Bioactive Protein Hydrolysates in The Functional Food Ingredient Industry Overcoming Current Challenges

Food Reviews International
ISSN: 8755-9129 (Print) 1525-6103 (Online) Journal homepage: https://www.tandfonline.com/loi/lfri20
Bioactive protein hydrolysates in the functional

food ingredient industry: Overcoming current
challenges
Tomas Lafarga & Maria Hayes
To cite this article: Tomas Lafarga & Maria Hayes (2017) Bioactive protein hydrolysates in the
functional food ingredient industry: Overcoming current challenges, Food Reviews International,
33:3, 217-246, DOI: 10.1080/87559129.2016.1175013
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FOOD REVIEWS INTERNATIONAL
2017, VOL. 33, NO. 3, 217–246
http://dx.doi.org/10.1080/87559129.2016.1175013
Bioactive protein hydrolysates in the functional food

ingredient industry: Overcoming current challenges
Tomas Lafarga and Maria Hayes
Food BioSciences Department, Teagasc Food Research Centre, The Irish Agricultural and Food Development
Authority, Ashtown, Dublin, Ireland
ABSTRACT KEYWORDS
Meat proteins and associated by-products can be used as a source of Allergenicity; bioactive
bioactive hydrolysates and peptides with potential for use as func- hydrolysates; bitter peptides;
tional food ingredients. Functional foods are foods that have a poten- functional foods; meat
tially positive effect on health, beyond basic nutrition. Numerous co-products; toxicity
bioactive peptides, including angiotensin-I-converting enzyme (ACE-
I, EC 3.4.15.1) and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) inhibi-
tors, have been generated from meat by-product proteins to date.
However, in order to use and commercialize bioactive hydrolysates
and peptides as food ingredients, a number of significant challenges
must first be overcome. This article gives an overview of the current
state-of-the-art of meat-derived bioactive hydrolysate and peptide
uses in the food industry. It also reviews frequent challenges faced
when developing biologically active hydrolysates and peptides as food
ingredients. These challenges include, but are not limited to, high
production costs, negative sensory attributes in end products, taste
modifications of carrier food products and compliance with, for exam-
ple, the European Food Safety Authority (EFSA), the Food and Drug
Administration (FDA), and other regulatory bodies in China, or Japan,
as well as potential toxicity or allergenicity. We suggest strategies that
may assist in overcoming these challenges, focusing on those that may
be used to improve the taste attributes of the end products.
Introduction
The human population is predicted to grow by two to four billion people by 2050.(1) This
expanded population is expected to consume twice as much animal protein than con-
sumed today and demand for animal protein is likely to continue to grow. Increased meat
production will certainly result in an increase in the amount of protein-rich by-products
generated during meat processing. Protein-rich resources may be used as a source of
bioactive hydrolysates and peptides that have potential for use as functional ingredients in
the prevention of diseases such as hypertension.(2–6)
Bioactive peptides are short sequences of amino acids that are inactive within the
sequence of the parent protein but have a positive health impact on systems of the body
once released.(7) Such peptides can be generated from natural sources by endogenous
enzymes during food processing, in vitro by microbial fermentation or by chemical and/or
CONTACT Maria Hayes maria.hayes@teagasc.ie Teagasc Food Research Centre, Ashtown, Dublin 15, Dublin,
Ireland.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lfri.
© 2017 Taylor & Francis
218 T. LAFARGA AND M. HAYES
enzymatic hydrolysis.(7) To date, numerous bioactive peptides and hydrolysates with

perceived health benefits, including angiotensin-I-converting enzyme (ACE-I, EC
3.4.15.1), renin (EC 3.4.23.15) and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) inhibi-
tory activities, have been reported.(3,4,8–11) Inhibition of enzymes including renin and
ACE-I within the salt-water, and blood pressure regulating system known as the renin–
angiotensin–aldosterone system (RAAS), plays a key role in the prevention of hyperten-
sion. Furthermore, inhibition of DPP-IV has potential for use in the prevention of type-2
diabetes, hypercholesterolemia and insulin resistance as demonstrated in a number of
studies to date.(12,13)
A number of functional food products containing bioactive hydrolysates and peptides
are currently commercialized and available worldwide. These include the ACE-I inhibitory
and antihypertensive product Calpis sour milk (Calpis Food Industry Co., Ltd., Tokyo,
Japan), which contains the tri-peptides IPP and VPP. This product was repeatedly found
to have in vivo blood pressure-lowering effects when administered to hypertensive
patients.(14–16) However, a review carried out by the European Food Safety Authority
(EFSA) concluded that a cause and effect relationship between consumption of IPP and
VPP, at the proposed dose of 5 mg/mL, and maintenance of normal blood pressure in
humans could not be established.(17) The rules set by EFSA and other regulatory agencies
are not the only challenge faced by food manufacturers when developing bioactive
compounds for the food industry. Developmental challenges include high production
costs and changes in the sensory and taste attributes of the final product. For example,
bioactive hydrolysates and peptides usually have a bitter taste(18) and Western consumers
do not seem to be willing to compromise taste for potential or proven health benefits.(19)
This article reviews bioactive protein hydrolysates and peptides, their generation,
perceived and proven health benefits, and potential for inclusion in food products. It
describes the existing challenges faced by the functional foods industry and proposes a
number of strategies that may overcome these challenges.
Bioactive peptides and metabolic syndrome

The term metabolic syndrome describes a combination of medical disorders including
obesity, hypertension and diabetes, all of which increase the risk of developing cardiovas-
cular disease.(20) Some of these factors can be treated and/or controlled by the inhibition
of key enzymes in regulation systems of the body by the use of chemically synthesized
drugs or the ingestion of functional food and nutraceutical products. Enzymes which may
be inhibited to regulate metabolic syndrome include DPP-IV, and those involved in the
RAAS such as renin and ACE-I.(21–24)
ACE-I inhibition
ACE-I is a monomeric glycoprotein naturally distributed in many tissues and biological
fluids of the human body. It acts by cleaving di-peptides from the free C-termini of two
typical substrates: angiotensin-I and bradykinin,(25) as shown in Fig. 1. The cleavage of
angiotensin-I results in the generation of angiotensin-II which stimulates signalling path-
ways in the heart, blood vessels, kidneys, adipose tissue, pancreas, and brain.(26) ACE-I
FOOD REVIEWS INTERNATIONAL 219
Figure 1. The renin–angiotensin–aldosterone system. Angiotensinogen is cleaved by renin to form

