Bioactive Compounds in Wine - Recent Advances and Perspe PDF

BIOCHEMISTRY RESEARCH TRENDS
BIOACTIVE COMPOUNDS
IN WINE
RECENT ADVANCES
AND PERSPECTIVES
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BIOACTIVE COMPOUNDS
IN WINE
RECENT ADVANCES
AND PERSPECTIVES
PEDRO ADRIN AREDES-FERNNDEZ
MARA JOS RODRIGUEZ-VAQUERO
GISSELLE RAQUEL APUD
AND
MARA GILDA STIVALA

EDITORS
New York
Copyright 2016 by Nova Science Publishers, Inc.

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CONTENTS
Preface
Chapter 1
Chapter 2
Chapter 3
Chapter 4
vii
Bioactive Peptides in Wine: Recent Advances
and Perspectives
Pedro A. Aredes-Fernndez, Gisselle R. Apud,
Mara G. Stivala and Mara J. Rodrguez-Vaquero
Wine Polyphenols: Biological Activities and Reuse
from Winery Waste
Mara J. Rodrguez-Vaquero,
Sofa M. Sosa-Marmol, Mara G. Stivala,
Gisselle R. Apud and Pedro A. Aredes-Fernndez
Factors Affecting Biogenic Amines Occurrence in
Wine: An Overview of Analytical Methods
Silvana C. Ledesma, Mara G. Stivala
and Pedro A. Aredes-Fernndez
Impact of Fungal Diseases in Grapes and Wine:
General Aspects and Recent Advances
Gisselle R. Apud, Pedro A. Aredes-Fernndez
and Diego A. Sampietro
35
61
91
Editors Contact Information
111
Index
113
PREFACE
Wine has been part of human culture for about 6000 years. From ancient
times, wine was used to treat fever as well as different diseases; howveer, its
benefitial effects related to human health are associated mainly with the
prevention of cardiovascular diseases, principally due to the high content of
bioactive compounds. Presently, there is consensus among the scientific
community that moderate wine consumption produces potentially beneficial
effects on the human body mainly due to its preventive properties on the
cardiovascular system. These beneficial effects are related to the presence of
different components with an antioxidant-promoting capacity against reactive
oxygen species produced naturally in the body, as well as antihypertensive
effects, lipid profile regulation and anti-inflammatory effects. The relationship
between wine consumption and cardiovascular disease prevention emerged in
1989 with the French paradox, which is based on countries like France where
many fatty foods are consumed, but the incidence of death from cardiovascular
disease was lower than in others countries like in Northern Europe. This is due
to the fact that wine is correlated with low incidence of cardiovascular disease,
indicating a protective effect of wine. In this sense, it is established that
moderate daily wine consumption (150 mL for women and 300 mL in men)
produces benefits on cardiovascular diseases due to the action of bioactive
compounds such as polyphenols. Recently, also has been shown that in the
prevention of hypertension have an important role the presence of bioactive
peptides generated by the metabolism of the microflora naturally present in the
fermentation process. On this subject, is a current and interest topic the
isolation and selection of wine microflora that possess advantageous
technological properties for vinification process, guaranteeing the wine quality
and sometimes incorporates an added value to final product. At present, the
study of the use of products and by-products generated in wine processes,
viii P. A. Aredes Fernndez, M. J. Rodriguez Vaquero, G. R. Apud et al.

which can be used in the pharmaceutical, food and cosmetic industries is an
interesting topic. The development of new processing technologies, accompanied by the evolution of scientific knowledge on bioactive comp-ounds
and the increasing consumer concern over for his health, has carried a growing
and sustained interest in wine components with beneficial biological activities.
However, some wine bioactive compounds generated under certain conditions
can modify the organoleptic properties and quality of wines. Biogenic
aminesnitrogen compounds generated mainly by the metabolism of
microorganisms associated to winemaking processcan exert negative effects
on consumers health. Another compound produced by contaminant filamentous fungi are mycotoxins, Ochratoxin A being the most relevant mycotoxin
in wine produced by Aspergillus carbonarius and Aspergillus niger that
contaminates grapes. This compound has nephrotoxic, hepatotoxic, teratogenic, genotoxic and immunotoxic properties on several animal species, and
causes kidney and liver tumors in mice and rats.
This book attempts to transfer scientific results and the most comprehensive and updated knowledge on bioactive compounds in general
particularly in wineand updates on the most recent advances in the field.
With this book, oenologists will be able to update their knowledge from a
deeper understanding of the importance of bioactive compounds in wine.
Moreover, researchers in oenology can expand their knowledge and conduct
their experiments in areas of growing interest.
In: Bioactive Compounds in Wine

Editors: P. A. Aredes Fernndez et al.
ISBN: 978-1-63482-765-2
2016 Nova Science Publishers, Inc.
Chapter 1
BIOACTIVE PEPTIDES IN WINE:

RECENT ADVANCES AND PERSPECTIVES
Pedro A. Aredes-Fernndez*, Gisselle R. Apud,
Mara G. Stivala and Mara J. Rodrguez-Vaquero
Facultad de Bioqumica, Qumica y Farmacia,
Universidad Nacional de Tucumn, Tucumn, Argentina
ABSTRACT
In recent years, a growing number of scientists have published
evidence, that many peptides from fermented beverages exhibit specific
biological activities. In view of the current trend to study the role of the
diet in the prevention and treatment of diseases, efforts are being put into
the production of foods with beneficial effects on human health.
Although most of the scientific literature links the benefits of wine
consumption on human health to the presence of phenolic compounds,
other compounds present in wine like peptides could also play a
significant role in the beneficial effects of wine on health, particularly in
the prevention of cardiovascular diseases. The purpose of this chapter is
to review the current literature regarding bioactive peptides in general and
recent findings regarding bioactive peptides in wine. Special attention is
paid to information in recent research papers with respect to the structureactivity relationships of angiotensin-converting enzyme (ACE) inhibitory
peptides, absorption and bioavailability in the human body, mechanisms
*
Corresponding Author address: Email: pedroaredes@fbqf.unt.edu.ar
Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

of action and the role of the vinification process in the occurrence of
bioactive peptides in wine.
Keywords: bioactive peptides, wine, antihypertensive activity, antioxidant

activity
INTRODUCTION
Our diet plays a crucial role in enhancing good health. Some foods contain
bioactive components that are beneficial to health and are able to reduce the
risks of chronic diseases. These foods are known as functional foods (TeckChai et al. 2013). The growing interest in disease prevention and health
promotion has led to the use of these functional foods because they may exert
positive health effects when present in a normal diet (Ruttarattanamongkol
2012). A wider definition of functional foods has been proposed by Diplock et
al. (1999), who described them as foods that beneficially affect one or more
target functions in the body in a way that is relevant to an improved state of
health and well-being, in addition to their adequate nutritional effects.
According to Sir et al. (2008), certain types of functional food like bioactive
peptides, are considered foods naturally containing increased content of
nutrients or components. Several authors have revealed scientific evidence
that food peptides exhibit specific biological activities on health aside from
their nutritional value (Hartmann and Meisel 2007; Tripathi and Vashishtha
2006; Yalcin 2006; Mller et al. 2008).
Bioactive peptides are considered functional components that have been
identified in different fermented foods and beverages. They have been defined
as specific protein fragments that have a positive impact on body functions or
conditions and may ultimately influence health (Kitts and Weiler 2003). They
can be produced through enzymatic hydrolysis of proteins presents in several
foods or through fermentation by proteolytic microorganisms (Korhonen and
Pihlanto 2006). Milk and dairy products are the most thoroughly studied
foodstuffs, and it has been found that they are a rich source of bioactive
peptides (Pihlanto 2011; Choi et al. 2012; Mandal et al. 2014). However, in
the past decade, bioactive peptides have been identified in other foods such as
meat (Balti et al. 2014), fish (Senevirathne and Kim, 2012), eggs (Majumder
et al. 2015), soybean (Singh et al. 2014), wheat (Cian et al. 2015), corn
(Zhuang et al. 2013) and wine (Takayanagui and Yokotsuka 1999; PozoBayn 2007; Alcaide-Hidalgo 2008).
Bioactive Peptides in Wine
Some food proteins can directly produce physiological effects in their

intact form. However, most peptides become bioactive after, hydrolysis or
breakdown during microbial fermentation. These peptides are often more
bioactive than the parent protein (Moughan and Rutherfurd-Markwick 2013).
Therefore, bioactive peptides can be generated by the starter and non-starter
microorganisms associated with fermented products. Lactic acid bacteria
(LAB), ubiquitous microorganisms involved in numerous fermentation
processes, exhibit proteolytic activity on proteins from natural environments
and they contribute to the release of bioactive peptides from dietary proteins
(Saavedra et al. 2013). The proteolytic system of many LAB species
associated with dairy products like Lactococcus lactis, Lactobacillus
helveticus and Lb. delbrueckii ssp. bulgaricus, has already been thoroughly
characterized. The proteolytic system in these LABs consists of a cell wallbound proteinase and a number of distinct intracellular peptidases, including
endopeptidases, aminopeptidases, tripeptidases and dipeptidases (Christensen
et al. 1999).
1. WINE BIOACTIVE PEPTIDES

The composition of the peptide fraction in wine is continuously affected
during the vinification process. Some wine peptides are derived from grapes
and then transferred to the must. The concentration of peptides generally
reduces during alcoholic fermentation. However, during the final stages of this
fermentation a maximum release of peptides takes place because of yeast
death and lysis (Usseglio-Tomasset and Bosia 1990). The presence of endo
and exocellular proteases has been observed during winemaking conditions
(Feuillat et al. 1980; Alexandre et al. 2001). The increase in wine peptides
could be the result of their release from the yeast cells or the action of endo
and exoproteases on proteins derived from yeast or grape juice (MorenoArribas and Polo 2005). This phenomenon has been evidenced in wine models
employing autolyzed yeasts (Martinez-Rodriguez et al. 2001; AredesFernndez et al. 2011).
During the vinification process, many LABs associated with wine are able
to carry out malolactic fermentation and peptides are released through their
proteolytic system. The exoproteases of certain LAB species involved in the
vinification process have been described (Faras et al. 1996, 2000; Folio et al.
2008) and their activity against wine and grape proteins has been documented
(Manca de Nadra et al. 2005; Aredes-Fernndez et al. 2004). Similarly, it has

been found that LABs associated with malolactic fermentation are able to
release bioactive peptides from yeast and wine proteins (Aredes-Fernndez et
al. 2011; Apud et al. 2013a, b).
2. FUNCTIONALITY OF BIOACTIVE PEPTIDES

Bioactive peptides are known to have antimicrobial, antioxidative,
antithrombotic, antihypertensive, anticarcinogenic, satiety regulating and
immunomodulatory activities and they may affect cardiovascular, immune,
nervous and digestive systems (Di Bernardini 2011; Mars et al. 2012).
Moreover, many known bioactive peptides are multifunctional and can present
two or more health promoting activities (Di Bernardini et al. 2011).
Bioactive peptides derived from milk proteins are known to exert diverse
effects, including opioid, mineral-binding, immunomodulatory, antimicrobial,
antioxidant, antithrombotic, hypocholesterolemic and antihypertensive activeities (Pihlanto 2011). They could also play an important role in the prevention
and treatment of metabolic syndrome and its complications through several
mechanisms: satiety response, regulation of insulin levels and blood pressure,
scavenging of free radicals and alteration of the lipid profile (Ricci-Cabello et
al. 2012). Pedersen et al. (2000) demonstrated that consumption of whey
proteins that contain bioactive peptides (mainly glycomacropeptide) leads to
appetite suppression by stimulation of the release of cholecystokinin, which
may promote satiety in rats. Several bioactive peptides isolated and purified
from soymilk, have been characterized by their angiotensin I-converting
enzyme inhibitory (ACE-I) activity (Vallabha and Tiku, 2013), as well as
hypocholesterolemic (Kobayashi et al. 2012), antioxidant (Park et al. 2010),
anti-obesity (Ascencio et al. 2004), immunomodulatory (Kong et al. 2008) and
anticancer (Hsieh et al. 2010) activities. Hydrolysis of whey proteins can also
generate bioactive peptides with many physiological effects such as
antioxidant, antimicrobial, antihypertensive, antidiabetic, immunomodulatory,
anticancer, opioid and hypocholesterolemic activities (Brandelli et al. 2015).
Fermented marine food (Harnedy and FitzGerald 2012) and fermented meat
products (Lafarga and Hanes 2014) are found to be rich sources of bioactive
peptides. Bioactive peptides derived from marine food have numerous
beneficial effects on health such as antioxidant, antihypertensive, antidiabetic
and anti-obesity activities (Ko and Jeon 2013).
Antioxidant and antihypertensive properties are the most important and

recognized biological activities of peptides.
2.1. Antioxidant Peptides

Oxidation is a vital process that provides the necessary energy for survival
to all organisms. Reactive oxygen species (ROS) are products generated by
oxygen metabolism and have a single unpaired electron in their outer orbit that
becomes highly reactive. They are produced in all aerobic organisms that carry
out cellular metabolisms. ROS comprise free radical and non-free radical
oxygen-containing molecules such as hydrogen peroxide (H2O2), ozone (O3),
superoxide (O2.-), singlet oxygen (1O2), hydroxyl radical (OH.), nitric oxide
(NO.), nitrogen dioxide (NO2.), peroxyl (ROO.) and lipid peroxyl (LOO.),
hypochlorous acid, nitrous acid (HNO2), peroxynitrite, dinitrogen trioxide and
lipid peroxide (Genestra 2007; Di Bernardini et al. 2011). Under normal
conditions, these products perform beneficial functions in the human body.
They act as intermediaries in phagocytosis, apoptosis, detoxification reactions,
executioners of precancerous cells and infections, etc. They also regulate many
metabolic and cellular processes including proliferation, migration, gene
expression, immunity and wound healing (Salganik 2001). In the human body,
an endogenous antioxidant system neutralizes reactive molecules and avoids
high ROS levels. This system includes enzymes such as catalase or superoxide
dismutase, non-enzymatic compounds like vitamin C as well as a number of
antioxidant peptides such as carnosine and anserine (Xiong 2010). However,
when the production of these reactive molecules exceeds the capacity of the
antioxidant defense mechanism of the organism, oxidative stress occurs. This
imbalance between ROS generation and antioxidant defense mechanisms
produces oxidative damage of biological macromolecules like proteins, lipids
and nucleic acids (Majzunova et al. 2013).
An antioxidant is a substance that, when present at a low concentration
compared with that of an oxidizable substrate, inhibits oxidation of the
substrate (Halliwell et al. 2007).
Peptides generated after digestion of certain proteins are reported to have
antioxidant properties and can be incorporated into food products to provide
antioxidant benefits. anldere Alolu and ner (2011) studied the antioxidant
effect of peptides released after microbial proteolysis from yogurt proteins.
They found that the total antioxidant activity of yogurt was low, but after
fractionation of peptides by HPLC one of the fractions showed high
antioxidant activity. Chen et al. (2012) confirmed antioxidant activity of

peptides released after enzymatic hydrolysis of walnut proteins.
2.2. Antihypertensive Peptides

Hypertension is a major risk factor for the development of cardiovascular
diseases, a key cause of global morbidity and mortality (Danaei et al. 2013).
The blood pressure in the human body is regulated by many factors and
the most important one is the balance between the renin-angiotensin system
(RAS) and kallikrein-kinin system (KKS). Angiotensin-converting enzyme
(ACE), a carboxyl-terminal dipeptidyl exopeptidase responsible for vasoconstriction, plays a crucial role in the regulation of the blood pressure as well as
cardiovascular functions (Li et al. 2007). ACE is a key enzyme of the RAS. In
this system, renin stimulates angiotensinogen to release a non-active peptide,
angiotensin I (decapeptide), which ACE catalyzes into angiotensin II
(octapeptide). The latter peptide performs a powerful vasoconstrictive action
and stimulates the secretion of aldosterone, favoring the retention of sodium
and water resulting in an increase in arterial blood pressure. In the KKS
system, ACE inactivates the bradykinin, a vasodilator, causing high blood
pressure. If ACE activity is inhibited by ACE-inhibiting compounds,
biosynthesis of angiotensin II is reduced and bradykinin is activated.
Consequently, the blood pressure goes down (Zhao and Li 2009; Majumder et
al. 2015).
Several synthetic ACE inhibitors such as captopril, enalapril, lisinopril,
and ramipril are effective in the treatment of hypertension in humans (Ondetti
et al. 1977). However, they also cause adverse side effects. Thus, the
development of safe and natural ACE inhibitors has gained attention in the
treatment of hypertension (Suzuki et al. 2006; Li et al. 2013).
Bioactive peptides released from food proteins have been widely used for
the treatment of hypertension, mostly based on their ability to inhibit ACE in
the physiological blood pressure-regulating RAS pathway (Udenigwe and
Mohan, 2014). Peptide fractions have been reported to decrease the blood
pressure in spontaneously hypertensive rats and in mild hypertensive human
volunteers (Pihlanto and Korhonen 2015).
Several scientific studies have demonstrated antihypertensive activity of
milk-protein derived peptides (FitzGerald et al. 2004; Lpez-Fandio et al.
2006; Contreras et al. 2009; Pihlanto et al. 2010). Recently, Kumar (2013)
showed that bioactive peptides from yak milk possess good antihypertensive
activity and Tomatsu et al. (2013) confirmed ACE-I activity of eight novel
peptides purified from soymilk using reversed-phase chromatography.
Figure 1. Diagram representing the blood pressure regulation mechanism by reninangiotensin and kallikrein-kinin systems.
With respect to wine peptides, Perrot et al. (2003) observed that the low
molecular weight fraction of Champagne wine exhibited antihypertensive
activity in hypertensive rats whereas it did not affect normotensive rats.
Alcaide-Hidalgo et al. (2007) found that peptides released during accelerated
autolysis of Saccharomyces cerevisiae in a wine model showed ACE-I activity
in addition to oxygen radical scavenging capacity. Aredes-Fernndez et al.
(2011) reported that sequential inoculation of the proteolytic X2L strain of
Oenococcus oeni in a synthetic wine medium increased the peptide nitrogen
concentration after accelerated yeast autolysis and improved antihypertensive
and antioxidant activities. Apud et al. (2013a) detected antihypertensive,
antioxidant and radical scavenging activities of peptides released from the
protein and polypeptide fraction of different Argentine wine varietals after
proteolytic activity of O. oeni m1. The authors showed that these activities
were higher when the peptide nitrogen source was derived from red wines.
Several authors have found that antihypertensive peptides with ACE-I
activity in fermented foods also present radical scavenging activity, suggesting
multifunctional activity of these compounds (Hernndez-Ledesma et al. 2005;
Aredes-Fernndez et al. 2011; Apud et al. 2013a).
3. STRUCTURE-ACTIVITY RELATIONSHIP
OF BIOACTIVE PEPTIDES
Bioactive peptides generally contain short chains of approximately 320
amino acids linked in specific sequence and derived from proteins with a
molecular mass less than 6 kDa (Mller et al. 2008; Shahidi and Zhong, 2008;
Di Bernardini et al. 2011).
The primary structure and amino acid composition of bioactive peptides
are closely related to their activity. The presence of aromatic or alkaline amino
acids in the N-terminus of peptides with ACE inhibitory (ACE-I) activity can
improve its activity. In addition, peptides containing leucine, isoleucine and
valine in the N-terminus exhibited good antihypertensive characteristics.
However, the presence of N-terminal proline diminished ACE-I activity
(Aleman et al. 2011; Pan et al. 2012). With respect to the C-terminus, Wu et
al. (2006) found that ACE-I activity was greatly affected by the threedimensional chemical properties and hydrophobicity of the C-terminal
tripeptide sequence. Jia et al. (2009) showed that the amino acids tyrosine,
proline, tryptophan, phenylalanine and leucine were more probable in peptides
with ACE-I activity. This means that the larger volume and the greater
hydrophobicity of these amino acids contribute to the antihypertensive effect
increasing ACE-I activity.
Alcaide-Hidalgo et al. (2007) demonstrated that a fraction from a yeast
autolysate obtained through liquid chromatography with high content of
hydrophobic peptides, exhibited high ACE-I activity. Some authors have
reported that the presence of hydrophilic amino acid residues in the peptide
sequence could negatively affect the inhibitory activity by blocking the entry
of the peptide to the active site of ACE (Li et al. 2004). On the other hand,
ACE-I activity did not improve with the increasing percentage of hydrophobic
amino acids in the peptide structure (Pripp et al. 2006; Otte et al. 2007).
Furthermore, it has been found that the presence of positively charged amino
acids like lysine and arginine in the C-terminus of peptides can promote ACEI activity (Ferreira et al. 2007). ACE is a zinc-dependent peptidase that
presents two homologous domains, the N-domain and the C-domain, each of
which contains an active site (Soubrier et al. 1988). The C-domain of ACE has
a short consensus sequence and zinc-binding motif, HEXXH, and it has
demonstrated to play a dominant role in the blood pressure control (He et al.
2014). Two histidine amino acids act as zinc ligands and the glutamate as
general base (Gomis-Rth, 2003). When the active site of the ACE C-domain
is occupied by inhibitory peptides that bind to specific amino acid residues, the
ACE activity is lost (He et al. 2014).
Presence of certain amino acids such as cysteine, methionine and histidine
that possess antioxidative activity is related to antioxidant activity in peptides.
It has been demonstrated that the activity of a single antioxidant amino acid is
much lower than the activity evidenced in a peptide containing that amino acid
(Zhu et al. 2012). In general, peptides with hydrophobic amino acids like
histidine, proline, cysteine, tyrosine, tryptophan, phenylalanine and methionine
display strong antioxidant activity whether they are present in the side chain or
C-terminus and/or N-terminus. The antioxidant activity is related to the delay
in the lipid peroxidation chain reaction by combining with oxygen or by
inhibition of hydrogen release (Cheng et al. 2009).
4. MODE AND MECHANISM OF INHIBITION OF ACE

