\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Current genetic engineering strategies for the production of
anti-hypertensive ACEI peptides}



\author[1]{Carolina Gomes}%
\author[2]{Diana  Ferreira}%
\author[2]{João Carvalho}%
\author[2]{Joana Fernandes}%
\author[2]{Marisol  Gouveia}%
\author[2]{Fernando  Ribeiro}%
\author[3]{Ana Duque}%
\author[2]{Sandra  Vieira}%
\affil[1]{Polish Academy of Science Institute of Plant Genetics}%
\affil[2]{Universidade de Aveiro Instituto de Biomedicina}%
\affil[3]{Universidade Nova de Lisboa Instituto de Tecnologia Química e Biológica}%


\vspace{-1em}



  
  \date{May 04, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Hypertension is a major risk factor for cardiovascular diseases, with
high prevalence in low- and high-income countries. Among the various
antihypertensive therapeutic strategies, synthetic Angiotensin
I-converting enzyme inhibitors (ACEI) are one of the most used
pharmacological agents. However, their use in hypertension therapy has
been linked to various side effects. In recent years considerable
research has been performed on the use of food-derived ACEI peptides
(ACEIp) as antihypertensive agents. Although promising, the industrial
production of these ACEIp through conventional methods, such as chemical
synthesis and enzymatic hydrolysis of food proteins, has been proven
troublesome and expensive. Limitations to the large-scale production of
ACEIp for functional foods and supplements can be overcome by producing
the precursors of these peptides in heterologous hosts. Bacterial hosts
have been the privileged choice, particularly to test the success of the
genetic engineering strategies, but new platforms based on plants and
microalgae have also been emerging. This work provides an overview of
the state of antihypertensive therapy, focusing on ACEI, illustrates the
latest advances on ACEIp research, and describes current genetic
engineer-based approaches for the heterologous production of ACEIp for
antihypertensive therapy.%
\end{abstract}%



\sloppy


\section*{Hypertension and anti-hypertensive
therapy}

{\label{hypertension-and-anti-hypertensive-therapy}}

\subsection*{Hypertension -- an overview}

{\label{hypertension-an-overview}}

Hypertension (HT) is defined as a systolic blood pressure (BP) of [?]140
mmHg and/or a diastolic BP of [?]90 mmHg in young, middle-aged and elderly
subjects (Williams et al., 2018). In 2004, HT was declared by the World
Health Organization (WHO) as the lead risk factor for death worldwide,
accounting for 7.5 million deaths (12.8\% of total deaths). Hypertension
is the leading risk factor for global disease burden (Lim et al., 2012)
, particularly cardiovascular diseases, and an important risk factor for
other diseases such as kidney failure. According to the Global Health
Observatory data repository 2015\textbf{,} HT affects 1.13 billion
people, with a global age-standardized prevalence of 22.1\% (24.1\% for
males). In low-income countries, the high prevalence (28.4\%) of
hypertension is mainly attributed to flawed health systems, resulting in
untreated and uncontrolled patients (Yeates et al., 2015). In
high-income countries, hypertension prevalence levels are slightly
decreased probably in response to public health actions (Lloyd-Jones and
Levy, 2013; World Health Organization, 2013), however HT prevalence is
expected to increase globally in coming years, due to the growth and
ageing of world's population, behavioral risk factors and lack of
efficient therapies.

Hypertension is classified as primary or secondary according to its
underlying cause. Most HT patients (\textasciitilde{}95\%) have primary
HT, which is defined as high BP without secondary causes. The remaining
5\% have secondary HT, deriving from a known medical condition
(`secondary cause') that is often reversible, (e.g. obstructive sleep
apnea, renovascular disease, renal failure) (Carretero and Oparil, 2000;
Oparil et al., 2003; Pullalarevu et al., 2014; Rimoldi et al., 2014).
Conversely, primary HT has no single causative agent - it seems to be
influenced by the presence of risk alleles and multiple other factors
such as body mass index, gender, insulin resistance, high alcohol and
salt intakes, low potassium and calcium intakes, stress, aging and
sedentary lifestyle (Carretero and Oparil, 2000; Pullalarevu et al.,
2014). Although still scarcely identified, genetic factors are thought
to play a major role in primary HT, and primary HT tends to be
associated with parental HT (Wang et al., 2008) - family studies
estimated the heritability of BP from 30 to 50\% (Ehret and Caulfield,
2013). However, this complex polygenic disorder seems to be influenced
by the interaction of genomic and environmental factors, challenging the
clear identification of susceptibility genes and epigenetically
modulated mechanisms. The advent of high-throughput genotyping
technologies provided some clues on the contribution of common genetic
variation on BP traits and primary HT (Ehret et al., 2016; Burrello et
al., 2017; Warren et al., 2017), although additional studies are needed
to confirm associations. Further, epigenetic marks (e.g. DNA
methylation, histone modifications and non-coding RNAs) might provide
missing links in BP traits variability (Richard et al., 2017).

\subsection*{Antihypertensive therapy}

{\label{antihypertensive-therapy}}

The central role of HT in the pathophysiology of many age-related
chronic diseases of high prevalence and socioeconomic burden, boosts
research on effective antihypertensive therapies. Their main goal is to
lower HT-associated complications, morbidity and mortality (Berryman,
2000; Kamath, 1990), related to target-organ damage such as
cardiovascular events, heart failure and kidney disease (Berryman,
2000). Controlled BP is the most feasible clinical end point, but
patients should be guided on both pharmacological therapy and lifestyle
modifications (`nonpharmacological therapy'), since BP reduction alone
may not guarantee prevention of future target-organ damage ( Wells et
al., 2009; Koda-Kimble and Alldredge, 2013;). There are many factors to
consider when choosing the right antihypertensive therapy, such as:
concomitant illnesses and medications, HT stage, age, compliance,
genetics and special populations (Wells et al., 2009; Clark et al.,
2011; Koda-Kimble and Alldredge, 2013). Nonpharmacological therapy can
include a combination of weight loss, restricted sodium intake,
increased aerobic exercise, moderation in alcohol consumption, and
stress relief (Brunton et al., 2005; Kasper et al., 2015; Gouveia et
al., 2017). Nonpharmacological therapy may suffice in cases of
prehypertension and, when combined with pharmacological regimens, can
increase the drug therapy efficacy, improving the quality of life and
longevity (Gouveia et al., 2017).

A great variety of pharmacological antihypertensive agents can
effectively lower BP. Current \emph{European Society of Cardiology and
European Society of Hypertension} (ESC/ESH) guidelines an initial
therapy with a combination of ACEI or angiotensin receptor blockers
(ARBs) with diuretics (including thiazides, chlorthalidone and
indapamide) or calcium channel blockers (CCB). A triple combination
could be prescribed but concomitant use of ACEi and ARB is discouraged
(Williams et al., 2018). As add-on therapy, alternative potent
antihypertensive drugs (e.g. spironolactone) can additionally lower BP
in patients with resistant hypertension although they have an high
incidence of adverse effects (Berryman, 2000). Supplementary Table S1
summarizes some of the first line antihypertensive agents according to
their subclasses, action mechanism, produced effects and clinical
applications. A list of antihypertensive drugs and common therapies is
also provided.

\subsection*{Angiotensin I-Converting Enzyme
Inhibitors}

{\label{angiotensin-i-converting-enzyme-inhibitors}}

Angiotensin I- Converting Enzyme (ACE) is a main target in hypertension
therapy given its key role in the Renin-Angiotensin system, the main
metabolic pathway regulating human BP and fluid homeostasis. The action
of ACE increases BP as a result of both increased vasoconstriction and
diminished vasodilation.

In the past twenty years, synthetic ACEI have been one of the preferred
first-line therapy for HT, especially in diabetic and chronic kidney
disease patients (TH et al., 2003; Jimsheena and Gowda, 2010; Zisaki et
al., 2015; Perico et al., 2017 ). ACEI act on both the Renin-Angiotensin
and the Kallikrein-Kinin systems (Figure 1). By blocking ACE activity,
ACEI prevent the conversion of angiotensin I into the potent
vasoconstrictor angiotensin II (Berryman, 2000). ACEI also block the
breakdown, by ACE, of the vasodilatory peptide bradykinin, responsible
for increasing the production at the blood vessels of two potent
vasodilators: prostacyclin and nitric oxide (Clark et al., 2011). By
reducing angiotensin II levels, ACEI also decrease the secretion of
aldosterone, and thus sodium and water retention (Berryman, 2000).
Overall, ACE inhibition decreases BP as a result of both increased
vasodilation (due to the action of bradykinin at blood vessels) and
diminished vasoconstriction (resulting from the lack of angiotensin II
and its target aldosterone) (Figure 1) (Clark et al., 2011).

ACE inhibitors have been divided in three categories: 1) captopril; 2)
prodrugs, such as enalapril and fosinopril; and 3) lisinopril, a
water-soluble and the only non-metabolized ACEI (Zisaki et al., 2014).
Most of ACEI are prodrugs, requiring hepatic conversion to
pharmacologically active metabolites. Captopril and lisinopril are
exceptions to this rule and are prescribed to patients with severe
hepatic impairment (Kelly and O'Malley, 1990; Zisaki et al., 2014). As
the majority of ACEI are eliminated primarily by the kidney, they may
require dose adjustments to varying degrees of renal impairment ( Weber,
1991; Piepho, 2000).

Although widely used for treating HT, congestive heart failure, and
diabetic neuropathy (Jimsheena and Gowda, 2010; Zisaki et al., 2014)
synthetic ACEI have been associated with various side effects including
cough, skin rashes, hypotension, loss of taste, angioedema, reduced
renal function and fetal abnormalities (Norris and FitzGerald, 2013). To
minimize the risk of side effects, synthetic ACEI have been combined
with other antihypertensive agents, such as calcium-channels blockers
(Egan, 2007) (Supplementary Table S1). Moreover, naturally occurring
ACEIp have gathered attention as potential antihypertensive agents to be
used alone or in combination with other non-pharmacological therapies,
for HT preventive measures and initial treatment of HT (Gouveia et al.,
2017).

\section*{ACEI peptides research}

{\label{acei-peptides-research}}

\subsection*{Food-derived ACEIp}

{\label{food-derived-aceip}}

ACEI peptides derived from proteins of food sources have attracted great
attention as antihypertensive agents. The major difference between these
ACEIp and synthetic ACEI is that the first do not cause significant BP
lowering effects on normotensive subjects, avoiding acute hypotensive
effects. Emerging evidence suggests that these food-derived peptides can
act through other mechanisms besides ACE inhibition, such as the
upregulation of ACE2 (an ACE homologue that counterbalances the
detrimental effect of elevated ACE), endothelial function improvement,
and reduced vascular oxidation and inflammation (Wu et al., 2017). Based
on these findings, ACEIp have been proposed for the initial treatment of
mildly hypertensive patients or as complementary treatment in
hypertensive patients (Rosales-Mendoza et al., 2013).

Food-derived ACEIp have great potential as food additives/ingredients
for novel functional foods/nutraceuticals, and as pharmaceutical
ingredients for novel drug formulations. ACEIp have been identified in
enzymatic hydrolysates of different food proteins, such as milk (Seppo
et al., 2003; Miguel et al., 2006; Ruiz-Gimenez et al., 2012), egg
(Asoodeh et al., 2012; Duan et al., 2014), plants (Marczak et al., 2006;
Jakubczyk and Baraniak, 2014; Orona-Tamayo et al., 2015; Rayaprolu et
al., 2015; V\selectlanguage{ngerman}ásquez-Villanueva et al., 2015; Wu et al., 2016), meat
(Castellano et al., 2013), fungi (Tran et al., 2014; Geng et al., 2015),
and marine source proteins (Fujita and Yoshikawa, 1999; Balti et al.,
2015; Ghanbari et al., 2015), and currently constitute the most
well-known class of bioactive peptides. ACEIp can be enzymatically
released from their precursor proteins during food processing and
gastrointestinal digestion. An extensive overview of ACEIp isolated from
food sources is given in Supplementary Table S2, while examples of
commercially available nutraceuticals or food ingredients containing
ACEIp are presented in Table 1. These nutraceuticals are normally
formulated in the form of beverages, capsules or powders that can be
directly consumed or used as ingredients for further food or
pharmaceutical applications. The main food sources of ACEIp are
fermented milk, bonito and other traditional foods with empirical health
benefits, consumed by humans long before the concepts of `functional
food' and `bioactivity' even existed. Hence, the most well-known natural
ACEIp-containing functional foods are fermented milk-derived, including
milk-derived beverages and sour milk tablets (Table 1). ACEIp from
marine sources, such as bonito, sardine and seaweeds, are also present
in food ingredients and functional foods in a broad variety of
formulations, including BP-lowering capsules and tablets (Table 1).
These ACEIp-containing products can be naturally obtained through
fermentation by specific microorganisms, or artificially through\emph{in
vitro} hydrolysis with gastrointestinal and commercial proteases
(Hartmann and Meisel, 2007; Pihlanto and Mäkinen, 2013; Hayes and
Tiwari, 2015)

\subsection*{Molecular determinants of ACEI
peptides}

{\label{molecular-determinants-of-acei-peptides}}

The discovery of novel ACEIp was traditionally based on the analysis of
whole food protein hydrolysates through wet chemistry techniques.
However, this workflow has since significantly evolved and \emph{in
silico} methods play an increasingly important role in ACEIp
research.\emph{In silico} methods can be coupled to \emph{in vitro}
techniques in the screening and characterization of novel ACEIp (Sun et
al., 2017). Particularly, the analysis of their ACE inhibitory activity
based on the peptide primary structure, termed quantitative
structure--activity relationship, has been widely used (Wu et al.,
2006).