angiotensin-I, which is then converted into angiotensin-II by the action of ACE-I, which is also
responsible for the inactivation of the vasodilatator bradykinin. Angiotensin-II exerts a potent vasocon-
strictive effect over the vessel walls by binding to the angiotensin-II type I receptor (AT1) and
stimulating the adrenal cortex to secrete aldosterone.
also degrades bradykinin, a peptide that reduces blood pressure and causes dilatation of
blood vessels.(22)
ACE-I inhibitors are used as the first-line treatment of hypertension, and their efficacy
is well proven.(24) There are over ten chemically synthesized ACE-I inhibitors currently
available on the market including the first such compound: captopril or D-3-mercapto-2-
methylpropanoyl-L-proline.(25) Side effects of captopril including dry cough, which
appears in 5–20% of patients, and angioedema, which affects 0.1–0.5% of patients, resulted
in the further development of two novel ACE-I inhibitors, enalaprilat and lisinopril.(25)
Although few studies have looked at the side-effects of naturally occurring ACE-I inhibi-
tors, their food-based source and their perceived lack of unwanted side-effects may
provide a better alternative to synthetic compounds.(27) It is well documented that peptidic
ACE-I inhibitors usually consist of short amino acid sequences with high concentration of
hydrophobic amino acids.(9,28) Cheung et al.(29) reported the amino acid residues trypto-
phan, tyrosine, proline and phenylalanine as the most effective for ACE-I inhibition when
they are present at the C-terminus of a di-peptide. Similar results were obtained in a
quantitative structure-activity relationships study (QSAR), which modelled the biological
activity of ACE-I inhibiting peptides as a function of molecular structure.(28) These
authors proposed that residues with large bulky chains as well as hydrophobic side chains
as found in the amino acids phenylalanine, tryptophan, and tyrosine present in a di-
peptide exhibit greater ACE-I inhibition. For active tri-peptides, amino acids with small,
bulky side chains as well as hydrophobic side chains such as valine, leucine, and isoleucine
were suggested for the N-terminus of the peptide.(28) In addition, it was suggested that
positively charged amino acids such as lysine and arginine for positions adjacent to the N-
terminus of the peptide, and residues with small bulk side chains and hydrophobic side
chains with high electronic properties as found in the amino acids proline, phenylalanine,
and tryptophan are most suitable at the C-terminal end of the peptide for effective ACE-I
inhibition.
To date, many ACE-I-inhibiting hydrolysates and peptides with in vitro ACE-I-inhibit-
ing properties have been generated from natural sources including meat and by-products
such as blood,(30,31) fish,(32) and milk.(33,34) However, only a few have been assessed for
their antihypertensive properties in vivo in human or animal studies. Katayama et al.(5)
identified and isolated the peptides KRQKYD and EKERERQ by enzymatic hydrolysis of
porcine muscle with pepsin (EC 3.4.23.1). These peptides inhibited ACE-I by half (IC50) at
a concentration of 26.2 and 552.5 µM, respectively. Moreover, the peptide KRQKYD
presented a temporary hypotensive activity between 3 and 6 h after oral administration
to spontaneously hypertensive rats (SHRs) at a dose of 10 mg/kg body weight. Muguruma
et al.(35) obtained the peptide KRVITY by hydrolysis of porcine skeletal myosin with
pepsin. This peptide presented an in vitro ACE-I-inhibitory IC50 value of 6.1 µM and was
found to significantly reduce blood pressure 6 h after oral administration, at a dose of
10 mg/kg body weight. Finally, Escudero et al.(2) investigated the in vivo antihypertensive
activity of the peptides RPR, KAPVA, and PTPVP in SHR. These peptides were derived
from porcine meat and presented in vitro ACE-I-inhibiting IC50 values of 382, 46.56 and
256.41 µM, respectively. All of them, but especially the tri-peptide RPR, showed significant
antihypertensive activity after oral administration at a dose of 1 mg/kg body weight.
Renin inhibition
The enzyme renin is an aspartyl protease produced mainly in the juxtaglomerular apparatus
of the kidney, but which is also found in other tissues including the brain, adrenal glands,
ovaries, the heart, and vascular tissue.(25,36) Renin is responsible for the conversion of
angiotensinogen into the deca-peptide angiotensin-I which subsequently undergoes pro-
teolytic cleavage by ACE-I to form the octa-peptide vasoconstrictor angiotensin-II(22,25)
(Fig. 1). Renin inhibition has long been a therapeutic goal because of the substrate specificity
of renin compared with that of ACE-I, which has actions in addition to the formation of
angiotensin-II.(37) In addition, cleavage of angiotensinogen by renin is the first and the rate-
limiting step of the RAAS.(38)
A number of renin inhibitors including Aliskiren (trade names Tekturna and Rasilez in
the USA and in the UK, respectively) are currently commercialized as antihypertensive
drugs. Aliskiren reduces blood pressure when used in mono-therapy as well as in
combination therapy.(39) However, a number of side-effects including fatigue, headache,
dizziness, and diarrhoea have been reported.(37) Renin can be also inhibited by the use of
natural compounds such as bioactive peptides. Di-peptides with hydrophobic residues at
the N-terminus and a bulky or aromatic group at the C-terminus were previously reported
to be the most effective inhibitors of renin.(40)
Although no renin inhibitors have been generated from meat proteins to date, several
bioactive hydrolysates from other food sources with renin-inhibitory properties have been
reported. For instance, Udenigwe et al.(41) obtained renin-inhibitory hydrolysates from flax-
seed protein that inhibited the activity of renin by half at concentrations ranging from 1.22 to
2.81 mg/mL. In a more recent study, Mundi and Aluko(6) generated an Alcalase hydrolysate
from kidney bean protein, which inhibited renin by 20–40% at a concentration of 1 mg/mL.
These values were lower, however, than those obtained from pancreatin hydrolysates of
Australian canola protein, which inhibited renin by 63.2% at a concentration of 1 mg/mL.(8)
These hydrolysates also presented antihypertensive properties when tested in SHR.
DPP-IV inhibition
DPP-IV is a serine protease that cleaves the amide bond X-A or X-P (where X represents an
amino acid) at the N-terminus of peptides and proteins.(21) The cleavage of a di-peptide
from a longer peptide can activate, deactivate, and increase or decrease the activity of such a
peptide. Indeed, DPP-IV degrades and inactivates glucagon-like peptide-1 (GLP-1) and
gastric-inhibitory peptide (GIP), two incretin hormones that contribute to the enhancement
of glucose-induced insulin secretion,(21) as shown in Fig. 2.
Inhibition of DPP-IV shows potential for use in the management of hyperglycemia in type-
2 diabetes.(42) DPP-IV inhibitors, known as gliptins, include sitagliptin, vildagliptin, saxaglip-
tin, linagliptin, and alogliptin, and are approved for the management of type-2 diabetes in
Canada, the European Union (EU), and the United States.(43) Their tolerance and safety have
been largely confirmed in fragile populations including the elderly.(23) The main differences
between gliptins include chemical structure, potency, target selectivity, and oral
bioavailability.(44) Vildagliptin and saxagliptin are both peptide-like inhibitors.(45) Although
the mechanisms of peptide-induced DPP-IV inhibition have not yet been fully elucidated,
peptides derived from food sources, including animal proteins, can be used to inhibit DPP-
IV.(45) Minkiewicz et al.(46) suggested bovine collagen and elastin as potential sources of DPP-
IV-inhibiting peptides based on in silico analysis. Recently, the interaction between sitagliptin
and bioactive peptides derived from milk proteins was studied.(47) Combinations of sitagliptin
and generated peptides resulted in an additive effect on DPP-IV inhibition. This means that in
combination with sitagliptin, all the concentrations of inhibitory peptides tested resulted in
higher or equal inhibition of DPP-IV compared to that of sitagliptin alone. DPP-IV-inhibiting
peptides isolated from food proteins to date consist of short sequences of between two and
nine amino acids in length.(48) Potent DPP-IV inhibitors contain proline or alanine residues in
the penultimate position at the N-terminus.(45) These peptides usually possess specific amino
acid sequences mainly comprised of hydrophobic residues together with proline, arginine, and
lysine.(48) However, di-peptides that do not contain proline have been shown to inhibit DPP-
IV.(43) Numerous di-peptides with a proline residue at the C-terminus have been identified as
DPP-IV inhibitors.(48) It has been shown that the sequence of a peptide rather than its
composition influences its DPP-IV-inhibiting potential.(45)
The number of DPP-IV-inhibiting hydrolysates and peptides derived from meat and
by-product proteins is still limited. Gallego et al.(49) recently identified several novel DPP-
IV-inhibiting peptides in Spanish dry-cured ham by size-exclusion chromatography
Figure 2. Role of DPP-IV, GIP, and GLP-1 in glycemic control. A complex sequence of physiological
responses, which include the release of GLP-1 and GIP, is activated after food intake. GIP and GLP-1 are
incretin hormones that have great effect on glucose and are secreted by K-cells in the duodenum and
jejunum and by L-cells in the ileum and colon respectively.(164) These hormones act on pancreatic β-
cells and stimulate insulin secretion.(165) Both GLP-1 and GIP exert actions well beyond the β cell. GIP
stimulates the activity of lipoprotein lipase and the modulation of fatty acid synthesis and promotes the
proliferation and survival of β-cells.(164) In addition, GLP-1 suppresses glucagon secretion, reduces
gastric emptying time, promotes satiety, and stimulates differentiation and proliferation of β-cells.
DPP-IV inhibitors act by inhibiting the degradation of GLP-1 and GIP and therefore stimulating the
secretion of insulin, suppressing the secretion of glucagon and promoting satiety.
(SEC). The peptides KA and AAATP showed the highest DPP-IV-inhibiting activities with
IC50 values of 6.27 and 6.47 mM, respectively. Moreover, Lafarga et al.(50) used in silico
analysis to identify the tri-peptides PPL and PPG, which, when chemically synthesized,
inhibited the activity of DPP-IV in vitro by half at a concentration of 0.39 and 2.25 mM,
respectively. DPP-IV inhibitors have also been isolated from food sources including
milk,(43,51) fish,(10) and plant proteins.(11) To date, numerous DPP-IV-inhibiting peptides
have been reported and these include the fish-derived peptides GPGA and GPAQ with
IC50 values of 49.6 and 41.9 µM, respectively.(10) Few in vivo studies have evaluated the
efficacy of DPP-IV peptide inhibitors. One study carried out by Uenishi et al.(52)
performed an oral glucose tolerance test with a cross-over design to evaluate the effect of
the DPP-IV-inhibiting peptide LPQNIPPL in vivo in rats. Plasma glucose concentrations
were found to be significantly lower in the group administrated this peptide, compared to
the control at 30 and 60 min after oral administration, at a dose of 30 mg/100 g rat weight.
Challenges for commercialization of peptides and hydrolysates