INHIBITORY PEPTIDES
Compared to synthetic drugs, bioactive peptides derived from food are
safe, effective and economical ACE inhibitors to prevent and treat
hypertension (Chen et al. 2007). The inhibition mode of ACE inhibitory
peptides can be explained by a hypothetic model proposed by Ondetti and
Cushman (1982). ACE has two active sites and each of them has three subsites, S1, S1 and S2, that interact with C-terminal tripeptide amino acid
residues of substrates or inhibitors (Cushman and Ondetti, 1991). The zinc ion
is located between S1 and S1 of the active site and participates in the
hydrolytic cleavage of the peptide bond. A hydrogen bond donating group
between sub-sites S1 and S2 is able to bind to the last peptide bond of the
substrate or the inhibitor molecule. The active site also possesses a positively
charged group that establishes ionic interactions with the negatively charged
carboxyl group of the C-terminal amino acid of the substrate or the inhibitor.
10 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Several studies of the inhibition mode of peptides with ACE-I activity
have demonstrated that they can act as competitive, noncompetitive and
uncompetitive inhibitors. Competitive inhibitors have a structure similar to
that of the substrate of the enzyme and when they bind to the active site, they
block it. They can also bind to the inhibitor-binding site that is distant from the
active site, resulting in a modification of the enzyme conformation and
consequently the substrate cannot bind to the active site (Hong et al. 2008).
This model was first proposed by Ondetti and Cushman (1982), who
demonstrated that the substitution of the carboxyl group by sulfhydryl group in
an ACE inhibitor produced a marked competitive inhibition, because it
enabled a strong ionic bond to the positively charged recognition site of ACE.
An example of such behavior is captopril.
In the noncompetitive inhibition system, both the inhibitor and the
substrate can bind to the enzyme at the same time. When this occurs, the
enzyme-substrate-inhibitor complex cannot form a product but can only be
converted back to the enzyme-substrate complex or the enzyme-inhibitor
complex (Si et al. 2009). However the noncompetitive inhibition mechanism
of ACE inhibitory peptides is not clear yet. When ACE is in the unbound
form, hypuril histidil leucine (HHL), an artificial substrate analogue to the
natural substrate of ACE, Angiotensin I, can enter the active site and is then
converted into hypuric acid (HA) (Ni et al. 2012). These authors found that the
presence of the inhibitory peptide TPTQQS does not block the active site of
ACE.
Consequently, TPTQQS does not compete with the substrate for the active
site, indicating that this peptide is a non-competitive ACE inhibitor. Even
though HHL would bind to the active site, it would be impossible to convert it
into HA, because of a change in the conformation of the active site of the
enzyme.
In the case of uncompetitive inhibition, the inhibitor is able to bind to the
substrate-enzyme complex but not to the free enzyme and because of this
inhibition, a decrease in the maximum enzyme activity occurs. Peptides such
as IW, FY and AW have been reported to act as uncompetitive ACE inhibitors
(Sato et al. 2002). Nevertheless, like the noncompetitive mechanism, the
inhibition mechanism of this system is not clear either.
11
5. ABSORPTION AND BIOAVAILABILITY OF ACE

INHIBITORY PEPTIDES
The physiological effects of bioactive peptides have been determined by
their ability to reach the target sites, which may involve absorption through the
intestinal epithelium to get to peripheral organs (Vermeirssen et al. 2004). In
general, digestion of proteins and peptides begins in the stomach through the
action of pepsine at a low pH. Then the polypeptides are hydrolyzed by
pancreatic enzymes like trypsin, -chymotrypsin, elastase and carboxypeptidases A and B. Absorption of peptides in the gastrointestinal tract takes
mainly place in the small intestine (approximately 90%) and in the colon
(Garcia-Redondo et al. 2010). Bioactive peptides can resist the digestive action
of acids and digestive enzymes. Korhonen and Pihlanto (2006) found
antihypertensive bioactivity of peptides after oral ingestion when absorbed in
an intact form. Some studies have also demonstrated that several peptides with
ACE-I activity were resistant to the digestive proteases after experiments
simulating gastrointestinal digestion (Choi et al. 2001; Ohsawa et al. 2008;
Ren et al. 2011). Other peptides were hydrolyzed into shorter but active forms
after simulation of gastrointestinal digestion (Shiozaki et al. 2010). Mizuno et
al. (2004) proved that antihypertensive peptides containing C-terminal proline
were resistant to proteolytic enzymes. Finally, tripeptides containing the Cterminus proline-proline have been reported to be resistant to proline-specific
peptidases (FitzGerald and Meisel, 2000).
The action of brush-border peptidases, recognition by intestinal peptide
transporters and the subsequent susceptibility to plasma peptidases determine
the physiological effect of bioactive peptides (Vermeirssen et al. 2004). The
mechanisms of intestinal absorption are mainly classified into three categories
according to Wada and Lnnerdala (2014): (1) the proton-dependent peptide
transporter, PepT1, present in intestinal epithelial cells that actively transports
di- and tripeptides. Peptides transported into the cell are generally hydrolyzed
into amino acids by cytoplasmic peptidases, but certain peptides may resist
hydrolysis, allowing absorption of particular bioactive di- and tripeptides via
this mechanism; (2) transcytosis through an intracellular vesicular transport
system. The vesicles are present in intestinal epithelial cells and enable the
traslocation of certain oligopeptides. However peptides are likely to be
hydrolyzed by cytoplasmic peptidases in this pathway; (3) paracellular
transport. This transport mechanism, which seems to be the more important
than transcytosis, is based on the passive diffusion of peptides between cells

and it does not possess specific transporters. Permeability between cells during
paracellular transport is regulated by tight junctions comprised of two proteins,
occludins occludin and cingulin that form small pores in the junction, enabling
transport of larger oligopeptides and di- and tripeptides by passive diffusion.
This paracellular pathway is non-digestive and it is considered to play an
important role in the absorption of bioactive peptides in an intact form. It is
known that di- and tripeptides may be transported through the intestinal
mucous membrane, although there is some evidence that larger peptides may
also be absorbed in the small intestine.
The half-life of peptides in plasma is generally very short. Two tripeptides
known for their antihypertensive bioactivity, IPP and VPP, have been detected
in human plasma at picomolar concentrations after oral administration. This
would mean that paracellular transport plays a crucial role in VPP transport
(Shimizu and Son 2007), since transport mediated by the PepT1 carrier leads
to a rapid hydrolysis of the peptide (Regazzo 2010). Once absorption of
peptides occurs, high peptidase concentrations in the blood cause fast
hydrolysis of peptides in plasma. After intravenous administration of the
tripeptide IVY to spontaneously hypertensive rats, this molecule was
metabolized by plasma peptidases to form a subsequent ACE inhibitor, VY
with has a lower in vivo antihypertensive effect (Matsui et al. 2002). SanchezRivera et al. (2014) recently published results obtained with peptides
containing four or more amino acids from dietary sources with antihypertensive activity. Based on pharmacokinetic parameters, the authors suggest a
considerable uptake of the antihypertensive pentapetide HLPLP in tissues after
oral or systemic administration in spontaneously hypertensive rats. This
peptide was characterized by its resistance to in vitro gastrointestinal digestion
and brush border peptidases (Quir s et al. 2008). After oral administration to
spontaneously hypertensive rats, the HLPLP peptide was rapidly absorbed and
biotransformed into the smaller and active LPLP and HLPL fragments, which
were distributed throughout the body by circulation (Snchez-Rivera et al.
2014).
6. ROLE OF FERMENTATIVE PROCESS AND OCCURRENCE

OF BIOACTIVE PEPTIDES IN WINE
Moderate consumption of red wine has been associated with reduced risk
of developing cardiovascular heart disease. This property is partly attributed to
13
peptides with biological characteristics, mainly antihypertensive and antioxidant activities. Some wine peptides originate from the must, but most of
them appear during the different stages of wine production either after yeast
autolysis or after proteolytic activity of lactic acid bacteria on wine proteins
(Covas et al. 2010).
The first study regarding bioactive peptides in wine was carried out by
Takayanagi and Yokotsuka (1999), who determined for the first time ACE
inhibitory (ACE-I) activity both in red and white wines. The authors also
demonstrated that red wines had higher ACE-I activity that white wines. They
hypothesized that peptides from the pulp of the grapes constitute the majority
of ACE inhibitory substances found in wine. This hypothesis is based on the
fact that ACE-I activity in the wines assayed declined during the fermentative
process. Considering that many phenolic compounds with ACE-I activity
could be extracted from the skin and seeds, the authors supposed that phenolic
compounds have a negligible incidence in the ACE-I activity. In studies
performed with normotensive and spontaneously hypertensive rats, Perrot et
al. (2003) found that the extract of the low molecular weight fraction from
Champagne wine exerted an antihypertensive effect on the hypertensive rats
but not on normotensive ones. The authors postulated that in view of the
complex composition of the extracted wine fraction it is unlikely that the
decrease in blood pressure can be attributed to the presence of a single
compound. In fact, since wine is rich in phenolic compounds and peptides,
both groups could comprise individual components with antihypertensive
activity (Sarr et al. 2006). Similarly, Pozo-Bayn et al. (2007) reported that red
wines showed higher ACE-I activity than white wines, confirming the results
by Takayanagi and Yokotsuka (1999). Furthermore, Pozo-Bayn et al. (2007)
postulated that phenolic compounds could have important participation in
wine ACE-I activity, although the authors only determined this activity in a
low peptide wine fraction and a total peptide wine fraction.
Wine is the product of complex interactions between yeasts and bacteria in
grape must (Costantini et al. 2009) and multiple metabolic reactions occur
during the fermentative process. Yeasts of the genus Saccharomyces, mainly
S. cerevisiae, develop during the alcoholic fermentation, and under anaerobic
conditions, they transform sugars present in the juice, mainly glucose and
fructose, into ethanol and carbon. During this process the medium is depleted
of assimilable nitrogenous compounds because the yeasts use these nutrients to
obtain energy and increase their population (Ribrau-Gayon et al. 2000). At
the end of the fermentation process, yeast autolysis can occur, resulting in the
release of intracellular compounds, thus increasing the concentration of

nitrogen compounds. After the fermentation process, special wines like
sparkling wine are exposed to an aging process in the presence of yeast cells.
Yeast autolysis occurs during this period, resulting in the release of
intracellular compounds that modify the organoleptic characteristics of the
wine (Charpentier et al. 2005; Nunez et al. 2005). The principal constituents
released after yeast lysis are peptides and to a lesser extent amino acids and
proteins (Alexandre and Guilloux- Benatier, 2006). Similarly, Alcaide-Hidalgo
et al. (2007) demonstrated that under accelerated autolysis conditions in a
synthetic wine, a commercial S. cerevisiae strain increased the release of high
molecular weight nitrogen compounds, mainly proteins, during the first stage
of autolysis. These high molecular weight nitrogen compounds are were
enzymatically hydrolyzed producing peptides and amino acids. This means
that peptides are the main autolysates, thus demonstrating their importance in
aged wines. The authors showed that under accelerated autolysis, peptides
released from yeast exhibited both ACE-I and antioxidant activities. They
concluded that these activities could be exclusively attributed to yeast
peptides, and highest activity was observed in a hydrophobic peptide isolated
fraction. Aredes-Fernndez et al. (2011) also demonstrated the release of
nitrogen compounds during accelerated autolysis of S. cerevisiae mc2 in a
synthetic wine medium. Yeast autolysis was confirmed by viable cell counts
and determination of dry weight before and after yeast autolysis. Under
accelerated autolysis conditions, yeast viability decreased dramatically after 24
hours, observing a reduction of 38% in dry weight. As a result, an increase in
the concentration of proteins, peptides and amino acids of 7.92, 3.66 and 5.97
mg N/L respectively, was detected (Aredes-Fernndez et al. 2011).
The nutrients released after yeast lysis become available for growth of
Oenococcus oeni, a lactic acid bacterium responsible for carrying out the
enzymatic conversion of L-malic acid to L-lactic acid in a process known as
malolactic fermentation (MLF) during the second stage of winemaking. MLF
is a significant process in wine making that affects operation efficiency and
product quality and safety. This fermentation produces stabilization, a
reduction in acidity and production of desirable wine aroma and flavor
compounds. O. oeni is the main bacterium responsible for this fermentation
process because of its ability to survive the harsh wine conditions (high
alcohol content, low pH, and low nutrient content) and production of desirable
sensory compounds (Bartowsky 2014).
During the first days of the alcoholic fermentation, the population of
O. oeni is limited to levels of about 104 CFU/mL. As the fermentation
advances, these values decrease to 102 CFU/mL owing to its sensitivity to
15
ethanol and the low pH of the medium. After the alcoholic fermentation, the
cells start multiplying and they can reach the necessary population of 106108
CFU/mL to start the MLF (Fleet et al. 1984), even though wine is an
unsuitable substrate for the growth of lactic acid bacteria. In general, wine is a
poor medium for bacterial growth because it has few available nutrients
(Guilloux- Benatier et al. 1985). Because lactic acid bacteria have complex
nutritional requirements, the release of peptides and amino acids plays an
important role in maintaining O. oeni growth in natural media. O. oeni has
complex free amino acid requirements to sustain growth, because it is unable
to synthesize certain amino acids (Saguir and Manca de Nadra 2007). To
overcome this inconvenience, it has developed complex enzyme systems,
producing small peptides and releasing free amino acids from larger peptides
into the immediate environment. Manca de Nadra et al. (1997, 1999) reported
proteolytic activity of O. oeni X2L on the macromolecular nitrogen fraction of
white and red wines, which favored peptide release. The authors also found
that the release of O. oeni proteases into the extracellular medium increased
under starvation conditions (Manca de Nadra et al. 2005). Faras and Manca de
Nadra (2000) partially purified and characterized an exoprotease from O. oeni.
In addition, Remize et al. (2005) confirmed the presence of extracellular
protease activity in O. oeni IOB84-13 during the growth phase in a poornitrogen medium. Moreover, Folio et al. (2008) demonstrated the presence of
extracellular proteins from O. oeni ATCC BAA-1163. One of them, EprA,
was isolated and the enzyme was able to hydrolyse several proteins.
O. oeni is able to use small peptides of up to eight amino acid residues
(Aredes-Fernndez et al. 2004; Ritt et al. 2008). Aredes-Fernndez et al.
(2004) exhaustively studied peptide utilization in O. oeni X2L, assaying the
individual and joint effect of different dipeptides as amino acids sources on the
growth of O. oeni X2L in a synthetic medium supplemented with L-malic acid.
They demonstrated that substitution of essential amino acids by dipeptides
resulted in a significant increase in the growth parameters of the
microorganism.
A previous report by No et al. (2008) evidenced that the increase in the
ACE-I activity takes place after alcoholic fermentation. The authors also
reported that the ACE inhibitors could be peptides. Aredes-Fernndez et al.
(2011) showed that after sequential inoculation of O. oeni in synthetic similwine medium bacterial proteolytic activity caused a decrease in the
concentration of proteins released after yeast autolysis. A concomitant increase
in peptide release also produced a significant increase in ACE-I activity

(36.5%) and antioxidant activity (430.67 mol FeSO4/L and 3.47% for FRAP
and DPPH scavenging, respectively).
Apud et al. (2013a) demonstrated that culturing O. oeni m1 in a synthetic
simil-wine medium supplemented with the protein and polypeptide fraction
with a molecular weight higher than 12 KDa (obtained after dialysis with a
cellulose membrane) from four varietals of Argentine wines from Cafayate,
(Cabernet Sauvignon, Malbec, Tannat and Torronts) allowed the release of
biologically active peptides. The authors also observed simultaneous protein
consumption. These results confirm that the release of bioactive peptides is a
result of bacterial proteolytic activity.
2.5
Proteolytic Activity [mmol/L]
2.0
1.5
1.0
0.5
0.0
0
24
48
Time [h]
72
96
Figure. 2. Proteolytic activity in synthetic simil-wine medium (SW) (open square) and
SW added with protein-polypeptide fraction of Cabernet Sauvignon (filled square);
Malbec (open circle); Tannat (filled circle) and Torronts (triangle) during 96 h
incubation.
Figure 2 shows the change in proteolytic activity of O. oeni m1 in

synthetic simil-wine medium (SW) supplemented with the high molecular
weight nitrogen fraction (HMN) obtained from different wine varietals. In
synthetic simil-wine medium (control medium), low proteolytic activity (0.396
mmol/L) of O. oeni was detected after 24 h of incubation. In SW
supplemented with Cabernet Sauvignon and Torronts HMN, maximum
17
proteolytic activity was detected after 24 h of incubation with values of 1.372

and 1.054 mmol/L, respectively. In SW supplemented with HMH from
Malbec and Tannat wines, proteolytic activity significantly increased after 48
h, reaching values of 0.992 and 1.536 mmol/L, respectively.
The highest increase in peptides in synthetic simil-wine medium
supplemented with Cabernet Sauvignon, Malbec, Tannat and Torronts HMN
occurred after 48 h incubation time with a release of 1.067, 0.397, 0.916 and
0.705 mg N/L of peptide nitrogen, respectively. Simultaneously with the
peptide release in synthetic simil-wine medium supplemented with HMN of
Cabernet Sauvignon, Malbec and Tannat wines, maximum ACE-I activity was
detected after 24 h of incubation showing an increase of 63.8, 36.4, and 70.0%
respectively. Nevertheless synthetic simil-wine medium supplemented with
Torronts HMN showed ACE-I activity that was lower than that in the red
wine varietals, presenting a maximum activity after 24 h incubation (18%).
With respect to antioxidant activities, evaluation of ferric reducing antioxidant
power (FRAP) and free radical scavenging ability (DPPH) showed that during
incubation of O. oeni in the presence of HMN of Cabernet Sauvignon and
Tannat wines, the highest increase in these activities was detected after 24 h
incubation. A synthetic simil-wine medium with O. oeni but without
supplement, did not exhibit significant changes in the two biological activities.
Figure 3 shows the relationship between the changes in peptide nitrogen and
biological activities in the presence of O. oeni m1 in SW added with each
HMN fraction obtained from the four different wine varietals. These results
are in agreement with those published by Apud et al. (2013b) who reported
that inoculation of O. oeni m1 in SW supplemented with HMN of Cabernet
Sauvignon and Syrah wines from Colalao del Valle, Tucumn, Argentina
produced a release of 1.247 and 1.373 mg N/L of peptide nitrogen,
respectively, after 48 h of incubation. The released peptides from Cabernet
Sauvignon and Syrah wines allowed an increase in the FRAP capacity, DPPH
activity and ACE-I activity.
Unpublished results presented at the XIV Congreso Latinoamericano de
Viticultura y Enologa showed a change in ACE-I activity after proteolytic
activity of O. oeni X2L on the HMN from Cabernet Sauvignon wine from
Tucuman, Argentina. The HMN was obtained after precipitation with
trichloroacetic acid in acetone at -20C. O. oeni X2L was inoculated in a
synthetic simil-wine medium supplemented with the protein fraction
previously obtained. The major increase in proteolityc activity was detected
after 96 h of incubation reaching a value of 0.300 mmol/L. At this time a
significant consumption of 215.93 mg N/L in protein nitrogen was observed

with a release of 77.73 mg N/L of peptide nitrogen (Table 1). Results obtained
in synthetic simil-wine medium without supplement of HMN, showed that O.
oeni did not grow and proteolytic activity was not detected. The peptides
released as a consequence of proteolytic activity in the presence of HMN from
Cabernet Sauvignon wine, increased ACE-I activity reaching the maximum
value at 96 h of incubation (17.73%).
Table 1. Changes in O. oeni X2L proteolytic activity and nitrogen
compounds in synthetic simil-wine medium supplemented with HMN
from Cabernet Sauvignon wine
Incubation
time
[h]
Proteolytic
activity
[mmol/L]
Protein
concentration
[mg N/L]
Peptide
concentration
[mg N/L]
Amino acid
concentration
[mg N/L]
0.000.02
289.2420.0
1.660.09
28.931.35
48
0.110.01
246.0521.52
7.030.20
26.732.23
96
0.300.04
73.315.60
79.395.97
27.121.78
Values are the means of three independent determinations carried out in duplicate.
Recently, Su et al. (2015) studied the antioxidant properties of intact cells

and cell-free extracts of different strains of O. oeni isolated from wine. The
authors suggest their possible use as probiotics, taking into account their
adaptation to the hostile wine environment, which mimics the acidic
conditions of the digestive tract. They demonstrated that the two fractions of
19 strains assayed presented different antioxidant activities. However the
authors did not establish the type of compounds involved in these activities.
7. FRACTIONATION AND ISOLATION OF

BIOACTIVE PEPTIDES
Peptides have many different physicochemical properties such as size,
charge, adsorption characteristics and solubility, making their fractionation
and isolation difficult. Successive fractionation steps are necessary for
peptides studies in order to eliminate high molecular weight compounds as
much as possible.
Figure 3. Changes in peptide release (lines) and biological activities (bars): FRAP: Ferric reducing antioxidant power (black bars);
DPPH scavenging: 2,2- diphenyl-1-picrylhydrazyl radical scavenging capacity (white bars) and ACE-I: angiotensin I-converting enzyme
inhibitory activity (gray bars) during 96 h incubation in synthetic wine (SW) and SW added with HMN from four different wines
varietals: ca: Cabernet Sauvignon; ma: Malbec; tn: Tannat and to: Torronts.