Considering results from analytical and chemometric studies, some rules
were established concerning the primary structure of ACEIp. Potent ACEIp
are generally short chain peptides (2-12 amino acids in length),
although some larger inhibitory peptides were identified in fertilized
egg (Duan et al., 2014), milk fermented with \emph{Enterococcus
faecalis} or the \emph{Lactobacillus casei} strain Shirota (Quiros et
al., 2007; Rojas-Ronquillo et al., 2012), koumiss (Chen et al., 2010),
tuna (Lee et al., 2010), bonito (Hasan et al., 2006) and rotifer (Lee et
al., 2009). Chain composition and amino acid position of ACEIp also play
a role on the ACE inhibition potential. The N-terminal of potent ACEIp
generally contains hydrophobic amino acids, especially those with
aliphatic chains such as Gly, Ile, Leu, and Val (Iwaniak et al., 2014).
The C-terminal tripeptide sequence of ACEIp (and ACE substrates)
strongly influences ACEI activity, since ACE cleaves the C-terminal
dipeptide of oligopeptide substrates with a wide specificity.
Hydrophobic amino acid residues with aromatic or branched side chains at
each of the C-terminal tripeptide positions are common features among
potent inhibitors (Soares de Castro and Sato, 2015). In general,
peptides showing higher activity against ACE have Pro, Tyr, Phe or Trp
at their C-terminus (Norris and FitzGerald, 2013). Indeed, many potent
food-derived ACEIp contain Pro residues at one or more positions in the
C-terminal tripeptide region (Iwaniak et al., 2014). This rule concerns
most particularly short-length peptides. Even though a similar pattern
has been observed for long chain milk-derived peptides, their activity
was generally not influenced by C-terminal Pro (Aluko, 2015),

The relationship between the peptides secondary structure and ACEI
activity has also been analyzed (Yu et al., 2011). The elucidation of
the crystal structure of a complex between human ACE and an inhibitor
(Natesh et al., 2003) has provided a platform for analyzing ACEIp
inhibitory mechanisms by molecular docking and molecular dynamics (MD)
simulation (Xie et al., 2015). Molecular docking, a fast technique, has
been coupled with accurate but time-consuming MD techniques in the
discovery of novel inhibitory peptides and their mechanism of
interaction with ACE at the molecular level (Alonso et al., 2006).

Finally, online \emph{in silico} tools as ToxinPred and PeptideCutter
can also be applied to predict the toxicity and enzymatic digestion of
novel ACEIp (Sun et al., 2017).

\subsection*{In vitro and physiological ACE inhibitory
activity}

{\label{in-vitro-and-physiological-ace-inhibitory-activity}}

Following predictions by \emph{in silico} techniques, the ACE inhibitory
activity of novel ACEIp needs to be confirmed. \emph{In vitro}inhibitory
activity assays generally rely on spectrophotometric, fluorometric,
colorimetric and radiochemical methods, as well as chromatography
techniques (Murray and FitzGerald, 2007). The measure of activity is
usually given as the IC50, defined as the concentration of peptide
required to inhibit ACE activity by 50\% (Donkor et al., 2005). However,
\emph{in vitro} ACE inhibitory activity does not always conduce to a BP
lowering effect, and ACE inhibitory activity assays must be performed
\emph{in vivo} . These assays generally consist in measuring the BP
response in spontaneously hypertensive rats (SHR), following intravenous
or intraperitoneal injection, or oral administration, of ACEIp or
related extracts.

Data from the ACE \emph{in vitro} inhibition and the \emph{in
vivo}BP-lowering effect have provided a basis to classify ACEIp into 3
categories (Ryan et al., 2011): 1) Inhibitor-type ACEI peptides
resistant to cleavage by ACE, hence their activity is not significantly
altered by binding to ACE; 2) Substrate-type ACEI peptides, showing a
decrease in ACEI activity due to their cleavage by ACE upon binding; 3)
Prodrug-type ACEI peptides, larger peptides that are converted to potent
ACE inhibitors following hydrolysis by ACE or gastrointestinal
proteases.

The susceptibility to cellular peptidases is another important factor
determining the physiological activity of ACEIp. Some peptides may be
deactivated by their susceptibility to degradation by gastrointestinal,
intestinal and kidney brush borders, serum and blood proteinases and
peptidases during transport to the target organ(s). Conversely,
oligopeptide sequences containing encrypted ACEIp may be activated and
their ACEI activity increased, following \emph{in vivo} proteinase and
peptidase activities (Tai et al., 2018). The intestinal absorption and
bioavailability of peptidic ACEI fragments is also of paramount
importance. ACEI should be able to cross the intestinal wall and
sequentially enter the blood circulation in order to exert an
antihypertensive effect. The Caco-2 cell (a human epithelial colorectal
adenocarcinoma cell line) has been generally used as a model to
investigate the stability of ACEIp during absorption/transepithelial
transport. Usually, di-/tripeptides are absorbed across the brush border
membrane in their intact forms, via specific peptide transporter
systems, while others can be prone to protease degradation (Shen and
Matsui, 2017).

\subsection*{Formulation/delivery
strategies}

{\label{formulationdelivery-strategies}}

During storage and/or food processing, the ACEIp activity may decrease
due to partial degradation or destabilization by proteases, extreme
conditions (e.g. of pH, temperature, oxygen) and/or by unwanted
interaction with other components (cations, lipids, proteins, etc.).
Protective strategies have been employed to maintain the bioavailability
and antihypertensive activity of ACEIp directly included in
food/nutraceuticals. Encapsulation solutions aiming at maintaining
peptide activity during their shelf life or even upon consumption
include the use of liposomes, chitosan particles, among others.

Encapsulation in food-grade liposomes, which can retain both hydrophilic
and hydrophobic peptides, can protect and help the transport of
bioactive peptides ( Mozafari et al., 2008; Malheiros et al., 2010).
Liposomes may even increase nutritional value if derived e.g. from
natural soy lecithin or partially purified phosphatidylcholine, with
high content in polyunsaturated fatty acid and low tocopherol content
(Taladrid et al., 2017). Peptide-loaded liposomes may be easily
introduced into functional foods via a film matrix, which helps avoiding
unwanted strong flavors or peptide interactions with other constituents
(da Silva Malheiros et al., 2010). Encapsulation of ACEIp into food
grade chitosan nanoparticles (CNP) ensures safe oral administration and
decreases gastrointestinal enzymatic degradation. Indeed, ACEIp
stabilized by CNP were shown to more efficiently reduce BP for extended
time periods in SHR (Auwal et al., 2017). Other encapsulation methods
may also improve the bioavailability and efficacy of the ACEIp (Huang et
al., 2017).

Finally, additives such as skim milk, sugars and sugar alcohols, soybean
(\emph{Glycine max} ) casein, casein hydrolysate, and others help to
mask the ACEIp's bitterness, and improve their applicability in
functional foods/nutraceuticals (Iwaniak et al., 2016a; Iwaniak et al.,
2016b; Pooja et al., 2017).

\section*{Genetic engineering ACEI peptides
production}

{\label{genetic-engineering-acei-peptides-production}}

Large-scale production of ACEIp for functional foods and food
supplements is still a challenge. Industrial production of ACEIp has
been mainly based on enzymatic proteolysis of whole food proteins to
release peptides with ACE inhibitory activity (Pihlanto and Mäkinen,
2013). However, such methods generate a complex mixture of compounds and
lead to challenging target peptide isolation processes (Losacco et al.,
2007). The high cost, low recovery and bioavailability of ACEIp produced
by such current methods pushed the need to develop alternative
approaches, as the production of ACEIp via heterologous expression
platforms. Most recombinant ACEIp have been produced in\emph{Escherichia
coli,} the most widely studied heterologous expression host. Emerging
platforms for ACEIp production include lactic acid bacteria (LAB),
plants (predominantly cereals and legumes) and the microalgae
\emph{Chlamydomonas reinhardtii} .

The main bottleneck in the heterologous expression of short peptide
chains as ACEIp is their susceptibility to proteolytic degradation
inside the host system. Accordingly, direct expression of genetically
engineered ACEIp has been proven troublesome. This section describes
strategies to tackle this problem (design of efficient expression
constructs, e.g. fusion proteins, tandem ACEIp; protein targeting
approaches; and use of protease-deficient hosts such as \emph{E.
coli}BL21 (Rao et al., 2009)) and demonstrates the potential of
heterologous expression systems to produce ACEIp at large-scale,
low-cost and in convenient formulations.

\subsection*{Recombinant ACEI peptide expression in
bacteria}

{\label{recombinant-acei-peptide-expression-in-bacteria}}

Bacteria are well-established expression systems for high-level
production of recombinant proteins and peptides, and have been the
preferred systems for heterologous ACEIp expression (Losacco et al.,
2007). \emph{E. coli} is particularly used in new ACEIp production
systems, and the use of LAB is an emerging alternative, given their
Generally Recognized as Safe (GRAS) status.

Four main strategies have been adopted for ACEIp production in bacterial
hosts: 1) generation of multimeric polypeptides containing tandem
repeats of an ACEIp; 2) fusion of ACEIp to proteins; 3) generation of
bioactive polypeptides containing multivariate ACEIp; and 4) mutation of
plant storage proteins to include ACEIp. In all these approaches, the
aim is to protect the ACEIp against degradation, as polypeptides and
fusion proteins are less susceptible to degradation by host peptidases
than single ACEIp. Table 2 presents several recombinant ACEIp produced
in bacterial platforms, their production strategy, yield and
bioactivity.

\subsubsection*{Tandem repeats of ACEIp}

{\label{tandem-repeats-of-aceip}}

The expression of chimeric configurations containing tandem repeats of a
desired ACEIp, flanked by protease recognition sequences that allow the
peptide release after \emph{in vitro} protease cleavage or
gastrointestinal digestion showed significant advantages at the
expression level (Kim et al., 2008).

One of the early examples of the use of ACEIp in tandem consisted of ten
amino acids derived from the yeast GAPDH enzyme (YG-1 gene) in 9, 18, or
27 tandem repeats of the gene separated by clostripain cleavage sites
(Park et al., 1998). Contrary to expectations, the highest expression
level of YG-1 was observed for the 9-mer (67\% of total proteins), a
fact related to the use of the T7 promoter (Park et al., 1998).

Fida et al. (2009) first reported the direct insertion of a small tandem
peptide multimer `gene' into an expression vector without a fusion
protein tag. In this example, the ACEIp `fragmented peptide B'
(PTHIKWGD), retrieved from thermal hydrolysates of tuna meat, was
produced in \emph{E. coli} as 6-mer. Although bypassing digestion steps
to remove the fusion tag, this approach required a prohibitively
expensive anti-peptide antibody affinity chromatography. Recently, the
expression levels of this peptide B in \emph{E. coli} have been improved
by multimerization of the tandem sequence. The original 4-mer peptide B
`genes' were further assembled as 1, 2, 4 and 8x tandem repeats and
fused to a his-tag. A correlation between the degree of multimerization
and the expression level of these multimer `genes' was observed, with
the 32 multimer (8 x 4mer) presenting the highest expression (45.2\% of
total protein). The purified peptide monomers presented antihypertensive
activity in SHR, decreasing systolic BP by 36.5 mm Hg upon 4 h of oral
administration (Li et al., 2015).

Another example is the expression of a sequence encoding IYPR, an ACEIp
isolated from sake and sake lees, as a 7-copy tandem repeat, linked by
trypsin cleavage sites. Following cleavage with trypsin and purification
by affinity chromatography, the resultant ACEIp presented
antihypertensive activity in 10-week old SHR, significantly reducing
systolic BP by 50 mm Hg, upon 4h of a single oral administration (Huang
et al., 2012). Similarly, the DNA-coding sequence for GVYPHK, derived
from a partially purified autolysate of bonito bowels, was linked by a
trypsin cleavage site to form a 10-mer tandem protein (Wang et al.,
2015). A single oral administration to SHR of the ACEIp obtained by
trypsin cleavage, significantly reduced systolic BP already after 2 h of
ingestion.

\subsubsection*{ACEI peptides fused to
proteins/polypeptides}

{\label{acei-peptides-fused-to-proteinspolypeptides}}

In a different approach, single or tandem ACEIp can be fused to proteins
and expressed as high molecular weight fusion proteins. The use of
fusion protein partners has several advantages: boosts peptide
expression by increasing mRNA translation; helps short polypeptides
stabilization; and facilitates the purification of the peptides by
affinity chromatography. An additional proteolytic step is usually
required after translation to release the ACEIp from the fusion partner.
The enzyme glutathione S-transferase (GST) has been the most commonly
used fusion partner, but ubiquitin, dihydrofolate reductase (DHFR) and
maltose-binding protein (MBP) have also been used.

The GST fusion protein system has been used both in single and tandem
ACEIp production. A 6-mer tandem of KVLPVP, derived from the milk
\selectlanguage{greek}β-\selectlanguage{english}casein hydrolysate, was linked by clostripain cleavage sites and fused
to GST (Liu et al., 2007). The pure peptide presented an
IC\textsubscript{50} of 4.6 \selectlanguage{greek}\emph{μ} \selectlanguage{english}M and showed antihypertensive
activity in SHR, dramatically decreasing systolic BP in a dose-dependent
manner. Another example comprises recombinant concatemers of multiple
IYVKY copies, fused to GST. Tandems of 2, 4 and 6-mer of the IYVKY
sequence, linked by the chymotrypsin cleavage site and fused
N-terminally to a His-tag and C-terminally to GST, were produced. Their
expression in \emph{E. coli} BL21 lead to a good yield production of
bioactive single ACEIp IY and VKY (Oh et al., 2002).

The GST expression system was also used to produce single peptides, such
as the ACEI BP1-3, derived from bovine \selectlanguage{greek}β-\selectlanguage{english}casein (Losacco et al., 2007),
and their precursors (Pro-BP1-3) (Losacco et al., 2007). Each Pro-BP or
BP-coding DNA sequences were cloned downstream the GST sequence and
flanked in both sides by the cleavage sites (3-5 aa long) of a membrane
proteinase from \emph{Lactobacillus helveticus} PR4. The use of this
membrane proteinase was strategic, given the common use of this LAB in
manufacturing dairy products that naturally contain similar ACEIp.
However, the IC50 of BP1-3 was relatively high (Table 2).

Besides GST, other fusion proteins have been used in recombinant ACEIp
production. BP1-3 and ProBP1-3 were also expressed in a probiotic strain
of \emph{Bifidobacterium pseudocatenulatum} , fused at the 5' end to a
Shine-Dalgarno ribosome binding site (RBS) consensus sequence (Losurdo
et al., 2013). Although the ACE-inhibitory activity increased in
cell-free extracts of all recombinant hosts, the expression of BP1 and
BP3 and their precursor forms was not detectable by RP-HPLC. This was
attributed to their hydrolysis by intracellular endopeptidases, such as
aminopeptidases and iminopeptidases. Other example is the production of
a single ACEIp from \selectlanguage{greek}α\selectlanguage{english}\textsubscript{s1}-casein FFVAPFPEVFGK (known as
CEI\textsubscript{12}), fused to DHFR. Nevertheless, relatively low
yields (0.5 mg.L\textsuperscript{-1}) of the CEI\textsubscript{12}-DHFR
fusion protein were obtained upon IPTG induction in \emph{E. coli} (Lv
et al., 2003).