In order to aid processing, extend shelf life, and to obtain low-cost and good-tasting foods,
the food industry has relied on the use of fats, sugars, chemical processing aids, and
plastics.(53) However, consumers are becoming more aware of the relationship between
food, diet, and health. This awareness is leading to increased interest in natural ingredi-
ents, including bioactive peptides, and in the consumption of food products that are not
only tasty but healthy.(19) The commercialization of bioactive peptides as food ingredients
must first overcome a number of challenges.
Production, purification, and characterization of bioactive peptides

Once a peptide has been selected for further development, it needs to be produced with
consistent quality.(54) The production of natural compounds such as peptides is achieved,
in the pharmaceutical industry, by using microbial or fungal strains or by chemical
synthesis.(54) Different strategies can be used for the synthesis of bioactive peptides and
these include solution-phase methods, solid-phase methods, and hybrid approaches.(55)
Solid-phase methodologies have been increasingly exploited for the manufacture of
commercial products including somatostatin and calcitonin, especially since purification
techniques such as reverse-phase high-performance liquid chromatography (RP-HPLC)
have become available.(55) Solution-phase methodologies can produce peptides in quan-
tities of up to hundreds of kilograms per year and are currently used for the synthesis of
ACE-I-inhibitors, aspartame, oxytocin, desmopressin, and goserelin.(55) The main chal-
lenge for the production of bioactive peptides in the food industry is the high cost
associated with their production as these methods are, in the majority of cases, not
economically viable. Most peptides manufactured by chemical synthesis are used as
peptide drugs and as peptides for diagnostic purposes(56) and not as food ingredients.
The most common way to produce bioactive peptides in the food industry is by
enzymatic hydrolysis of whole protein molecules using food-grade enzymes.(7) ACE-I,
renin, and DPP-IV peptide inhibitors have been generated by enzymatic hydrolysis from
varied sources including seaweed,(57) blood,(58) muscle,(5,35) and dairy proteins.(51) This
strategy results in protein hydrolysates containing the desired peptides together with many
other unwanted peptides. In order to identify the peptide causing the observed bioactivity,
the generated hydrolysate is usually fractionated based on size by ultrafiltration.(59) The
first step in the enrichment of bioactive peptide containing fractions is usually to use at
least one or several membrane filtration procedures to fractionate the hydrolysate into 1–
10 kDa fractions. For example, Mundi and Aluko(6) recently obtained four different
peptide fractions, using 1, 3, 5, and 10 kDa ultrafiltration membranes, which presented
significant differences in their antioxidant and ACE-I-inhibiting properties. Downstream
processing is a key step in the manufacture of peptides as it involves product isolation and
purification. A combination of different methods, including HPLC, flash chromatography,
and mass spectrometry (MS), as well as amino acid analysis, are currently used to establish
the identity of a peptide.(60) MS is an analytical technique for the determination of the
composition of a compound and can also be used for the elucidation of the chemical
structure of molecules such as peptides.(61) It is based on the ionization of a compound to
generate charged molecules or fragments and the measurement of their mass-to-charge
ratio.(61) MS together with database search engines including SEQUEST or Mascot play an
increasing role in the characterization of bioactive peptides from hydrolysates.
Enzymatic hydrolysis of proteins may result in the generation of unwanted peptides
that could limit the use of the obtained hydrolysate in the food industry. The complete
chemical characterization of a food ingredient, such as enzymatic hydrolysates with ACE-
I, renin and DPP-IV inhibitory properties, is required for the assessment of claims on
foods in Europe.(62) In vitro biological characterization of inherent health effects is also
carried out prior to animal and human studies and may involve the use of cell or tissue
culture techniques.
Resistance of ACE-I, renin, and DPP-IV inhibitors to food processing

Bioactive peptides used as functional food ingredients must undergo processing, which
can include subjection to high pressure, high and low temperatures, and strong pH
variations, as shown in Fig. 3. Heat- and pH-sensitive amino acids can be destroyed by
high pH and temperature variations, which affect the physical and structural properties of
Figure 3. Bioavailability of bioactive peptides: resistance to food processing, gastrointestinal degrada-

tion, and absorption into the blood stream. Bioactive hydrolysates and peptides used as functional
ingredients may undergo food processing which can include subjection to high pressure, high/low
temperature, and strong pH variations. Moreover, in order to exhibit a beneficial effect in vivo, peptides
must survive gastrointestinal digestion and must reach the target site intact and in sufficient quantities.
a protein or a peptide.(63) Acidic treatments can destroy glutamine and aspargine, and
alkaline conditions can produce lysinoalanine and D-amino acids and can destroy
cysteine, serine and threonine. In addition, lysine bioavailability can be reduced by high
pH and temperature variations because of an increase in the occurrence of Maillard
reactions.(63) Low temperatures and freezing can also alter the structure of a protein and
its activity.(64) Moreover, the amount of water that remains available for chemical reac-
tions in a frozen food product depends on the temperature to which the product is frozen
and depends also on the temperature and the freezing duration at which proteolytic
reactions can occur.(64) High-pressure processing (HPP) is a non-thermal process with
potential to provide microbiologically safe, nutritionally intact, and organoleptically high-
quality products.(65) HPP can also be used to improve nutritional and/or functional
properties such as gelation, emulsification, and foaming and to create novel foods,
textures, and tastes.(66,67) It is known that conformational changes in proteins can occur
during or after pressure treatment. Depending on the pressure applied and on the
duration of the treatment, HPP can enhance the susceptibility of a protein to hydrolysis,
digestion and, may reduce bioavailability.(66)
Bioavailability of ACE-I, renin, and DPP-IV peptide inhibitors