Proteins are removed from samples by precipitation with different
precipitants like 7% Trichloroacetic acid (TCA) (Yokotsuka et al. 1975) or
95% ethanol (Moreno-Arribas et al. 1996, 1998a; Martnez-Rodrguez et al.
2002).
Ultrafiltration (UF) is especially useful to remove proteins during
fractionation of peptides. This size-exclusion pressure-driven separation
process uses membranes with a certain pore size that will retain particles or let
them through according to their size. This process retains proteins but not
small peptides or carbohydrates (Berk 2013). Desportes et al. (2000) and
Pozo-Bayn et al. (2007) used this technique in the firt stage of isolation of
wine peptides.
A more recent technology, electrodialysis with ultrafiltration membranes
(EDUF), has been developed to fractionate peptides from complex mixtures
based on their electrical charge, size, or molecular weight. It is essentially a
batch process which one or more filtration membranes stacked inside a
conventional electrodialysis cell. This technique allows separation of
molecules according to their charge and molecular size in an electric field.
EDUF has been successfully used to separate bioactive peptides from various
food protein hydrolysates (Suwal et al. 2014). Lately, Roblet et al. (2014)
isolated soybean peptides using electrodialysis with an ultrafiltration
membrane. Doyen et al. (2011, 2012) successfully fractionated antimicrobial
and anticancer peptides from a snow crab using EDUF.
After precipitation or ultracentrifugation of proteins, traditional
chromatographic methods are generally used to separate peptide mixtures
because of their high selectivity. The most commonly used chromatographic
methods for separation and purification of peptides is gel filtration or sizeexclusion chromatography (SEC), ion-exchange chromatography (IEC),
affinity chromatography (AC) and hydrophobic interaction chromatography
(HIC). Of these methods, SEC is the technique of choice to separate wine
peptides (Desportes et al. 2000, Pozo-Bayn et al. 2007). SEC allows
separation of peptides according to their molecular size. The pore size is
determined by the molecular weight range of the peptides in the sample.
High Performance Liquid Chromatography (HPLC) is a widely used
technique to separate, identify, and purify bioactive peptides. Reverse-phase
HPLC (RP-HPLC), which uses hydrophobic interactions as the main
separation principle, is considered the most powerful method to purify
peptides. It is characterized by the use of a stationary phase and an aqueous
mobile phase containing an organic solvent, such as acetonitrile or an alcohol.
21
Mandal et al. (2014) recently fractionated and purified twenty-four peptides

from human milk using RP-HPLC.
Peptides are difficult to isolate from wine because they are together with
non-peptidic compounds in a complex mixture (Desportes et al. 2000). This is
the reason that not many studies have been carried out to separate these
compounds from wine.
Desportes et al. (2000) separated peptides from wine using ultrafiltration
followed by gel-filtration chromatography with Sephadex columns and the
fractions obtained were subjected to RP-HPLC in order to isolate small
peptides. Pozo-Bayn et al. (2007) isolated peptides with antihypertensive
activity from red and white wines using ultrafiltration and SEC with a
Sephadex LH-20 column. Alcaide-Hidalgo et al. (2008) fractionated peptides
of from red wines using SEC and HPLC with Sephadex LH-20 and Cosmosil
140 C18-OPN columns respectively.
Peptides are generally detected at an absorbance between 200 and 220 nm,
but many compounds present in wine could interfere in the ultraviolet
detection of peptides when low wavelengths are used. Therefore, it is better to
apply sensitive and selective detection methods. A solution is to synthesize
peptide derivatives because these are detectable at higher and more specific
wavelengths. Peptides that contain fluorescent amino acids, like tyrosine and
tryptophan, may be detected using fluorescence. Peptides without this property
can be derivativatized using special fluorescent agents, a technique that has
shown to be very useful (Moreno-Arribas et al. 1998a).
Determination of the amino acid sequence can be carried out using mass
spectrometry or Edman degradation sequencing. Edman degradation is a
chemical method based on the cleavage of one amino acid at a time from the
N-terminus of the peptide chain. This terminal amino acid is then separated
and identified. The cleavage reaction is repeated until the complete peptide
sequence is known.
A complication is that this method requires highly purified samples. This
technique can be carried out manually or automatically using special
automated peptide sequencers (Gouda et al. 2006; Kuba et al. 2009; Rho et al.
2009).
Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) are
powerful techniques widely employed for the characterization of bioactive
peptides. With MS, an unknown peptide undergoes fragmentation and the
fragments (ions) are subsequently registered in a peptide mass spectrum. With
MS/MS these ions are called precursor ions and they break into two parts
generating one fragment containing the N-terminus of the original peptide

sequence and a complementary fragment containing the C-terminus. Then,
computational methods deduce the peptide sequence from its spectrum.
MS/MS and MS spectra are similar to one another, with the difference that in
the former technique, the peaks correspond to fragment ions of a peptide and
in the later one, the peaks correspond to complete peptide ions (Steen and
Mann 2004; Menschaert et al. 2010; Costa et al. 2013).
Junfeng et al. (2009) determined the amino acid sequence of a peptide
derived from fermented soybean food with ACE-I activity applying Edman
degradation. Boutrou et al. (2013) identified milk-protein bioactive peptides
with opioid and antihypertensive activity using tandem mass spectrometry.
8. PERSPECTIVES
The most challenging task in the study of bioactive peptides from wine is
identification of peptides with biological activity produced after microbial
metabolism during the vinification process and evaluation of their in vitro and
in vivo activity. Current studies should focus on the isolation and selection of
wine microflora with advantageous technological vinification properties that
guarantee the wine quality and may even add additional value to the final
product. More studies on this topic are necessary to ensure that orally ingested
bioactive peptides present in wine pass through the digestive tract and are
subsequently absorbed through the intestinal epithelium. Finally, the beneficial
effect of these peptides on the target organs or tissues should be assessed.
Another current topic of interest is the supplement of bioactive peptides as
therapeutic agents in food and beverages. They are used as natural ingredients
in functional and novel foods, dietary supplements and even pharmaceuticals
with the purpose of delivering specific health benefits. In this way, it is
necessary to investigate strategies for increasing the resistance of digestive
enzymes and cellular permeability of bioactive peptides.
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ISBN: 978-1-63482-765-2
Chapter 2
WINE POLYPHENOLS:
BIOLOGICAL ACTIVITIES
AND REUSE FROM WINERY WASTE
Mara J. Rodrguez-Vaquero*, Sofa M. Sosa-Marmol,
Mara G. Stivala, Gisselle R. Apud
and Pedro A. Aredes-Fernndez
Universidad Nacional de TucumnTucumn, Argentina
ABSTRACT
Argentina is in the fifth position of world production of wine; the
68.5% of national production is located in Mendoza. Wine contains a
number of polyphenolic constituents which determine important sensorial
characteristics, such as color, mouthfeel, astringency and bitterness. The
phenolic compounds profile and concentration in wines depend on
several factors, such as the grape variety, climate, soil, as well as on the
oenological practices applied for winemaking and aging and storage
conditions. The beneficial properties of phenolic compounds from
different sources were reported as potent antioxidants, free radical
scavengers and metal chelators; lipid peroxidation inhibitors and exhibit
*
Corresponding author: Mara J. Rodrguez-Vaquero. Facultad de Bioqumica, Qumica y

Farmacia Universidad Nacional de Tucumn, Ayacucho 471 CONICET. Tucumn.
Argentina. E-mail: mariajo@fbqf.unt.edu.ar.
36
M. J. Rodrguez-Vaquero, S. M. Sosa-Marmol, M. G. Stivala et al.

various physiological activities including anti-inflammatory, antiallergic,
anticarcinogenic, antihypertensive, antiarthritic and antimicrobial activities. A big problem of wine industry is the large amount of waste
generated during the production of wines. Polyphenols from winery
wastes can exhibit different activities of interest, like antibacterial and
antifungal. So, the reuse of phenolic compounds from winery waste could
be proposed as alternative to control several microorganisms.
Keywords: phenolic compounds, wine, biological activities, winery waste
INTRODUCTION
Wine production is one of the most important agricultural activities
throughout the world. The most competitive wineproducing countries are the
United States, Australia and Chile, followed by Italy, Spain, Argentina and
South Africa, with France and Germany also producing important quantities of
wine (Hussain et al. 2008). Wine contains polyphenolic constituents which
determine important sensorial characteristics, such as color, mouthfeel,
astringency and bitterness. They are the main components responsible for the
differences between red and white wines, especially for the color, taste, and
mouthfeel sensations of red wines (Ivanova-Petropulos et al. 2015).
Phenolic compounds are phytochemicals, secondary metabolites widely
distributed in most vegetables and fruits. They are molecules containing a
benzene ring bearing one or more hydroxyl groups. In plant tissues, phenolic
compounds play important roles in growth, reproduction, warding off or
restricting the attack of pathogenic microorganisms. Polyphenols exhibit a
huge variety of structures in nature; they consist of simple phenols, benzoic
and cinnamic acid, coumarins, tannins, lignins, lignans and flavonoids.
Phenolic acids could be divided into two groups: hydroxybenzoic acids
with C6-C1 structures and hydroxycinnamic acids with C6C3 squeleton.
Whereas flavonoids have a C6-C3-C6 base structure, formed by a series of
condensation reactions between a hydroxycinnamic acid and malonyl residues.
Flavonoids commonly occur as flavonoid O-glycosides, in which one or
more hydroxyl groups of the aglycone are bounded to a sugar with formation
of an acid labile glycosidic OC bond. Glucose is the most commonly
encountered sugar, galactose, rhamnose, xylose and arabinose are not
uncommon, and mannose, fructose, glucuronic and galacturonic acids are rare
(Iwashina 2000). Disaccharides are also often found in association with
flavonoids, the most common ones being rutinose and neohesperidose.
Wine Polyphenols
37
Flavonoids are a widespread family of phytochemicals with diverse

biological functions in plants. Variations in the heterocyclic ring C give rise to
flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavonoids.
The basic structure of flavonoids allows a multitude of substitution patterns in
the benzene rings A and B. Over 4000 different naturally occurring flavonoids
have been described (Middleton and Kandaswami, 1994).
Flavan-3-ols are the other important group of wine phenolics that could be
present as monomers giving the bitter character, and oligomers and polymers
contributing to wine astringency (Sarni-Manchado et al. 1999).
Wine is a complex mixture of several hundred compounds present at
different concentrations, some originating from the grapes and some metabolic
by-products of yeast activity during fermentation (Soleas et al. 1997). Wine is
composed of water, ethanol, glycerol, polysaccharides, different types of acids
and phenolic compounds. The phenolic compounds of wine can be divided
into flavonoids and non-flavonoids. Flavonoids, which account for over 85%
of the phenolic components in red wine, include different molecular families
like flavonols, flavones and anthocyanidins. Non-flavonoid compounds
include hydroxycinnamic acids, hydroxybenzoic acids, stilbenes, and
hydrolysable tannins (Stockley et al. 2012). Red wine is known to contain 10foldmore phenolic compounds than white wine.
The total polyphenol concentration in Argentinean wine is around 2500
mg/L (Rodrguez-Vaquero et al. 2007a), being flavonoids the majority
fraction, but the phenolic profile is related with the grape variety used in wine
elaboration. The grape polyphenols belong to different classes distributed in
every part of fruit but skin contains the highest amount of polyphenols and in
particular of condensed tannins (Souquet et al. 1996), monomeric flavanols
and flavonols, phenolic acids and resveratrol (Man et al. 2007; Pinelo et al.
2006). The major constituents of pulp are phenolic acids and monomeric
flavonoids, such as flavanols, although at lower concentrations than in the skin
(Man et al. 2007). The seeds contain polymeric condensed tannins with minor
quantities of procyanidins and monomeric flavonoids (Pinelo et al. 2006).
As widely accepted by the scientific community, wine is one of the most
important sources of dietary polyphenolic antioxidants including a large
variety of both flavonoid (flavonol, flavan-3-ol and anthocyanin) and nonflavonoid compounds (phenolic acids, phenolic alcohols, stilbene,
hydroxycinnamic acid) (Bravo 1998; Burns et al. 2000; Fernndez-Pachn et
al. 2006; Monagas et al. 2005).
The ageing process produces the maturation of wines and improve their
sensorial characteristics. In this way, the ageing produces wines with more
38
elegant and stable colours, a more complex aroma and better taste due to the
loss of sensations of astringency and bitterness (Puech et al. 1999).
1. FACTORS AFFECTING WINE PHENOLIC COMPOSITION

The polyphenolic profile of red wines differs essentially from that of
white wines by effect of differences in composition between red and white
grapes, and also in the vinification technology used. White grapes are used to
obtain white wine; these grapes are green in colour and derived from the red
grapes. Mutations in two regulatory genes of white grapes turn off production
of anthocyanins, which are responsible for the colour of red grapes.
Chardonnay is the most famous wine variety.
The phenolic compounds profile and concentration in wines depends on
several factors, such as the grape variety, climate, soil, as well as the
oenological practices applied for winemaking and aging and storage
conditions (Ivanova et al. 2009; 2011a,e; Koyama et al. 2007; Gil-Muoz et al.
2009; Kostadinovic et al. 2012). The phenolic composition of wine is
determined initially by the phenolic composition of the grapes used for making
the wine (Ribreau-Gayon et al. 1998) and exposure to sunlight and
temperature are the main factors influencing the phenolic composition of
grapes. It is generally accepted that the final quality of fruit wine is
significantly influenced by the raw materials (Gonzalez-Mas et al. 2009).
Therefore, the variety and cultivar of raw materials may have important effects
on wines, including with regard to sensory properties and antioxidant activity
(Mikami-Konishide et al. 2013).
During the winemaking process, the polyphenol composition was
changing, for example, the anthocyanins reach a maximum level after few
days of maceration, followed by decrease of the content during the
fermentation, stabilization and storage as a result of co-precipitation with
tartaric acid salts in a form of colloidal material, adsorption on yeast cell walls,
elimination during filtration and fining or their participation in numerous
chemical reactions forming numerous novel monomeric, oligomeric and
polymeric compounds (He et al. 2006; Rentzsch et al. 2007; Rentzsch et al.
2010; Oliveira et al. 2010; Blanco-Vega et al. 2011).
The contact of the wine with the wood foments the transfer of
ellagiotannins and other molecules to the wine.
The ellagiotannins take part in polymerisation, condensation and
precipitation reactions which, apart from agglomerating colloids and
Wine Polyphenols
39
improving the clarity of the wine, contribute to producing wines with more
stable colours and complex aromas (Ortega-Heras et al. 2004).
2. BIOLOGICAL PROPERTIES AND PERSPECTIVES

One important subject of research concerning the beneficial effects of
polyphenols is their bio-availability. The bio-availability of a dietary
compound is dependent upon its digestive stability, its release from the food
matrix and the efficiency of its transepithelial passage. Bio-availability differs
greatly from one polyphenol to another, and for some compounds it depends
on dietary source (Manach et al. 2005). Some authors reported that the
absorption of polyphenols happens through passive diffusion across the
membranes of the gut epithelial cells. In this contest, most polyphenols are
probably too hydrophilic to penetrate the gut wall by passive diffusion
(Manach et al. 2004). The absorption of phenolic compounds is considered to
be low due to the chemical structures (Manach et al. 2005). Respect to
polyphenols that are present in food in the form of esters, glycosides, or
polymers, these cannot be absorbed in native form (Li et al. 2013), so they
must be hydrolyzed by intestinal enzymes of by colonic microflora. PrezVicente et al. (2002) reported that the very low bio-availability of anthocyanins can be attributed, at least partially, to the high instability of these
molecules in the mild alkaline condition of the small intestine.
Phenolic compounds molecules are known for their antioxidant properties.
Polyphenols are reducing agents, and similarly to vitamins C or E, protect
body tissues against oxidative stress and associated pathologies such as cancer,
coronary heart disease, and inflammation.
White and, especially, red wines are considered as rich sources of
antioxidant polyphenolic compounds. In clinical studies, moderate wine
consumption decreased the risk of certain cancers, such as lung (Chao et al.
2011), colon (Anderson et al. 2005), upper digestive tract (Pandeya et al.
2009) and skin (Freedman et al. 2003). Zell and colleagues found that regular
wine consumption had favorable effects on stage at presentation and survival
in familial colorectal cancer cases (Zell et al. 2007).
Epidemiological evidence indicates that the moderate consumption of
wines reduces the incidence of coronary heart disease (CHD), atherosclerosis
and platelet aggregation (Tedesco et al. 2000).
40
This greater protection may be due to the phenolic components of wines,

which are particularly abundant in the red wine, since they behave as reactive
oxygen species-scavengers and metal-chelators.
The heart health benefits of polyphenols can be explained by the French
paradox, a phrase popularized by Renaud and Lorgeril in the 1990s. It
revealed that while France is traditionally one of the highest consumers of
saturated fats and cholesterol, it has one of the lowest rates of CHD and
mortality maybe due to high consumption of red wine in this country (Mennen
et al. 2003).
3. ANTIBACTERIAL ACTIVITY
The beneficial properties of phenolic compounds from different sources
were extensible reported. The total phenolic content in three varieties of
Argentinean wines, Cabernet Sauvignon, Malbec and Merlot and their relation
with the antimicrobial activity against pathogenic bacteria as study for
Rodrguez-Vaquero et al. (2007a,b, 2008).
The authors demonstrated that phenolic compounds concentrations in
Malbec and Merlot wines were higher than in Cabernet Sauvignon wine;
Cabernet Sauvignon failed to show any activity against E. coli and P.
aeruginosa ATCC 27853, although all Malbec and Merlot wine samples were
active against the tested bacteria.
The lowest antimicrobial activity showed with samples of Cabernet
Sauvignon wine that could be related with its lower phenolic concentration.
Pr. mirabilis was the bacterium most sensitive to Cabernet Sauvignon and
Malbec wine samples, whereas E. coli was the bacterium most sensitive to
Merlot wine samples followed by Pr. mirabilis. The largest inhibition zone
diameter was 10.0 mm against E. coli for Merlot wine fourfold concentrated,
no such effect was found with the others wine samples.
Vallejo et al. (2013) demonstrated that phenolic compounds produce the
inhibition of biofilm formation by pathogenic bacteria. The effect of different
concentrations of phenolic compounds could be beneficial to growth of lactic
acid microorganisms (Reguant et al. 2000) or inhibitory (Campos et al. 2003).
Wine Polyphenols
41
4. ANTIMICROBIAL ACTIVITY AGAINST SPOILAGE

WINE BACTERIA
The studies conducted about the inhibitory activity of wine phenolics
compounds against LAB were performed utilizing pure compounds in
different growth conditions (Garca-Ruiz et al. 2009; 2011; Campos et al.
2009). Several studies reported inhibitory activity against a number of LAB,
including oenological strains of Lactobacillus, Pediococcus and Oenococcus,
of phenolic extracts from different origins, such as eucalyptus extract (GarcaRuiz et al. 2012) and other aromatic plant extracts (Garca-Ruiz et al. 2012),
and of pure phenolic acids at significantly higher concentrations than that
detected in wines (Campos et al. 2009). Garca-Ruiz et al. (2012) reported that
in general, phenolic extracts have antimicrobial activity against strains of L.
hilgardii and P. pentosaceus (IC50 > 1000 mg/L). Garca-Ruiz et al. (2009),
demonstrated that p-coumaric acid has antimicrobial effect on P. pentosaceus
at 200 mg/L. Campos et al. (2009), indicated that phenolic acids like gallic
acid and p-coumaric acid affect viability of deteriorating wine bacteria (like
Lactobacillus hilgardii and Oenococcus oeni) by increase of cell membrane
permeability at concentrations of 2200 mg/L.
The authors of this chapter have characterized by first time the low
molecular weight fraction (LMF) containing phenolic compounds from
Malbec (M), Cabernet Sauvignon (CS) and Tannat (T) wines produced in
Cafayate, Salta, Argentina (Stivala et al. 2014). The total concentration of
phenolic compounds determined by HPLC of LMF were higher in LMF-M
wine than that detected in T and CS varietals, as well as from Shiraz and
Tempranillo varietals from Mendoza, another province of Argentina, and then
that reported in italian wines, like Sangiovese varietal from Siena and
Cabernet Sauvignon, Syrah, Petit-Verdot, Nero and Merlot from Sicilia
(Ghiselli et al. 1998; Fanzone et al. 2011; La Torre et al. 2006).
Under uncontrolled conditions, the production of bacterial extracellular
polysaccharides increases viscosity and produce the visually ropy character of
wines (Walling et al. 2005). A wine with a polysaccharide concentration of 95
mg/L is considered not ropy, as opposed to a wine with a polysaccharide
concentration of 300 mg/L (Ribreau-Gayon et al. 2006a). Lonvaud-Funel et
al. (1993) considered that a polysaccharide production around 100 mg/L is
enough to give the wine an abnormal and unacceptable viscosity. In addition
ropy Pediococci are very tolerant to hostile conditions present in wine, like
SO2 and ethanol concentration.
42
Manca de Nadra and Strasser de Saad (1995) reported by first time the
production of exopolysaccharides by P. pentosaceus strains isolated from
Argentinean wines. These authors mentioned that the strains 12p and E2p of P.
pentosaceus increase the exopolysaccharides production in presence of ethanol
and SO2 in MRS culture medium.
The antimicrobial activity of the LMF against P. pentosaceus 12p, a
spoilage wine lactic acid bacterium that overproduce exopolysaccharides, was
study by first time by Stivala et al. (2014).
In a synthetic simile-wine medium (SWM) supplemented with LMF-M,
LMF-CS and LMF-T at concentration adjusted to the same value detected in
the original wine (1X) produces a reduction of viable cell count of P.
pentosaceus in 1.84, 0.75 and 0.64 logarithmic units, compared to the control
medium (SWM unsupplemented). In addition, these authors determined that
all LMF adjusted at concentration four times higher than wines (4X) reduced
dramatically the viable cell count of P. pentosaceus 12p, being LMF-T the
most effective fraction, producing a reduction in 6.46 logarithmic units
compared to the control (Figure 1). In SWM control medium, the exopolysaccharides production of P. pentosaceus 12p reach a value of 24.12
mg/L. The supplementation of SWM with all LMF at concentration 1X and
4X produces a significantly decrease in the EPS production by P. pentosaceus
12p reaching values closed to zero.
In previous reports, alterations in the membrane produced by phenolic
compounds have been described in the scientific literature (Campos et al.
2009; Johnston et al. 2003).
Figure 1. Effect on P. pentosaceus 12p growth after 96 h incubation in synthetic likewine medium (SWM) (hatched bars) and SWM supplemented with phenolic fraction
of Malbec (LMF-M), Cabernet Sauvignon (LMF-CS) and Tannat (LMF-T) wine at 1X
(gray bars) and 4X (black bars).
Wine Polyphenols
43
In recent studies, Stivala et al. (2014) described that the presence of LMF
from Argentinean wines induced modifications in cell morphology with
alterations in the microbial cell integrity of the strain 12p of P. pentosaceus.
Figure 2 shows that the LMF extracted from Malbec, Cabernet Sauvignon and
Tannat wine varietals from Cafayate, at concentration four times higher than
wines produces changes in cell morphology/integrity respect to control
medium after 96 h incubation in synthetic-simile wine medium.
The study of the mechanisms by which polyphenols inhibit the growth of
LAB are in the first stages nowadays, however several authors reported that
polyphenols disturb the cell membrane structure producing leakage of
intracellular constituents (Johnston et al. 2003; Rodrguez et al. 2009) as well
as enhanced the proton influx and the potassium and phosphate efflux
(Campos et al. 2009) by hydroxycinnamic and hydroxybenzoic acids in
Oenococcus oeni and Lactobacillus hilgardii suspensions. Other authors
demonstrated that the incubation of P. pentosaceus and O. oeni with
kaempferol, ethyl gallate, ferulic acid and trans-resveratrol produced a
breakdown of the cell membrane and the subsequent release of cytoplasm
material into the medium (Garca-Ruiz et al. 2009, 2011).
Figure 2. Electron micrographs of ultrathin sections of P. pentosaceus 12p after 96 h