These last reports demonstrate the limitations of single ACEIp
expression approaches, which may be partially overcome by using tandem
ACEIp. The synthetic gene coding for the ACEIp HHL, derived from a
Korean soybean paste, was tandemly multimerized to a 40-mer and ligated
to ubiquitin as a fusion gene (UH40). HHL monomers were recovered at 6.2
mg. L\textsuperscript{-1} yield (Jeong et al., 2007), showing the
advantages of fusing proteins with ACEI tandem multimers, in comparison
to single peptide approaches. Sixteen tandem repeats of the
\selectlanguage{greek}α-\selectlanguage{english}lactalbumin-derived ACEIp IW were N-terminally fused to MBP, and
expressed in \emph{E. coli} BL21 (Michelke et al., 2018). This MBP-IW
fusion protein was recovered at low yield (0.52 mg soluble protein/g
of\emph{E. coli} ) and, after hydrolysis with \selectlanguage{greek}α-\selectlanguage{english}chymotrypsin, only 50.78
\selectlanguage{greek}µ\selectlanguage{english}g of IW monomers were released. Still, the ACEI activity of the
recombinant IW was indistinguishable from that of the chemically
synthesized dipeptides.

Despite the moderate success of fusion protein approaches in \emph{E.
coli} , most require expensive and time-consuming purification steps to
remove protein tags and the use of non-food-grade inducer molecules,
such as IPTG. A recent study, based on the initial
CEI\textsubscript{12}expression in \emph{E. coli} (Lv et al., 2003) and
\emph{Streptococcus thermophilus} (Renye and Somkuti, 2008; Renye and
Somkuti, 2015) attempted to surpass these shortcomings by employing
nisin-induced CEI\textsubscript{12} expression in three LAB strains:
\emph{S. thermophilus} ST128, \emph{Lactococcus lactis} subsp.
\emph{lactis} ML3, and \emph{L. casei} C2. A synthetic
CEI\textsubscript{12}-coding gene was cloned under the nisA promoter,
in-frame with the pediocin leader peptide to direct the secretion of the
resultant fusion peptide by LAB hosts. Both \emph{L. lactis} ML3 and
\emph{L. casei} C2 secreted the recombinant peptide, as confirmed by
SDS-PAGE (Renye and Somkuti, 2015). Although recovered recombinant
peptide yields were low, this study is a landmark, as it first reported
the use of GRAS LAB species and of nisin as ``food-grade'' inducer,
prospecting the use of LAB as ACEIP production platforms for the
functional foods industry.

\subsubsection*{Multivariate ACEI peptides in bioactive
polypeptides}

{\label{multivariate-acei-peptides-in-bioactive-polypeptides}}

A third strategy for bacterial expression of bioactive ACEIp relies on
engineering synthetic genes with different ACEIp in tandem. Rao et al.
(2009) described the design and production of a tandem antihypertensive
peptide multimer (AHPM), as a precursor of 11 different ACEIp, joined by
1-3 aa-long linkers corresponding to cleavage sites of gastrointestinal
proteases. The recombinant AHPM polypeptide, fused to a GST tag, was
expressed in \emph{E. coli} mostly as inclusion bodies, and reached a
maximal production of 35\% of total intracellular protein. Although the
yield of multimer peptide recovery was low, simulated gastrointestinal
digestion confirmed the release of highly active fragments from AHPM
(Rao et al., 2009). This research team has also reported the design of a
new polypeptide (BPP-1) composed of several ACEI and antioxidant
peptides, tandemly linked by gastrointestinal proteases cleavage sites.
The BPP-1 precursor consisted of a tandem multimer of the ACEIp: MRW,
WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR, IKPVA, AKF, IW, VAF,
VSV, IQY and IVY, and the antioxidant peptides DTHK, YPIL, FLEPDY,
YLEPFR, YLEPDY, YDEPEW, HYRPFW, YEPDY and IWAPFY. To improve the yield
of soluble form, BPP-1 was further fused to the tag `cationic
elastin-like polypeptide and SUMO' (cELP-SUMO). This tag both enhances
the solubility of fusion proteins and allows its cleavage to efficiently
release peptides (Rao et al., 2016). As a result, cELP-SUMO-BPP-1 was
highly expressed in a soluble form in \emph{E. coli} , consisting of
approximately 52\% of the total soluble proteins, with more than 70\% of
the fusion protein being expressed in a soluble form. After simulated
gastrointestinal digestion of the purified BPP-1 the resulting
hydrolysates exhibited notable \emph{in vitro} ACE inhibitory and
antioxidant activities (Rao et al., 2016).

\subsubsection*{Modified plant storage proteins containing ACEI
peptides}

{\label{modified-plant-storage-proteins-containing-acei-peptides}}

The feasibility of modifying subunits of plant storage proteins to
contain ACEIp has been widely tested in \emph{E. coli} as primary
evaluation step, envisaging their application in food crops. Two plant
storage proteins have been used for ACEIp production: the soybean
\selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit and the Amaranthus amarantin acidic
subunit\emph{.}

\selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit (Soybean). Matoba et al. (2001) introduced
Novokinin (the RPLKPW ACEIp, a potent analogue of ovokinin) into three
homologous sites of the soybean \selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit, by
site-directed mutagenesis. This modified RPLKPW-containing \selectlanguage{greek}α\selectlanguage{english}' subunit
was first expressed in \emph{E. coli} and recovered in a soluble
fraction at yields of 15\% of total protein (Matoba et al., 2001a). When
orally administered, the undigested RPLKPW-containing \selectlanguage{greek}α\selectlanguage{english}' subunit
demonstrated a potent antihypertensive effect and a time-course behavior
similar to the one of free RPLKPW peptide, denoting the rapid release of
the peptide from the protein after ingestion. This study first attested
the \emph{in vivo} functionality of a modified storage protein
containing ACEIp. Nevertheless, the release of the RPLKPW sequences from
the modified subunit by gastrointestinal digestion was only about 30\%
in SHR. To overcome this limitation, Onishi and colleagues (Onishi et
al., 2004) optimized the aa residues surrounding the three RPLKPW
peptide units, to facilitate \emph{in vivo} release. Furthermore, a
fourth RPLKPW sequence was also introduced to improve RPLKPW peptides
yield. This new modified RPLKPW-containing \selectlanguage{greek}α\selectlanguage{english}' subunit was efficiently
expressed in \emph{E. coli} as \textasciitilde{}25\% of total bacterial
proteins (a 10\% yield increased relative to previously reported).
Further, it significantly lowered systolic BP in SHR 4h after oral
administration of a 2.5 mg.kg\textsuperscript{-1} dose, one-fourth of
the previously reported dose, proving the benefits of inserting a
supplementary RPLKPW unit. Finally, a more potent antihypertensive
protein was produced in \emph{E. coli} , as an extension domain
corresponding to residues 1-143 of the modified \selectlanguage{greek}α\selectlanguage{english}' subunit, containing
four RPLKPW sequences, with 1.0 mg.kg\textsuperscript{-1} as the minimum
effective dose.

Acidic subunit of amarantin (Amaranthus). Another plant storage protein
that has been modified to produce active ACEIp is amarantin, the main
seed storage protein of \emph{Amaranthus hypochondriacus} . This
modified amarantin acidic subunit has been extensively evaluated in
\emph{E. coli} , using different site directed mutagenesis strategies.
Luna-Su\selectlanguage{ngerman}árez and colleagues (Luna-Suárez et al., 2010) first reported the
insertion of 4 tandem repeats of the ACEIp VY into the third
hypervariable region of the amarantin acidic subunit, and named this
chimeric protein `bioamarantin'. The same group improved bioamarantin
design by further inserting one copy of RIPP (Castro-Martínez et al.,
2012) or IPP (Medina-Godoy et al., 2013) into the forth hypervariable
region of the amarantin acidic subunit. All modified subunits showed
higher ACEI activities than the native protein. Furthermore, the
enzymatic hydrolysates of AMC3-containing ACEIp sequences (4xVY and IPP)
presented a significant antihypertensive action at 100
mg.kg\textsuperscript{-1} dose, 4.5h after oral administration in SHR
(Medina-Godoy et al., 2013). Finally, this team modified the acidic
subunit of amarantin by inserting 4 VY peptides into the fourth
hypervariable region of the acidic subunit, (`AACM.4') (Morales-Camacho
et al., 2016). AACM.4 showed highest expression levels, greater \emph{in
vivo} or kinetic stability and higher percentage of soluble form
(\textasciitilde{}5\% of total protein), compared to the native protein
or previously reported variants (Luna-Suárez et al., 2010;
Castro-Martínez et al., 2012). Moreover, AACM.4 was also the most
thermostable protein, suggesting that the fourth hypervariable region
modification improves the subunit thermal stability. These results
confirmed AACM.4 as a potential target for future plant transformation
and food additive production. Importantly, the improvement of thermal
stability can be a critical factor during functional food processing
procedures.

\subsection*{Recombinant ACEI expression in plants and
microalgae}

{\label{recombinant-acei-expression-in-plants-and-microalgae}}

Plant proteins are precursors of numerous ACEIp that can be released
during gastrointestinal digestion or plant crops processing. However,
given their low content in natural ACEIp, plant/plant-derived food
consumption is usually insufficient to significantly lower BP.
Furthermore, the industrial production of ACEIp, through enzymatic
hydrolysis of plant proteins, can be troublesome and economically
unviable due to high process costs and low ACEIp yield. The use of plant
biotechnology (Figure 2 and Table 3) can thus expand the ACEI properties
of plant crops, envisaging the establishment of novel plant-derived
functional foods and food supplements.

Until now, rice (\emph{Oryza sativa} ) and soybean were the favored
plant expression hosts for producing ACEIp. Some advantages include the
existence of well-established transformation systems, high level of
recombinant proteins production and high grain yield. Furthermore, the
possibility of ACEIp' accumulation in rice and soybean seeds is highly
beneficial for peptide stability and storage, providing a direct peptide
delivery route (Twyman et al., 2003). Other reported systems of ACEIp
production include cell suspension cultures and transplastomic platforms
based on \emph{C. reinhardtii} .

Figure 2 summarizes the main strategies for ACEIp production in plants
and algae, and Table 3 provides detailed information. In Table 3 three
main approaches are presented by chronological order of report: 1) the
modification of storage proteins to carry ACEIp; 2) the generation of
chimeras containing tandem repeats of ACEIp; 3) the generation of
bioactive polypeptides containing multivariate ACEIp (Kim et al., 2008;
Rosales-Mendoza et al., 2013).

\subsubsection*{Modified plant storage proteins containing ACEI
peptides}

{\label{modified-plant-storage-proteins-containing-acei-peptides-1}}

Expression of ACEIp in plant storage proteins has the advantage of
ensuring long-term protein stability and storage. Storage proteins are
generally located in specialized compartments, such as protein bodies
and vacuoles, which provide appropriate biochemical environments for
protein/peptide accumulation, protecting those from proteolytic
degradation (Twyman et al., 2003). ACEIp-coding sequences are generally
introduced into homologous sites of the protein's variable regions to
minimize changes in protein folding, productivity and localization in
plants. Examples are the modified subunits of glutelin, \selectlanguage{greek}β-\selectlanguage{english}conglycinin
and amarantin.

Glutelin. Modified subunits of the rice storage protein glutelin,
containing the potent ACEIp novokinin (RPLKPW), have been expressed in
transgenic rice (Yang et al., 2006). Fusion proteins were expressed
under the control of endosperm-specific glutelin promoters and
specifically accumulated in seeds. Oral administration in SHR of either
the RPLKPW-glutelin fraction or the transgenic rice seeds significantly
reduced systolic BP, confirming the potential as valid nutraceutical
delivery systems for antihypertensive peptides.

\selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit (Soybean). Based on the findings above
described for \emph{E. coli} (Matoba et al., 2001a; Onishi et al.,
2004), a modified \selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit carrying 4 novokinin peptide
units has been expressed in soybean (Nishizawa et al., 2008). However,
the chimeric protein was only 0.2\% of the total extracted protein from
the transgenic soybean seeds, a low value to assess the seeds' \emph{in
vivo} effects. More recently, novokinin was expressed in transgenic
soybean seeds as a 4 tandem multimer of novokinin fused to the
\selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit. The chimeric protein produced in transgenic
soybean seeds comprised 0.5 \% of total soluble protein and 5 \% of
total \selectlanguage{greek}β-\selectlanguage{english}conglycinin \selectlanguage{greek}α\selectlanguage{english}' subunit. This chimeric protein was shown to
possess antihypertensive activity, reducing systolic BP in SHR after
0.15 g.kg\textsuperscript{-1} protein extract oral administration. A
similar effect was attained following administration of 0.25
g.kg\textsuperscript{-1} dose of defatted flour (Yamada et al., 2008).

Acidic subunit of amarantin (Amaranthus). Based on previous studies in
\emph{E. coli} (Luna-Su\selectlanguage{ngerman}árez et al., 2010), bioamarantin was also
expressed in cell suspension cultures of\emph{Nicotiana tabacum L.} NT1.
Protein hydrolysates of transgenic\emph{calli} showed high ACEI
activity, with 3.5 \selectlanguage{greek}μ\selectlanguage{english}g.ml\textsuperscript{-1} IC50 value , 10-fold lower
than protein extracts of wild-type cells (IC50 of 29.0
\selectlanguage{greek}μ\selectlanguage{english}g.ml\textsuperscript{-1}) (Santos-Ballardo et al., 2013). This was the
first report of the production of a chimeric protein comprising ACEIp in
plant cell suspension cultures. More recently, bioamarantin was also
expressed in transgenic tomato fruits, and stably accumulated at levels
up to 12.71\% of total protein content. Furthermore, a remarkable
increase (5--22 \%) in total protein content was also observed in
transgenic tomato fruits, when compared to non-transformed ones. Protein
hydrolysates from transgenic tomato fruits showed 0.376 to 3.241
\selectlanguage{greek}μ\selectlanguage{english}g.ml\textsuperscript{-1}\emph{in vitro} IC50 values, corresponding to
an increase of up to 13-fold in ACEI activity, when compared to the
non-transformed fruits (Germ\selectlanguage{ngerman}án-Báez et al., 2014). Positive reports of
the expression of modified amarantin variants in \emph{E. coli}
(Luna-Suárez et al., 2010; Medina-Godoy et al., 2013; Morales-Camacho et
al., 2016) along with their sustained expression in tomato (Germán-Báez
et al., 2014) and tobacco (Santos-Ballardo et al., 2013), prospect the
high scale production of ACEIp-containing modified amarantin in
heterologous hosts.

\subsubsection*{Tandem repeats of ACEI
peptides}

{\label{tandem-repeats-of-acei-peptides}}

Following the first attempts of novokinin production in modified storage
proteins in soybean (Nishizawa et al., 2008; Yamada et al., 2008) and
rice (Yang et al., 2006); Wakasa et al. (2011) aimed to generate
transgenic rice seeds that would accumulate higher amounts of novokinin.
Their strategy comprised the expression of 10 or 18 tandemly repeated
novokinin sequences with a KDEL endoplasmic reticulum-retention signal
at the C-terminus, using the glutelin promoter and its signal peptide.
Although the chimeric protein was unexpectedly accumulating in the
nucleolus and at a low level, significant antihypertensive activity was
detected after a single oral dose in SHR. More importantly, this effect
was observed over 5-wk at doses as low as 0.0625
g.kg\textsuperscript{-1}.

