-administration; activity
continued over a 4-h period
have roles (directly or indirectly) in promoting CVD health, there
are prospects for using microalgae-derived peptides as anticancer
and immunomodulatory agents (Kim & Wijesekara, 2010; Morris
et al., 2007). Clinically proven health benets, food regulations,
and development of feasible technologies to the food industry are
essential components in bringing the microalgae-derived bioactive
peptide-rich functional foods into the food market.
9. Conclusion
Functional foods containing microalgae-derived peptides have
the potential to ameliorate CVD risk factors, particularly hyper-
tension and oxidative stress, but more research is needed prior to
implementing large-scale incorporation of the peptides in the hu-
man diet. Further studies on establishing the efcacy of the
microalgal peptides when incorporated into foods, their suscepti-
bility to digestion, and whether or not these peptides interact with
other components of the food is required. Furthermore, the
development of large-scale production and extraction facilities for
optimizing microalgae growth, protein production and processing
are also necessary. Due to its rapid growth, high content and
diverse prole of proteins, microalgae is well-positioned for use as
a sustainable source of proteins for industry-scale production of
peptide-based functional foods.
Acknowledgements
The research program of C.C.U. is supported by the Natural
Sciences and Engineering Research Council of Canada (NSERC)
Discovery Grant RGPIN 435865-2013.
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