incubation in synthetic like-wine medium (SWM) (A); and SWM supplemented with
the phenolic fraction at four times concentrated (4X) from Malbec (B); Cabernet
Sauvignon (C) and Tannat (D) wines.
44
5. ANTIOXIDANT ACTIVITY
Reactive oxygen species (ROS) are highly reactive molecules produced by
living organisms as a result of normal cellular metabolism and environmental
factors. Aerobic organisms have an antioxidant system with enzymatic and
non-enzymatic antioxidants that protect them of harmful effects of ROS.
However, an imbalance between the production of ROS and the antioxidant defense system of body generates an oxidative stress which conduces
to oxidative damage of biological macromolecules like proteins, lipids and
nucleic acids causing aging and several diseases (Birben et al. 2012).
Li et al. (2009) demonstrated that red wines have higher phenolic content
levels than white or rose wines and the same result is obtained for antiradical
activity and antioxidant capacity. The amount of phenolic materials and
antioxidant activity is considerably different between the wines, depending on
the grape variety, environmental factors in the vineyard and the wine
processing techniques. Because of a relatively tight coupling of the ORAC,
DPPH, ABTS and CUPRAC methods, any of these methods can be used for
the quick evaluation of antioxidant capacity of wines. In this way,
Xanthopoulou et al. (2010) studied the activity of red and white wine extracts
with different classes of phenolic compounds against soybean lipoxygenase,
free radical formation and linoleic acid oxidation. They demonstrated that red
wine extracts are more potent scavengers and inhibitors of lipid peroxidation
than white wine extracts.
Mudnic et al. (2010) determined the antioxidative capacity of nine
phenolic acids from wine: p-hydroxybenzoic, protocatechuic, vanillic, gallic,
and syringic acids, as derivatives of hydroxybenzoic acid, and p-coumaric,
caffeic, ferulic, and sinapic acids, as derivatives of hydroxycinnamic acid by
ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant
capacity (TEAC) methods and they demonstrated that gallic acid had the
higher activity.
6. ANTIHYPERTENSIVE ACTIVITY
Cardiovascular disease (CVD) is a class of diseases that affects the
cardiovascular system involving the heart and the blood vessels (arteries,
capillaries, and veins) or both.
Wine Polyphenols
45
Hypertension is the most common cause of CVD and it characterized by a

sustained increase in blood pressure levels. This illness is the major risk factor
for stroke and renal dysfunction and affects more than 25% of the population
in the worldwide (approximately 1 billion) (Kearney et al. 2005). Renin
angiotensin system (RAS) is a hormone system that regulates blood pressure
and fluid balance and its over-activation is the major causative factor in the
development of hypertension (Brunner et al. 1972; Hammoud et al. 2007).
Angiotensin-converting enzyme plays a significant role in RAS. Plasma
renin is responsible for the conversion of angiotensinogen released by the liver
into angiotensin I, which subsequently by proteolytic cleavage by angiotensin
I-converting enzyme (ACE), release angiotensin II in the lungs. Angiotensin II
is a hormone with potent vasoconstriction action and their formation increases
blood pressure, while angiotensin II also triggers aldosterone secretion, a
sodium retaining steroid, and thereby increases blood pressure (Hsueh and
Wyne, 2011). Furthermore, ACE also can degrade the peptide bradykinin
which has vasodilatation properties.
The modulation of RAS activity has been shown to cause significant
decrease in cardiovascular mortality (7%) along with successful blood
pressure reduction (Ferrari, 2013).
Several synthetic ACE inhibitors like captopril, enalapril, lisinopril and
ramipril are currently used in the treatment of hypertension in humans (Ondetti
et al. 1977; Patchett et al. 1980) showing a significant reduction in the arterial
pressure. However they also cause adverse side effects, such as cough, allergic
reactions, taste disturbances, and skin rashes. Thus, the exploration of safe and
natural ACE inhibitors like phytochemicals including polyphenols has gained
attention as antihypertensive agents (Li et al. 2013; Suzuki et al. 2006).
A large number of polyphenols including phenolic acids (Zhao et al. 2011)
have been assessed for their antihypertensive and ACE inhibitory potential.
Actis-Goretta et al. (2006) demonstrated certain ACE inhibitory potential of
various wine phenolic compounds including gallic acid, chlorogenic acid and
caffeic acid.
Bhullar et al. (2014) reported that chlorogenic acid and quercetin exhibit
inhibitory action on renin enzyme with an in vitro IC50 value of 730.83 and
971.78 M, respectively demonstrating that these compounds have more
inhibitory activity on renin that the commercial drug captopril (IC50 = 1108.04
M). The inhibitory activity of ACE (ACE-I) of chlorogenic acid (IC50 =
21.53 M) and quercetin (IC50 = 9.85 M) are lower than a specific ACE-I
drug, like captopril (IC50 = 1.02 M).
46
On the other hand the ACE-I activity of these compounds are much higher
compared with a caffeic acid (IC50 = 430.01 M), another phenolic acid
commonly found in wine.
Another compounds present in wines are procyanidins, that are
represented by a group of polymeric polyphenols composed of the flavan-3-ol
units, (3)-epicatechin and (+)-catechin. Actis-Goretta et al. (2003) reported
that from a kinetic analysis that procyanidins inhibit the ACE by competing
with the substrate for the active sites.
A recent study based on clinical trials evidence (Onakpoya et al. 2014),
reported the evaluation of the effectiveness of chlorogenic acids on blood
pressure, using data from published randomized clinical trials. This metaanalysis revealed a statistically significant reduction in systolic blood pressure
of chlorogenic acid (-4.31 mm Hg) as well as a significant reduction in
diastolic blood pressure (-3.68 mm Hg), without evidence of adverse events.
Vallejo et al. (2013) investigated the antihypertensive activity of three
concentrations of chlorogenic acid in vitro. They reported all concentrations
tested possess ACE-I activity that increased with the concentration.
Al-Awwadi et al. (2004) studied the effects of a red wine polyphenolic
extract, ethanol, or both combined in a model of insulin resistance associated
with hypertension, the fructose-fed rats, and confirmed that a red wine
polyphenolic extract (100 mg/kg), ethanol (1 mL/kg), or the combination of
both prevented the development of high blood pressure.
Considerable evidence suggests that oxidative stress caused by an
excessive production of reactive oxygen species (ROS), plays a key role in the
development of hypertension. This phenomenon leads to endothelial
dysfunction as a consequence of an imbalance between endothelium-derived
relaxing factors, such as nitric oxide (NO), and contracting factors, such as
angiotensin-II and endothelin. In this way, polyphenols with antioxidant
properties play a beneficial role in prevention and treatment of hypertension,
because they acting as free radical scavengers, metal chelators, and in enzyme
modulation and expression (Rodrigo et al. 2012). Considering the antioxidant
properties of resveratrol, De Oliveira et al. (2012) investigated the effects of
the chronic treatment with resveratrol on cardiovascular system from renal
hypertensive rats and they have demonstrated that the hypertensive levels were
significantly reduced after six weeks of treatment with resveratrol.
It has been demonstrated that some polyphenols present anti-inflammatory
properties. In this way, resveratrol, curcumin, quercetin, and others were
shown to inhibit NF-kB activation in cellular cultures (Lim et al. 2007;
Devarajan 2007; Bauer et al. 2008).
Wine Polyphenols
47
NF-kB is a transcription factor that is involved in immune and

inflammatory responses. The activation of this factor causes the expression of
inflammatory and immune genes and the secretion of pro-inflammatory
chemokines and cytokines increasing the risk of inflammatory diseases.
Resveratrol also inhibits the activation of AP-1, other transcriptional factor
involved in the regulation of cytokines.
Prostaglandins, lipids derived from arachidonic acid, play a key role in the
inflammatory responses. The enzymes that produce prostaglandins are called
cyclooxygenase (COX). There are two types of COX enzymes, COX-1 and
COX-2. Both enzymes produce prostaglandins that promote inflammation,
pain, and fever. However, only COX-1 produces prostaglandins that activate
platelets and protect the stomach and intestinal lining (Smith et al. 2000;
Ricciotti and FitzGerald 2012). Many polyphenols, such as resveratrol,
catechin, and epigallocatechin gallate (EGCG), inhibit COX-2 activity and
their expression (Arlinghaus et al. 2008; Leng et al. 2008; Tong et al. 2008).
Leukotrienes are also lipids derived from arachidonic acid involved in
inflammatory responses produced by the 5-Lipoxygenase enzyme (5-LOX).
Flavonols, including kaempferol, quercetin, morin, and myricetin, were found
to be 5-LOX inhibitors Devireddy et al. 2001) and Curcumin was demonstrated to be both COX and LOX inhibitor (Miharada et al. 2005).
In other hand, the production of nitric oxide (NO) by inducible nitric
oxide synthase (iNOS) is also activates during inflammation. Polyphenols,
such as quercetin (Miharada et al. 2008), EGCG (Bolignano et al. 2010) and
resveratrol (Xu and Venge, 2000), were found to inhibit iNOS. Moreover, if
the simultaneous suppression of COX-2 and iNOS expression by polyphenols
ocurrs, the NF-kB will be also inhibited (Zerega et al. 2000).
7. WINERY WASTES
Wine production generates huge amounts of waste. Before the 1990s, the
most economical option for waste removal was the payment of a disposal fee
usually being of around 3000 Euros. Vinification involves all of the steps
carried out during the elaboration of wine from grapes. Winemaking generates
different residues (Asselin and Delteil 2003) characterized by high contents of
biodegradable compounds and suspended solids (Navarro et al. 2005). The
residues consist of plants remains derived from the destemmed grapes, the
sediments obtained during clarification, bagasse from pressing, and lees,
which are obtained after different decanting steps.
48
The wastewater generated from vinification lees contains grape pulp,

skins, seeds and dead yeasts used in the alcoholic fermentation. Baydar et al.
(2007) studied the antiradical and antioxidant activities of grape seed and
bagasse extract from different mixed solvent extractions and determined the
total phenolic compounds of the extracts to find out the relationship between
antioxidant activities and total phenolic content. The authors demonstrated that
grape seed extracts contained a higher amount of total phenolic content than
the bagasse extract. These extracted compounds could be used as easily
accessible source of natural antioxidants.
Argentina is the first producer of lemon and Tucuman is responsible of
89.8% of country lemon production; one problem in this activity is citrus
canker caused by Xanthomonas citri subsp. citri. The symptoms consist of
erumpent lesions on fruit, leaves and young stems, reducing fruit quality and
production. The control of bacterial pathogens is a serious problem in
citriculture, and the management strategies include the use of diseasefree
seedlings, resistant cultivars, windbreaks to hinder inoculums dispersal, and
bactericides containing copper (Schubert and Sun 2003).
But, these strategies are not always effective, especially when the
environmental conditions are optimal for disease dissemination or inoculum
density is high (Sahin and Miller 1996).
Some compounds have already been tested but were not effective against
citrus canker (Graham and Leite 2004), thus, further studies are needed with
new compounds. Rodrguez-Vaquero et al. (2015) developed techniques for
phenolic compounds recovery from winery wastes and investigated the effect
of phenolic compound extracted on the viability of Xanthomonas citri subsp.
citri in vitro and in vivo assays.
Their results showed that winery wastes were effective to inhibit the
growth of X. citri, and a bactericide effect was shown with the high
concentration tested. In vivo and in vitro assays demonstrated that grape skin
and grape seeds possess antibacterial activity against X. citri. The authors
demonstrated that the use of winery waste, as source of phenolic compounds,
is a promising environmentally friendlier method to control the presence of X.
citri in citrus.
Some of by-products formed during winemaking can be used for different
purposes, such as alternative natural antioxidants to the synthetic antioxidants
used in food industry to prolong the shelf life of food, in canker treatment
(Sosa-Mrmol et al. 2014).
However, lees (basically remains of dead yeasts) have been considered for
use as a supplement in animal feed (Maugenet 1973).
Wine Polyphenols
49
Some authors investigated about the antifungal activity of three

agroindustrial subproducts: sugar beet, sugar cane and wine vinasse against
Fusarium populations in several soils (Santos, 2008). Their results were in
agreement with those found by Dinez et al. (2007) for grape marc compost,
where suppressiveness was detected against nine phytopathogenic fungi.
Vizoso-Pinto et al. (2014) demonstrated the antifungal activity of phenolic
fraction of grape skin and seed against Candida albicans, C. glabrata and C.
krusei. The possibility of reutilization of these wastes is a new alternative for
the final disposal of the waste.
REFERENCES
Actis-Goretta, L., Ottaviani, J. I., Fraga, C. G., (2006). Inhibition of
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ISBN: 978-1-63482-765-2
Chapter 3
FACTORS AFFECTING BIOGENIC AMINES

OCCURRENCE IN WINE: AN OVERVIEW
OF ANALYTICAL METHODS
Silvana C. Ledesma, Mara G. Stivala
and Pedro A. Aredes-Fernndez*
ABSTRACT
Biogenic amines (BAs) are nitrogen compounds that can exert
negative effects on human health. They are normally present in fermented
beverages like wine and beer. The presence of biogenic amines is
principally attributed to microbial decarboxylation of the corresponding
amino acids, although some of them may come directly from grapes.
Therefore, BAs have been associated with a lack of hygiene during the
winemaking process and hence they have been suggested as indicators of
quality control or poor sanitary conditions. The main BAs present in wine
are putrescine, cadaverine, histamine, tyramine and phenylethylamine.
BA formation in wines is frequently associated with the presence and
metabolism of lactic acid bacteria involved in the winemaking process.
*
Corresponding author: Pedro A. Aredes-Fernndez. Facultad de Bioqumica, Qumica y

Farmacia Universidad Nacional de Tucumn, Ayacucho 471 CONICET. Tucumn.
Argentina. E-mail: pedroaredes@fbqf.unt.edu.ar.
62
S. C. Ledesma, M. G. Stivala and P. A. Aredes-Fernndez

Consequently, it can be expected that bacterial proteolytic activity, in
addition to their relevance in bacterial nutrition, plays an important role in
the formation of precursors like amino acids and peptides. This chapter
provides an overview of the most recent information about wine biogenic
amines and the main factors affecting their presence. In addition, a
special paragraph deals with methods for their detection in wine.
Keywords: biogenic amines, wine, lactic acid bacteria, detection methods
INTRODUCTION
During malolactic fermentation (MLF) certain microorganisms produce
secondary metabolites that may affect the quality of the wine and/or the health
of its consumers. Biogenic amines (BAs) are an example of such compounds.
BAs are organic compounds of low molecular weight endowed with
biological activity, with at least one amino group in its structure and a basic
character conferred by the amino group. BAs are frequently found in
fermented foods and beverages, usually produced as a consequence of
decarboxylation of amino acids during the normal fermentation process carried
out by microbial metabolism. Lactic acid bacteria (Lactobacillus, Leuconostoc, Pediococcus and Oenococcus spp.) are the main microorganisms
responsible for BA synthesis in wine (Landete et al. 2007; Sebastian et al.
2011). Some amines in wines come from the grapes, and their level may vary
according to the grape variety and degree of ripening, as well as
agroecological conditions. Therefore, geographical characterization based on
the BA content has been proposed as a criterion to discriminate between wines
from different countries or regions (Soufleros et al. 1998). Determination of
BAs in wines is of great importance because, when they are present at high
concentrations, they can be directly or indirectly toxic. Consequently,
regulation of the biogenic amines in wine is a growing concern in countries
that are committed to keep track of these compounds, especially histamine,
which is used as a safety and quality marker of wines (Lehtonen, 1996). The
fact that there are more and more people diagnosed with diamine oxidase
(DAO) deficiency may be a very good reason to accept the challenge to
consume food with very low content of histamine and other biogenic amines
and implement appropriate measures during different stages of the production
process. Regarding fermented products, a so-called "technology of low
histamine" has been proposed, which is based on ensuring the hygienic quality
Factors Affecting Biogenic Amines Occurrence in Wine
63
of raw materials, addition of properly selected starter cultures and the use of
specific production techniques that inhibit formation of histamine. This
technology has already been successfully implemented in the production of
certain wines in countries like Switzerland and Spain (Vidal-Carou and
Latorre Moratalla, 2014).
Commission regulation (EC) No. 2073/2005 on microbiological criteria
for foodstuffs sets a lower limit for biogenic amines of 100 mg/kg and a
maximum limit of 200 mg/kg. In wines, the limit allowed for histamine by the
OIV (Organisation Internationale de la Vigne et du Vin) is 12 mg/L although
some countries have other limits. In Switzerland and Austria wines that
contain more than 10 mg/L are rejected, and even lower limits have been
recommended: in Germany 2 mg/L, Holland 3 mg/L, Finland 5 mg/L, Belgium
6 mg/L and France 8 mg/L (Lehtonen 1996; Smit et al. 2008; Hidalgo Torres
2011). In Argentina there is no law in force yet that regulates the maximum
levels of biogenic amines in wine. However, for the export of Argentine
wines, the content of biogenic amines is controlled according to the legislation
of the buyers country. According to their chemical structure, BAs can be
classified into three groups: aliphatic (putrescine, cadaverine, spermine and
spermidine), aromatic (tyramine and phenylethylamine) or heterocyclic
(histamine and tryptamine) and aliphatic volatile amines (ethylamine, methylamine, isoamylamine). In accordance with the number of amine groups, they
can be divided into monoamines (tyramine and phenylethylamine), diam-ines
(putrescine and cadaverine) or polyamines (spermine and spermidine) (Mafra
et al. 1999; Lonvaud-Funel, 2001; Spano et al. 2010).
The main BAs present in wine are putrescine, cadaverine, histamine,
tyramine and phenylethylamine (Smit et al. 2008; Komerl et al. 2013) (Figure
1), and putrescine is the most abundant compound (Soufleros et al. 1998;
Mangani et al. 2005).
Figure 1. Chemical structure of the biogenic amines most frequently found in wines.
64
The occurrence of BAs have been associated with a lack of hygiene during
the winemaking process and they have been suggested as an indicator of
quality or poor sanitary conditions of grapes or bad manufacturing practices
(Soufleros et al. 1998; Leito et al. 2005; Moreno-Arribas and Polo 2008).
1. PHYSIOLOGICAL ROLE AND TOXICOLOGICAL ASPECTS

The normal physiological functions of biogenic amines in humans include
regulation of the body temperature, stomach volume, stomach pH and brain
activity (ten Brink et al. 1990). Usually, when a low concentration of biogenic
amines is ingested, they are efficiently broken down by mono- and diamine
oxidase enzymes in the intestinal tract (Moreno-Arribas and Polo, 2009).
Amine oxidases catalyze the oxidative deamination of biogenic amines
producing aldehydes, hydrogen peroxide and ammonia (Gardini et al. 2005).
However, when an excessive amount of biogenic amines is ingested or when
the normal catabolic routes are inhibited or genetically deficient, several
physiological disorders can occur (ten Brink et al. 1990). In alcoholic
beverages, especially wine, BAs have received more attention because ethanol,
acetaldehyde and other biogenic amines can enhance the toxic effects by direct
or indirect inhibition of the enzymes responsible for detoxification of these
compounds (Maynard and Schenker, 1996; Smit et al. 2008). BAs may
amplify the harmful effects of histamine and tyramine by inhibiting their
normal metabolism in humans (Landete et al. 2006).
The best known reactions are those caused by histamine. Histamine is
often described as the most important biogenic amine since it is one of the
most biologically active ones (Halsz et al. 1994). Histamine is present at low
levels in the human body and it is involved in key functions such as allergic
response, neurotransmission and vascular permeability (Ohtsu and Watanabe,
2003). Histamine causes dilation of peripheral blood vessels, capillaries and
arteries, thus resulting in hypotension, palpitation, edema, rash, headaches and
heart problems (Silla Santos, 1996; Ladero et al. 2010). It can also cause
contraction of the intestinal smooth muscle, causing abdominal cramps,
diarrhea and vomiting (Taylor, 1986).
Putrescine is the most abundant biogenic amine in wine. Along with
cadaverine, putrescine aggravates the adverse effects of histamine, tyramine
and phenylethylamine through interference with the enzymes that metabolize
them (Shalaby 1996; Silla Santos 1996) or by inhibition of histamine
metabolism.
65
This latter act favors the passage of histamine from the intestines into the
systemic circulation by competition for binding sites in the gastrointestinal
tract (Kanny et al. 2001). Moreover, it can have negative effects on wine
aroma, producing flavors of putrefaction or rotten flesh (Moreno-Arribas and
Polo, 2009), and it can reduce sensorial quality at concentrations of 15 to 20
mg/L in white wine and 20 to 30 mg/L in red wine (Lehtonen 1996; Arena and
Manca de Nadra 2001).
Tyramine and phenylethylamine can produce hypertension through the
release of noradrenalin and norepinephrine, respectively, which are
vasoconstrictors.
Apart from an allergic response, other serious human pathologies caused
by biogenic amines include carcinogenesis and tumor invasion (ornithinederived polyamines and histamine), immune response and neurological
disorders (histamine), formation of carcinogenic nitrosamines by reaction
between nitrite and secondary amines (putrescine, cadaverine, agmatine),
migraine and hypertension (tyramine and phenylethylamine) and Parkinsons
disease, schizophrenia and mood disorders (tyramine) (Smith 1980; ten Brink
et al. 1990; Silla Santos 1996; Medina et al. 1999).
The toxic level of biogenic amines depends on the body tolerance for
these compounds, the total amine concentration and the consumption of
ethanol and/or drugs. Soufleros et al. (1998) suggested that wines with
histamine concentrations between 8 and 20 mg/L may have toxic effects when
consumed frequently. In the case of tyrosine, wines with concentrations
between 25 and 40 mg/L should be consumed with precaution, while as little
as 3 mg/L phenylethylamine can cause negative physiological effects.
2. ORIGIN AND OCCURRENCE