More recently, a synthetic gene containing tandem repeats of the VLPVP
ACEIp has been expressed in a transplastomic \emph{C.
reinhardtii}strain. VLPVP-coding sequences were linked by cleavage sites
of pepsin, trypsin, and chymotrypsin. Biomass of recombinant \emph{C.
reinhardtii}was \emph{in vitro} digested, and the VLVPV peptide was
identified and quantified by HPLC. The highest expression line produced
0.292 mg recombinant protein/mg freeze-dried biomass. Intragastric
administration to SHR, at a dose of 30 mg.kg\textsuperscript{-1},
significantly reduced rats systolic BP (Ochoa-Mendez et al., 2016). This
\emph{in vivo}antihypertensive effect first provided a functional
validation of using ACEIp-producing microalgae as food supplements for
hypertension patients.

\subsubsection*{Multivariate ACEI peptides in bioactive
polypeptides}

{\label{multivariate-acei-peptides-in-bioactive-polypeptides-1}}

Campos-Quevedo et al. (2013) reported the design and production of a
milk-derived chimeric protein containing sequences for multifunctional
bioactive peptides, including peptides with hypocholesterolemic,
antihypertensive, opioid, and antimicrobial activities. The precursor
chimeric protein contained 20 different bioactive peptides linked by
gastrointestinal protease cleavage sites. The synthetic gene coding for
the milk-derived chimeric protein was transferred to \emph{C.
reinhardtii} using biolistic bombardment. Transplastomic transformants
containing the target synthetic gene were identified, and ELISA
quantification assay revealed that the expressed chimeric protein
accumulated at levels ranging between 0.16 and 2.4\% of total soluble
protein. Although no functional studies (\emph{in vitro} ACEI activity
or \emph{in vivo} antihypertensive effect) were performed, this study
first showed the potential of \emph{C. reinhardtii} as expression
platform for the production of ACEIp in multifunctional bioactive
polypeptides.

\section*{The future of ACEI
production}

{\label{the-future-of-acei-production}}

ACEI are a major first line treatment in managing hypertension, but
synthetic ACEI carry side-effects.

Food-derived ACEIp have been effective in BP reduction without adverse
effects and can be used as an alternative to synthetic ACEI. Potent
ACEIp have been isolated from whole-food protein hydrolysates of both
animal and plant origin. However, protein hydrolysis-based methods
generate complex peptidic mixtures from which ACEIp must be purified,
increasing production costs. Besides, the resulting recovery yields and
ACEIp bioavailability are low, rendering the process economically
unviable.

The use of recombinant technology for the production of ACEIp can extend
its application for functional foods and pharmaceutical purposes as it
allows the large-scale and low-cost production. \emph{E. coli} has been
the preferred host for recombinant ACEIp production, but lactic acid
bacteria (LAB), plants (predominantly cereals and legumes) and the
microalgae \emph{C. reinhardtii} have also emerged as ACEIp production
hosts.

Bacteria, such as \emph{E. coli} have the advantage of growing fast and
not requiring complex culture systems. A diverse set of molecular tools
available for ACEIp production in \emph{E. coli} including, a wide range
of vectors, promotors and inducers, provide also an important
competitive advantage. Furthermore, LAB species with its GRAS status and
extensive use in the dairy industry are important ACEIp production hosts
for the functional foods industry.

Nevertheless, in recent years, the potential use of plant biotechnology
for the large-scale production of pharmaceutically relevant proteins and
peptides has significantly increased. Given their low-price and safety,
plants offer many advantages for producing valuable recombinant proteins
and peptides, as ACEIp, when compared to mammalian cell cultures (Gomes
et al., 2019). Further, plants are very versatile, encompassing a wide
range of production platforms, from transgenic plants to cell suspension
cultures. The use of rice and soybean, for example, allows ACEIp
accumulation in edible seeds, a direct delivery vehicle with improved
protein stability and storage (Twyman et al., 2003), with soybeans seeds
accumulating up to 40\% protein (dry weight). Legumes as soybeans
produce more proteins than other plants, being promising host systems
for molecular farming. Alternatively, tomato fruits are palatable as raw
tissue and can be lyophilized and stored for a long time (Lico et al.,
2012). The possibility of using plant tissues as direct oral delivery
means, is a major differentiating factor of bioactive peptide production
in plant platforms, compared to mainstream production platforms, such
as\emph{E. coli} . In bacteria, the produced peptides must undergo
complex purification procedures, and processed into a consumable
product. Plant cell suspension cultures are alternatives to transgenic
plants; tobacco\emph{calli} cultures proliferate rapidly, and
technologies for gene transfer and expression are well-established for
this species (Pires et al., 2012; Schillberg et al., 2013). Further,
plant cell suspension cultures grown in sterilized contained
environments provide a cGMP-compatible production environment,
advantageous to the pharmaceutical industry and regulatory authorities
(Paul et al., 2013; Spök et al., 2008). Chloroplast-based expression
platforms also attract great interest in mass scale production of ACEIp,
given its high recombinant protein expression levels (Fletcher et al.,
2007). The algae model \emph{C. reinhardtii} has fast growth rates, GRAS
designation, and can be grown in contained environments (González-Ortega
et al., 2015). Direct oral delivery of ACEIp in this host is also an
option, as green algae are edible and do not contain endotoxins, human
viral or prion contaminants (Mayfield et al., 2007). All these
expression platforms are compatible with the above described genetic
engineering strategies to improve ACEIp expression, including the
multimerization of small peptides in tandem repeats, their fusion with
(or insertion in) highly expressed proteins - with the plus of
targeting/ accumulating the ACEIp-carrying chimeric proteins in seeds -
and the use of similar chimeras, assembling different ACEI and other
peptides into multifunctional bioactive polypeptides/proteins. Together
with the use of gastrointestinal proteases cleavage sites to flank the
peptides, these approaches are perfect for the adequate release of
pharmacologically relevant peptides in edible platforms, for preventive
or therapeutic purposes.

\textbf{Acknowledgments}

Authors acknowledge financial support from Fundação para a Ciência e
Tecnologia (FCT), Portugal2020/Centro2020, and COMPETE, through Duque AS
DL57 research contract, research funding via pAGE program
(Centro-01-0145-FEDER-000003), Research units GREENit ``Bioresources for
Sustainability'' (UID/Multi/04551/2013) and Institute for
Biomedicine/iBiMED (UID/BIM/04501/2013, UID/BIM/04501/2019). Carolina
Gomes and Marisol Gouveia acknowledge their PhD scholarships in the
frame of the Narodowe Centrum Nauki (NCN) grant Sonata-bis
UMO-2015/18/E/NZ2/00694 (PurpleWalls Project), and FCT
SFRH/BD/128893/2017, respectively. Angelini Pharmaceuticals and
Premivalor are also acknowledged for Angelini University Award
2012/2013, as well as all the members of the winner group of the AUA
4\textsuperscript{th} Ed.

\textbf{Authors' declaration}

The authors have no conflict of interest to declare.



\textbf{Figure captions}

\textbf{Figure 1} . The renin-angiotensin and the kallikrein-kinin
systems in the regulation of blood pressure. The intricate interplay
between these two systems, dependent on the ACE, is the basis of the
control of blood pressure. While the renin-angiotensin mechanism leads
to vasoconstriction and water retention, increasing blood pressure, the
kallikrein-kinin system has opposite effects by promoting vasodilation.

\textbf{Figure 2.} Overview of cloning and expression of ACEIp-coding
synthetic genes in plants and algae. Three main cloning strategies are
used: 1) tandem repeats of a target ACEIp sequence; 2) introduction of
the ACEIp in the hypervariable region of storage proteins, and the 3)
assembly of ACEI polypeptides with others bioactive peptides. These
strategies can be applied in different plant/algae expression platforms,
such as whole transgenic plants/algae, transplastomic plants/algae and
plant/algae cell suspension cultures. The resulting ACEIp-enriched
plants/algae can be used for direct human consumption (direct oral
delivery) or processed by hydrolysis and purified to be used as food
supplements. Images used are from Adobe Stock.

\textbf{Tables}

\textbf{Table 1.} Commercially available functional foods or food
ingredients containing ACE inhibitory peptides.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Peptides\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Food\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Product (name)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
FFVAPFPEVFGK\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Milk-derived ingredient (casein-derived dodecapeptide)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
C12 Peption, Casein DP Peptio Drink\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
LKPNM\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Bonito-derived capsules; Bonito-derived tablets; Bonito-derived powder;
Bonito-derived ingredients\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Peptide ACE 3000, PeptACE; Vasotensin\selectlanguage{ngerman}®; Peptide Tea; Levenorm®\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
AKYSY\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Seaweed (\emph{Porphyra yezoensis}) powder; Seaweed (\emph{P.
yezoensis}) beverage\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Mainichi Kaisai Nori; Peptide Nori S\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
IPP and VPP\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Sour milk tablet, beverage; Fermented milk enriched in calcium\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Ameal S, Ameal S 120; Evolus\selectlanguage{ngerman}®\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
LVY\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Sesame-derived beverage\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Goma Pepucha\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
VY\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Sardine-derived ingredient; Sardine-derived beverage; Sardine-derived
tablet\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Valtyron\selectlanguage{ngerman}®; Lapis Support Sato Marine Super P\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
VY, IY and FY\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Seaweed (\emph{Undaria pinnatifida}) jelly\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Wakame Jelly\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
VY, IY and IVY\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Royal jelly beverage\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
StayBalance RJ\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Bonito peptides\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Bonito-derived soup\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Peptide Soup\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Whey peptides\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
b-LG hydrolysate\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
BioZate\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Mushroom peptides\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Mushroom-derived powder\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Bunaharitake\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} ACEI peptides expressed as recombinant chimeric
constructs in bacterial platforms. \emph{Escherichia coli} strains
BL21(DE3), JM109, HMS174(DE3), Origami (DE3) were the mainly used hosts.
Other hosts include LAB species such as \emph{Lactococcus lactis}
,\emph{Lactobacillus casei} (Renye and Somkuti, 2015) and the
probiotic\emph{Bifidobacterium pseudocatenulatum} (Losurdo et al.,
2013). `{[}peptide{]}': yield in amount of purified peptide (mg per L of
culture); `ACEI IC50': ACE inhibitory in vitro activity (IC50, in \selectlanguage{greek}μ\selectlanguage{english}M);
`Anti-HT': antihypertensive activity (mmHg); `Dose': dose of peptides in
mg per Kg of body weight.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Peptide(s)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Construct strategy\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
{[}peptide{]} (mg.L\textsuperscript{-1})\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
ACEI IC\textsubscript{50} (\selectlanguage{greek}μ\selectlanguage{english}M)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Anti-HT (mmHg)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Dose (mg.kg\textsuperscript{-1})\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Ref.\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
YG-1 (GHKIATFQER)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem repeats} 9, 18 or 27x YG-1 linked by the cleavage site of
clostripain;\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
105 (5h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
= natural YG-1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Park et al., 1998)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
KVLPVP\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem repeats} 6x KVLPVP linked by the cleavage site of
clostripain; GST tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
170 (5h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-21.4 (4h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Liu et al., 2007)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Tuna AI (PTHIKWGD)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem repeats} 6x Tuna AI\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
105--115 (8h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Fida et al., 2009)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem repeats} 4, 8, 16 or 32x Tuna AI; N and C- terminal His
tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
218.9 (5h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-36.5 (4h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Li et al., 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IYPR\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem repeats} 7xIYPR linked by the cleavage site of trypsin;
N-terminal enterokinase cleavage site; N-terminal His tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.8 (7h)\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
111.3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-50 (4h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Huang et al., 2012)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
GVYPHK\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem repeats} 10x GVYPHK linked by the cleavage site of
trypsin; N-terminal His tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
13.8 (5h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-42.6 (2h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Wang et al., 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
HHL\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem + Fusion with Ubiquitin} 40xHHL; C- terminal His
tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6.2 (4h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Jeong et al., 2007)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
CEI\textsubscript{12} (FFVAPFPEVFGK)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Fusion with DHFR} 1xCEI\textsubscript{12}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.5 (5h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Lv et al., 2003)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Fusion with pediocin leader peptide}
1xCEI\textsubscript{12}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Renye and Somkuti, 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IW\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem + Fusion with MBP} 16xIW; N-terminal MBP affinity
tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.51 (7h)\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.72\textsuperscript{3} 5.15\textsuperscript{4}
11.08\textsuperscript{5}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Michelke et al., 2018)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IY and VKY\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Tandem + Fusion with GST} 2, 4 or 6x IYVKY linked by the
cleavage site of chymotrypsin; N-terminal His tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
38 (4h)\textsuperscript{6}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
18.53\textsuperscript{6}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Oh et al., 2002)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BP1 (SLVYPFPGPI) BP2 (NIPPLTQTPV) BP3 (DKIHPF)\textsuperscript{7}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Single peptides + Fusion with GST} 1xBP1, 1xBP2 or 1xBP3 flanked
by the cleavage sites of a proteinase from \emph{L. helveticus}
PR4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.5 (6h)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BP1 467.03 BP2 807.18 BP3 1729.21\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Losacco et al., 2007)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BP1, BP2 and BP3 Pro-BP1, Pro-BP2, Pro-BP3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Single peptides + Fusion with RBS} 1xBP1, 1xBP2 or 1xBP3 and
their precursor forms, fused to Shine-Dalgarno RBS sequence for \emph{B.
pseudocatenulatum} at the 5' end\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
NR\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND (increased in extracts)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Losurdo et al., 2013)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
VWIS, VW, RIY, IY, LW, IKW, LKPNM, LKP, RPLKPW, NMAINPSK, IPP\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Multimer of ACEIPs} tandem multimer of 11 ACEIP linked by
cleavage sites of gastrointestinal proteases; GST tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
399 (5h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
33\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Rao et al., 2009)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
MRW, WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR, IW, IKPVA, AKF,
IQY VAF, VSV, IVY\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Multimer of bioactive peptides} tandem multimer of 18 ACEIP and
9 antioxidant peptides linked by cleavage sites of gastrointestinal
proteases; N-terminal cELP-SUMO tag\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
167.2 (20h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.28\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Rao et al., 2016)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Novokinin (RPLKPW)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{Modified β-conglycinin α' subunit}\selectlanguage{english} 1x Novokinin in 3 homologous
sites of the core domain flanked by trypsin and/or chymotrypsin cleavage
sites\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.2 (42h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-25 (4h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Matoba et al., 2001a)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{Modified β-conglycinin α' subunit}\selectlanguage{english} 1x Novokinin in 3 homologous
sites of the core domain plus 1x Novokinin in a homologous site of the
extension domain; flanked by gastrointestinal proteases cleavage
sites\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-18 (4h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.5\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Onishi et al., 2004)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
VY\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Modified amarantin acidic subunit} 4xVY in the third variable
region (VR3); C-terminal His tag \textbf{AACM.3}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
60 (3h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.0\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Luna-Su\selectlanguage{ngerman}árez et al., 2010)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Modified amarantin acidic subunit} 4xVY in the fourth variable
region (VR4); C-terminal His tag \textbf{AACM.4}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
560 (6h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Morales-Camacho et al., 2016)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
VY and RIPP\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Modified amarantin acidic subunit} 4xVY in VR3 and 1xRIPP in
VR4; C-terminal His tag \textbf{AACM3.4}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
50 (3h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.41\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Castro-Mart\selectlanguage{ngerman}ínez et al., 2012)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
VY and IPP\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Modified amarantin acidic subunit} 4xVY in VR3 and 1xIPP in VR4;
C-terminal His tag \textbf{AMC3}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5640\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-58 (4.5h)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
100\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Medina-Godoy et al., 2013)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}T7 promoter was used in all reports, except in (Lv et
al., 2003) (T5 promoter), (Liu et al., 2007) and (Rao et al., 2009) (tac
promoter) and (Renye and Somkuti, 2015) (nisA promoter)

\textsuperscript{b}The presented values correspond to the higher peptide
expression levels obtained. Peptide expression was induced with IPTG,
excepting (Renye and Somkuti, 2015), where nisin was used instead.