Many biologically active peptides identified have never become clinically useful because of
a number of impacting factors including poor stability, low permeability through biolo-
gical barriers, and low water solubility.(68) The solubility of a protein or a peptide depends
on several factors including pH, temperature and the amino acid composition and
sequence of the peptide.(69) Although di- and tri-peptides are usually soluble in water,
ACE-I, renin, and DPP-IV inhibitory peptides may present poor aqueous solubility at high
concentrations due to the high proportion of hydrophobic amino acids within their
sequences.(28,48,70) Poor solubility and low dissolution rates often cause insufficient bioa-
vailability and these drugs usually require high doses in order to reach therapeutic
concentrations in blood after oral administration.(71)
Moreover, in order to exhibit a beneficial effect in vivo, peptides must survive gastro-
intestinal digestion.(72) Poor stability of peptides against gastrointestinal degradation is
one of the main challenges for the use of bioactive peptides as health-promoting ingre-
dients in food. Proteins and peptides are subjected to severe changes after ingestion by the
action of enzymes, acids, and strong pH variations within the gastrointestinal tract.(73)
Trypsin, carboxypeptidase, and chymotrypsin are secreted from the pancreas into the
small intestine and are responsible for 20% of the total enzymatic degradation of the
ingested proteins and peptides.(73) Although DPP-IV and ACE-I inhibitors have been
suggested to act locally in the gastrointestinal tract by inhibiting intestinal DPP-IV and
ACE-I,(74) other bioactive peptides such as prolyl endopeptidase (PEP; EC 3.4.21.26)
inhibitors, must not only survive degradation but also must be absorbed into the blood-
stream and reach the target sites intact and in sufficient quantities.(75) The absorption
mechanisms for peptides through the intestinal epithelial cell monolayers are complex and
two main mechanisms have been suggested. Although di- and tri-peptides can be hydro-
lysed into amino acids in the epithelial cells, they can also be transported via a specific
transporter named PepT1.(76) Moreover, peptides may also be passively transported via the
paracellular route, which has been suggested as the main mechanism for transport of
intact peptides across the monolayer of epithelial cells in the small intestine.(76)
In order to reach their target site intact and in sufficient quantities, ACE-I and renin
inhibitory peptides must be resistant to enzymatic degradation, and to degradation by
acids and plasma peptidases. Indeed, Satake et al.(77) studied the mechanism for the
intestinal transport of the lacto-peptide VPP by using monolayer-cultured human intest-
inal Caco-2 cells. In their study, a significant amount of the peptide was found to be
transported across the Caco-2 cell layer. However, no intact peptide was detected in the
cells and the authors suggested that the peptide VPP may have been hydrolysed into the
amino acids valine and proline by intracellular peptidases.
Safety challenges
Most of the studies on bioactive peptides carried out to date focus on their efficacy and
there has been little work carried out regarding the safety of peptides and peptide
hydrolysates.(78) It is therefore of the upmost importance that all peptide-containing
hydrolysates and peptides are subjected to the correct toxicity and allergenicity assess-
ment, and are then labelled carefully if they are found to contain allergic or toxic peptides/
proteins, in accordance with current EU legislation.(79)
Allergenicity of peptides
Allergenicity of protein-rich products should be considered as many people can show
local or generalized reactions after ingestion, or even contact with certain foods. The
weight of evidence, case by case approach, is usually adopted when assessing a food
product for potential allergenicity.(69) The most common food allergens are cow milk,
egg, peanut, soy, shellfish and wheat.(80) Although pork allergy has not been well
characterized, beef allergy has been demonstrated in numerous studies.(81)
Hypersensitivity to red meat is not common and has been related to cross-reactivity
between beef and cow milk.(82) The major cow allergens belong to the casein fraction of
proteins and to whey proteins including α- and β-lactoglobulin.(83) Food allergy is
usually mediated by immunoglobulin E (IgE) antibodies against specific protein
allergens.(84) However, over the last decade, allergic reactions to mammalian meat
have been associated with IgE antibodies directed against oligosaccharides found in
mammalian cells including galactose-alpha 1,3-galactose.(82) Several techniques have
been used to decrease or eliminate the number of allergens in food and these include
heat treatment, bacterial fermentation, and enzymatic digestion.(81,85,86) There are no
confirmed cases of allergic reactions caused by the intake of enzyme-treated foods.(87)
However, peptides can potentially cause allergic reactions in humans as peptides can be
used as allergens in specific allergen immunotherapy and have been suggested as a safe
approach in the management of allergic diseases.(88)
The toxicity of peptides

Peptides can be toxic.(89) For example, in wheat gluten, toxic components for celiac disease
patients belong to the closely related proline- and glutamine-rich proteins known as
gliadins.(90) Well-known toxic peptides include melittin, a peptide of 26 amino acids in
length which is the principal active component of bee venom.(91) This peptide can target
live cells such as red blood cells and bind to their membrane leading to lipid bilayer
disruption and to haemolysis.(91) In addition, the most important toxic substances in
inedible mushrooms are peptides that can be divided into two main groups: phallotoxins
and amatoxins.(92) Peptides with ACE-I, renin, and DPP-IV inhibitory properties can also
be toxic, although none have been reported to date. Toxic peptides contain higher
concentrations of histidine, proline, and asparagine residues compared to nontoxic pep-
tides, and the composition of cysteine was found to be exceptionally higher in toxic
peptides.(89) Moreover, the amino acid residues valine, arginine, threonine, glutamine,
methionine, leucine, lysine, isoleucine, phenylalanine, and alanine are dominant in non-
toxic compared to toxic peptides.(89) Although the amino acid residues phenylalanine,
arginine, leucine, isoleucine, and tryptophan are dominant in ACE-I, renin, and DPP-IV
inhibitors, the high occurrence of proline residues within their sequences suggests that
these peptides can be potentially toxic. A nontoxic nature is a key parameter for bioactive
peptides or hydrolysates used as food ingredients and the safety of a peptide product must
be assessed in suitable in vitro and in vivo animal models before use in human interven-
tion studies.
Legislation and health claims

Foods for Specified Health Issues (FOSHU)
The functional foods concept started in Japan in 1984 with the launch of large-scale
government-funded research programmes on “Systematic analyses and development of
functional foods”.(93) A number of policy issues have arisen regarding the regulatory
environment for the approval of new functional food products and supplements and how
health claims for these new products are formulated, approved, and communicated.(94) The
Japanese Ministry of Health and Welfare established a policy for approving some selected
functional food products as Foods for Specified Health Uses (FOSHU) whose health claims
are legally permitted.(93) In 2005, FOSHU-foods were classified into four groups on the basis
of the strength of the evidence behind a substance or a product and a disease or health-
related condition.(95) Despite the fact that there are currently over 1000 FOSHU products in
the market, the variety of health claims approved by the Japanese Ministry of Health and
Welfare are still limited and these include reduction of blood pressure, reduction of blood
glucose, bone health and dental health.(95) Future FOSHU categories such as anti-fatigue
foods or skin-health-promoting foods are expected by Japanese consumers.(96)
European regulation on health claims and functional foods

Products containing bioactive hydrolysates with ACE-I, renin, and DPP-IV inhibitory
activities have potential to achieve a health claim under European regulation. In Europe
and the US, functional foods are regulated by EFSA and the Food and Drug
Administration (FDA), respectively. A health claim is defined as “any claim that states,
suggests or implies that a relationship exists between a food category, a food or one of its
constituents and health”.(97) Moreover, a reduction of disease risk claim is defined as “any
health claim that states, suggests, or implies that the consumption of a food category, a
food or one of its constituents significantly reduces a risk factor in the development of a
human disease”.(97) Although Qualified FOSHU and qualified health claims exist in Japan
and in the USA, respectively, there is no such claim in Europe for those health claims with
weaker levels of scientific evidence.(98) Substantial scientific validation of the health-

promoting effects of bioactive ingredients is required before a health claim can be made
for EFSA approval. EFSA’s Panel on Dietetic Products, Nutrition and Allergies is respon-
sible for the verification of the scientific substantiation of the health claims. In the EU,
health claims made in relation to food products require authorization under the nutrition
and health claims Regulation EC 1924/2006 before they can be used in the labelling and
marketing of these products. This regulation ensures a high level of protection for
consumers and harmonizes the provisions laid down by law, regulation, or administrative
action in Member States, which relate to nutrition and health claims.(79) Health claims
should only be authorized for use in the Community after a scientific assessment of the
highest possible standard. A claim should be scientifically validated by taking into con-
sideration the totality of the available scientific data and by weighing the evidence.
Negative sensory and taste attributes: The bitter taste of peptides