OF BIOGENIC AMINES IN WINE
The study of BAs in wines is important for two reasons: the first reason is
the toxicological risk associated with BA content and the second one is the
evaluation of hygienic-sanitary conditions during the winemaking process.
BAs may be present in the grape or occur during winemaking. The main
BAs in grapes are putrescine and spermidine (20 and 45 mg/kg of fresh fruit,
respectively), whereas ethanolamine, agmatine, cadaverine, spermidine,
histamine and tyramine have been found at lower amounts (Ough 1971; Rivas-
66
Gonzalo et al. 1983; Broquedis et al. 1989; Radler and Fth 1991; Baucom et
al. 1996).
BA production in wine depends on the presence of amino acid precursors
and microorganisms that have amino-decarboxylase enzymes able to
decarboxylate amino acids, as well as suitable environmental conditions (pH,
temperature, O2, SO2, etc.,). Putrescine, cadaverine, spermine, spermidine,
agmatine, tyramine, tryptamine, histamine and 2- phenylethylamine are of
microbial origin. The microbial enzymes are inducible and therefore they are
produced in response to the presence of amino acid precursors (Marcobal et al.
2006). Cadaverine, histamine, 2- phenylethylamine, tyramine and tryptamine
are derived from decarboxylation of the corresponding precursor amino acids
(lysine, histidine, phenylalanine, tyrosine, and tryptophan, respectively), while
putrescine can be derived from decarboxylation of ornithine or arginine.
Another BA found in wine is ethanolamine, but its metabolic origin is not
well defined yet. Ethanolamine was initially considered the precursor of 1,2ethanediol, and Choi et al. (2004) reported that ethanolamine would be a
precursor of phosphatidylcholine, the most abundant phospholipid in
membranes of eukaryotic cells. Ethylamine, methylamine and isoamylamine
are possibly originated through amination of non-nitrogen compounds such as
aldehydes and ketones (Bauza et al. 2007).
2.1. Grape and Viticulture Procedures

The type of BA and its concentration in wines vary according different
factors. Several amines are normal constituents of grapes. According to
Broquedis et al. (1989), amines such as putrescine and spermidine are found at
high concentrations in the pericarp of Cabernet Sauvignon berries (20 and 45
kg/L, respectively). The BA concentrations in grapes vary with the variety,
soil type and composition (viticulture area), fertilization, climatic conditions
during the grape growth and the degree of ripeness of the grapes (Glria et al.
1998; Moreno-Arribas et al. 2000; Souza et al. 2005; Ferreira and Pinho,
2006). Potassium deficiency in the soil has been linked to an increase in
putrescine content in plants in response to stress conditions (Konigshofer,
1989, 1990, 1991; Adams, 1991; Dohmen et al. 1990; Santerre et al. 1990;
Tenter and Wild, 1991), while the lack of water does not seem to affect the BA
content in grape berries and hence in the wine (Bover-Cid and Holzapfel,
1999). Nitrogen fertilization treatments can cause an increase in grape amino
acid and amine concentrations (Soufleros et al. 2007). In addition, grapes are
67
considered to be the main source of volatile amines and ammonia in wines

(Ough et al. 1981). The BA content of grapes can vary among the different
varieties. In fact, Hajs et al. (2000) reported significant differences in amine
levels in grapes and wines of three grape varieties from the Tokaj region in
Hungary. Other authors have reported that the average contents of BAs in
grapes, except for cadaverine, significantly vary over vintages (Martn-lvarez
et al. 2006). These authors partially explained this behavior by the variation in
the content of most of the precursor amino acids over the years. Furthermore,
differences in BA content among vintages could also be caused by the
diversity of yeast and bacterial strains that are present on the grapes each year.
The storage of grapes prior to crushing under non-sterile conditions could also
affect biogenic amine concentrations (Cecchini and Morassut, 2010).
Kalkan Yildirim et al. (2007) reported on BA levels in wines from
different organic and non-organic grapes of Vitis vinifera varieties, showing
that the content of putrescine and ethylamine was higher in organic wines than
in non-organic wines. This difference could be explained by the fact that
organic grapes are absolutely natural and they grow without the use of any
pesticides, insecticides or synthetic chemicals. Herbicides are not allowed
either, and altered or damaged grapes are never used. Furthermore, the
vinification process does not employ yeasts or enzymes that result from
bioengineering or synthetic chemicals for wine clarification and it does not use
high temperatures as a biological stabilization technique. Del Prete et al.
(2009) studied seven different cultivars of organic grapes and only found
ethanolamine, ethylamine and putrescine (about 5 mg/L each) in all samples
assayed showing a grape variety dependence in the amount of BA.
2.2. Alcoholic Fermentation

During the winemaking process, all groups of wine microorganisms
probably participate in the production of BAs. Yeasts generally have less
participation than lactic acid bacteria in the production of amines in wine.
Previous studies have shown no relationship between alcoholic fermentation
and the amount of BAs (Marcobal et al. 2006). However, certain authors have
attributed, at least partly, the production of BAs to yeasts (Caruso et al. 2002;
Torrea Goi and Ancn-Azpilicueta, 2002). Caruso et al. (2002) demonstrated
that of a total of 50 yeast strains the species Saccharomyces cerevisiae,
Kloeckera apiculata, Candida stellata, Metschnikowia pulcherrima and
Brettanomyces bruxellensis, isolated from grapes or during grape must
68
fermentations, showed low or non-detectable production of histamine.

However, all the species assayed formed methylamine and agmatine. B.
bruxellensis and S. cerevisiae produced higher total BA concentrations (15 and
12 mg/L, respectively).
Yeasts may also indirectly contribute to the production of BAs in wines
through alteration of the grape must composition, because of their metabolic
activity during alcoholic fermentation and/or autolysis. This could result in an
increase in the wine amino acid concentration, which could be used by other
microorganisms in the later fermentation stages (Soufleros et al. 1998).
Few studies have focused on the formation of biogenic amines by yeasts,
and most of them only compared different yeast species and only assayed
histamine (Torrea Goi and Ancn-Azpilicueta, 2002).
Somavilla et al. (1986) used six yeast strains and demonstrated that small
amounts of histamine were produced during alcoholic fermentation. VidalCarou et al. (1990) did not detect formation of histamine during alcoholic
fermentation, but they observed tyramine formation at very low concentrations
(0.60 mg/L). Torrea Goi and Ancn-Azpilicueta (2001) examined the
concentration of BAs produced by different strains of S. cerevisiae in ros
wines, and they found a slight increase in the concentration of certain biogenic
amines (putrescine, spermine, spermidine, tyramine and phenylethylamine),
observing a strain-dependent behavior in the amine production. Granchi et al.
(2005) reported a decrease in biogenic amines, especially putrescine, during
alcoholic fermentation. Landete et al. (2007) assayed 36 strains of different
yeast species isolated from must and wines on the production of BAs
(Aureobasidum, Candida, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia and Saccharomyces cerevisiae), but with negative results.
Moreno-Arribas and Polo (2008) did not detect production of histamine,
tyramine and putrescine in 50 yeast strains isolated from grapes and wine from
Spain. From the current research it can be concluded that yeasts are most
likely not directly involved in the production of most amines found in wine.
2.3. Malolactic Fermentation

Oenococcus oeni is normally the dominant species at the end of the
alcoholic fermentation and it is the main species responsible for malolactic
fermentation (MLF). Although this lactic acid bacterium (LAB) adds desirable
characteristics to the wine, it also forms questionable, undesirable metabolites
(Bartowsky, 2009). Usually, BA production results from the presence of
69
bacteria that are able to decarboxylate amino acids (Gale, 1946). In addition to
O. oeni, several other LAB species belonging to the genera Lactobacillus,
Leuconostoc and Pediococcus may develop during the winemaking and are
frequently involved in BA production. For a long time enologists thought that
only Pediococcus strains were responsible for histamine production (Aerny,
1985). Delfini (1989) compared the ability of several strains of Leuconostoc
spp., Lactobacillus spp. and Pediococcus spp. to produce histamine, and
observed that only P. damnosus was able to produce significant amounts of
histamine while Leuconostoc oenos (O. oeni) strains were poor histamine
producers. In 1990, Choudhury et al. showed that a strain of O. oeni was
capable of producing tyramine in a synthetic medium, and Straub et al. (1995)
reported on an O. oeni strain capable of producing histamine through histidine
decarboxylase. However, some authors found that O. oeni significantly
contributed to the overall histamine content in wines and that the ability of the
species to produce this amine was strain-dependent (Coton et al. 1998;
Guerrini et al. 2002; Garcia-Moruno and Muoz, 2012). Different strains of
Lactobacillus hilgardii, L. brevis, L. buchneri and L. mali have been found to
produce a variety of BAs in wine (Moreno-Arribas and Lonvaud-Funel, 1999;
Moreno-Arribas et al. 2000, 2003; Constantini et al. 2006; Landete et al.
2007). Marcobal et al. (2004) isolated and identified an O. oeni strain from a
Spanish wine that produced putrescine using gene expression.
The capability of lactic acid bacteria to produce amines seems to be straindependent, and not a specific characteristic of the species. Gale (1946)
suggested that some strains might possess more than one amino acid
decarboxylase activity under certain conditions. This means that particular
LAB strains would have the ability to simultaneously produce different amines
(Coton et al. 1998; Moreno-Arribas et al. 2000; Guerrini et al. 2002).
As the formation of BAs in wines is frequently associated with MLF, it
could be expected that O. oeni possesses the necessary enzymes for protein
and peptide breakdown and decarboxylation of amino acids present in wine
(Leito et al. 2000). O. oeni only expresses proteolytic and decarboxylase
activities when there is no alternative strategy for cell survival (Molenaar et al.
1993). Decarboxylase activity is also expressed when the cells require
additional energy produced during amino acid transport. Konings (2006)
demonstrated that amino acid decarboxylation reactions generate additional
metabolic energy or regulate intracellular pH, which allows bacterial survival
in poor or acid environments.
Lactic acid bacteria from wine are able to hydrolyze and metabolize
proteins and peptides and use the released amino acids as nutrients or energy
70
source (Manca de Nadra et al. 1999; Aredes Fernndez et al. 2006, 2010).
Several scientific reports have demonstrated that peptides of low molecular
weight are more favorable to wine LAB growth than free amino acids (Feuillat
et al. 1985; Aredes Fernndez et al. 2003, 2004). Consequently, it can be
assumed that proteolysis and peptide release play an important role in
sustaining growth and viability of LAB in wine. However, the released amino
acids and peptides may include precursors for biogenic amines (LonvaudFunel and Joyeux, 1994). Leito et al. (2000) assayed a total of 220 O. oeni
isolates for decarboxylase and proteolytic activity in wine, and found that only
six isolates showed both activities. These results suggest that the ability of O.
oeni to use wine peptides and produce BAs is not a constant characteristic of
this species, but instead depends on the strain and on the nutritional and
energetic composition of the medium. BA production generally increases
when high-energy nutrients are exhausted in the late exponential growth
phase.
Previous results have revealed that pure cultures of L. hilgardii 5w
isolated from wine were able to produce histamine from histidine through
expression of histidine decarboxylase (Faras et al. 1993, 1995). Landete et al.
(2005a) reported on the presence of the hdc gene in this strain. L. hilgardii 5w
was assayed in a synthetic medium for its ability to utilize histidine-containing
dipeptides as sole histidine source.
L. hilgardii 5w was unable to grow in a synthetic medium without
histidine or glycine and lost viability, confirming that these amino acids are
essential for growth of this bacterium (Table 1). When the dipeptide
histamine-glycine was incorporated to the medium as sole source of glycine
and histidine, growth of the microorganism was restored, reaching a bacterial
cell count similar to that in synthetic basal medium (control medium) and with
a slight diminution in growth rate after 72 h of incubation (Table 1). These
results suggest that L. hilgardii 5w has the potential to utilize dipeptides
containing histidine to sustain growth. Consequently, internalization of
histidine using peptide transport could play an important role in histamine
production. Suzzi and Gardini (2003) reported that a Lactobacillus curvatus
strain produced tyramine from di- and tripeptides containing tyrosine in dry
fermented sausages.
Aredes Fernndez et al. (2010) reported the ability of L. hilgardii 5w to
produce histamine in pure and in mixed cultures with the proteolytic strain O.
oeni X2L in a complex growth medium.
71
Table 1. Effect of dipeptides containing histidine on Lactobacillus hilgardii

5w growth parameters
Growth parameters
[h-1]
A [Log CFU/mL]
0.39 a
2.41 a
Glycine
0.00 d
0.02 d
Glycine
His-Gly
0.27 c
2.22 a
Histidine
0.00 d
0.05 d
Histidine
His-Gly
0.31 c
2.23 a
A: difference in cell concentration (Log CFU/mL) between the inoculum and the
stationary phase (72 h).
Values with the same letter in the same column are not signicantly different (p <
0.05).
Absent amino acid
Incorporated dipeptide
The authors informed that in a pure culture, L. hilgardii 5w reached a

consumption of 0.044 mg N/L of histidine with a concomitant release of 0.033
mg N/L of histamine at the end of the incubation period. In the mixed culture,
a significant increase in histamine was detected in the medium with respect to
the pure culture of L. hilgardii 5w, reaching a maximum release of 0.044 mg
N/L at the end of the incubation period. The authors also reported that they did
not observe a quantitative correlation between histamine production and
histidine consumption in the mixed culture. They hypothesized that this
behavior could be caused by the utilization of other histidine precursors like
histidine-containing peptides present in the complex medium or through
histidine production by bacterial proteolytic activity.
2.4. Winemaking Procedures and Aging

One of the most important steps in winemaking is aging to enhance the
quality of the final product. This process can be carried out in bottles or in
barrels. Gerbaux and Monamy (2000) found that the concentration of
histamine increased between four and eight months after malolactic fermentation in Pinot Noir and Chardonnay wine. Furthermore, Landete et al.
(2005b) noticed a further increase in histamine during the first six months of
storage in bottles, while putrescine and tyramine content in red wines seemed
to increase immediately after MLF.
72
Bauza et al. (1995) reported an increase in putrescine and histamine

during aging in oak barrels. With regard to the evolution of non-volatile
amines, several authors have reported that during aging of wine in barrels the
concentration of these moieties did not change, while a decrease in
dimethylamine and isobutylamine was observed (Gonzlez-Marco and AncnAzpiculeta, 2006; Alcaide-Hidalgo et al. 2007). It has been shown that the
type of oak used to make the barrel (American, French, etc.,) to conduct wine
aging does not affect accumulation of BAs in the final product (JimnezMoreno et al. 2003). On the other hand, the type of container used for MLF
seems to affect the final BA content. Significantly higher BA contents were
detected in wines undergoing MLF in stainless steel tanks compared with
MLF carried out in oak barrels (Alcaide-Hidalgo et al. 2007). Because of an
increase in pH during aging, SO2 effectivity diminishes and therefore, the
amount of biogenic amines increases (Gerbaux and Monamy, 2000).
Another factor that may increase the BA content is aging of wine in
contact with yeast lees. Martn-lvarez et al. (2006) left wines in contact with
the lees for two months after alcoholic fermentation but before aging in
barrels, and they found that the contents of methylamine and putrescine were
higher in the wines aged with lees. Marcobal et al. (2004) explained that this
increase was caused by the presence of the ornithine decarboxylase gene in
putrescine-producing O. oeni. Jimenez-Moreno et al. (2003) did not observe a
decrease in putrescine during wine aging in the presence or absence of lees.
Moreover, Marques et al. (2003) found higher levels of tyramine and
cadaverine in wines stored with lees compared with wines stored without lees,
while putrescine concentrations remained stable. Traditionally, only few wines
were left in contact with lees during aging, while nowadays, wine aging with
lees is a common habit in most viticultural areas.
The pH is another factor that plays an important role in the accumulation
of biogenic amines during aging. In general, wines with higher pH have higher
amine concentrations. This relationship may be explained by the fact that at
higher pH a greater number of bacteria can develop, increasing the probability
of the development of strains able to form amines (Vzquez-Lasa et al. 1998).
Normally, the storage temperature affects the wine quality, because the
reactions which take place in the bottled product intensify with increasing
temperature. Although wine should never be kept at a temperature above
20C, climatic or transport conditions may cause an increase in the wine
temperature of up to 30C. However, it has been found that the wine storage
temperature has little effect on the amine concentration. Vidal-Carou et al.
(1991) found no increase in biogenic amines (histamine or tyramine) at
73
temperatures ranging from 4C to 35C in wines stored for a period of 93 to

125 days. Gonzlez Marco and Ancn Azpilicueta (2006) reported a minor
influence of the temperature on the BA evolution in wine. In particular, in
wines stored at different temperatures (4, 20 and 35C) for a period of up to
105 days, the recorded spermidine level decreased to undetectable values,
while putrescine, tyrosine and cadaverine concentrations did not show
perceptible variations throughout the assay period.
3. METHODS TO REDUCE BIOGENIC

AMINE CONTENT IN WINE
In order to reduce the content of biogenic amines in wine it is highly
recommended to inhibit growth of indigenous decarboxylase-positive bacteria
and other microorganisms responsible for production of these N compounds.
SO2 can prevent growth of these bacteria. An alternative is the use of SO2
together with lysozyme to delay or inhibit LAB growth. Lysozyme is an
enzyme that can cause lysis of the cell wall of Gram-positive bacteria, but the
pH of grape must or wine must be suitable to maintain lysozyme activity
(Lpez et al. 2009). Clarification is another oenological treatment to decrease
the BA content. It can be carried out by physical methods (sedimentation,
flotation, centrifugation and filtration), by addition of fining agents (gelatin,
albumin, casein) or by addition of pectolytic enzymes (Ribreau-Gayon et al.
1998). Other authors have mentioned that bentonite, an oenological coadjuvant
used in wine clarification, is a very effective tool to reduce BA content in wine
(Mannino et al. 2006). Kallay and Body-Szalkai (1996) observed that after
addition of 80 g/hL of bentonite in red wines, histamine content was reduced
with 60%.
Other authors have reported that the incidence of BA could be reduced
after inoculation with commercial LAB starter cultures compared with
spontaneous MLF in wines (Martn-lvarez et al. 2006; Schneider et al. 2011).
Van der Merwe (2007) proposed co-inoculation of O. oeni and starter cultures
during the alcoholic fermentation, because it could curb biogenic amine
formation more than inoculation of O. oeni after alcoholic fermentation. The
natural wine flora, present in any cellar, is probably one of the main causes of
histamine formation. Pasteurization can reduce the natural flora sufficiently so
as to eliminate histamine in wines with direct inoculation of starter cultures.
74
The International Organization of Vine and Wine (OIV, 2011) adopted a

code for good vitivinicultural practices in order to minimise the presence of
biogenic amines in vine-based products (Resolution OENO 4/97), establishing
actions in vineyards and wineries to reduce the risks related to the presence of
biogenic amines in wines. These techniques include prevention of an increase
in the pH of the must. The organization also recommends to carry out selective
harvest in order to eliminate bunches or parts of bunches that are damaged by
fungi, since these could increase the formation of biogenic amines in the wine.
Transport and maceration times should also be minimized. With respect to
fermentative operations, the OIV recommends that alcoholic fermentation
preferably be carried out using yeast of the Saccharomyces type, which have
low predisposition to amine formation. Malolactic fermentation should be
carried out by inoculation after the alcoholic fermentation or, if possible, by
co-inoculation with lactic bacteria during alcoholic fermentation.
4. ANALYSIS AND DETECTION METHODS

Analysis of biogenic amines, individually or together, is important
because of their toxicity potential and their potential to be applied as indicators
of wine spoilage. Biogenic amines can be identified qualitatively, semiquantitatively or quantitatively.
4.1. Qualitative and Semi-Quantitative Methods

Screening Methods Using Selective Media
One of the first methods developed for qualitative biogenic amine
detection (generally histamine) is microbiological screening that involves the
use of a differential agar medium with a pH indicator (e.g., bromocresol purple
or cresol red). With this technique an increase in pH as a result of amine
formation can easily be observed through a change in color. Amino acid
precursors must be contained in the decarboxylase assay medium (Moeller,
1954; Nivel et al. 1981). Various researchers have introduced modifications of
the screening plate medium to make it more suitable for growth of lactic acid
bacteria and activity of decarboxylase enzymes. Improvements have also led
to greater sensitivity and reliability of the screening plate method, and the
technique has shown good correlation with other chemical analytical methods
(Choudhury 1990; Maijala 1993; Bover-Cid and Holzapfel 1999; Landete et
75
al. 2005a; Shiling et al. 2015). However, it is necessary to confirm the results
of qualitative screening methods with other analytical methods such as highpressure liquid chromatography (HPLC), because false positives may occur
due to the formation of other alkaline compounds such as ammonia, as well as
false negatives (Moreno-Arribas et al. 2003).
Thin-Layer Chromatography (TLC)