\textsuperscript{c}Peptides were orally administered, excepting
(Medina-Godoy et al., 2013), where recombinant protein hydrolysates were
administered by gastric gavage.

ND- Not determined

NR- Not recovered

\textsuperscript{1}mg of peptide per g of wet weight cells

\textsuperscript{2} Value for the chimeric protein

\textsuperscript{3} Value for Rabbit lung ACE

\textsuperscript{4} Value for Human plasma ACE

\textsuperscript{5} Value for Human umbilical vein endothelial cells
(HUVECs) ACE

\textsuperscript{6}Value for the peptide multimer

\textsuperscript{7}BP1-3 complete amino acid sequences (the bioactive
sequences are underlined): BP1, QTQ\emph{SLVYPFPGPI} PNS; BP2,
LPQ\emph{NIPPLTQTPV} VVP; and BP3, EDELQ\emph{DKIHPF} AQTQS. Correspond
to fragments 57--66, 73--82, and 47--52 of bovine \selectlanguage{greek}β-\selectlanguage{english}casein
(A\textsuperscript{2} allelic variant), respectively.

\textbf{Table 3.} ACEI peptides expressed as recombinant constructs in
plant and algae platforms. `Anti-HT': antihypertensive activity (mmHg);
`Dose': dose of peptides in g per Kg of body weight. HVR, hypervariable
region; VR, variable region.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{System}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{Host (accumulation region)}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{ACEI peptide (s)}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{Construct strategy}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{Administration mode}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{Anti-HT (mmHg)}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{Dose (g.kg\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\textbf{Ref.}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Whole transgenic plant}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Rice (seeds)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Novokinin (RPLKPW)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Fusion with glutelin B1 (pGluB1-HRP)} 2x Novokinin in 2 HVR of
GluB1 acidic subunit plus 1x Novokinin in the C terminal VR of GluB1
basic subunit\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
oral administration of crude glutelin B1 fraction\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
-28\selectlanguage{ngerman}±7 (4h)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.03\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Yang et al., 2006)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
oral administration of unpolished pulverized GluB1-HRP rice seeds\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Not significant\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Fusion with glutelin A2 and C (pGluA2-HRP/GluC-HRP)} 2x
Novokinin in 2 HVR of GluA2 and GluC acidic subunits\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
oral administration of unpolished pulverized GluA2-HRP/GluC-HRP rice
seeds\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
-15.6\selectlanguage{ngerman}±4.8 (2h)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Tandem structure} 10x Novokinin + KDEL; 18x Novokinin +
KDEL\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
oral administration of transgenic seeds containing 18x novokinin\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
-15.6\selectlanguage{ngerman}±1.1 (4h)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Wakasa et al., 2011)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Soybean (seeds)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Novokinin (RPLKPW)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{Fusion with β-conglycinin α' subunit}\selectlanguage{english} 1x Novokinin in 4
sites\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
oral administration of \selectlanguage{greek}β-\selectlanguage{english}conglycinin fraction\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
-16.0\selectlanguage{ngerman}±1.5 (8h)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.15\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Yamada et al., 2008)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
oral administration of defatted flour\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-16.4\selectlanguage{ngerman}±4.3 (6h)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.25\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{Fusion with β-conglycinin α' subunit}\selectlanguage{english} 4x Novokinin\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Nishizawa et al., 2008)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Tomato (fruits)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
VY\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Fusion with acidic subunit of amarantin} 4x VY + KDEL\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Germ\selectlanguage{ngerman}án-Báez et al., 2014)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Cell suspension cultures}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Tobacco (\emph{calli)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
VY\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Fusion with acidic subunit of amarantin} 4x VY + KDEL\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Santos-Ballardo et al., 2013)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Transplastomic}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\emph{C. reinhardtii} (chloroplasts)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
VLPVP\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Tandem structure} 6x VLPVP\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Oral administration of freeze-dried biomass\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
-20 (6h)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.30\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Ochoa-Mendez et al., 2016)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GLDIQK VAGTWY LDAQSAPLRLLQK VLVLDTDYK CMENSA ALPMHIR\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Multivariate chimeric peptide}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(Campos-Quevedo et al., 2013)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{References}

Alonso H, Bliznyuk AA, Gready JE. 2006. Combining docking and molecular
dynamic simulations in drug design. \emph{Med. Res. Rev.}\textbf{26}
:531--568.

Aluko RE. 2015. Antihypertensive Peptides from Food Proteins.\emph{Annu.
Rev. Food Sci. Technol.} \textbf{6} :235--262.

Asoodeh A, Yazdi MM, Chamani J. 2012. Purification and characterisation
of angiotensin I converting enzyme inhibitory peptides from lysozyme
hydrolysates. \emph{Food Chem.} \textbf{131} :291--295.

Auwal SM, Zarei M, Tan CP, Basri M, Saari N. 2017. Improved in vivo
efficacy of anti-hypertensive biopeptides encapsulated in chitosan
nanoparticles fabricated by ionotropic gelation on spontaneously
hypertensive rats. \emph{Nanomaterials} \textbf{7} .

Balti R, Bougatef A, Sila A, Guillochon D, Dhulster P, Nedjar-Arroume N.
2015. Nine novel angiotensin I-converting enzyme (ACE) inhibitory
peptides from cuttlefish (Sepia officinalis) muscle protein hydrolysates
and antihypertensive effect of the potent active peptide in
spontaneously hypertensive rats. \emph{Food Chem.}\textbf{170}
:519--525.
http://www.sciencedirect.com/science/article/pii/S0308814613004366.

Berryman LY. 2000. Pharmacotherapy Handbook. 2nd Edition. \emph{Ann.
Pharmacother.} Vol. 34 1490--1490 p.

Brunton L, Lazo J, Parker K. 2005. Goodman \& Gilman's the
pharmacological basis of therapeutics 13th ed. McGraw-Hill Education.

Burrello J, Monticone S, Buffolo F, Tetti M, Veglio F, Williams TA,
Mulatero P. 2017. Is there a role for genomics in the management of
hypertension? \emph{Int. J. Mol. Sci.} \textbf{18} :1131.

Campos-Quevedo N, Rosales-Mendoza S, Paz-Maldonado LMT, Mart\selectlanguage{ngerman}ínez-Salgado
L, Guevara-Arauza JC, Soria-Guerra RE. 2013. Production of milk-derived
bioactive peptides as precursor chimeric proteins in chloroplasts of
Chlamydomonas reinhardtii. \emph{Plant Cell, Tissue Organ
Cult.}\textbf{113} :217--225.
http://dx.doi.org/10.1007/s11240-012-0261-3.

Carretero OA, Oparil S. 2000. Essential hypertension. Part I: Definition
and etiology. \emph{Circulation} \textbf{101} :329--335.

Castellano P, Aristoy MC, Sentandreu MÁ, Vignolo G, Toldrá F. 2013.
Peptides with angiotensin I converting enzyme (ACE) inhibitory activity
generated from porcine skeletal muscle proteins by the action of
meat-borne Lactobacillus. \emph{J. Proteomics} \textbf{89} :183--190.

Castro-Martínez C, Luna-Suárez S, Paredes-López O. 2012. Overexpression
of a modified protein from amaranth seed in Escherichia coli and effect
of environmental conditions on the protein expression. \emph{J.
Biotechnol.} \textbf{158} :59--67.
http://www.sciencedirect.com/science/article/pii/S0168165611006687.

Chen Y, Wang Z, Chen X, Liu Y, Zhang H, Sun T. 2010. Identification of
angiotensin I-converting enzyme inhibitory peptides from koumiss, a
traditional fermented mare's milk. \emph{J. Dairy Sci.}\textbf{93}
:884--892.

Clark MA, Harvey RA, Finkel R, Rey JA, Whalen K. 2011. Pharmacology.
Wolters Kluwer Health. Illustrated Reviews.

Donkor ON, Henriksson A, Vasiljevic T, Shah NP. 2005. Probiotic Strains
as Starter Cultures Improve Angiotensin-converting Enzyme Inhibitory
Activity in Soy Yogurt. \emph{J. Food Sci.} \textbf{70} :m375--m381.
https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-2621.2005.tb11522.x.

Duan X, Wu F, Li M, Yang N, Wu C, Jin Y, Yang J, Jin Z, Xu X. 2014.
Naturally occurring angiotensin I-converting enzyme inhibitory peptide
from a fertilized egg and its inhibitory mechanism. \emph{J. Agric. Food
Chem.} \textbf{62} :5500--5506.

Egan BM. 2007. Combination therapy with an angiotensin-converting enzyme
inhibitor and a calcium channel blocker. \emph{J. Clin. Hypertens.
(Greenwich).} \textbf{9} :783--789.

Ehret GB, Caulfield MJ. 2013. Genes for blood pressure: An opportunity
to understand hypertension. \emph{Eur. Heart J.} \textbf{34} :951--961.

Ehret GB, Ferreira T, Chasman DI, Jackson AU, Schmidt EM, Johnson T,
Thorleifsson G, Luan J, Donnelly LA, Kanoni S, Petersen AK, Pihur V,
Strawbridge RJ, Shungin D, Hughes MF, Meirelles O, Kaakinen M,
Bouatia-Naji N, Kristiansson K, Shah S, Kleber ME, Guo X, Lyytikäinen
LP, Fava C, Eriksson N, Nolte IM, Magnusson PK, Salfati EL, Rallidis LS,
Theusch E, Smith AJP, Folkersen L, Witkowska K, Pers TH, Joehanes R, Kim
SK, Lataniotis L, Jansen R, Johnson AD, Warren H, Kim YJ, Zhao W, Wu Y,
Tayo BO, Bochud M, Absher D, Adair LS, Amin N, Arking DE, Axelsson T,
Baldassarre D, Balkau B, Bandinelli S, Barnes MR, Barroso I, Bevan S,
Bis JC, Bjornsdottir G, Boehnke M, Boerwinkle E, Bonnycastle LL, Boomsma
DI, Bornstein SR, Brown MJ, Burnier M, Cabrera CP, Chambers JC, Chang
IS, Cheng CY, Chines PS, Chung RH, Collins FS, Connell JM, Döring A,
Dallongeville J, Danesh J, De Faire U, Delgado G, Dominiczak AF, Doney
ASF, Drenos F, Edkins S, Eicher JD, Elosua R, Enroth S, Erdmann J,
Eriksson P, Esko T, Evangelou E, Evans A, Fall T, Farrall M, Felix JF,
Ferrières J, Ferrucci L, Fornage M, Forrester T, Franceschini N, Franco
OH, Franco-Cereceda A, Fraser RM, Ganesh SK, Gao H, Gertow K, Gianfagna
F, Gigante B, Giulianini F, Goel A, Goodall AH, Goodarzi MO, Gorski M,
Gräßler J, Groves CJ, Gudnason V, Gyllensten U, Hallmans G, Hartikainen
AL, Hassinen M, Havulinna AS, Hayward C, Hercberg S, Herzig KH, Hicks
AA, Hingorani AD, Hirschhorn JN, Hofman A, Holmen J, Holmen OL, Hottenga
JJ, Howard P, Hsiung CA, Hunt SC, Ikram MA, Illig T, Iribarren C, Jensen
RA, Kähönen M, Kang HM, Kathiresan S, Keating BJ, Khaw KT, Kim YK, Kim
E, Kivimaki M, Klopp N, Kolovou G, Komulainen P, Kooner JS, Kosova G,
Krauss RM, Kuh D, Kutalik Z, Kuusisto J, Kvaløy K, Lakka TA, Lee NR, Lee
I Te, Lee WJ, Levy D, Li X, Liang KW, Lin H, Lin L, Lindström J, Lobbens
S, Männistö S, Müller G, Müller-Nurasyid M, Mach F, Markus HS, Marouli
E, McCarthy MI, McKenzie CA, Meneton P, Menni C, Metspalu A, Mijatovic
V, Moilanen L, Montasser ME, Morris AD, Morrison AC, Mulas A, Nagaraja
R, Narisu N, Nikus K, O'Donnell CJ, O'Reilly PF, Ong KK, Paccaud F,
Palmer CD, Parsa A, Pedersen NL, Penninx BW, Perola M, Peters A, Poulter
N, Pramstaller PP, Psaty BM, Quertermous T, Rao DC, Rasheed A, Rayner
NW, Renström F, Rettig R, Rice KM, Roberts R, Rose LM, Rossouw J, Samani
NJ, Sanna S, Saramies J, Schunkert H, Sebert S, Sheu WHH, Shin YA, Sim
X, Smit JH, Smith A V., Sosa MX, Spector TD, Stan\selectlanguage{polish}č\selectlanguage{ngerman}áková A, Stanton A V.,
Stirrups KE, Stringham HM, Sundstrom J, Swift AJ, Syvänen AC, Tai ES,
Tanaka T, Tarasov K V., Teumer A, Thorsteinsdottir U, Tobin MD, Tremoli
E, Uitterlinden AG, Uusitupa M, Vaez A, Vaidya D, Van Duijn CM, Van
Iperen EPA, Vasan RS, Verwoert GC, Virtamo J, Vitart V, Voight BF,
Vollenweider P, Wagner A, Wain L V., Wareham NJ, Watkins H, Weder AB,
Westra HJ, Wilks R, Wilsgaard T, Wilson JF, Wong TY, Yang TP, Yao J,
Yengo L, Zhang W, Zhao JH, Zhu X, Bovet P, Cooper RS, Mohlke KL,
Saleheen D, Lee JY, Elliott P, Gierman HJ, Willer CJ, Franke L, Hovingh
GK, Taylor KD, Dedoussis G, Sever P, Wong A, Lind L, Assimes TL,
Njølstad I, Schwarz PEH, Langenberg C, Snieder H, Caulfield MJ, Melander
O, Laakso M, Saltevo J, Rauramaa R, Tuomilehto J, Ingelsson E, Lehtimäki
T, Hveem K, Palmas W, März W, Kumari M, Salomaa V, Chen YDI, Rotter JI,
Froguel P, Jarvelin MR, Lakatta EG, Kuulasmaa K, Franks PW, Hamsten A,
Wichmann HE, Palmer CNA, Stefansson K, Ridker PM, Loos RJF, Chakravarti
A, Deloukas P, Morris AP, Newton-Cheh C, Munroe PB. 2016. The genetics
of blood pressure regulation and its target organs from association
studies in 342,415 individuals. \emph{Nat. Genet.}\textbf{48}
:1171--1184.