Enzymatic hydrolysis of food proteins, including meat proteins, has been shown to
increase biological activity by, for example, the release of antioxidant and ACE-I and
DPP-IV inhibitory peptides.(4,8,31) However, a major disadvantage of fermentation and
hydrolysis processes in the food industry is the release of bitter-tasting peptides.(18)
Although humans accept bitterness to some extent in certain products such as coffee or
beer, there is a natural preference for sweet and fat tastes.(99) Flavor can be defined as a
combination of aroma and taste. Aroma compounds are volatile and are perceived by the
olfactory system and taste compounds, which can be volatile or non-volatile, are detected
by the gustatory system.(100) Mammalian taste receptor cells are clustered in taste buds on
the surface of the tongue and palate.(101) Humans can distinguish between five basic tastes:
sweet, sour, salty, unami, and bitter.(102) Each taste modality is thought to be mediated by
different transduction pathways expressed in different subsets of receptors.(101) Bitter taste
was suggested to be mediated by G-proteins and a family of approximately 30 different G-
protein-coupled receptors (Type-2 Receptor [T2R]) that interact with bitter-tasting com-
pounds and initiate signalling cascades that culminate in neurotransmitter release.(103)
Many substances, including peptides, are responsible for the flavor of a product (18) Bitter-
taste inhibitors can act peripherally or cognitively. At the peripheral level, bitter-taste
inhibitors interact with the T2R receptors preventing the interaction between these taste
receptor cells and the bitter-tasting compound.(104) At the cognition level, inhibition
occurs in the central nervous system (CNS) via taste-taste interactions.(104)
A number of methods have been used to predict the bitterness of a peptide. The ‘Q rule’
formulated by Ney(105) quantifies the bitterness of a peptide based on its amino acid
composition, where by a Q-value is calculated from the solubility data of the individual
amino acids. According to this method, when the Q-value of a peptide with a molecular
weight (MW) under 6 kDa exceeds 1400 cal/mol this peptide will be almost certainly
bitter. Although this rule can be applied to the majority of known peptides, there are
exceptions. For instance, Cho et al.(106) characterized the flavor, chemical properties and
hydrophobicity of a number of peptides derived from two commercial enzymatic hydro-
lysates of soy protein. In their study, the hydrophobicity data based on Q-values did not
support Ney’s rule as a predictor of bitterness. The application of Fourier transform
Raman spectrometry was also shown as a useful tool for bitterness predictions of peptides
and amino acids.(107) However, this method is expensive and time-consuming, and the
currently accepted method for quantification of bitterness is to use sensory evaluation
panels.(108)
Peptides and bitter taste

Different parameters impact the intensity of the bitter taste of a peptide including the native
protein amino acid and peptide sequence, the processing/enzymatic treatment and the
hydrophobicity of the peptide.(104) The total solid content in the hydrolysis reaction mixture
can also affect bitterness generation. Spellman et al.(108) compared the bitterness of two
whey protein hydrolysates with equivalent degrees of hydrolysis and different total solid
contents previously. These hydrolysates were significantly less bitter when generated at a
higher concentration of solids. In general, bitterness was reported to increase as the overall
hydrophobicity of the peptide molecules increased.(109–111) Bitter taste was previously
observed in peptides containing the amino acids leucine, tyrosine, and phenylalanine, and
it was found to be more intense when hydrophobic amino acids with the L-configuration
were located at the C-terminus of the peptide.(109,110,112) A QSAR study that modelled
bitterness as a function of molecular structure revealed that hydrophobic amino acids at the
C-terminus and bulky basic amino acids at the N-terminus of a peptide were highly
correlated to bitterness.(111) Moreover, a QSAR study on bitter di- and tri-peptides revealed
that hydrophobic amino acids at the C-terminus and bulky amino acids adjacent to the C-
terminus are the major determinants of the intensity of bitterness in di- and tri-peptides.(113)
The presence of hydrophobic residues within a peptide has been related to bitterness and
also to ACE-I, renin, and DPP-IV inhibition. However, Wu and Aluko(113) reported no
significant correlation between the bitterness of di- and tri-peptides and their ACE-I-
inhibitory properties. No studies regarding the relationship of renin and DPP-IV inhibition
and bitterness of a peptide have been carried out to date.
Bitter-tasting hydrolysates and peptides

Peptides with a distinct bitter taste, such as those listed in Table 1, have been identified in a
variety of foods and are responsible in some cases for the taste qualities of the product.(99)
No studies have been carried out regarding the isolation and characterization of specific
bitter peptides from meat muscle and by-product proteins. However, a number of studies
showed a relationship between bitter taste in meat products and low MW peptides. For
instance, Aubes-Dufau et al.(114) identified a bitter fraction corresponding to MW below
5 kDa hydrophobic amino acids from an enzymatic hydrolysate of bovine haemoglobin.
Moreover, peptide generation during ham processing was highly correlated to flavor for-
mation and bitterness was related to hydrophobic peptides.(115)
In contrast to meat products, the development of bitter taste in the dairy industry has been
widely studied. For example, Karametsi et al.(100) identified by tandem mass spectrometry and
de novo peptide sequencing the bitter peptides GPVRGPFPIIV, YQEPVLGPVRGPFPI,
MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF from β-casein.
Liu et al.(116) identified the bitter peptides YGLF, IPAVF, LLF, and YPFPGPIPN in a commercial
whey protein hydrolysate by LC-TOF(time of flight)-MS/MS analysis. Spellman et al.(117)
identified the bitter peptide VEELKPTPEGDLEIL, which corresponded to f(43–57) of bovine
β-lactoglobulin, after enzymatic hydrolysis of a commercial whey protein concentrate. This
Table 1. Bitter peptides identified from animal sources.

Sequence Source MW (Da) Reference
GPVRGPFPIIV f(214–224) of bovine β-casein 1151.42 (100)
YQEPVLGPVRGPFPI f(208–222) of bovine β-casein 1668.96 (100)
MPFPKYPVEP f(124–133) of bovine β-casein 1204.45 (100)
MAPKHKEMPFPKYPVEPF f(117–134) of bovine β-casein 2173.62 (100)
APHGKEMPFPKYPVEPF f(118–134) of bovine β-casein 1971.30 (100)
YGLF f(69–72) of bovine α-lactalbumin 498.58 (116)
IPAVF f(94–98) of bovine β-lactoglobulin 545.68 (116)
LLF f(71–73) of bovine serum albumin 391.51 (116)
YPFPGPIPN f(75–83) of bovine β-casein 1001.15 (116)
VEELKPTPEGDLEIL f(43–57) of bovine β-lactoglobulin 1681.90 (117)
VVYPWTQR f(32–39) of the β-chain of bovine hemoglobin 1048.21 (114)
VVYPW f(32–36) of the β-chain of bovine hemoglobin 662.79 (114)
peptide was further digested by the action of a glutamyl endopeptidase and the breakdown
peptides LKPTPE and IL were found to present higher Q-values than the parent peptide.
Overcoming negative challenges

Bioactive peptide-containing hydrolysates with ACE-I inhibitory properties are currently
commercialized worldwide, especially in Japan. In recent years, efforts have been made to
overcome the challenges faced by the inclusion of bioactive ACE-I, renin, and DPP-IV
inhibitors into food products. For example, the Japanese company Senmi Ekisu Co. Ltd.
obtained generally recognized as safe (GRAS) status for its hydrolysed sardine muscle
protein from the FDA, and EFSA issued a positive opinion regarding the safety of
hydrolysed sardine muscle protein, for use as a novel food ingredient in 2010.(118) This
product, labelled as Valtyron, is rich in the di-peptide VY with known ACE-I inhibitory
and anti-hypertensive benefits.(119) However, a cause and effect relationship between
consumption of this peptide and reduced blood pressure has not yet been established.
Moreover, the peptide LKPNM has been shown to exhibit antihypertensive effects in
hypertensive subjects after oral administration.(120) This peptide is currently being used as
an ingredient in a number of FOSHU-approved, commercially available, functional food
products (Table 2). In Europe, following an application from Valio Ltd. submitted
pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent
Authority of Finland, the Panel on Dietetic Products, Nutrition and Allergies of EFSA
concluded that a cause-and-effect relationship has not yet been established between the
consumption of IPP and VPP and maintenance of normal blood pressure and questioned
the dietary intervention studies carried out and used in the dossier submission. Work is
currently being carried out by Valio Ltd. regarding this.
Food processing: Selection of suitable food vehicles

Resistance to food processing temperature and pH variations are viewed as drawbacks when
formulating peptides into carrier foods. However, the bioactivity of an ACE-I-inhibiting
peptide can resist food processing. Indeed, Jang et al.(121) studied the stability of the ACE-I-
inhibiting peptides GFHI, DFHINQ, FHG, and GLSDGEWQ during treatment at different
Table 2. Products containing bioactive hydrolysates and peptides currently available.