One of the first techniques used for the determination of biogenic amines
in foods was thin-layer chromatography (TLC) (Halsz et al. 1994). TLC
represents a good alternative to HPLC, although it is a semi-quantitative
method. This technique does not require heavy and/or costly equipment,
allows simultaneous analysis of several samples, is fast and it easy to
implement. At present, improvements in the systems for image capture and
analysis allow the use of TLC for quantitative analysis with satisfactory results
(Poole, 2003).
TLC methods have been proposed for the determination of biogenic
amines in fish, meat and cheese (Shalaby, 1995, 1999; Jeya Shakila et al.
2001; Lapa-Guimares and Pickova, 2004; OIV, 2011).
TLC has been successfully applied in the detection and/or semiquantification of biogenic amines produced by bacteria in different culture
media (Garcia-Moruno et al. 2005; Latorre-Moratalla et al. 2009). Romano et
al. (2012) determined the four main biogenic amines in wine (i.e., histamine,
tyramine, putrescine and cadaverine) by means of TLC/densitometry in a wine
from Apulia, Italy. This represents a valid alternative to HPLC and a useful
analytical tool for all laboratories that are not adequately equipped for
instrumental analysis.
4.2. Quantitative Methods

Biogenic amines are present in wine at low concentrations complicating
their determination, and they are also difficult to detect because they do not
exhibit ultraviolet (UV) absorption characteristics, fluorescent properties or
electrochemical activity.
The lack of these properties make direct detection of biogenic amines, for
example by spectrophotometric or fluorimetric methods, impossible. To solve
this problem, biogenic amines have to be derivatized to enable their detection
at higher absorbance wavelengths.
76
Enzymatic Methods
Enzymatic methods to quantify histamine were first applied to fish by
Lerke et al. (1983). The methods were then modified by Lpez-Sabater et al.
(1993) and Rodriguez-Jerez et al. (1994), and afterwards applied to musts and
wines. Landete et al. (2004) developed a direct enzymatic assay for the use in
wine based on the sequential activity of two enzymes. The first enzyme, DAO,
catalyzes the breakdown of histamine into imidazole acetaldehyde, ammonia
and hydrogen peroxide. The second one, peroxidase (HRP), produces a change
in color in the chromogen (colorless in the reduced form and colored in the
oxidized form) in the presence of hydrogen peroxide. The advantages of this
method are a limited sample preparation and short assaying time, and it does
not require expensive or sophisticated equipment.
The enzyme-linked immunosorbent assay (ELISA) is commonly used for
quantitative analysis of histamine. This assay was for the first time applied to
wine samples by Marcobal et al. (2005b). This fast and easy method could be
used for screening in laboratories that do not have HPLC equipment, in order
to distinguish between wines with a histamine content of approximately 10
mg/L.
Liquid Chromatography
HPLC is at present the most commonly used method to confirm presence
of biogenic amines. This method usually includes pre- or post-column
derivatization and subsequent fluorimetric detection of the corresponding
derivatives. Modifications and improvements are still effected to reduce the
preparation and analysis time and to improve resolution of biogenic amine
peaks in the chromatogram (Soleas et al. 1999; Marcobal et al. 2005b).
One of the most frequently applied derivatization agents is ophthaldialdehyde (OPA) producing highly fluorescent derivatives. Dansyl
chloride is another popular derivatizing reagent that reacts with primary,
secondary and tertiary amino groups under selected conditions. Fluorenylmethyl chloroformate (FMOC) can be employed as derivatization reagent to
determine spermine and spermidine in wine along with simultaneous detection
of their precursor amino acids (Bauza et al. 1995). Yet another derivatization
reagent that can react with primary, secondary and aromatic amino groups is
1,2-naphthoquinone-4-sulfonate (NQS). 8-phenyl-(4-oxy-acetic acid N hydroxysuccinimide ester)- 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacene (TMPAB-OSu) is a recently synthesized fluorescent reagent that can
be used in rapid pre-column derivatization to analyze primary amines and
secondary biogenic amines in wine by HPLC (Li et al. 2006). A different
77
HPLC method, using a fluorescence detector, suggests direct injection of

samples previously derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate (AQC). In contrast to the above reagents, the latter technique does
not require pre-treatment such as extraction, and allows analysis of histamine,
putrescine, cadaverine and tyramine in musts and wines (Hernndez-Orte et al.
2006).
Gmez-Alonso et al. (2007) used diethylethoxymethylenemalonate
(DEEMM) as derivatizing agent to form amino enones that can be detected in
the UV-visible region. This method allows simultaneous analysis of biogenic
amines, amino acids and ammonium with a single injection using reversed
phase-HPLC.
Loukou and Zotou (2003) presented the first HPLC-fluorescence method
to simultaneously assay 11 important biogenic amines.
Another method to determine biogenic amines is HLPC with a diode array
detector coupled to a system of atmospheric pressure chemical ionization and
mass spectrometry (HPLC-DAD-APCI-MS). This detection method was for
the first time used in wine and other alcoholic beverages by Loukou and Zotou
(2003) to characterize dansyl amides after pre-treatment with polyvinylpyrrolidone (PVP). Pre-treatment is necessary to remove substances from the
matrix that can interfere in the derivatization and quantification. mo Dugo et
al. (2006) described a similar method (reversed phase HPLC-DAD) but this
technique does not require any sample treatment prior to derivatization with
dansyl chloride. Micellar liquid chromatography (MLC) uses a surfactant
solution (SDS as mobile phase) instead of aqueous-organic solvents. This
method described by Gil-Agust et al. (2007) does not require pre-treatment of
samples (other than filtration) or an extraction procedure prior to analysis, and
can be performed with a single direct injection. This method allows
simultaneous determination of tryptamine and tyramine and their precursors,
tryptophan and tyrosine, in wine samples.
Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) is a method characterized by its short
analysis time and high resolution. A disadvantage is the lack of sensitivity, but
this can be overcome by coupling CE to mass spectrometry (MS) detection
instead of UV detection (Mao et al. 2002). An alternative to this method is
coupling CE to HPLC, called high-performance capillary electrophoresis
(HPCE) (Kovcs et al. 1999). Acre et al. (1998) described an automated
technique to determine biogenic amines in wine. This method uses a
minicolumn for solid-phase extraction (SPE) to simultaneously clean-up and
78
concentrate the samples prior to analysis by capillary electrophoresis coupled

to indirect UV detection. The method allows separation and determination of a
wide range of biogenic amines present in wine in less than 15 min., compared
to 25 min. with traditional HPLC procedures. Santos et al. (2004) described an
automated method for biogenic amine determination in white and red wines,
by means of capillary electrophoresis-electrospray ionization coupled to mass
spectrometry (CE-ESI-MS). Kvasnicka and Voldrich (2006) developed a CE
method using conductometric detection which requires no derivatization or
sample cleaning steps.
Gas Chromatography (GC)

Gas chromatography (GC) has also been used to determine amines. In
combination with various detectors it is one of the most valuable and common
methods for precise quantitative analysis of biogenic amines (Fernandes and
Ferreira, 2000). However, this technique is more suitable for analysis of
volatile compounds and not so much for nonvolatile molecules.
Gas-chromatography/mass spectrometry is a highly sensitive method
developed by Fernandes and Ferreira (2000), with a run time of only 18 min.
This is the first GC-MS method that allowed simultaneous determination of
diamines, polyamines and aromatic amines in grape juice and various wines.
The conversion of biogenic amines into their corresponding volatile (oheptafluorobutyryl) derivatives allows determination with gas chromategraphy. The GC method may have several disadvantages such as a suitable
sample preparation, which could include an extraction step, a cleaning or
concentration process and derivatization.
The main disadvantage of gas chromatography appears to be the
derivatization procedure, due to the complexity of the reactions and types of
reagents required. Derivatization speed differs from one amine to another, and
reproduction of strict reaction conditions is essential for all samples. The
technique also requires careful extraction and sample pre-concentration which
should be taken into account when ensuring the reliability of the final results
(Herbert et al. 2000).
Detection of the Responsible Enzymes: Polymerase Chain

Reaction (PCR)
Polymerase chain reaction (PCR) is used for the detection of specific
lactic bacteria strains that possess genes coding for enzymes involved in
79
biogenic amine production in wine. By targeting the suitable gene, PCR can be
performed to detect their presence in the strains (conventional PCR) (Coton et
al. 1998). Although these results do not determine final BA concentrations, the
risk of BA spoilage is linked to the presence of the genes in the bacterial
population (Lucas et al. 2008). Another PCR method is multiplex PCR that
can be used to detect the presence of several genes. This method reduces the
quantity of the reagent, labor costs and time, because genes are detected
simultaneously. This is a suitable technique for routine screening of BAproducing lactic bacteria in wine. Multiplex PCR assays for the detection of
decarboxylases in fermented food and beverages, including wine, have been
described by Coton and Coton (2005), Marcobal et al. (2005a), Constantini et
al. (2006) and De las Rivas et al. (2006). For instance, Marcobal et al. (2005a)
selected three pairs of primers for a multiplex PCR assay for the simultaneous
detection of lactic bacteria strains with potential production of histamine,
tyramine and putrescine. Quantitative PCR or qPCR is a method to detect and
quantify histamine-, tyramine- and putrescine-forming bacteria in wines
(Lucas et al. 2008; Nannelli et al. 2008).
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ISBN: 978-1-63482-765-2
Chapter 4
IMPACT OF FUNGAL DISEASES IN GRAPES

AND WINE: GENERAL ASPECTS
AND RECENT ADVANCES
Gisselle R. Apud, Pedro A. Aredes-Fernndez
and Diego A. Sampietro*
ABSTRACT
Fungal diseases cause important economic losses in the production of
fresh fruits and vegetables at the field, and during storage and transportation. Some of their ethiological agents contaminate the agricultural
products with mycotoxins which can be toxic for human and animals. In
the context of the vineyards, fungal diseases reduce yield and quality of
grapes. This problem often affects chemical and sensory properties of
wine. The purpose of this chapter is to describe the main fungal diseases
affecting grapes and wine quality, the mycotoxigenic risk associated to
wine production and strategies currently performed to prevent and control
fungal contamination. Special attention was paid to ochratoxin A, the
main mycotoxin present in grapes, must and wine.
Keywords: fungal diseases, mycotoxins; wine

*
Corresponding author address: Email: dasampietro2006@yahoo.com.ar
92
G. R. Apud, P. A. Aredes-Fernndez and D. A. Sampietro
INTRODUCTION
Fungi are the most important and prevalent plant pathogens. They infect a
wide range of host plants and cause economic losses in the production of fresh
fruits and vegetables both at the field and during storage and transportation
(Huang et al. 2010). Quantitative direct damage generated by phytopathogenic
fungi on crop yields can reach 12% or more at pre-harvest. A 20-25% of the
harvested fruits and vegetables are lost due to post-harvest fungal diseases
even in developed countries (Spadaro and Gullino, 2004). In developing
countries, post-harvest losses are often more severe due to inadequate storage
and transportation facilities (Sharma et al. 2009). The low pH, high moisture
contents and nutrient compositions do fruits and vegetables highly susceptible
to pathogenic fungi. The indirect damage in fruits includes the reduction of
nutritive value, changes in desirable organoleptic properties, and contamination with mycotoxins (Bezerra da Rocha et al. 2014). The last ones are
low molecular weight secondary metabolites produced by filamentous fungi
that commonly growth in foods or food crops throughout the food chain. The
intake of mycotoxins above certain levels can produce a variety of toxic
effects in humans and animals, from allergic responses to immunosuppression
and cancer. Some mycotoxins are present only in the fungus whereas others
are excreted in foods and feeds (Filtenborg et al. 1996). The contaminaion of
agricultural products with mycotoxins can occur in the field, after the harvest,
or during transportation and storage (Sforza et al. 2006). Once mycotoxins are
found in the food, they generally persist during the processing and storage. For
this reason, strategies to overcome the mycotoxigenic risk often are focused to
minimize or prevent the entrance of mycotoxins to the food chain (Scott et al.
1992). More than 400 of these compounds are currently known (Bennet and
Klich, 2003), but the real number of existing mycotoxins likely could be
higher than thousands. In all crop productions, mycotoxin problems vary from
year to year depending on favorable conditions for the development of the
organisms that produce them. Grapes and its derivatives (i.e., wine) are into
the context explained above. Fungal diseases reduce yield and quality of
grapes and affect chemical and sensory properties of wine (Scott, 2010). In
this chapter, we depict the main fungal diseases affecting grapes and wine
quality, the mycotoxigenic risk associated to wine production and strategies
currently performed to prevent and control fungal contamination.
Impact of Fungal Diseases in Grapes and Wine
93
1. FUNGAL DISEASES OF GRAPES AND THEIR INCIDENCE

ON WINE QUALITY
Grapes are exposed to fungal contamination during pre-harvest, harvest
and further grape processing. The main fungal species that contaminate and
cause grape spoilage are Erysiphe necator, Plasmopara viticola, Botrytis
cinerea, Aspergillus spp, Alternaria spp, Cladosporium spp, Penicillium spp
and Rhizopus spp (Magnoli et al. 2003; Valero et al. 2005; Hocking et al.
2007):
Powdery mildew: it is also known as oidium. This disease is caused by
Erysiphe necator. Hyphae and conidia of this fungus look as a grey-white
powder that covers the surface of leaves and young berries. The fungus can
penetrate the fruit skin and draw the nutrients causing splitting and dehydration of berries. Although this infection does not rot the berries, the skin
damage can aid the infection of other pathogenic fungi. Grapes with powdery
mildew are smaller than healthy grapes. Moreover, infected leaves may dry
out and prematurely drop (Pearson and Gadoury 1992; Emmett et al. 1992). In
seasons with favorable weather conditions, the disease can be severe and can
result in yield reduction and loss of grape and wine quality (Pool et al. 1984).
Downy mildew: Plasmopara viticola, an oomycete pathogen, is the main
ethiological agent of Downy mildew. This disease is worldwide distributed
and affects both the vine leaves and berries. It occurs during the growing
season wherever the weather is humid and rainy (Hewitt et al. 1988). The first
symptoms include yellow areas visible on the organ surfaces with an oily
appearance. But, as the lesions progress, they expand rapidly, change to brown
color and dry. Berries infected lose turgidity and rapidly dry out (Wan et al.
2007).
Botrytis bunch rot: it is also known as grey mould. This disease is caused
by the fungus B. cinerea and occurs in the vineyards of all over the world. It is
characterized by a white moldy growth that can be observed on the surface of
the infected fruit. The presence of high humidity on the surface of berries and
a temperature of 18C are optimal conditions for the germination and mycelial
growth of the fungus (Ribreau-Gayon et al. 2006a). Botrytis cinerea alters the
chemical composition of the infected grapes. The fungus oxidizes glucose to
produce gluconic acid and glycerol, and degrades tartaric and malic acids as
well as the proteins and amino acids (Ribreau-Gayon et al. 2006a). In
addition, it secretes lytic enzymes like pectinases, cellulases and hemicellulases that break components of the plant cell walls (Kars and Van Kan,
94
2004). Botrytis cinerea is characterized by the secretion of the laccase enzyme

that, at the pH of wine, oxidizes phenolic compounds to quinones, which then
suffer polymerization to form brown colored compounds (Slomczynski et al.
1995). The activity of this enzyme causes browning of the white wines, the
loss of color in red wines and the production of oxidized precipitates
(Ribreau-Gayon et al. 2006a; Claus, 2009). For these reasons, grey mould
cause damage and a significant yield loss in grapes (Emmett et al. 1992;
Flaherty et al. 1992) and affects the wine quality due to the oxidation, changes
of colour and difficulties in filtration and clarification (Ribreau-Gayon et al.
2006a).
Black rot: it is a grape disease characteristic of warm and hot climates
caused by members of Aspergillus Section Nigri, mainly Aspergillus niger and
A. carbonarius that can appear prior to harvest, when the grapes are ripe, in
post-harvest, during storage or in the dried process under sun or artificial light.
The infection is characterized by an intense black spore production on the
surface of the berries skin. In optimal conditions the spores germinate and the
fungus can invade and consume the nutrients conferring to berries a
completely empty and dry appearance. The primary reservoir of black
Aspergillus spp. is the soil at a deep of 0-5 cm below the surface (Leong et al.
2006b). These pathogens are considered secondary invaders because they
infect grapes that have been previously damaged by insects, other fungi like
Erysiphe necator or Plasmopara viticola, pest and ambient factors like
excessive rain, hail, wind and sunburn (Emmett et al. 1992).
Alternaria rot: The genus Alternaria is a cosmopolitan group of fungi that
include saprophytic, endophytic and pathogenic species, widely distributed in
soil and organic matter in decomposition (Pavn et al. 2012; Polizzotto et al.
2012). Among them, A. alternata causes grape rots under very high humidity.
It can enter berries through wounds, and can cause important pre and postharvest losses. Lesions are tan at first and become brown or black with the
time (Swart and Holz, 1991; Bernadoviov and Ivanov , 2011). It has been
demonstrated that this fungus can remain latent at harvest and the infection is
not detected, playing an important role in post-harvest rot, when the symptoms
are only evident at the end of a prolonged cold storage (Swart and Holz, 1991).
On the other hand, Musetti et al. (2005) reported that A. alternata produces
metabolites which can inhibit sporulation of Plasmopara viticola, the fungus
that causes downy mildew disease.
Cladosporium rot: Cladosporium cladosporioides and Cladosporium
herbarum are the main ethiological agents of this disease which is very
common in grapevines, particularly red vines that are harvested very late to
95
obtain a complete phenolic maturity of the berries, to ensure aroma and flavor
development for optimal wine quality (Saint-Cricq et al. 1998; Briceo and
Latorre, 2007, 2008). However, the occurrence of this disease reduces the
yield and affects the quality of wines (Briceo et al. 2009; Pszczlkowski et al.
2001). These moulds invade the surface of the grape skin and develop dark
green lesions with a velvety appearance. Although the disease begins in the
vineyard, also it can occur during post-harvest after a long period of cold
storage. Mycelium growth of Cladosporium can be considerably diminished,
but not totally inhibited at 0C (Briceo and Latorre, 2008).
Blue mold rot: it is a post-harvest disease caused by Penicillium, mainly
Penicillium expansum which usually infects wounded grapes that have been
stored at 0C for a long time. The disease begins with the growth of a white
mycelium which produces greenish blue powdery spores over the grapes, few
days after the infection (Donoso and Latorre, 2006).
Rhizopus rot: is other post-harvest disease caused mainly by Rhizopus
nigricans under warm and moist conditions during the storage. The spores of
this fungus can be found in vineyards at hot or humid locations, either in the
soil or on the berries. The disease starts at the base of mature berries as a soft
and watery rot that damages the berry skin. As a consequence, longitudinal
fissures are produced and a black mold develops rapidly along the fissures.
Thus, the skin of the berry changes its colour to light gray. Rhizopus rot can be
inhibited totally if grapes are stored at 4C (Latorre et al. 2002b).
2. OCURRENCE OF MYCOTOXINS IN WINE AND THEIR