Fida HM, Kumada Y, Terashima M, Katsuda T, Katoh S. 2009. Tandem
multimer expression of angiotensin I-converting enzyme inhibitory
peptide in Escherichia coli. \emph{Biotechnol J} \textbf{4} :1345--1356.

Fletcher SP, Muto M, Mayfield SP. 2007. Optimization of Recombinant
Protein Expression in the Chloroplasts of Green Algae. In: León, R,
Galván, A, Fernández, E, editors. \emph{Transgenic Microalgae as Green
Cell Factories} . New York, NY: Springer New York, pp. 90--98.
http://dx.doi.org/10.1007/978-0-387-75532-8\_8.

Fujita H, Yoshikawa M. 1999. LKPNM: a prodrug-type ACE-inhibitory
peptide derived from fish protein. \emph{Immunopharmacology}\textbf{44}
:123--127.
http://www.sciencedirect.com/science/article/pii/S0162310999001186.

Geng X, Tian G, Zhang W, Zhao Y, Zhao L, Ryu M, Wang H, Ng TB. 2015.
Isolation of an Angiotensin I-Converting Enzyme Inhibitory Protein with
Antihypertensive Effect in Spontaneously Hypertensive Rats from the
Edible Wild Mushroom Leucopaxillus tricolor. \emph{Molecules}\textbf{20}
:10141--10153. http://www.mdpi.com/1420-3049/20/6/10141/pdf.

Germán-Báez L, Cruz-Mendívil A, Medina-Godoy S, Milán-Carrillo J,
Reyes-Moreno C, Valdez-Ortiz A. 2014. Expression of an engineered
acidic-subunit 11S globulin of amaranth carrying the antihypertensive
peptides VY, in transgenic tomato fruits. \emph{Plant Cell, Tissue Organ
Cult.} \textbf{118} :305--312.
http://dx.doi.org/10.1007/s11240-014-0483-7.

Ghanbari R, Zarei M, Ebrahimpour A, Abdul-Hamid A, Ismail A, Saari N.
2015. Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Oxidant
Activities of Sea Cucumber (Actinopyga lecanora) Hydrolysates.\emph{Int.
J. Mol. Sci.} \textbf{16} :28870--28885.
http://www.mdpi.com/1422-0067/16/12/26140/pdf.

Gomes C, Oliveira F, Isabel Vieira S, Sofia Duque A. 2019. Prospects for
the Production of Recombinant Therapeutic Proteins and Peptides in
Plants: Special Focus on Angiotensin I-Converting Enzyme Inhibitory
(ACEI) Peptides. In: . \emph{Genet. Eng. {[}Working Title{]}} .
IntechOpen.
https://www.intechopen.com/online-first/prospects-for-the-production-of-recombinant-therapeutic-proteins-and-peptides-in-plants-special-focu.

González-Ortega O, López-Limón AR, Morales-Domínguez JF, Soria-Guerra
RE. 2015. Production and purification of recombinant hypocholesterolemic
peptides. \emph{Biotechnol. Lett.} \textbf{37} :41--54.
http://dx.doi.org/10.1007/s10529-014-1657-4.

Gouveia M, Xia K, Colón W, Vieira SI, Ribeiro F. 2017. Protein
aggregation, cardiovascular diseases, and exercise training: Where do we
stand? \emph{Ageing Res. Rev.} \textbf{40} :1--10.

Hartmann R, Meisel H. 2007. Food-derived peptides with biological
activity: from research to food applications. \emph{Curr. Opin.
Biotechnol.} \textbf{18} :163--169.

Hasan F, Kumada Y, Hashimoto N, Katsuda T, Terashima M, Katoh S. 2006.
Fragmentation of angiotensin-I converting enzyme inhibitory peptides
from bonito meat under intestinal digestion conditions and their
characterization. \emph{Food Bioprod. Process.} \textbf{84} :135--138.

Hayes M, Tiwari BK. 2015. Bioactive carbohydrates and peptides in foods:
An overview of sources, downstream processing steps and associated
bioactivities. \emph{Int. J. Mol. Sci.} \textbf{16} :22485--22508.
http://www.mdpi.com/1422-0067/16/9/22485/pdf.

Huang GQ, Xiao JX, Hao LQ, Yang J. 2017. Microencapsulation of an
Angiotensin I-Converting Enzyme Inhibitory Peptide VLPVP by Membrane
Emulsification. \emph{Food Bioprocess Technol.} \textbf{10} :2005--2012.

Huang L, Ma H, Li Y, Li S. 2012. Antihypertensive activity of
recombinant peptide IYPR expressed in Escherichia coli as inclusion
bodies. \emph{Protein Expr. Purif.} \textbf{83} :15--20.
http://www.sciencedirect.com/science/article/pii/S1046592812000538.

Iwaniak A, Minkiewicz P, Darewicz M. 2014. Food-originating ACE
Inhibitors, including antihypertensive peptides, as preventive food
components in blood pressure reduction. \emph{Compr. Rev. Food Sci. Food
Saf.} \textbf{13} :114--134.

Iwaniak A, Minkiewicz P, Darewicz M, Hrynkiewicz M. 2016a. Food
protein-originating peptides as tastants - Physiological, technological,
sensory, and bioinformatic approaches. \emph{Food Res. Int.}\textbf{89}
:27--38.

Iwaniak A, Minkiewicz P, Darewicz M, Sieniawski K, Starowicz P. 2016b.
BIOPEP database of sensory peptides and amino acids. \emph{Food Res.
Int.} \textbf{85} :155--161.

Jafar TH, Stark PC, Schmid CH, Landa M, Maschio G, De Jong PE, De Zeeuw
D, Shahinfar S, Toto R, Levey AS. 2003. Progression of Chronic Kidney
Disease: The Role of Blood Pressure Control, Proteinuria, and
Angiotensin-Converting Enzyme Inhibition. A Patient-Level
Meta-Analysis.\emph{Ann. Intern. Med.} \textbf{139} :244--252.

Jakubczyk A, Baraniak B. 2014. Angiotensin i Converting Enzyme
Inhibitory Peptides Obtained after in Vitro Hydrolysis of Pea (Pisum
sativum var. Bajka) Globulins. \emph{Biomed Res. Int.}\textbf{2014}
:438459.

Jeong DW, Shin DS, Ahn CW, Song IS, Lee HJ. 2007. Expression of
antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia
coli. \emph{J. Microbiol. Biotechnol.} \textbf{17} :952--959.

Jimsheena VK, Gowda LR. 2010. Arachin derived peptides as selective
angiotensin I-converting enzyme ( ACE) inhibitors: Structure-activity
relationship. \emph{Peptides} \textbf{31} :1165--1176.

Kamath BL. 1990. Applied Therapeutics. The Clinical Use of
Drugs.\emph{J. Pharm. Sci.} Wolters Kluwer/Lippincott Williams \&
Wilkins. Vol. 79 279 p.

Kasper DL, Fauci AS, Hauser SL, Longo DL. 2015. Harrison's PRINCIPLES OF
INTERNAL MEDICINE.

Kelly JG, O'Malley K. 1990. Clinical Pharmacokinetics of the Newer ACE
Inhibitors: A Review. \emph{Clin. Pharmacokinet.} \textbf{19} :177--196.

Kim JM, Jang SA, Yu BJ, Sung BH, Cho JH, Kim SC. 2008. High-level
expression of an antimicrobial peptide histonin as a natural form by
multimerization and furin-mediated cleavage. \emph{Appl. Microbiol.
Biotechnol.} \textbf{78} :123--130.
http://dx.doi.org/10.1007/s00253-007-1273-5.

Lee JK, Hong S, Jeon JK, Kim SK, Byun HG. 2009. Purification and
characterization of angiotensin I converting enzyme inhibitory peptides
from the rotifer, Brachionus rotundiformis. \emph{Bioresour.
Technol.}\textbf{100} :5255--5259.

Lee SH, Qian ZJ, Kim SK. 2010. A novel angiotensin I converting enzyme
inhibitory peptide from tuna frame protein hydrolysate and its
antihypertensive effect in spontaneously hypertensive rats. \emph{Food
Chem.} \textbf{118} :96--102.

Li Y, Wang B, Zhang H, Wang Z, Zhu S, Ma H. 2015. High-level expression
of angiotensin converting enzyme inhibitory peptide Tuna AI as tandem
multimer in Escherichia coli BL21 (DE3). \emph{Process
Biochem.}\textbf{50} :545--552.
http://www.sciencedirect.com/science/article/pii/S1359511315000501.

Lico C, Santi L, Twyman RM, Pezzotti M, Avesani L. 2012. The use of
plants for the production of therapeutic human peptides. \emph{Plant
Cell Rep.} \textbf{31} :439--451.

Lim SS, Vos T, Flaxman AD, Danaei G, Shibuya K, Adair-Rohani H, Amann M,
Anderson HR, Andrews KG, Aryee M, Atkinson C, Bacchus LJ, Bahalim AN,
Balakrishnan K, Balmes J, Barker-Collo S, Baxter A, Bell ML, Blore JD,
Blyth F, Bonner C, Borges G, Bourne R, Boussinesq M, Brauer M, Brooks P,
Bruce NG, Brunekreef B, Bryan-Hancock C, Bucello C, Buchbinder R, Bull
F, Burnett RT, Byers TE, Calabria B, Carapetis J, Carnahan E, Chafe Z,
Charlson F, Chen H, Chen JS, Cheng ATA, Child JC, Cohen A, Colson KE,
Cowie BC, Darby S, Darling S, Davis A, Degenhardt L, Dentener F, Des
Jarlais DC, Devries K, Dherani M, Ding EL, Dorsey ER, Driscoll T, Edmond
K, Ali SE, Engell RE, Erwin PJ, Fahimi S, Falder G, Farzadfar F, Ferrari
A, Finucane MM, Flaxman S, Fowkes FGR, Freedman G, Freeman MK, Gakidou
E, Ghosh S, Giovannucci E, Gmel G, Graham K, Grainger R, Grant B,
Gunnell D, Gutierrez HR, Hall W, Hoek HW, Hogan A, Hosgood HD, Hoy D, Hu
H, Hubbell BJ, Hutchings SJ, Ibeanusi SE, Jacklyn GL, Jasrasaria R,
Jonas JB, Kan H, Kanis JA, Kassebaum N, Kawakami N, Khang YH,
Khatibzadeh S, Khoo JP, Kok C, Laden F, Lalloo R, Lan Q, Lathlean T,
Leasher JL, Leigh J, Li Y, Lin JK, Lipshultz SE, London S, Lozano R, Lu
Y, Mak J, Malekzadeh R, Mallinger L, Marcenes W, March L, Marks R,
Martin R, McGale P, McGrath J, Mehta S, Mensah GA, Merriman TR, Micha R,
Michaud C, Mishra V, Hanafiah KM, Mokdad AA, Morawska L, Mozaffarian D,
Murphy T, Naghavi M, Neal B, Nelson PK, Nolla JM, Norman R, Olives C,
Omer SB, Orchard J, Osborne R, Ostro B, Page A, Pandey KD, Parry CDH,
Passmore E, Patra J, Pearce N, Pelizzari PM, Petzold M, Phillips MR,
Pope D, Pope CA, Powles J, Rao M, Razavi H, Rehfuess EA, Rehm JT, Ritz
B, Rivara FP, Roberts T, Robinson C, Rodriguez-Portales JA, Romieu I,
Room R, Rosenfeld LC, Roy A, Rushton L, Salomon JA, Sampson U,
Sanchez-Riera L, Sanman E, Sapkota A, Seedat S, Shi P, Shield K,
Shivakoti R, Singh GM, Sleet DA, Smith E, Smith KR, Stapelberg NJC,
Steenland K, Stöckl H, Stovner LJ, Straif K, Straney L, Thurston GD,
Tran JH, Van Dingenen R, Van Donkelaar A, Veerman JL, Vijayakumar L,
Weintraub R, Weissman MM, White RA, Whiteford H, Wiersma ST, Wilkinson
JD, Williams HC, Williams W, Wilson N, Woolf AD, Yip P, Zielinski JM,
Lopez AD, Murray CJL, Ezzati M. 2012. A comparative risk assessment of
burden of disease and injury attributable to 67 risk factors and risk
factor clusters in 21 regions, 1990-2010: A systematic analysis for the
Global Burden of Disease Study 2010. \emph{Lancet}\textbf{380}
:2224--2260.

Liu D, Sun H, Zhang L, Li S, Qin Z. 2007. High-Level Expression of
Milk-Derived Antihypertensive Peptide in Escherichia coli and Its
Bioactivity. \emph{J. Agric. Food Chem.} \textbf{55} :5109--5112.
http://dx.doi.org/10.1021/jf0703248.

Lloyd-Jones DM, Levy D. 2013. Epidemiology of
Hypertension.\emph{Hypertens. A Companion to Braunwald's Hear. Dis.
Second Ed.}\textbf{2} :1--11.

Losacco M, Gallerani R, Gobbetti M, Minervini F, De Leo F. 2007.
Prodution of active angiotensin-I converting enzyme inhibitory peptides
derived from bovine \selectlanguage{greek}β-\selectlanguage{english}casein by recombinant DNA
technologies.\emph{Biotechnol. J.} \textbf{2} :1425--1434.
http://dx.doi.org/10.1002/biot.200700092.

Losurdo L, Quintieri L, Caputo L, Gallerani R, Mayo B, De Leo F. 2013.
Cloning and expression of synthetic genes encoding angiotensin-I
converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium
pseudocatenulatum. \emph{FEMS Microbiol. Lett.} \textbf{340} :24--32.
http://femsle.oxfordjournals.org/femsle/340/1/24.full.pdf.