Bioactive Product
peptide Activity Product name Manufacturer type Source
LKPNM Antihypertensive PeptACE Natural Factors Capsules Bonito
Nutritional Products Ltd.,
Canada
LKPNM Antihypertensive Vasotensin Metagenics, USA Tablet Bonito
LKPNM Antihypertensive Levenorm Ocean Nutrition Canada N/A Bonito
Ltd., Canada
LKPNM Antihypertensive Peptide ACE Nippon Supplement Inc., Capsules Bonito
3000 Japan
LKPNM Antihypertensive Peptide Tea Nippon Supplement Inc., Powder Bonito
Japan
VY Antihypertensive Lapis Support Tokiwa Yakuhin Co., Ltd., Beverage Sardine
Japan
VY Antihypertensive Valtyron Senmi Ekisu Co., Ltd., Ingredient Sardine
Japan
FY, VY and IY Antihypertensive Wakame Jelly Riken Vitamin, Japan Jelly Undaria pinnatifida
(seaweed)
AKYSY Antihypertensive Peptide Nori S Riken Vitamin, Japan Beverage Porphyra yezoensis
(seaweed)
AKYSY Antihypertensive Mainichi Kaisai Shirako Co., Ltd., Japan Powder Porphyra yezoensis
Nori (seaweed)
IPP and VPP Antihypertensive Ameal S 120 Calpis Co., Ltd., Japan Beverage Milk
IPP and VPP Antihypertensive Ameal S Calpis Co., Ltd., Japan Tablet Milk
IPP and VPP Antihypertensive Evolus Valio Ltd., Finland Beverage Milk
VY Antihypertensive Sato Marine Sato Pharmaceutical Co., Tablet Sardine
Super P Ltd., Japan
FFVAPFPEVFGK Antihypertensive Casein DP Kracie Pharmaceutical, Beverage Milk
Peptio Drink Japan
FFVAPFPEVFGK Antihypertensive C12 Peption DMV International, The Ingredient Milk
Netherlands
LVY Antihypertensive Goma Pepucha Suntory Beverage & Food Beverage Sesame
Ltd., Japan
IY Antihypertensive Bunaharitake Yakult Health Foods Co., Powder Mycoleptodonoides
Ltd., Japan aitchisonii
(mushroom)
VY, IY, IVY Antihypertensive StayBalance RJ Api Co., Ltd., Japan Beverage Royal jelly
VVYP Weigth Seishou-sabou Moringa & Co., Ltd., Beverage Blood (bovine and
management Japan porcine)
CSPHP Cholesterol- Remake Kyowa Hakko, Japan Beverage Soy
lowering CholesterolBlock
YLGYLEQLLR Stress-relief Lactium Ingredia, France Beverage Milk
and
capsules
N/A Stress-relief Stabilium 200 Yalacta, France Capsules Fish
N/A Stress-relief AntiStress 24 Forte Pharma Capsules Fish
Laboratories, France
N/A Stress-relief Protizen Copalis Sea Solutions, Powder Fish
France
N/A Joint health CH-Alpha Gelita Health Products Beverage Bovine collagen
GmbH, Germany
N/A Joint health Peptan Rousselot SAS, France Powder Bovine collagen
N/A Joint health Collagen HM Copalis Sea Solutions, Powder Fish collagen
France
N/A Joint health Glycollagen Copalis Sea Solutions, Powder Skate collagen
France
N/A Immunomodulatory PeptiBal InnoVactiv Inc., Canada Capsules Shark
N/A Gastrointestinal Seacure Proper Nutrition, USA Capsules Fish
health
temperatures (70–100 ºC) and pH conditions (6.0–8.0). These peptides were found to be pH-
stable, heat-stable, and resistant to proteinases found in the gastrointestinal tract and showed
potential for use in the food industry. In addition, Reunanen and Saris(122) studied the
resistance of an antimicrobial peptide to heat treatment in sausages. The peptide was found
to be resistant to degradation when cooked at 70 ºC. However, a partial loss of activity was
observed when this peptide was autoclaved at 115 ºC for 20 min.(123) In a more recent study,
Contreras et al.(124) scaled up the process to produce the antihypertensive peptides RYLGY
and AYFYPEL from casein. The stability of these peptides to processing was studied and
results suggested that these peptides are stable to atomization, homogenization and pasteur-
ization. Moreover, the peptides were incorporated into yoghurt and no significant reduction of
either peptide or their associated bioactivities were detected during the shelf life of the
product.
Appropriate foods for use as bioactive peptide delivery vehicles include those extensively
and frequently consumed with which peptides are compatible. The selection of a suitable food
vehicle is important not only to facilitate the consumption of the product but to aid resistance
of bioactive peptides to food processing. For example, the activity of bioactive ACE-I, renin or
DPP-IV inhibitory hydrolysates or peptides may be altered after their inclusion into fermented
products such as cheese as these can be cleaved during milk fermentation with the starter
cultures employed by the dairy industry for the manufacturing of cheese or fermented
milks.(7) Baked products, for example, are not usually exposed to high pressure or strong
pH variations and have been previously used as food vehicles for nutritive compounds
including folic acid, chitosan, lutein, and protein hydrolysates.(125–127) Moreover, besides
being encouraged as part of a healthy diet, bread and other baked products are of particular
interest due to their popularity and widespread consumption and the addition of peptide
hydrolysates rich in the amino acid lysine is especially favorable as cereal products are usually
deficient in this essential amino acid.(127)
Increasing bioavailability of ACE-I, renin, and DPP-IV peptide inhibitors

As previously mentioned, poor stability against enzymatic degradation, low permeability,
and low water solubility are the main factors limiting the bioavailability of bioactive ACE-
I, renin, and DPP-IV inhibitory hydrolysates and peptides. Effective ACE-I, renin, and
DPP-IV inhibitory peptides are generally short sequences of amino acids of between two
to four residues in length that are usually soluble in water.(128) In order to overcome
problems related to poor peptide solubility in water, numerous strategies have been
studied. These include fusion of the target peptide to a solubilizing protein fusion partner
and glycosylation with hydrophobic carbohydrates.(129) These strategies, however, intro-
duce large molecules to the test peptide increasing its MW and may alter their structure
and bioactivity.(129) Novel approaches to overcome the poor aqueous solubility of ACE-I,
renin, and ACE-I inhibitors as well as other non-peptidic drugs are being investigated and
are one of the main challenges in pharmaceutical development. These include salt forma-
tion, particle size reduction, nanosuspensions, and solid-lipid nanoparticle.(130)
Stability of peptides against gastrointestinal degradation can be enhanced in various
ways. Modification of a peptide by masking the free carboxyl and amino terminus without
loss of bioactivity can make a peptide resistant to hydrolysis by exopeptidases.(68)
One class of structural modification under study is cyclization, which is currently used
to improve the delivery of cyclosporine, a fungal-derived peptide containing 11 amino
acids in length with a cyclic backbone and a single D-amino acid, which is used as an
immune system modulator.(73) Other modifications that can be made to peptides include
PEGylation, which is FDA approved, and substitution of natural L-amino acids with D-
amino acids.(73)
The use of polymeric reservoirs, protecting the encapsulated compound and controlling
the site and speed of release, are promising methods for bioactive peptide delivery.(131)
Different encapsulation techniques including liposomes, double emulsions and solid lipid
nanoparticles have been used to date. Goto et al.(132) evaluated the potential of fusogenic
liposomes to improve the mucosal absorption of insulin in rats. Fusogenic liposomes are
unique delivery vehicles prepared by fusing conventional liposomes with inactivated
Sendai virus particles. Results suggested that fusogenic liposomes are useful carriers for
improving the absorption of poorly absorbable proteins such as insulin, via the intestinal
tract. Furthermore, Zhang et al.(133) designed and characterized lectin-modified solid lipid
nanoparticles containing insulin and evaluated their potential as carriers for oral admin-
istration of peptides and proteins. The studied nanoparticles were prepared following
three different methods, and were found to promote oral absorption of insulin in rats. As
mentioned previously, ACE-I-, renin-, and DPP-IV-inhibiting peptides usually consist of
di- and tri-peptides. Di- or tri-peptides, especially those with proline or hydroxyproline
residues at their C-terminus, are generally resistant to degradation by digestive enzymes
and are expected to be absorbed directly from the gastrointestinal tract into the blood
circulatory system.(72) For instance, Iwai et al.(134) identified several food-derived collagen
peptides in human blood after oral ingestion of gelatine hydrolysates. In this study, a
significant increase in the amount of hydroxyproline and small peptides including P-Hyp,
A-Hyp, P-Hyp-G and P-Hyp in blood was observed after oral ingestion. Moreover, Foltz
et al.(135) assessed the bioavailability of the lacto-peptide IPP and seven other ACE-I-
inhibiting peptides present in an enriched yogurt beverage. The peptide IPP was detected
in blood in nanomolar amounts after oral ingestion and was proved to selectively escape
from intestinal degradation. In this regard, it would be desirable to study the bioactive
effect of small di- and tri-peptides. However, not only di- and tri-peptides can cross the
gastrointestinal system. Quirós et al.(136) studied the resistance of the casein-derived
peptide LHLPLP to brush border peptidases and examined its mechanism for transepithe-
lial transport using Caco-2 cells. The peptide LHLPLP was found to be resistant to
simulated gastrointestinal digestion, but it was hydrolysed to a shorter form, HLPLP by
cellular peptidases before transport across the intestinal epithelium. This penta-peptide
was also observed in human plasma after oral administration.(137) Dia et al.(138) assessed
the presence of lunasin, a peptide with 43 amino acid residues in length, in the blood of
men fed with soy protein products. Five males aged 18–25 years old consumed 50 g of soy
protein during five days, and the authors estimated that an average of over 4% of lunasin
from the total ingested was absorbed. Moreover, in this same study, a peptide with
relatively high MW, and similar to lunasin, was present in plasma samples of people
who consumed soy protein while absent in the baseline plasma samples from the same
individuals.
In addition to structural modifications and to the use of encapsulation techniques,
bioavailability of a peptide can be enhanced in the pharmaceutical industry by the use of
enzyme inhibitors, such as trypsin inhibitors, and by the administration of absorption