IMPACT ON HEALTH
The ochratoxin A (OTA) is the most relevant mycotoxin in grapes and
wine. The main source of OTA in wine is the contamination of grapes with
Aspergillus carbonarius and Aspergillus niger (Serra et al. 2003). Currently,
other Aspergillus species included in section Nigri are not considered to be
OTA producers (Esteban et al. 2004). The accumulation of OTA in grapes are
produced between veraison and harvesting time or during winemaking as a
consequence of several factors such as geographical area, climatic conditions,
mycoflora composition, grape management, damage in berries and winemaking techniques (Cozzi et al. 2006; Ponsone et al. 2007; Visconti et al.
2008). The OTA showed nephrotoxic, hepatotoxic, teratogenic, genotoxic and
immunotoxic properties on several animal species. It also caused kidney and
96
liver tumors in mice and rats (Walker and Larsen, 2005). The IARC
(International Agency for Research on Cancer) has classified OTA in the
group 2B as a possible carcinogen to humans (Beardall and Miller, 1994). The
Codex Alimentarius Commission suggests that 15% of the total intake of this
toxin in Europe is due to wine (Visconti et al. 2008). Therefore, the OIV
established that the maximum concentration of OTA in wine is 2 g/L (OIV,
2002).
Majerus et al. (2000) suggested that red and white wines have different
concentration of OTA due to the different vinification techniques. The
winemaking process of red wines, which is carried out with maceration and
fermentation temperatures higher than those of white wines, usually produce
wines with a high concentration of OTA.
Ponsone et al. (2007) investigated the occurrence and toxigenicity of
Aspergillus section Nigri species in vineyards from Mendoza province
(Argentina). All vineyards evaluated were contaminated with OTA-producing
species at harvest stage. However, the berries collected were OTA free
irrespective of the growth stage considered, the variety and the cropping
system. The ability of the species studied to produce OTA was evaluated on
yeast extract sucrose agar (YES) medium. The cultures were incubated at 30C
for 10 days in darkness and the OTA content of the grapes was determined by
HPLC. The analysis of 246 strains indicated that 24% of them were OTA
producers. In this regard, it is important to notice that the presence of toxigenic
strains not always imply OTA contamination. Ecological conditions like pH,
water activity (aw) and temperature, which favour growth and subsequent high
fungal contamination, are different from those which allow optimum OTA
biosynthesis (Esteban et al. 2004, 2006; Romero et al. 2007). Esteban et al.
(2004) investigated the effects of temperature and incubation time on fungal
growth and OTA production by A. carbonarius and A. niger cultured on
Czapek yeast agar (CYA) and on YES media. They showed that A. niger
achieved maximum OTA levels mainly at 20 and 25C after 5 days of
incubation in YES medium. However, at 15C, maximum OTA concentration
was achieved after 10-20 days of incubation. Aspergillus carbonarius
produced the highest amounts of OTA at 15 and 20C after 5 days of
incubation in CYA medium. When the temperature was 15C, the maximum
level was obtained after 30 days of incubation.
Other authors determined the effect of temperature and aw on growth and
OTA production by strains of A. carbonarius and A. niger isolated from
Australian vineyards and cultured on a synthetic grape juice medium. They
demonstrated that the maximum growth for A. carbonarius occurred at 0.97 aw
97
and 30C, and for A. niger, at 0.98 aw and 35C. The optimum temperature for
OTA production was 15C and the optimum aw was 0.95-0.98 for A.
carbonarius and 0.95 for A. niger (Leong et al. 2006b). Spadaro et al. (2010)
examined the effect of temperature, aw and pH on OTA production by three
strains of A. carbonarius isolated from Italy and they demonstrated that the
optimal conditions for growth and production of OTA by A. carbonarius
strains were 30C, aw 0.98 and pH 4.0, confirming that the environmental
conditions in which A. carbonarius can grow also favour a strong OTA
production.
The fumonisins are important mycotoxins produced mainly by fungi of the
Fusarium genus which cause ear rot in corn (Sampietro et al. 2009, 2011).
However it has been reported that A. niger is also able to produce fumonisin
B2 (FB2) in grapes and wines (Frisvad et al. 2007, Logrieco et al. 2009;
Mogensen et al. 2010b). The fumonisins are known to cause human and
animal toxicoses by the consumption of contaminated food and feeds
(Sydenham and Savolainen, 1991). The fumonisins are structurally similar to
sphingolipids and have shown to inhibit the sphingolipid biosynthesis via the
ceramide synthase pathway (Stockmann-Juvala et al. 2008). They have shown
to induce leukoencephalomalacia in horses and pulmonary edema and
hydrothorax in pigs (Sydenham and Savolainen 1991; Yazar and Omurtag
2008).
The patulin is other mycotoxin produced in grapes by contamination with
Penicillium expansum that cause blue mold rot. Currently this fungus is
considered the most efficient patulin producer. The patulin causes gastrointestinal problems, skin rashes, and is known to be mutagenic neurotoxic,
immunotoxic, genotoxic, teratogenic and carcinogenic in humans and animals
(Bragulat et al. 2008; Puel et al. 2010). The world Health Organization has
established a daily dose of 0.4 mg/kg of body weight as the maximum intake
for this mycotoxin. The patulin is relatively stable in grapes and grapes juice
(Ough and Corison, 1980). However, Moss and Long (2002) reported that
patulin has not been detected in wine mainly due to enzymatic degradation by
Saccharomyces cerevisiae during alcoholic fermentation. Daz et al. (2011)
studied the presence of patulin in wines of Chile made with grapes infected
with P. expansum and they determined that concentration of this mycotoxin
significantly decreased during alcoholic fermentation with the addition of 30
mg/L of sulfur dioxide (SO2). In this way, Pohland and Allen (1970) have
reported previously that SO2 destroys patulin by the opening of the lactone
ring.
98
Fungi of the genus Alternaria, mainly A. alternata, produce several

mycotoxins including alternariol (AOH), alternariol monomethyl ether
(AME), altertoxin (ATX) and L-tenuazonic acid. These toxins induce harmful
effects in animals, including fetotoxic and teratogenic mutagenic and
genotoxic effects (Weidenbrner, 2001; Wollenhaupt et al. 2008; Fehr et al.
2009). It has been demonstrated that grapes are good substrates for the
production of AOH and AME by A. alternata (Tournas and Stack, 2001).
Scott et al. (2006) found levels of 0.03-5.2 ng/mL of AOH in Canadian red
wines while AME was found at lower concentrations. Other study based on
the occurrence of these mycotoxins in white wines of Germany showed that
AOH was present at levels of 1.3 and 1.5 g/mL (Ackermann et al. 2011).
Broggi et al. (2013) studied the presence of AOH and AME in wines from
Entre Rios (Argentina). They found that 4 of 53 white wine samples and 6 of
56 samples of red wine were contaminated with a maximum of 18 ng/mL and
13 ng/mL of AOH, respectively. Maximum permitted levels in foods were not
yet legislated for Alternaria mycotoxins (Juan-Garca and Fernndez-Blanco
2014).
3. PREVENTION AND CONTROL OF FUNGAL

CONTAMINATION
Strategies for fungal control in grapes are performed at harvestand postharvest (Kabak et al. 2006; Amzqueta et al. 2009). Prevention of fungal
infection is the first step to avoid or minimize grapes damage and mycotoxin
contamination. Grapes must be protected against mechanical and insect
damages. Wounded grapes can be removed. Neverthless, the weeds, the agricultural residues or the dirty agricultural materials also should be eliminated
because they can act as source of fungal inoculum (Park et al. 1999; Codex
Alimentarius Commission, 2003). Chemical control with fungicides at preharvest is a traditional strategy to minimize fungal damage. The effect of
fungicides on mold growth and mycotoxin production is affected by several
factors, including their chemical nature, rate of application, crop type, fungal
species, and storage conditions (Kabak et al. 2006).
Control of powdery mildew and downy mildew is generally based on the
use of properly selected and timed fungicide sprays. In addition, cultural
practices may reduce the severity of the disease and can increase the
effectiveness of chemical controls. Fungicides used to control powdery
99
mildew disease are Calaxin (0.07%), Karathane EC (0.04%), Myclobutanil

(Systhane at 0.05%), Triademifon (Bayleton at 0.1%) and Penconazol (Topas
at 0.025%). Downy mildew disease is controlled by fungicides Bordeaux
mixture (1%), Copper Oxychloride (0.2%), Mancozeb (0.2%), Metalaxyl
(0.2%) or Fosetyl Al (0.2%) (Srinivasa Naidu, 2008).
It has been reported that fungicides such as dinocap, captan, tebuconazole,
azoxystrobin and penconazole applied to grapes before harvest reduce
infections generated by Aspergillus section Nigri (Lo Curto et al. 2004; Bell
et al. 2006). Others like carbendazim, Switch (25% fludioxonil and 37.5%
cyprodinil) and Chorus (cyprodinil) have also been studied for their activity
against Aspergillus (Cabras et al. 1997; Cabras and Angioni 2000; Tjamos et
al. 2004). Switch (25% fludioxonil and 37.5% cyprodinil) is a broad spectrum
fungicide that provides high-level control of B. cinerea, Aspergillus,
Alternaria, Penicillium, Cladosporium and Rhizopus in grapes.
The timing of wine grape harvest is also very critical. Grapes can be too
much acid in early harvests and can lack the acidity or suffer a stronger rot
damage if the growers harvest too late.Even in cold post-harvest storage (0C),
grapes are affected by fungi like Penicillium spp., Aspergillus spp., A.
alternata and B. cinerea (Karabulut et al. 2003; Thakur and Saharan, 2008;
Senthil et al. 2011; Romanazzi et al. 2012) and suffer yield losses of about
39%.The early harvests in areas with high mycotoxigenic risk, the shortening
of storage times and the use of sulphur dioxide during cold storage prevent
these yield losses before the processing of grapes for winemaking (Soto, 1973;
Singh et al. 1985; Lichter et al. 2005). In the context of grape for wine
production, the International Organisation of Vine and Wine (acronym OIV,
from its french language name L'Organisation Internationale de la Vigne et du
Vin) recommends to eliminate the grapes damaged or contaminated and the
fast transportation of healthy grapes to the winery. In addition, it is important
to maintain clean the containers after each load of grapes, especially in the
case of rotten harvests. Spoilage of grapes intended for direct human
consumption can be performed by exposures to SO2 fumigation for several
weeks. In contrast, the SO2 applications for wine grapes are only allowed for a
few hours prior to fermentation (Considine and Foyer, 2015).
The concentration of OTA is high in winemaking process at the start of
the alcoholic fermentation and decreases at the end (Lasram et al. 2008). The
reasons of this lowering are not enterily known. Meca et al. (2010) showed
that yeast cells adsorb mycotoxins during alcoholic fermentation. The OTA
concentration is also reduced during clarification of wine because of its
adsorption to the suspended solids that are later removed (Fernandes et al.
100
2007). Racking is other step in the winemaking process in which the OTA
concentration is reduced (Leong et al. 2006b). Storage of wine bottles and
ageing of wine is also considered another process that reduces the OTA
concentration (Lasram et al. 2008).
4. DETERMINATION OF OTA IN WINES

The high-pressure liquid chromatography coupled to fluorescence
detection (HPLC-FL) is the most common analytical method used for OTA
quantification in wine (Hernndez et al. 2006; Gonzlez-Osnaya et al. 2008;
Tessini et al. 2010). It is a rapid, sensitive, precise, efficient and reproducible
method (Tafuri et al. 2008). The use of fluorescence detection is due to the fact
that OTA possesses natural fluorescence (Monbaliu et al. 2010; Tessini et al.
2010). The method uses an isocratic mobile phase of acetonitrile, water, and
acetic acid (49:49:2, vol/vol/vol, respectively) and a monolithic C18 column
(Tafuri et al. 2008). It is characterized by direct injection of the wine into the
HPLC apparatus, with no need of extraction or cleanup. Optionally, clean-up
of sample and pre-concentration steps are used to remove matrix components
and increase the sensitivity before the chromatographic analysis (Prelle et al.
2013). An alternative detection method is the liquid chromatography tandem
mass spectrometry (LC-MS/MS). Reinsch et al. (2005) proposed a method for
OTA quantification in red wine based on a combined anion exchange/
reversed-phase clean-up before an analysis by liquid chromatography coupled
with tandem mass spectrometry.
Capillary electrophoresis (CE) with DAD detection has also been
developed for the OTA quantification in wine (Gonzlez-Peas et al. 2006).
This chromatographic technique was compared with HPLC-FL. The most
important advantage of CE was the use of the economical and ecological
aqueous borate buffer in the separation process.Another chromatographic
method used in the OTA quantification is the thin layer chromatography
(TLC) (Teixeira et al. 2010; Welke et al. 2010). It is usually combined with
immunoaffinity columns (IAC) for the cleaning of sample extracts and the
isolation of specific mycotoxin in foodstuff (Valero et al. 2008; Anli and
Bayram, 2009; Welke et al. 2010). Teixeira et al. (2010) evaluated the
presence of OTA using thin layer chromatography (TLC) with a chargecoupled detector (CCD) in red wine samples from Brazil. The CCD is a two
dimensional detector containing an array of sensors that can image an area in
fraction of seconds. The output from each sensor pixel on the CCD is a
101
voltage, which is proportional to the intensity of light falling on the sensor and
the exposure time. These series of voltages are digitized and transferred to a
computer for storage and data processing (Lancaster et al. 2005; 2006). Other
researchers used a novel and advanced technology on solid phase extraction
(SPE) column previous to ultra high performance liquid chromatography
(UHPLC) coupled to tandem mass spectrometry for the determination of OTA
in red wine samples (Mario-Repizo et al. 2015).
Several methods for determination of OTA are expensive and timeconsuming. For this reason, rapid screening tests such as biosensors (Liu et al.
2009; Alonso-Lomillo et al. 2010) and enzyme-linked immunosorbent assays
(ELISA) (Klari et al. 2009) have emerged. Samples subjected to immunobased assays usually require a previous cleanup to eliminate matrix interferences, specially in the analysis of red wines. These immunoassays are rapid,
easy to perform, and inexpensive. Nevertheless, they also show poor analytical
performances in term of accuracy and reproducibility compared with
chromatographic techniques. Longobardi et al. (2013) developed an analytical
method for the quantification of OTA in red wine using a double-extract
cleanup and fluorometry. Wine samples were diluted with a solution of
polyethylene glycol and sodium hydrogen carbonate, filtered, and purified by
immunoaffinity and then in an aminopropyl solid-phase column. The OTA
contents of the cleaned sample were determined in a spectrofluorometer.
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EDITORS CONTACT INFORMATION

Pedro Adrin Aredes-Fernndez
Associate Researcher,
National Council of Scientific and Technical Investigations (CONICET)
Professor, Institute of Biotechnology,
Faculty of Biochemistry, Chemistry and Pharmacy,
National University of Tucumn,
Ayacucho 471 (4000), Tucumn, Argentina
pedroaredes@hotmail.com
pedroaredes@fbqf.unt.edu.ar
Mara Jos Rodriguez-Vaquero
Assistant Researcher,
National Council of Scientific and Technical Research (CONICET)
Professor, Institute of Biotechnology,
Faculty of Biochemistry, Chemistry and Pharmacy
National University of Tucumn
mariajo@fbqf.unt.edu.ar
Gisselle Raquel Apud
Fellow, National Council of Scientific and Technical Research (CONICET)
PhD Student, Faculty of Biochemistry, Chemistry and Pharmacy,
112
Editors Contact Information
Mara Gilda Stivala

Fellow, National Council of Scientific and Technical Research (CONICET)
PhD Student, Faculty of Biochemistry, Chemistry and Pharmacy,
INDEX
A
acetaldehyde, 64, 76
acetic acid, 76, 85, 100
acetone, 17
acetonitrile, 20, 100
acid, 3, 5, 9, 10, 14, 15, 17, 18, 20, 21, 33,
36, 37, 38, 41, 43, 44, 45, 46, 47, 50, 53,
54, 55, 58, 62, 66, 69, 74, 81, 88, 93, 98,
99
acidic, 18
acidity, 14, 99
active centers, 32
active site, 8, 9, 10, 46
adaptation (s), 18, 83
adenocarcinoma, 56
adsorption, 18, 38, 99
adverse effects, 64
adverse event, 46
aflatoxin, 102
Africa, 90
agar, 74, 85, 96
age, 29
aging process, 14
agmatine, 65, 66, 68
albumin, 73
alcohol consumption, 50
alcohols, 37
aldehydes, 64, 66
aldosterone, 6, 45, 50, 52, 53, 56
allergic reaction, 45
alters, 93
AME, 98
amine(s), 61, 62, 63, 64, 65, 66, 68, 69, 72,
73, 74, 76, 78, 79, 80, 82, 83, 86, 87, 88,
90
amine group, 63
amino, 8, 9, 11, 12, 14, 15, 21, 22, 23, 24,
28, 34, 61, 62, 66, 68, 69, 70, 71, 76, 77,
79, 80, 81, 82, 83, 84, 87, 90, 93
amino acid(s), 8, 9, 11, 12, 14, 15, 21, 22,
23, 24, 28, 34, 61, 62, 66, 67, 68, 69, 70,
71, 76, 77, 79, 80, 81, 82, 83, 84, 87, 90,
93
amino groups, 76
ammonia, 64, 67, 75, 76
ammonium, 77, 82
anemia, 50
angiogenesis, 58
angiotensin converting enzyme, 24, 25, 49
angiotensin II, 6, 32, 45
anthocyanin, 37, 54
antihypertensive agents, 30, 45, 56
antihypertensive effects, vii
anti-inflammatory effects, vii
antioxidant, vii, 2, 4, 5, 7, 9, 13, 14, 16, 17,
18, 19, 23, 24, 27, 30, 32, 38, 39, 44, 46,
48, 49, 50, 53, 54, 55
antioxidant-promoting capacity, vii
antioxidative activity, 9
apoptosis, 5, 31, 51
114
Index
appetite, 4
apples, 85
Argentina, 1, 17, 35, 36, 41, 48, 51, 57, 61,
63, 91, 96, 98, 102, 106, 108
arginine, 9, 66
aromatic compounds, 102
arteries, 26, 44, 64
Aspergillus carbonarius, viii, 95, 96, 102,
108, 109, 110
Aspergillus niger, viii, 94, 95, 103, 105, 110
Aspergillus terreus, 86
assessment, 101
assimilation, 31
atherosclerosis, 39, 52, 54
atmosphere, 105
atmospheric pressure, 77, 85
Austria, 63
autolysis, 7, 13, 15, 23, 24, 29, 68
B
bacteria, 3, 13, 15, 24, 26, 33, 40, 41, 50,
51, 52, 53, 57, 58, 61, 62, 67, 69, 72, 73,
74, 75, 78, 80, 81, 84, 85, 86, 87, 88
bacterial pathogens, 48
bacterial strains, 67
bactericides, 48
bacterium, 14, 40, 42, 68, 70
base, 9, 36, 95
beer, 61, 82, 107
Belgium, 63
beneficial effect, vii, 1, 4, 22, 39
benefits, vii, 1, 5, 22, 31, 40
benzene, 36, 37
beverages, 1, 2, 22, 50, 55, 61, 62, 64, 77,
79, 85, 102
bicarbonate, 104
bioactive compounds, vii, viii
bioavailability, 1, 54
biochemistry, 29, 87
bioinformatics, 29
biological activities, viii, 1, 2, 5, 17, 19, 24,
36
biological activity, 22, 26, 62
biological control, 108, 109
biological markers, 50
biological responses, 50
biosensors, 101
biosynthesis, 6, 96, 97
biotechnology, 103
blood, 4, 6, 7, 9, 12, 13, 28, 29, 44, 45, 46,
55
blood pressure, 4, 6, 7, 9, 13, 28, 45, 46, 55
blood pressure reduction, 45
blood vessels, 44
body weight, 97
bradykinin, 6, 45
brain, 64
brain activity, 64
Brazil, 100, 109
breakdown, 3, 43, 69, 76
breast cancer, 49
brevis, 69, 87
buyer, 63
by-products, vii, 25, 37, 48, 54
C
cancer, 31, 39, 55, 86, 92
capillary, 77, 79, 84, 88, 104, 109
carbohydrates, 20
carbon, 13
carbon dioxide, 13
carboxyl, 6, 9, 10
carcinogen, 96
carcinogenesis, 65
carcinoma, 51
cardiovascular disease, vii, 1, 6
cardiovascular function, 6
cardiovascular risk, 25
cardiovascular system, vii, 44, 46
carnosine, 5
cascades, 26
casein, 24, 29, 30, 32, 34, 73
catabolism, 24
cellulose, 16
ceramide, 97
charge coupled device, 105
cheese, 75
Index
chemical, 8, 21, 28, 38, 39, 63, 74, 77, 85,
91, 92, 93, 98, 110
chemical properties, 8
chemical reactions, 38
chemical structures, 39
chemicals, 67
chemokines, 47
Chile, 36, 97, 102, 103, 105
China, 54
cholesterol, 27, 40
chromatographic technique, 100, 101, 108
chromatography, 7, 20, 21, 52, 75, 78, 81,
82, 84, 85, 88, 100, 105, 109, 110
chronic diseases, 2
chymotrypsin, 11
circulation, 12, 65
clarity, 39
classes, 37, 44
cleaning, 78, 100
cleanup, 100, 101, 106
cleavage, 9, 21, 45
climate(s), 35, 38, 94
clinical trials, 46, 56, 57
cloning, 32
coding, 78
coffee, 107
colon, 11, 39
color, 35, 36, 74, 76, 93, 94
colorectal cancer, 39, 59
commercial, 14, 45, 73, 85, 109
community, vii, 37
competition, 65
complex interactions, 13
complexity, 78
complications, 4
composition, 3, 8, 13, 29, 38, 66, 68, 70, 93,
95, 102, 110
compost, 49, 51
compounds, vii, viii, 1, 5, 6, 8, 13, 14, 18,
21, 27, 30, 33, 35, 36, 37, 38, 39, 40, 41,
45, 46, 47, 48, 52, 53, 54, 56, 57, 62, 64,
65, 73, 75, 78, 89, 92, 94
computer, 101
condensation, 36, 38
configuration, 33
115
congress, 57
consensus, vii, 9, 25
constituents, 14, 35, 36, 37, 43, 66
consumers, viii, 40, 49, 62
consumption, vii, 1, 4, 12, 16, 17, 25, 39,
40, 51, 55, 56, 58, 59, 65, 71, 97, 99
containers, 99
contaminant, viii
contaminated food, 97
contamination, 91, 92, 93, 95, 96, 97, 98,
104, 109
copper, 48
coronary heart disease, 39
correlation(s), 71, 74, 83, 89
cosmetic, viii
cough, 45
coumarins, 36
crop(s), 55, 92, 98, 109
crop production, 92
CST, 87, 106
cultivars, 48, 52, 67
cultivation, 55
cultural practices, 98
culture, vii, 42, 71, 75
culture media, 75
culture medium, 42
curcumin, 46
CVD, 44, 45
cyclooxygenase, 47
cysteine, 9
cytokines, 47, 51
cytoplasm, 43
D
damages, 95, 98, 103
data processing, 101
decomposition, 94
decontamination, 101
defense mechanisms, 5
deficiency, 62, 66, 79
degradation, 21, 22, 26, 97, 103
dehydration, 93
deprivation, 51
116
Index
derivatives, 21, 44, 76, 78, 82, 83, 85, 86,