Luna-Su\selectlanguage{ngerman}árez S, Medina-Godoy S, Cruz-Hernández A, Paredes-López O. 2010.
Modification of the amaranth 11S globulin storage protein to produce an
inhibitory peptide of the angiotensin I converting enzyme, and its
expression in Escherichia coli. \emph{J. Biotechnol.}\textbf{148}
:240--247.
http://www.sciencedirect.com/science/article/pii/S0168165610002695.

Lv GS, Huo GC, Fu XY. 2003. Expression of Milk-Derived Antihypertensive
Peptide in Escherichia coli. \emph{J. Dairy Sci.}\textbf{86}
:1927--1931.
http://www.sciencedirect.com/science/article/pii/S0022030203737795.

Marczak ED, Ohinata K, Lipkowski AW, Yoshikawa M. 2006. Arg-Ile-Tyr
(RIY) derived from rapeseed protein decreases food intake and gastric
emptying after oral administration in mice. \emph{Peptides}\textbf{27}
:2065--2068.
http://www.sciencedirect.com/science/article/pii/S0196978106001409.

Matoba N, Doyama N, Yamada Y, Maruyama N, Utsumi S, Yoshikawa M. 2001a.
Design and production of genetically modified soybean protein with
anti-hypertensive activity by incorporating potent analogue of
ovokinin(2--7). \emph{FEBS Lett.} \textbf{497} :50--54.
http://www.sciencedirect.com/science/article/pii/S0014579301024346.

Matoba N, Yamada Y, Usui H, Nakagiri R, Yoshikawa M. 2001b. Designing
potent derivatives of ovokinin(2-7), an anti-hypertensive peptide
derived from ovalbumin. \emph{Biosci. Biotechnol. Biochem.}\textbf{65}
:736--739.

Mayfield SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, Muto M,
Marin-Navarro J. 2007. Chlamydomonas reinhardtii chloroplasts as protein
factories. \emph{Curr. Opin. Biotechnol.} \textbf{18} :126--133.

Medina-Godoy S, Rodríguez-Yáñez SK, Bobadilla NA, Pérez-Villalva R,
Valdez-Ortiz R, Hong E, Luna-Suárez S, Paredes-López O, Valdez-Ortiz A.
2013. Antihypertensive activity of AMC3, an engineered 11S amaranth
globulin expressed in Escherichia coli, in spontaneously hypertensive
rats. \emph{J. Funct. Foods} \textbf{5} :1441--1449.
http://www.sciencedirect.com/science/article/pii/S1756464613001400.

Michelke L, Deussen A, Kettner K, Dieterich P, Hagemann D, Kriegel TM,
Martin M. 2018. Biotechnological production of the
angiotensin-converting enzyme inhibitory dipeptide
isoleucine-tryptophan. \emph{Eng. Life Sci.} \textbf{18} :218--226.

Miguel M, Recio I, Ramos M, Delgado MA, Aleixandre MA. 2006.
Antihypertensive effect of peptides obtained from Enterococcus
faecalis-fermented milk in rats. \emph{J. Dairy Sci.}\textbf{89}
:3352--3359.
http://www.sciencedirect.com/science/article/pii/S0022030206723724.

Morales-Camacho JI, Paredes-López O, Espinosa-Hernández E, Fernández
Velasco DA, Luna-Suárez S. 2016. Expression, purification and thermal
stability evaluation of an engineered amaranth protein expressed in
Escherichia coli. \emph{Electron. J. Biotechnol.} \textbf{22} :44--51.
http://www.sciencedirect.com/science/article/pii/S0717345816300264.

Mozafari MR, Khosravi-Darani K, Borazan GG, Cui J, Pardakhty A, Yurdugul
S. 2008. Encapsulation of food ingredients using nanoliposome
technology. \emph{Int. J. Food Prop.} \textbf{11} :833--844.
https://www.tandfonline.com/doi/full/10.1080/10942910701648115.

Murray B, FitzGerald R. 2007. Angiotensin Converting Enzyme Inhibitory
Peptides Derived from Food Proteins: Biochemistry, Bioactivity and
Production. \emph{Curr. Pharm. Des.} \textbf{13} :773--791.

Natesh R, Schwager SLU, Sturrock ED, Acharya KR. 2003. Crystal structure
of the human angiotensin-converting enzyme-lisinopril
complex.\emph{Nature} \textbf{421} :551--554.
https://doi.org/10.1038/nature01370.

Nishizawa K, Kita A, Doi C, Yamada Y, Ohinata K, Yoshikawa M, Ishimoto
M. 2008. Accumulation of the bioactive peptides, novokinin, LPYPR and
rubiscolin, in seeds of genetically modified soybean. \emph{Biosci.
Biotechnol. Biochem.} \textbf{72} :3301--3305.
https://www.jstage.jst.go.jp/article/bbb/72/12/72\_80468/\_pdf.

Norris R, FitzGerald RJ. 2013. Antihypertensive peptides from food
proteins. \emph{Bioact. Food Pept. Heal.
Dis.}http://www.intechopen.com/books/export/citation/EndNote/bioactive-food-peptides-in-health-and-disease/antihypertensive-peptides-from-food-proteins.

Ochoa-Mendez CE, Lara-Hernandez I, Gonzalez LM, Aguirre-Banuelos P,
Ibarra-Barajas M, Castro-Moreno P, Gonzalez-Ortega O, Soria-Guerra RE.
2016. Bioactivity of an antihypertensive peptide expressed in
Chlamydomortas reinhardtii. \emph{J. Biotechnol.} \textbf{240} :76--84.
http://ac.els-cdn.com/S0168165616315905/1-s2.0-S0168165616315905-main.pdf?\_tid=a9fa3f72-ec61-11e6-8c63-00000aacb361\&acdnat=1486381669\_04731dc4c06a041c70d82ed1f530e768.

Oh KS, Park YS, Sung HC. 2002. Expression and purification of an
ACE-inhibitory peptide multimer from synthetic DNA in Escherichia
coli.\emph{J. Microbiol. Biotechnol.} \textbf{12} :59--64.

Onishi K, Matoba N, Yamada Y, Doyama N, Maruyama N, Utsumi S, Yoshikawa
M. 2004. Optimal designing of \selectlanguage{greek}β-\selectlanguage{english}conglycinin to genetically incorporate
RPLKPW, a potent anti-hypertensive peptide. \emph{Peptides}\textbf{25}
:37--43.
http://www.sciencedirect.com/science/article/pii/S0196978103003711.

Oparil S, Zaman MA, Calhoun DA. 2003. Pathogenesis of
Hypertension.\emph{Ann. Intern. Med.} \textbf{139} :761--776.

Orona-Tamayo D, Valverde ME, Nieto-Rend\selectlanguage{ngerman}ón B, Paredes-López O. 2015.
Inhibitory activity of chia (Salvia hispanica L.) protein fractions
against angiotensin I-converting enzyme and antioxidant
capacity.\emph{LWT - Food Sci. Technol.} \textbf{64} :236--242.

Park CJ, Lee JH, Hong SS, Lee HS, Kim SC. 1998. High-level expression of
the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem
multimers in Escherichia coli. \emph{Appl. Microbiol.
Biotechnol.}\textbf{50} :71--76.
http://dx.doi.org/10.1007/s002530051258.

Paul M, Teh A, Twyman R, Ma J. 2013. Target Product Selection - Where
Can Molecular Pharming Make the Difference? \emph{Curr. Pharm. Des.}

Perico N, Ruggenenti P, Remuzzi G. 2017. ACE and SGLT2 inhibitors: the
future for non-diabetic and diabetic proteinuric renal
disease.\emph{Curr. Opin. Pharmacol.} \textbf{33} :34--40.

Piepho RW. 2000. Overview of the angiotensin-converting-enzyme
inhibitors. \emph{Am. J. Heal. Pharm.} \textbf{57} :S3-7.

Pihlanto A, Mäkinen S. 2013. Antihypertensive properties of plant
protein derived peptides. \emph{Bioact. Food Pept. Heal.
Dis.}http://www.intechopen.com/books/export/citation/EndNote/bioactive-food-peptides-in-health-and-disease/antihypertensive-properties-of-plant-protein-derived-peptides.

Pires AS, Rosa S, Castanheira S, Fevereiro P, Abranches R. 2012.
Expression of a recombinant human erythropoietin in suspension cell
cultures of Arabidopsis, tobacco and Medicago. \emph{Plant Cell. Tissue
Organ Cult.} \textbf{110} :171--181.

Pooja K, Rani S, Prakash B. 2017. In silico approaches towards the
exploration of rice bran proteins-derived angiotensin-I-converting
enzyme inhibitory peptides. \emph{Int. J. Food Prop.}\textbf{20}
:2178--2191. https://doi.org/10.1080/10942912.2017.1368552.

Pullalarevu R, Akbar G, Teehan G. 2014. Secondary Hypertension, Issues
in Diagnosis and Treatment. \emph{Prim. Care - Clin. Off.
Pract.}\textbf{41} :749--764.

Quiros A, Ramos M, Muguerza B, Delgado MA, Miguel M, Aleixandre A, Recio
I. 2007. Identification of novel antihypertensive peptides in milk
fermented with Enterococcus faecalis. \emph{Int. Dairy J.}\textbf{17}
:33--41.

Rao SQ, Su YJ, Li JH, Xu ZZ, Yang YJ. 2009. Design and Expression of
Recombinant Antihypertensive Peptide Multimer Gene in Escherichia coli
BL21. \emph{J. Microbiol. Biotechnol.} \textbf{19} :1620--1627.

Rao SQ, Zang XY, Yang ZQ, Gao L, Yin YQ, Fang WM. 2016. Soluble
expression and purification of the recombinant bioactive peptide
precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion
system. \emph{Protein Expr. Purif.} \textbf{118} :113--119.
http://ac.els-cdn.com/S1046592815300978/1-s2.0-S1046592815300978-main.pdf?\_tid=c64c1932-1c43-11e6-b14f-00000aacb360\&acdnat=1463498990\_c56f26b96fca15b3535af97d2b0a7125.

Rayaprolu S, Hettiarachchy N, Horax R, Satchithanandam E, Chen P,
Mauromoustakos A. 2015. Amino Acid Profiles of 44 Soybean Lines and
ACE-I Inhibitory Activities of Peptide Fractions from Selected
Lines.\emph{J. Am. Oil Chem. Soc.} \textbf{92} :1023--1033.
http://download.springer.com/static/pdf/189/art\%253A10.1007\%252Fs11746-015-2655-y.pdf?originUrl=http\%3A\%2F\%2Flink.springer.com\%2Farticle\%2F10.1007\%2Fs11746-015-2655-y\&token2=exp=1455304249\textasciitilde{}acl=\%2Fstatic\%2Fpdf\%2F189\%2Fart\%25253A10.1007\%25252Fs11746-015-265.

Renye JA, Somkuti GA. 2008. Cloning of milk-derived bioactive peptides
in Streptococcus thermophilus. \emph{Biotechnol. Lett.}\textbf{30}
:723--730.
http://download.springer.com/static/pdf/609/art\%253A10.1007\%252Fs10529-007-9600-6.pdf?originUrl=http\%3A\%2F\%2Flink.springer.com\%2Farticle\%2F10.1007\%2Fs10529-007-9600-6\&token2=exp=1450235258\textasciitilde{}acl=\%2Fstatic\%2Fpdf\%2F609\%2Fart\%25253A10.1007\%25252Fs10529-007-960.

Renye JA, Somkuti GA. 2015. Nisin-induced expression of a recombinant
antihypertensive peptide in dairy lactic acid bacteria.\emph{Biotechnol.
Lett.} \textbf{37} :1447--1454.

Richard MA, Huan T, Ligthart S, Gondalia R, Jhun MA, Brody JA, Irvin MR,
Marioni R, Shen J, Tsai PC, Montasser ME, Jia Y, Syme C, Salfati EL,
Boerwinkle E, Guan W, Mosley TH, Bressler J, Morrison AC, Liu C,
Mendelson MM, Uitterlinden AG, van Meurs JB, Heijmans BT, 't Hoen PAC,
van Meurs J, Isaacs A, Jansen R, Franke L, Boomsma DI, Pool R, van
Dongen J, Hottenga JJ, van Greevenbroek MMJ, Stehouwer CDA, van der
Kallen CJH, Schalkwijk CG, Wijmenga C, Zhernakova A, Tigchelaar EF,
Slagboom PE, Beekman M, Deelen J, van Heemst D, Veldink JH, van den Berg
LH, van Duijn CM, Hofman A, Uitterlinden AG, Jhamai PM, Verbiest M,
Suchiman HED, Verkerk M, van der Breggen R, van Rooij J, Lakenberg N,
Mei H, van Iterson M, van Galen M, Bot J, van 't Hof P, Deelen P, Nooren
I, Moed M, Vermaat M, Zhernakova D V., Luijk R, Bonder MJ, van Dijk F,
Arindrarto W, Kielbasa SM, Swertz MA, van Zwet EW, Franco OH, Zhang G,
Li Y, Stewart JD, Bis JC, Psaty BM, Chen YDI, Kardia SLR, Zhao W, Turner
ST, Absher D, Aslibekyan S, Starr JM, McRae AF, Hou L, Just AC, Schwartz
JD, Vokonas PS, Menni C, Spector TD, Shuldiner A, Damcott CM, Rotter JI,
Palmas W, Liu Y, Paus T, Horvath S, O'Connell JR, Guo X, Pausova Z,
Assimes TL, Sotoodehnia N, Smith JA, Arnett DK, Deary IJ, Baccarelli AA,
Bell JT, Whitsel E, Dehghan A, Levy D, Fornage M. 2017. DNA Methylation
Analysis Identifies Loci for Blood Pressure Regulation. \emph{Am. J.
Hum. Genet.} \textbf{101} :888--902.

Rimoldi SF, Scherrer U, Messerli FH. 2014. Secondary arterial
hypertension: When, who, and how to screen? \emph{Eur. Heart
J.}\textbf{35} :1245--1254.

Rojas-Ronquillo R, Cruz-Guerrero A, Flores-Najera A, Rodriguez-Serrano
G, Gomez-Ruiz L, Reyes-Grajeda JP, Jimenez-Guzman J, Garcia-Garibay M.
2012. Antithrombotic and angiotensin-converting enzyme inhibitory
properties of peptides released from bovine casein by Lactobacillus
casei Shirota. \emph{Int. Dairy J.} \textbf{26} :147--154.

Rosales-Mendoza S, Paz-Maldonado LMT, Govea-Alonso DO, Korban SS. 2013.
Engineering production of antihypertensive peptides in
plants.\emph{Plant Cell Tissue Organ Cult.} \textbf{112} :159--169.

Ruiz-Gimenez P, Salom JB, Marcos JF, Valles S, Martinez-Maqueda D, Recio
I, Torregrosa G, Alborch E, Manzanares P. 2012. Antihypertensive effect
of a bovine lactoferrin pepsin hydrolysate: Identification of novel
active peptides. \emph{Food Chem.} \textbf{131} :266--273.