enhancers.(73)
Overcoming production challenges

The use of inexpensive protein sources, enzymes, and novel strategies are necessary to
reduce production costs and to establish viable industrial-scale production processes for
bioactive peptides. The use of inexpensive, protein-rich sources, such as by-products from
the meat and marine industry, as raw materials for the generation of bioactive peptides
can reduce costs with the added advantage of a reduction of by-products for disposal or
incineration. Moreover, the use of microbial proteases, instead of proteases derived from
other sources such as plants or animals, present a number of advantages including lower
costs for cultivation, harvesting, and purification.(139) Novel technologies such as the use
of in silico analysis can predict the bioactivity of a peptide, aid in the selection of a protein
and enzyme for use in hydrolysis and reduce costs in terms of time and money.(139) ACE-
I- and DPP-IV-inhibiting peptides have been recently generated by the use of in silico
analysis from various sources including proteins from meat muscle and by-products,(50)
cereals,(140) and milk.(141)
Chemical synthesis requires the consumption of large amounts of solvents and leads to
the generation of waste, making this technology an environmentally unfriendly method.(142)
Recombinant production of peptides has become an excellent option for the production of
long peptides with 25 or more amino acids in length.(142) However, this strategy is not
suitable for the generation of short sequences of amino acids. Enzymatic hydrolysis of
proteins is the most common method for the generation of ACE-I, renin, and DPP-IV
inhibitors in the pharmaceutical and food industries because of the lack of residual solvents
or chemicals in the final products.(139) From a production point of view, the use of
immobilized enzymes and membrane separation techniques, including ultrafiltration,
were previously suggested for the large-scale production of bioactive peptides. The employ-
ment of immobilized enzymes would permit reuse of enzymes and the possibility of working
with milder conditions. Working using continuous hydrolysis/fermentation methods
instead of conventional batch methods, as usually used with lab-scale hydrolysis, can offer
several advantages including higher efficiency and lower operating costs.(139) Continuous
processes are widely used for the hydrolysis of proteins in the food industry.(7) Moreover,
novel, non-enzymatic methods for the generation of bioactive peptides show potential to
reduce costs and to generate high-quality peptides from food proteins and by-products.
Alvarez et al.(143) published a novel non-enzymatic technique for the breakdown of porcine
hemoglobin at high temperatures (120–180 ºC) and low pressures (4 MPa) under a nitrogen
stream. Peptides generated in this study presented an average size of 3.2 kDa and antiox-
idant and functional properties.
The separation and purification of bioactive peptides by chromatographic technologies
is prohibitive for large-scale applications in the food industry.(139) Wu et al.(144) recently
suggested the use of an enzymatic ultrafiltration membrane reactor in conjunction with
chromatography as a potential method for the industrial production of bioactive peptides.
Electro-membrane filtration combines the separation mechanisms of electrophoresis and
membrane filtration and this technique was recently suggested as a promising alternative
method for the isolation of bioactive peptides.(145) The development of novel
methodologies to generate and isolate peptides using inexpensive and food-grade proce-
dures are on-going and will certainly broaden the use of bioactive peptides in food
production.
Safety of biologically active peptides

The processes used for the generation of protein hydrolysates are common in the food
industry and usually use food-grade source materials, processing aids and equipment.
Meat by-products such as blood are utilized in numerous food products including
morcilla sausage and sangre frita (Spain), Thuringian sausage (Germany) and black
pudding (Ireland and the UK).(31) Enzymes used in the generation of peptides are
generally obtained from edible parts of plants or animals, which are considered as posing
no health risk.(87) Moreover, after ingestion of food, proteins are naturally hydrolysed in
the gastrointestinal tract. This often results in the release of di-peptides, tri-peptides and
free amino acids, which are transported across the intestine wall.(146) Protein hydrolysates
and peptides with low MW are known to be less allergenic than their native protein and
are widely used in the formulation of hypoallergenic infant foods.(147) However, the
nontoxic nature of the product must be confirmed before ingestion. Toxicity of peptides
can be predicted in silico using ToxinPred available at http://www.imtech.res.in/raghava/
toxinpred/.(89) In silico prediction of the toxicity of peptides is not enough and toxicity
should be assessed in vivo before human consumption. The in vivo assessment of the
toxicity of food products and their safety for consumption must be carried out following
the guidelines proposed by international authorities. This requires large quantities of
scientific evidence and tests, which are usually carried out on vertebrate models, on
vertebrate cell lines and unicellular microbial species.(148) Numerous peptide toxicity
studies are carried out in animal models. Ponstein-Simarro Doorten et al.(146) conducted
three in vitro genotoxicity studies, including the bacterial reverse mutation test, mamma-
lian cell gene mutation test and mammalian chromosomal aberration test and an in vivo
90-day repeated-dose oral toxicity study in rats. The in vitro tests confirmed that
Tensguard, a milk protein hydrolysate containing the peptide IPP, was not mutagenic
or clastogenic. Moreover, no observed adverse effect level resulted for an overall intake of
40 mg IPP/kg body weight/day, 141-fold higher than the maximal anticipated intake. Dent
et al.(149) carried out a 90-day repeated-dose oral gavage toxicity development study in rats
and embryofetal rabbit to ensure the safety of the tri-peptides IPP and VPP. This study
investigated renal development and if bioactive peptides were associated with the same
type of fetopathy exhibited by ACE-I-inhibiting synthetic drugs. Results showed that there
were no adverse effects of treatment at the highest doses tested. Moreover, Jauhiainen et
al.(150) evaluated the safety and antihypertensive effect of a fermented milk product rich in
tri-peptides on hypertensive subjects using 24 h ambulatory measurements. This product
was found to have an antihypertensive effect in hypertensive subjects and no difference in
the sum of the adverse effects was observed between the control and the fermented milk
groups.
The zebrafish (Danio rerio) larvae assay has gained much attention recently for preclinical
drug discovery applications(151) and although it has not been validated yet, it shows promise
for use in assessment of the toxicity of a bioactive hydrolysate. Indeed, a papain hydrolysate of
Palmaria palmata was recently assessed for toxicity using this assay.(152)
Overcoming negative sensory and taste attributes