92
detectable, 21, 68
detection, 21, 53, 62, 74, 75, 76, 77, 78, 79,
80, 81, 82, 84, 85, 86, 87, 89, 100, 104,
105, 106, 109, 110
detoxification, 5, 64
developed countries, 58, 92
developing countries, 92
diabetes, 52
diabetic kidney disease, 56
dialysis, 16
diamines, 63, 78
diarrhea, 64
diastolic blood pressure, 46
diet, 1, 2, 25, 27
diffusion, 11, 39
digestibility, 30
digestion, 5, 11, 12, 30, 56
digestive enzymes, 11, 22
dilation, 64
dipeptides, 15, 23, 70, 71, 80
diseases, vii, 1, 44, 91, 92, 103, 104, 107,
108, 109
distribution, 53, 81
diversity, 67, 108
DNA, 54
douchi, 27
drugs, 9, 27, 65
E
E. coli, 40
economic losses, 91, 92
edema, 64
egg, 26
elaboration, 22, 37, 47
electric field, 20, 33
electrodes, 101
electron, 5
electrophoresis, 77, 79, 84, 86, 88, 100, 103,
104
enantiomers, 109
endothelial dysfunction, 32, 46
endothelium, 46
energy, 5, 13, 69
engineering, 24
England, 31
environment, 15, 18, 55, 85
environmental conditions, 48, 66, 97
environmental factors, 44
environments, 3, 69, 83
enzyme(s), 1, 4, 5, 6, 10, 11, 15, 19, 22, 24,
26, 27, 28, 29, 30, 31, 32, 33, 34, 39, 45,
46, 47, 56, 64, 66, 67, 69, 73, 74, 76, 78,
93, 94, 101, 105
enzyme inhibitors, 56
enzyme-linked immunosorbent assay
(ELISA), 76, 86, 101, 105
epithelial cells, 11, 39
epithelium, 11, 22
EPS, 42
equipment, 75, 76
ESI, 50, 78
esophagus, 56
ester, 76, 85
ethanol, 13, 15, 20, 28, 37, 41, 42, 46, 49,
64, 65, 86, 104, 105
eucalyptus, 41
eukaryotic, 66
eukaryotic cell, 66
Europe, vii, 25, 96, 102
evidence, 1, 2, 12, 25, 26, 39, 46, 51
evolution, viii, 56, 72, 73, 88
exclusion, 20
exopolysaccharides, 42
experimental design, 54
exposure, 38, 81, 101
extraction, 53, 54, 56, 77, 78, 100, 101, 106
extracts, 18, 24, 41, 44, 48, 49, 52, 55, 57,
59, 100
extrusion, 31
F
false negative, 75
false positive, 75
families, 37
fatty foods, vii
Index
fermentation, vii, 2, 3, 13, 14, 15, 23, 24,
26, 29, 33, 37, 38, 48, 52, 59, 62, 67, 68,
71, 72, 73, 74, 79, 81, 83, 89, 90, 96, 97,
99, 106
fertilization, 66
fever, vii, 47, 79
fibrinolytic, 29
filtration, 20, 21, 38, 73, 77, 94
Finland, 30, 63
fish, 2, 32, 75, 76, 83, 84, 85
flavonoids, 36, 37, 53, 55
flavonol, 37
flavor, 14, 24, 95
flora, 73
flotation, 73
fluid, 31, 45
fluid balance, 45
fluorescence, 21, 77, 85, 100, 104, 109, 110
food, viii, 2, 3, 4, 5, 6, 9, 20, 22, 26, 27, 28,
32, 33, 39, 48, 54, 62, 79, 82, 88, 89, 92,
103, 104, 105, 108, 110
food additive, 103
food chain, 92, 110
food industry, 48
food poisoning, 89
food products, 5
food safety, 88
food spoilage, 103
force, 63, 87
Ford, 54
formation, 36, 40, 45, 61, 63, 65, 68, 69, 73,
74, 89, 90
fragments, 2, 12, 21
France, vii, 36, 40, 63
free radicals, 4, 51
fructose, 13, 36, 46, 49
fruits, 36, 91, 92, 108, 109, 110
functional food, 2, 25, 34
fungal infection, 98
fungi, viii, 49, 57, 74, 92, 93, 94, 97, 99,
102
fungus, 92, 93, 94, 95, 97, 106
117
G
gastrointestinal tract, 11, 30, 65
gel, 20, 21
gene expression, 5, 69
genes, 38, 47, 78
genus, 13, 94, 97, 98, 107
Germany, 36, 63, 98, 103
germination, 93
globalization, 52
glucose, 13, 31, 93
glutamate, 9
glutathione, 81
glycerol, 37, 93
glycine, 70
glycol, 101
glycosaminoglycans, 86
grapes, viii, 3, 13, 34, 37, 38, 47, 54, 61, 62,
64, 65, 66, 67, 68, 80, 81, 82, 88, 91, 92,
93, 94, 95, 96, 97, 98, 99, 102, 103, 105,
106, 107, 108, 109, 110
Greece, 110
growth, 14, 15, 23, 31, 36, 40, 41, 42, 43,
48, 50, 52, 54, 55, 57, 66, 70, 71, 73, 74,
79, 80, 84, 92, 93, 95, 96, 98, 102, 104,
107, 108, 109
growth factor, 54
growth rate, 70
H
half-life, 12
harmful effects, 44, 64, 98
harvesting, 95
hazards, 31
headache, 89
health, viii, 1, 2, 4, 22, 29, 30, 32, 40, 57,
62, 102
health effects, 2, 29
health promotion, 2, 30
heart attack, 50
heart disease, 12
heart failure, 55
hemoglobin, 31
118
Index
high blood pressure, 6, 46

histamine, 61, 62, 63, 64, 65, 66, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 79, 81, 82, 83,
84, 85, 86, 87, 88, 90
histidine, 9, 66, 69, 70, 71, 84, 85, 87
histidine-containing dipeptides, 70
homocysteine, 55
hormone, 45
host, 92
human, vii, 1, 5, 6, 12, 21, 23, 28, 29, 31,
32, 33, 49, 55, 57, 61, 64, 65, 88, 91, 97,
99, 102, 107, 109
human body, vii, 1, 5, 6, 64
human health, vii, 1, 23, 61, 88
human milk, 21, 28, 33
humidity, 93, 94
Hungary, 67
hydrogen, 5, 9, 64, 76, 101
hydrogen peroxide, 5, 64, 76
hydrolysis, 2, 3, 6, 11, 12, 25, 30
hydrophobicity, 8
hydroxyl, 5, 36
hydroxyl groups, 36
hygiene, 61, 64
hypertension, vii, 6, 9, 28, 32, 33, 45, 46,
49, 50, 51, 52, 53, 58, 65
hypertrophy, 49
hypotension, 64
hypotensive, 26, 32
hypothesis, 13
I
identification, 22, 24, 31, 34, 54, 84, 88
image(s), 75, 87, 100, 105
immobilization, 105
immune response, 65
immunity, 5, 29, 55
immunomodulatory, 4
immunosuppression, 92
improvements, 75, 76
in vitro, 12, 22, 25, 27, 30, 45, 46, 48, 51,
86, 104
in vivo, 12, 22, 48, 51
incidence, vii, 13, 39, 73
incubation period, 71
incubation time, 17, 96, 103
India, 109
industries, viii
industry, 36, 52, 55, 57
infection, 93, 94, 95
inflammation, 29, 39, 47, 51, 55, 59
inflammatory disease, 47
inflammatory responses, 47
ingestion, 11
ingredients, 22, 25, 27
inhibition, 9, 10, 30, 32, 33, 40, 50, 51, 53,
58, 64, 86
inhibitor, 9, 10, 12, 22, 24, 31, 32, 47, 55
inoculation, 7, 15, 17, 73, 74
inoculum, 48, 71, 98
insects, 94
insulin, 4, 23, 46, 49
insulin resistance, 46
integrity, 43
interference, 64
intermediaries, 5
internalization, 70
intestinal tract, 64
ion-exchange, 20
ionization, 77, 78, 85
ions, 21
Iowa, 34
isoflavonoids, 37
isolation, vii, 18, 20, 22, 27, 85, 100
isoleucine, 8
Israel, 102
issues, 110
Italy, 36, 75, 97, 103, 107
J
Japan, 55
jejunum, 24
K
kaempferol, 43, 47
ketones, 66
Index
kidney, viii, 95
kinetics, 24, 32, 59
L
lactic acid, 13, 14, 15, 24, 26, 27, 33, 40, 42,
50, 51, 52, 57, 61, 62, 67, 68, 69, 74, 80,
81, 84, 85, 86, 87, 88
Lactobacillus, 3, 31, 41, 43, 50, 51, 62, 69,
70, 71, 80, 81, 87
LC-MS, 100, 106
LC-MS/MS, 100, 106
leakage, 43
legislation, 63
lesions, 48, 93, 95
leucine, 8, 10
leukemia, 49
light, 94, 95, 101
lignans, 36
linoleic acid, 44
lipid peroxidation, 9, 35, 44
lipid profile regulation, vii
lipids, 5, 44, 47
liquid chromatography, 8, 53, 75, 77, 80,
82, 85, 100, 101, 104, 106, 107, 109
Listeria monocytogenes, 57
liver, viii, 23, 45, 96
lung cancer, 51
Luo, 59
lysine, 9, 66
lysis, 3, 14, 73
lysozyme, 73, 85
M
Macedonia, 53
macromolecules, 5, 44
majority, 13, 37
management, 48, 95, 104
manufacturing, 64
marketing, 32
Mars, 4, 28
mass, 21, 22, 28, 77, 78, 85, 88, 100, 101,
106, 107, 108
119
mass spectrometry, 21, 22, 77, 78, 85, 100,

101, 106, 107, 108
materials, 38, 44, 98
matrix, 39, 77, 100, 101
measurements, 105
meat, 2, 4, 23, 75
media, 15, 96
membrane permeability, 41
membranes, 20, 25, 31, 39, 66
meta-analysis, 46, 50, 56
metabolic, 103
metabolic syndrome, 4, 27, 31
metabolism, vii, 5, 22, 31, 44, 50, 61, 62, 64
metabolites, 36, 62, 68, 92, 94, 105
metabolized, 12
mice, viii, 87, 96
microorganism(s), viii, 2, 3, 15, 36, 40, 62,
66, 67, 68, 70, 73, 82, 103
migration, 5
mild hypertensive, 6
mildew, 93, 94, 98, 104, 107, 110
minicolumn, 77
model system, 23, 82
models, 3
modifications, 43, 57, 74, 80
moisture, 92
moisture content, 92
mold, 27, 95, 97, 98, 103, 107
molecular biology, 57
molecular mass, 8
molecular weight, 7, 13, 14, 16, 18, 20, 30,
41, 62, 70, 92
molecules, 5, 20, 36, 38, 39, 44, 78
monomers, 37
mood disorder, 65
morbidity, 6
morphology, 43
mortality, 6, 40, 45, 51
motif, 9
mRNA, 23
mucous membrane, 12
mycelium, 95
mycology, 105
mycotoxins, viii, 91, 92, 97, 98, 99, 102,
104, 106, 107, 110
120
Index
N
national income, 25
national product, 35
negative effects, viii, 61, 65
Netherlands, 105
neurotransmission, 64
nitric oxide, 5, 46, 47
nitric oxide synthase, 47
nitrite, 65
nitrogen, viii, 5, 7, 14, 15, 16, 17, 18, 23,
31, 61, 66, 79, 80
nitrogen compounds, viii, 14, 18, 61, 66, 79,
80
nitrogen dioxide, 5
nitrosamines, 65
non-enzymatic antioxidants, 44
norepinephrine, 65
Norway, 81
Norway spruce, 81
nucleic acid, 5, 44
nucleotides, 24
nutrient(s), 2, 13, 14, 15, 69, 92, 93, 94
nutrition, 62
O
obesity, 4
Ochratoxin A, viii, 101, 105, 106, 110
oenologists, viii
oligomers, 37
OPA, 76, 83, 85
operations, 74
opportunities, 88
orbit, 5
organ(s), 11, 93
organic compounds, 62
organic matter, 94
organic solvents, 77
organism, 5
ornithine, 65, 66, 72, 86
ovarian cancer, 54
oxidation, 5, 44, 55, 94
oxidative damage, 5, 25, 44
oxidative stress, 5, 25, 26, 39, 44, 46

oxygen, 5, 7, 9, 44
oyster, 32
ozone, 5, 81
P
pain, 47
pancreatic cancer, 58
pathogenesis, 105
pathogens, 92, 94, 108
pathophysiology, 28
pathway, 6, 11, 31, 97
PCR, 78, 81, 86, 87
peptidase, 9, 12
peptide(s), vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34,
45, 62, 69, 70, 71, 79
peptide chain, 21
peripheral blood, 64
permeability, 22, 64
peroxide, 5, 76
peroxynitrite, 5
pesticide, 105
pH, 11, 14, 15, 24, 64, 66, 69, 72, 73, 74,
92, 94, 96, 97, 103, 109
phagocytosis, 5
pharmaceutical(s), viii, 22
pharmacology, 51
phenolic compounds, 1, 13, 35, 36, 37, 38,
39, 40, 41, 42, 44, 45, 48, 51, 53, 55, 56,
57, 58, 94
phenotype, 85
phenylalanine, 8, 9, 66
phosphate, 43
phosphatidylcholine, 66
phospholipids, 80
physicochemical properties, 18
physiology, 56
plant growth, 104
plants, 37, 47, 53, 66, 92
platelet aggregation, 39
platelets, 47
playing, 94
Index
poison, 103
polyamine(s), 63, 65, 78, 80, 81, 83, 86, 88,
89
polymerization, 94
polymers, 37, 39
polypeptide(s), 7, 11, 16, 28
polyphenols, vii, 32, 37, 39, 40, 43, 45, 46,
47, 49, 50, 52, 54, 57, 58
polysaccharide(s), 37, 41
population, 13, 14, 31, 45, 49, 79, 110
potassium, 43, 79
precipitation, 17, 20, 38
preparation, 27, 76, 78
prevention, vii, 1, 2, 4, 31, 46, 74, 105
preventive properties, vii
probability, 72
probe, 54
probiotics, 18
producers, 69, 95, 96, 107, 108
prognosis, 49
pro-inflammatory, 47
proliferation, 5
proline, 8, 9, 11, 29
prostaglandins, 47
protection, 40, 103
protective mechanisms, 31
protein hydrolysates, 20, 24, 30
proteinase, 3
proteins, 2, 3, 4, 5, 6, 8, 11, 12, 13, 14, 15,
20, 27, 28, 29, 30, 31, 32, 33, 34, 44, 54,
57, 69, 93, 110
proteolysis, 5, 30, 70
proteolytic enzyme, 11
pulmonary edema, 97
pulp, 13, 37, 48
purification, 20, 30
PVP, 77
121
R
radical formation, 44
radicals, 26
RAS, 6, 45
rash, 64
raw materials, 38, 63
reactions, 5, 13, 36, 38, 64, 69, 72, 78
reactive oxygen, vii, 40, 46, 49
reagents, 77, 78
recognition, 10, 11, 104
recommendations, 52
recovery, 27, 48, 54
red blood cells, 58
red wine, 8, 12, 13, 15, 17, 21, 22, 28, 29,
30, 36, 37, 38, 39, 40, 44, 46, 50, 51, 52,
53, 58, 65, 71, 73, 78, 79, 83, 84, 86, 94,
96, 98, 100, 101, 102, 106, 110
relevance, 62
reliability, 74, 78
renal dysfunction, 45
renin, 6, 7, 30, 45, 50, 52, 55
reproduction, 36, 78
requirements, 15, 31, 34
researchers, viii, 74, 101
residues, 8, 9, 15, 23, 36, 47, 98, 102
resistance, 12, 22, 26, 52, 108, 110
resolution, 76, 77
response, 4, 64, 65, 66, 80, 81
resveratrol, 37, 43, 46, 47, 51
rhizopus, 93, 95, 99, 105
rings, 37
risk(s), 2, 6, 12, 25, 39, 45, 47, 49, 50, 51,
56, 65, 74, 79, 91, 92, 99, 106, 110
risk assessment, 110
risk factors, 25
room temperature, 108
routes, 64
Q
quality control, 61
quantification, 52, 75, 77, 88, 100, 101, 104,
105, 106
quercetin, 45, 46, 47
quinones, 94
S
safety, 14, 62
salts, 38
saturated fat, 40
122
Index
scavengers, 35, 40, 44, 46

schizophrenia, 65
science, 25, 109
scientific knowledge, viii
SDS, 77
secretion, 6, 30, 45, 47, 94
sedimentation, 73
sediments, 47
seed, 48, 49, 57
seedlings, 48
selectivity, 20, 33
sensations, 36, 38
sensitivity, 14, 74, 77, 100
sensor, 100
sequencing, 21, 33
serum, 23
shelf life, 48, 110
shellfish, 26
shoot, 83
showing, 17, 45, 67, 79
side chain, 9
side effects, 6, 45
simulation, 11
skin, 13, 37, 39, 45, 48, 49, 93, 94, 95, 97
small intestine, 11, 12, 39
smoking, 51
smooth muscle, 64
society, 104
sodium, 6, 45, 101, 104
soil type, 66
solid phase, 101, 106
solubility, 18
solution, 21, 77, 101
South Africa, 36, 90, 109
soymilk, 4, 7, 27
Spain, 36, 49, 63, 68
species, vii, 3, 5, 40, 44, 46, 49, 67, 68, 69,
70, 93, 94, 95, 96, 98, 102, 103, 104,
105, 107, 108, 110
spore, 94
squamous cell, 56
squamous cell carcinoma, 56
stability, 39
stabilization, 14, 38, 67
starvation, 15
state, 2, 80
steel, 72
sterile, 67
stimulant, 26
stimulation, 4
stomach, 11, 47, 64
storage, 26, 35, 38, 53, 67, 71, 72, 80, 91,
92, 94, 95, 98, 99, 101, 108, 109
stress, 66
stroke, 45, 50
structure, 1, 8, 10, 24, 34, 36, 37, 43, 53, 62,
63
substitution, 10, 15, 37
substrate, 5, 9, 10, 15, 30, 46, 98
sucrose, 96
sugar beet, 49
sulfur, 97, 103
sulfur dioxide, 97, 103
sulphur, 99
Sun, 24, 48, 53, 57
supplementation, 42, 55
suppression, 4, 47, 55
surfactant, 77
survival, 5, 39, 69
susceptibility, 11
suspensions, 43
Switzerland, 63
symptoms, 48, 79, 93, 94
synthesis, 62
systolic blood pressure, 46
T
tanks, 72
tannins, 36, 37, 56, 57
target, 2, 11, 22
target organs, 22
techniques, 21, 44, 48, 52, 56, 63, 74, 75,
95, 96, 109
technologies, viii
technology, 20, 24, 38, 62, 101, 109
temperature, 38, 52, 53, 64, 66, 72, 93, 96,
103, 108, 109
therapeutic agents, 22
time use, 77
Index
toxic effect, 64, 65, 92, 109
toxicity, 74
toxicology, 89
toxin, 96
transcription, 47
transformations, 24
translation, 110
transport, 11, 12, 31, 69, 70, 72, 83
transportation, 91, 92, 99
treatment, 1, 4, 6, 31, 45, 46, 48, 73, 77, 109
triggers, 45
trypsin, 11
tryptophan, 8, 9, 21, 66, 77
tumor, 65
tumor invasion, 65
tumorigenesis, 27, 54
tumors, viii, 96
tyramine, 61, 63, 64, 65, 66, 68, 69, 70, 71,
72, 75, 77, 79, 81, 82, 85, 86, 87, 88, 90
tyrosine, 8, 9, 21, 65, 66, 70, 73, 77, 87
viscosity, 41
vitamin C, 5, 56
vitamins, 39, 51, 55
vomiting, 64
W
waste, 26, 36, 47, 48, 49, 86
wastewater, 48
water, 6, 37, 66, 96, 100, 108, 109
wavelengths, 21, 75
well-being, 2
WHO, 106
wine consumption, vii, 1, 25, 39, 59
wine microflora, vii, 22
winemaking process, viii, 38, 61, 64, 65, 67,
90, 96, 99
wood, 38, 56
worldwide, 45, 53, 93
wound healing, 5
U
United Kingdom, 23, 55, 107
United States (USA), 36, 102
urbanization, 25
123
X
xylem, 83
Y
V
valine, 8
variations, 73
varieties, 40, 51, 54, 67
vasoconstriction, 6, 45
vasodilator, 6
vegetables, 36, 91, 92, 108, 109
vessels, 64
vinasse, 49, 54
vinification process, vii, 2, 3, 22, 67, 85, 88
yeast, 3, 4, 7, 8, 13, 14, 15, 23, 24, 28, 29,

37, 38, 67, 68, 72, 74, 80, 84, 90, 96, 99,
106
yield, 91, 92, 93, 94, 95, 99, 107
Z
zinc, 9

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BIOCHEMISTRY RESEARCH TRENDS

BIOCHEMISTRY RESEARCH TRENDS

Additional e-books in this series can be found on Novas website

BIOCHEMISTRY RESEARCH TRENDS

MARA GILDA STIVALA

Copyright 2016 by Nova Science Publishers, Inc.

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Library of Congress Control Number: 2015948678

Published by Nova Science Publishers, Inc. New York

Editors Contact Information

viii P. A. Aredes Fernndez, M. J. Rodriguez Vaquero, G. R. Apud et al.

In: Bioactive Compounds in Wine

BIOACTIVE PEPTIDES IN WINE:

Corresponding Author address: Email: pedroaredes@fbqf.unt.edu.ar

Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Keywords: bioactive peptides, wine, antihypertensive activity, antioxidant

Bioactive Peptides in Wine

Some food proteins can directly produce physiological effects in their

1. WINE BIOACTIVE PEPTIDES

Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

(Manca de Nadra et al. 2005; Aredes-Fernndez et al. 2004). Similarly, it has

2. FUNCTIONALITY OF BIOACTIVE PEPTIDES

Bioactive Peptides in Wine

Antioxidant and antihypertensive properties are the most important and

2.1. Antioxidant Peptides

Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

antioxidant activity. Chen et al. (2012) confirmed antioxidant activity of

2.2. Antihypertensive Peptides

Bioactive Peptides in Wine

Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

4. MODE AND MECHANISM OF INHIBITION OF ACE

10 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

5. ABSORPTION AND BIOAVAILABILITY OF ACE

12 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

6. ROLE OF FERMENTATIVE PROCESS AND OCCURRENCE

Bioactive Peptides in Wine

14 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

16 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Proteolytic Activity [mmol/L]

Figure 2 shows the change in proteolytic activity of O. oeni m1 in

Bioactive Peptides in Wine

proteolytic activity was detected after 24 h of incubation with values of 1.372

18 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Recently, Su et al. (2015) studied the antioxidant properties of intact cells

7. FRACTIONATION AND ISOLATION OF

20 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

Mandal et al. (2014) recently fractionated and purified twenty-four peptides

22 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

Alcaide-Hidalgo, J.M., Pueyo, E., Polo, M.C., Martnez-Rodrguez, A.J.,

24 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

26 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

28 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.

Bioactive Peptides in Wine

30 Pedro A. Aredes-Fernndez, Gisselle R. Apud, Mara G. Stivala et al.