Ryan JT, Ross RP, Bolton D, Fitzgerald GF, Stanton C. 2011. Bioactive
peptides from muscle sources: Meat and fish. \emph{Nutrients}\textbf{3}
:765--791.

Santos-Ballardo D, Germán-Báez L, Cruz-Mendívil A, Fuentes-Gutiérrez C,
Milán-Carrillo J, Reyes-Moreno C, Valdez-Ortiz A. 2013. Expression of
the acidic-subunit of amarantin, carrying the antihypertensive
biopeptides VY, in cell suspension cultures of Nicotiana tabacum
NT1.\emph{Plant Cell, Tissue Organ Cult.} \textbf{113} :315--322.
http://dx.doi.org/10.1007/s11240-012-0271-1.

Schillberg S, Raven N, Fischer R, Twyman R, Schiermeyer A. 2013.
Molecular Farming of Pharmaceutical Proteins Using Plant Suspension Cell
and Tissue Cultures. \emph{Curr. Pharm. Des.}

Seppo L, Jauhiainen T, Poussa T, Korpela R. 2003. A fermented milk high
in bioactive peptides has a blood pressure-lowering effect in
hypertensive subjects. \emph{Am. J. Clin. Nutr.} \textbf{77} :326--330.

Shen W, Matsui T. 2017. Current knowledge of intestinal absorption of
bioactive peptides. \emph{Food Funct.} \textbf{8} :4306--4314.

da Silva Malheiros P, Daroit DJ, Brandelli A. 2010. Food applications of
liposome-encapsulated antimicrobial peptides. \emph{Trends Food Sci.
Technol.} \textbf{21} :284--292.

Soares de Castro RJ, Sato HH. 2015. Biologically active peptides:
Processes for their generation, purification and identification and
applications as natural additives in the food and pharmaceutical
industries. \emph{Food Res. Int.} \textbf{74} :185--198.

Spök A, Twyman RM, Fischer R, Ma JKC, Sparrow PAC. 2008. Evolution of a
regulatory framework for pharmaceuticals derived from genetically
modified plants. \emph{Trends Biotechnol.} \textbf{26} :506--517.

Sun H, Chang Q, Liu L, Chai K, Lin G, Huo Q, Zhao Z, Zhao Z. 2017.
High-Throughput and Rapid Screening of Novel ACE Inhibitory Peptides
from Sericin Source and Inhibition Mechanism by Using in Silico and in
Vitro Prescriptions. \emph{J. Agric. Food Chem.}\textbf{65}
:10020--10028.

Tai HM, Li CC, Hung CY, Yin LJ. 2018. Production of functional peptides
with inhibition ability against angiotensin I-Converting enzyme using P.
pastoris expression system. \emph{J. Food Drug Anal.}\textbf{26}
:1097--1104. https://doi.org/10.1016/j.jfda.2018.02.001.

Taladrid D, Marín D, Alemán A, Álvarez-Acero I, Montero P, Gómez-Guillén
MC. 2017. Effect of chemical composition and sonication procedure on
properties of food-grade soy lecithin liposomes with added
glycerol.\emph{Food Res. Int.} \textbf{100} :541--550.

Tran HB, Yamamoto A, Matsumoto S, Ito H, Igami K, Miyazaki T, Kondo R,
Shimizu K. 2014. Hypotensive effects and angiotensin-converting enzyme
inhibitory peptides of reishi (Ganoderma lingzhi) auto-digested
extract.\emph{Molecules} \textbf{19} :13473--13485.
http://www.ncbi.nlm.nih.gov/pubmed/25178067.

Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. 2003.
Molecular farming in plants: Host systems and expression
technology.\emph{Trends Biotechnol.} \textbf{21} :570--578.

Vásquez-Villanueva R, Marina ML, García MC. 2015. Revalorization of a
peach (Prunus persica (L.) Batsch) byproduct: Extraction and
characterization of ACE-inhibitory peptides from peach stones. \emph{J.
Funct. Foods} \textbf{18} :137--146.

Wakasa Y, Zhao H, Hirose S, Yamauchi D, Yamada Y, Yang L, Ohinata K,
Yoshikawa M, Takaiwa F. 2011. Antihypertensive activity of transgenic
rice seed containing an 18-repeat novokinin peptide localized in the
nucleolus of endosperm cells. \emph{Plant Biotechnol. J.}\textbf{9}
:729--735. http://dx.doi.org/10.1111/j.1467-7652.2010.00576.x.

Wang NY, Young JH, Meoni LA, Ford DE, Erlinger TP, Klag MJ. 2008. Blood
pressure change and risk of hypertension associated with parental
hypertension: The Johns Hopkins precursors study. \emph{Arch. Intern.
Med.} \textbf{168} :643--648.

Wang XL, Ma SN, Yuan YH, Ding Y, Li DS. 2015. Expression and
purification recombinant antihypertensive peptide ameliorates
hypertension in rats with spontaneous hypertension. \emph{Protein Expr.
Purif.} \textbf{113} :30--34.
http://www.sciencedirect.com/science/article/pii/S1046592815001011.

Warren HR, Evangelou E, Cabrera CP, Gao H, Ren M, Mifsud B, Ntalla I,
Surendran P, Liu C, Cook JP, Kraja AT, Drenos F, Loh M, Verweij N,
Marten J, Karaman I, Segura Lepe MP, O'Reilly PF, Knight J, Snieder H,
Kato N, He J, Shyong Tai E, Abdullah Said M, Porteous D, Alver M,
Poulter N, Farrall M, Gansevoort RT, Padmanabhan S, Mägi R, Stanton A,
Connell J, Bakker SJL, Metspalu A, Shields DC, Thom S, Brown M, Sever P,
Esko T, Hayward C, van der Harst P, Saleheen D, Chowdhury R, Chambers
JC, Chasman DI, Chakravarti A, Newton-Cheh C, Lindgren CM, Levy D,
Kooner JS, Keavney B, Tomaszewski M, Samani NJ, Howson JMM, Tobin MD,
Munroe PB, Ehret GB, Wain L V., Barnes MR, Tzoulaki I, Caulfield MJ,
Elliott P, Wain V, Vaez A, Jansen R, Joehanes R, van der Most PJ, Mesut
Erzurumluoglu A, O'Reilly P, Rose LM, Verwoert GC, Hottenga JJ,
Strawbridge RJ, Esko T, Arking DE, Hwang SJ, Guo X, Kutalik Z, Trompet
S, Shrine N, Teumer A, Ried JS, Bis JC, Smith A V., Amin N, Nolte IM,
Lyytikäinen LP, Mahajan A, Wareham NJ, Hofer E, Joshi PK, Kristiansson
K, Traglia M, Havulinna AS, Goel A, Nalls MA, Sõber S, Vuckovic D, Luan
J, Fabiola Del Greco M, Ayers KL, Marrugat J, Ruggiero D, Lopez LM,
Niiranen T, Enroth S, Jackson AU, Nelson CP, Huffman JE, Zhang W, Gandin
I, Harris SE, Zemonik T, Lu Y, Shah N, de Borst MH, Mangino M, Prins BP,
Campbell A, Li-Gao R, Chauhan G, Oldmeadow C, Abecasis G, Abedi M,
Barbieri CM, Batini C, Blake T, Boehnke M, Bottinger EP, Braund PS,
Brumat M, Campbell H, Cocca M, Collins F, Cordell HJ, Damman JJ, Davies
G, de Geus EJ, de Mutsert R, Deelen J, Demirkale Y, Doney ASF, Dörr M,
Ferreira T, Frånberg M, Giedraitis V, Gieger C, Giulianini F, Gow AJ,
Hamsten A, Harris TB, Hofman A, Holliday EG, Jarvelin MR, Johansson Å,
Johnson AD, Jousilahti P, Jula A, Kähönen M, Kathiresan S, Khaw KT,
Kolcic I, Koskinen S, Langenberg C, Larson M, Launer LJ, Lehne B,
Liewald DCM, Lin L, Lind L, Mach F, Mamasoula C, Menni C, Milaneschi Y,
Morgan A, Morris AD, Morrison AC, Munson PJ, Nandakumar P, Nguyen QT,
Nutile T, Oldehinkel AJ, Oostra BA, Org E, Palotie A, Paré G, Pattie A,
Penninx BWJH, Pramstaller PP, Raitakari OT, Rice K, Ridker PM, Riese H,
Ripatti S, Robino A, Rotter JI, Rudan I, Saba Y, Saint Pierre A, Sala
CF, Sarin AP, Schmidt R, Scott R, Seelen MA, Siscovick D, Sorice R,
Stott DJ, Sundström J, Swertz M, Taylor KD, Tzourio C, Uitterlinden AG,
Völker U, Vollenweider P, Wild S, Willemsen G, Wright AF, Yao J,
Thériault S, Conen D, John A, Debette S, Mook-Kanamori DO, Zeggini E,
Spector TD, Palmer CNA, Vergnaud AC, Loos RJF, Polasek O, Starr JM,
Girotto G, Lindgren CM, Vitart V, Tuomilehto J, Gyllensten U, Knekt P,
Deary IJ, Ciullo M, Elosua R, Keavney BD, Hicks AA, Scott RA, Gasparini
P, Laan M, Liu YM, Watkins H, Hartman CA, Salomaa V, Toniolo D, Perola
M, Wilson JF, Schmidt H, Zhao JH, Lehtimäki T, van Duijn CM, Gudnason V,
Psaty BM, Peters A, Rettig R, James A, Wouter Jukema J, Strachan DP,
Palmas W, Ingelsson E, Boomsma DI, Franco OH, Bochud M, Morris AP. 2017.
Genome-wide association analysis identifies novel blood pressure loci
and offers biological insights into cardiovascular risk. \emph{Nat.
Genet.} \textbf{49} :403--415.

Weber MA. 1991. Overview of Fosinopril: A Novel ACE Inhibitor.\emph{Drug
Investig.} \textbf{3} :3--11.

Williams B, Mancia G, Spiering W, Rosei EA, Azizi M, Burnier M, Clement
DL, Coca A, De Simone G, Dominiczak A, Kahan T, Mahfoud F, Redon J,
Ruilope L, Zanchetti A, Kerins M, Kjeldsen SE, Kreutz R, Laurent S, Lip
GYH, McManus R, Narkiewicz K, Ruschitzka F, Schmieder RE, Shlyakhto E,
Tsioufis C, Aboyans V, Desormais I, De Backer G, Heagerty AM, Agewall S,
Bochud M, Borghi C, Boutouyrie P, Brguljan J, Bueno H, Caiani EG,
Carlberg B, Chapman N, Cífková R, Cleland JGF, Collet JP, Coman IM, De
Leeuw PW, Delgado V, Dendale P, Diener HC, Dorobantu M, Fagard R,
Farsang C, Ferrini M, Graham IM, Grassi G, Haller H, Hobbs FDR,
Jelakovic B, Jennings C, Katus HA, Kroon AA, Leclercq C, Lovic D, Lurbe
E, Manolis AJ, McDonagh TA, Messerli F, Muiesan ML, Nixdorff U, Olsen
MH, Parati G, Perk J, Piepoli MF, Polonia J, Ponikowski P, Richter DJ,
Rimoldi SF, Roffi M, Sattar N, Seferovic PM, Simpson IA, Sousa-Uva M,
Stanton A V., Van De Borne P, Vardas P, Volpe M, Wassmann S, Windecker
S, Zamorano JL. 2018. 2018 ESC/ESH Guidelines for themanagement of
arterial hypertension. \emph{Eur. Heart J.} \textbf{39} :3021--3104.
https://doi.org/10.1093/eurheartj/ehy339.

World Health Organization. 2013. A global brief on Hypertension - World
Health Day 2013. \emph{World Heal. Organ.} Geneva, World Health
Organization 1--40 p.

Wu J, Aluko RE, Nakai S. 2006. Structural Requirements of Angiotensin
I-Converting Enzyme Inhibitory Peptides:~ Quantitative
Structure\selectlanguage{english}-Activity Relationship Study of Di- and Tripeptides. \emph{J.
Agric. Food Chem.} \textbf{54} :732--738.

Wu J, Liao W, Udenigwe CC. 2017. Revisiting the mechanisms of ACE
inhibitory peptides from food proteins. \emph{Trends Food Sci.
Technol.}\textbf{69} :214--219.

Wu Q, Du J, Jia J, Kuang C. 2016. Production of ACE inhibitory peptides
from sweet sorghum grain protein using alcalase: Hydrolysis kinetic,
purification and molecular docking study. \emph{Food Chem.}\textbf{199}
:140--149.
http://ac.els-cdn.com/S0308814615302909/1-s2.0-S0308814615302909-main.pdf?\_tid=cfd75606-1c43-11e6-bfba-00000aab0f01\&acdnat=1463499005\_23eac51ea42ac2856762f238dbb0352b.

Xie C liang, Choung S young, Cao G ping, Lee KW, Choi YJ. 2015. In
silico investigation of action mechanism of four novel angiotensin-I
converting enzyme inhibitory peptides modified with Trp. \emph{J. Funct.
Foods} \textbf{17} :632--639.

Yamada Y, Nishizawa K, Yokoo M, Zhao H, Onishi K, Teraishi M, Utsumi S,
Ishimoto M, Yoshikawa M. 2008. Anti-hypertensive activity of genetically
modified soybean seeds accumulating novokinin.
\emph{Peptides}\textbf{29} :331--337.

Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Takaiwa F. 2006. A
transgenic rice seed accumulating an anti-hypertensive peptide reduces
the blood pressure of spontaneously hypertensive rats. \emph{FEBS
Lett.}\textbf{580} :3315--3320.
http://www.sciencedirect.com/science/article/pii/S0014579306005746.

Yeates K, Lohfeld L, Sleeth J, Morales F, Rajkotia Y, Ogedegbe O. 2015.
A Global Perspective on Cardiovascular Disease in Vulnerable
Populations. \emph{Can. J. Cardiol.} \textbf{31} :1081--1093.

Yu Z, Yin Y, Zhao W, Yu Y, Liu B, Liu J, Chen F. 2011. Novel peptides
derived from egg white protein inhibiting alpha-glucosidase. \emph{Food
Chem.} \textbf{129} :1376--1382.
http://www.sciencedirect.com/science/article/pii/S0308814611007709.

Zisaki A, Miskovic L, Hatzimanikatis V. 2014. Antihypertensive Drugs
Metabolism: An Update to Pharmacokinetic Profiles and Computational
Approaches. \emph{Curr. Pharm. Des.} \textbf{21} :806--822.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1-RAKK-systems/Figure-1-RAKK-systems}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2-ACEI-peptides-production/Figure-2-ACEI-peptides-production}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