Preference for food is influenced not only by sensory attributes but also by a number of
circumstances, including those shown in Fig. 4.(153) In Japan, for example, functional foods are
considered a distinct class product and the importance of their health-promoting activity
often exceeds the importance of their sensory attributes.(19) However, Western cultures have a
different attitude towards functional foods and acceptance of a product is more conditional,
especially in respect to taste.(154) Taste expectations and experiences were shown to be critical
factors when selecting functional foods in numerous studies.(155,156) Different methods can be
applied to protein hydrolysates in order to reduce their bitter taste. These include the removal
of hydrophobic peptides, which are the key determinants of bitter taste.(104) However, most of
the essential amino acids that contribute to the bioactivity of a peptide are hydrophobic, and
the removal of such peptides is likely to alter the bioactivity of the hydrolysate. The identifica-
tion of effective bitter taste-inhibiting or masking agents would assist in broadening the use of
bioactive peptides and hydrolysates as functional food ingredients.
The use of exopeptidases, including amino- and carboxy-peptidases has been successfully
used for reducing the bitter taste of food protein hydrolysates.(157) To the best of our knowledge,
there are no studies addressing the inhibition of the bitter taste of hydrolysates and peptides
derived from meat or meat by-product proteins. However, many studies have used bitter taste-
inhibiting agents including salts, sweeteners, amino acids, and nucleotides, for the reduction of
bitter taste in a wide variety of bitter-tasting compounds. For example, Ogawa et al.(158) studied
the bitterness depressant effect of L-arginine, L-lysine, L-Ornithine, and L-citrulline on Quinine
and suggested L-arginine as an efficiend bitterness inhibitor when used as a concentration of 5
mM. In addition, Leksrisompong et al.(104) studied the effect of different bitter-taste suppressors
on whey protein hydrolysates with two different degrees of hydrolysis. Results showed that the
inhibitors tested did not inhibit the bitter taste of quinine, which was used as a positive control
and of the two hydrolysates in the same way. A number of bitter-taste inhibitors including
Figure 4. Factors affecting preference for food. Preference for food is influenced by numerous
characteristics as well as taste and appearance.
sucralose, fructose, sucrose, sodium acetate, monosodium glutamate, AMP, adenosine 5’ mono-
phosphate disodium (5’ AMP Na2) and sodium gluconate inhibited both whey protein hydro-
lysates. However, sodium chloride only inhibited the bitter taste of the hydrolysate with higher
degree of hydrolysis. Moreover, the amino acids l-lysine and l-arginine inhibited the bitter taste
of the control but not of the protein hydrolysates. Sharafi et al.(159) determined the bitter
response of 37 adults to vegetables with bitter-masking agents including sodium acetate,
aspartame and sodium chloride. Results suggested that aspartame was the most effective at
suppressing bitterness and improving hedonic responses and that bitter-masking agents, mainly
sweeteners, can suppress bitterness if they are matched to perceived vegetable bitterness or
vegetable disliking. In a more recent study, Wilkie et al.(160) studied the effect of sodium chloride
on the bitterness of vegetables and quinine. The addition of sodium chloride to quinine
significantly reduced bitterness ratings. However, reported bitterness and liking of the vegetables
did not change with the addition of sodium chloride.
Although the additional processing cost of encapsulation is likely to be prohibitive in a
food product, a number of compounds have been tested as delivery systems of bitter-
tasting compounds in foods.(161) These bitter-tasting compounds include protein hydro-
lysates from natural sources. Favaro-Trindade et al.(162) reduced the bitter taste of a casein
hydrolysate using spray-drying and different mixtures of gelatin and soy protein isolate as
carriers. In addition, Yang et al.(163) recently encapsulated whey protein hydrolysate by
spray-drying using maltodextrin or maltodextrin/β-cyclodextrin mixture as wall materials
to attenuate the bitter taste and enhance the stability of a whey protein hydrolysate.
Results suggested that the bitterness of both the maltodextrin-encapsulated and the
maltodextrin/β-cyclodextrin-encapsulated hydrolysate was significantly lower than that
of the original non-encapsulated. In addition, the encapsulation process did not lead to
any negative effect on the solubility of whey protein hydrolysate.
Conclusions
Meat muscle and by-product-derived peptides possess a wide variety of potential
health-promoting benefits. The inhibition of enzymes such as ACE-I, renin and
DPP-IV with enzymatic hydrolysates of natural sources may play an important role
in the prevention and treatment of diseases associated with metabolic syndrome and
other health disorders. The use of bioactive peptides is being actively considered by
food and pharmaceutical companies for both human nutrition and health promotion.
Indeed, a few functional, peptide-based products such as those shown in Table 2 have
already been launched on the market. This trend is likely to continue to grow as
knowledge about the bioactivities and properties of peptides increases and as consumer
demand for personalised nutrition for health continues. However, the development of
a novel functional food is not an easy task for a number of reasons, including changes
in the sensory and taste attributes of the final product. The lack of economically viable,
large-scale purification and characterization techniques is limiting the development
and large-scale production of meat-derived bioactive peptides and hydrolysates. New
production and separation technologies must be developed and are necessary in order
to reduce costs. Numerous methods can be applied to protein hydrolysates in order to
reduce their bitter taste. These include encapsulation, and the use of bitter-taste
inhibiting or masking agents such as salts, sweeteners, amino acids, and nucleotides.
Taste is a key aspect in the acceptance of a product, especially in Western cultures.
The development of novel, bitter-taste inhibiting agents that do not alter the bioactiv-
ity of the product is required in order to promote and broaden the use of meat-derived
hydrolysates in the functional foods industry.
Funding
Tomas Lafarga was supported by a Teagasc Walsh Fellowship. This work forms part of the
ReValueProtein Research Project [grant number 11/F/043] which is supported by the Irish
Department of Agriculture, Food and the Marine (DAFM) and the Food Institutional Research
Measure (FIRM) both funded by the Irish Government under the National Development Plan
2007–2013.
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Bioactive Protein Hydrolysates in The Functional Food Ingredient Industry Overcoming Current Challenges

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Food Reviews International

ISSN: 8755-9129 (Print) 1525-6103 (Online) Journal homepage: https://www.tandfonline.com/loi/lfri20

Bioactive protein hydrolysates in the functional

Tomas Lafarga & Maria Hayes

To link to this article: https://doi.org/10.1080/87559129.2016.1175013

Published online: 17 Jun 2016.

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Bioactive protein hydrolysates in the functional food

enzymatic hydrolysis.(7) To date, numerous bioactive peptides and hydrolysates with

Bioactive peptides and metabolic syndrome

Figure 1. The renin–angiotensin–aldosterone system. Angiotensinogen is cleaved by renin to form

Challenges for commercialization of peptides and hydrolysates

Production, puriﬁcation, and characterization of bioactive peptides

Resistance of ACE-I, renin, and DPP-IV inhibitors to food processing

Figure 3. Bioavailability of bioactive peptides: resistance to food processing, gastrointestinal degrada-

Bioavailability of ACE-I, renin, and DPP-IV peptide inhibitors

The toxicity of peptides

Legislation and health claims

European regulation on health claims and functional foods

weaker levels of scientiﬁc evidence.(98) Substantial scientiﬁc validation of the health-

Negative sensory and taste attributes: The bitter taste of peptides

Peptides and bitter taste

Bitter-tasting hydrolysates and peptides

Table 1. Bitter peptides identiﬁed from animal sources.

Overcoming negative challenges

Food processing: Selection of suitable food vehicles

Table 2. Products containing bioactive hydrolysates and peptides currently available.

Increasing bioavailability of ACE-I, renin, and DPP-IV peptide inhibitors

enzyme inhibitors, such as trypsin inhibitors, and by the administration of absorption

Overcoming production challenges

Safety of biologically active peptides

Overcoming negative sensory and taste attributes

75. Segura-Campos, M.; Chel-Guerrero, L.; Betancur-Ancona, D.; Hernandez-Escalante, V.M.

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