Blueberries Harvesting Methods, Antioxidant Properties and Health Effects

NUTRITION AND DIET RESEARCH PROGRESS
BLUEBERRIES
HARVESTING METHODS,
ANTIOXIDANT PROPERTIES
AND HEALTH EFFECTS
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NUTRITION AND DIET RESEARCH PROGRESS
BLUEBERRIES
HARVESTING METHODS,
ANTIOXIDANT PROPERTIES
AND HEALTH EFFECTS
MALCOLM MARSH
EDITOR
New York
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CONTENTS
Preface vii
Chapter 1 Blueberries: Market, Cultivars, Chemical
Composition and Antioxidant Capacity 1
Paula Becker Pertuzatti, Isidro Hermosín-Gutiérrez
and Helena Teixeira Godoy
Chapter 2 Bioactive Compounds, Color and Physicochemical
Parameters of Blueberries 31
Paula Becker Pertuzatti, Milene Teixeira Barcia,
Andressa Carolina Jacques and Rui Carlos Zambiazi
Chapter 3 Blueberries: Antioxidant Properties, Health and
Innovative Technologies 55
Guillermo Petzold, Jorge Moreno, Pamela Zúñiga,
Karla Mella and Patricio Orellana
Chapter 4 Blueberry Anti-Inflammatory Effects over Metabolic
Diseases Associated with Obesity 71
J. Soto-Covasich, M. Reyes-Farias, A. Ovalle-Marin,
C. Parra-Ruiz and D. F. Garcia-Diaz
Chapter 5 Blueberry Extracts Protect against Gross Mouse
Fetal Defects Induced by Alcohol Toxicity 89
Zach S. Gish, Sharang Penumetsa, Diana J. Valle
and Roman J. Miller
Index 111
PREFACE
According to Food and Agriculture Organization (FAO) statistics, from

1970 to 2011 the world production of blueberries increased approximately 7
times (FAOSTAT, 2012). According to the National Agricultural Statistics
Service (NASS) of the United States Department of Agriculture (USDA), the
United States is the world leading producer of blueberries, being the second
most produced and commercialized small fruit in the country after strawberry.
There are three main groups of blueberries commercialized and produced in
the world: the lowbush, the highbush and the Rabbiteye (RASEIRA e
ANTUNES, 2004). The high amount of bioactive compounds present in both
the pulp and in the peel of the blueberries, makes it a fresh fruit rich in natural
antioxidants. This book discusses the harvesting methods, antioxidant
properties and health effects of blueberries.
Chapter 1 - The production of blueberry in Brazil began at the decade of
80’s and its commercialization during the decade of 90’s of the XXth century.
Despite being a new crop in the country, it is observed that each day the fruit
has been gaining ground, which led to an increase in the number of producers
and cultivars marketed. The growing in blueberry cultivation in Brazil is
mainly attributable to the privileged climatic conditions with temperate
regions in four states, Rio Grande do Sul, Santa Catarina, Paraná and São
Paulo. Thus, the country has production potential over all the year. In addition,
another economically important aspect is that Brazilian production of this fruit
mainly occurs from December to February, which happens between the
harvest seasons in the United States and that of the European Union that are
the main consumer centers. The health benefits of blueberries became widely
accepted after Prior reported that blueberries had the highest antioxidant
capacity of more than forty fruits and vegetables evaluated. In addition, most
viii Malcolm Marsh
researches have correlated the high antioxidant capacity with the phenolic
content in the fruit, especially flavonoids, because these kinds of compounds
are the major pigments found in these fruits. Among the phenolic compounds,
the blueberries are rich in flavonoids, namely anthocyanins, and flavanols, and
hidroxycinamic acids, which are associated in the literature with beneficial
health effects due to their ability to act as antioxidants, helping to protect the
body against free radicals and thus to avoid various types of cancer.
Roopchand et al. observed that the polyphenols present in blueberry may be
useful for the dietary management of diabetes, because lowered fasting blood
glucose levels, lowered serum cholesterol and reduced weight gain in mice.
Studies dealing with blueberries highlighted that, besides the presence of
phenolic compounds, other interesting compounds such as carotenoids,
tocopherols and ascorbic acid, also are present in this fruit, being sometimes
found in high levels. These compounds may also impact on the antioxidant
capacity of this fruit, once the antioxidant capacity of carotenoids has been
reported in the literature, as well as for tocopherols and ascorbic acid.
However, the composition in bioactive compounds of blueberries can be
highly variable, depending on cultivar, stage of maturation and harvesting and
storage conditions, usually because of its non climacteric nature with regard to
their production and responsiveness to ethylene. Therefore, this chapter aimed
to discuss about the blueberry producer and consumer market, the importance
of blueberry production in Brazil and in the world, which are the cultivars
produced in Brazil, in addition to its chemical and bioactive composition and
its influence in antioxidant capacity.
Chapter 2 - According to Food and Agriculture Organization (FAO)
statistics, from 1970 to 2011 the world production of blueberries increased
approximately 7 times (FAOSTAT, 2012). According to the National
Agricultural Statistics Service (NASS) of the United States Department of
Agriculture (USDA), the United States is the world leading producer of
blueberries, being the second most produced and commercialized small fruit in
the country after strawberry. There are three main groups of blueberries
commercialized and produced in the world: the lowbush, the highbush and the
Rabbiteye (RASEIRA e ANTUNES, 2004). The high amount of bioactive
compounds present in both the pulp and in the peel of the blueberries, makes it
a fresh fruit rich in natural antioxidants. Therefore, the identification and
quantification of the major bioactive compounds and some physicochemical
parameters in the peel, pulp and entire fruit of six blueberry cultivars
belonging to the Rabbiteye group are discussed in this chapter. Phenolic
compounds, anthocyanins, color, hydrolyzed and condensed tannins,
Preface ix
carotenoids and physicochemical analyses were done. There were evaluated

Powderblue, Briteblue, Bluebelle, Climax, Delite and Woodard cultivars. The
blueberry fruits showed as rich sources of phenolic compounds and
anthocyanins, besides to have considerable amounts carotenoids and tannins.
All phytochemicals analyzed were found at the highest levels in the peels of
blueberry cultivars tested.
Chapter 3 - Blueberries are a soft and small fruit native to North America
with an attractive blue color. In addition, blueberries are very popular because
they have low calories, high nutritional value and important antioxidant
properties. Blueberries have an interesting content of phenolic compounds
with high antioxidant capacity against free radicals and reactive species, such
that blueberry consumption may have a potential beneficial effect on human
health. Innovative technologies in the food industry are new technologies
based to develop more efficient process or products, reduction of energy and
water. Innovative technologies, such as freeze concentration, osmotic
dehydration and vacuum impregnation at mild temperatures, are considered
minimal processing techniques because they preserve the fresh characteristics
of fruits such as blueberries. Freeze concentration is an innovative technology
for producing a blueberry concentrate juice in a process at low temperatures
where no vapor/liquid interface exists. On the other hand, osmotic dehydration
and vacuum impregnation of blueberries preserves different valuable attributes
of the fruit, providing products with an extended shelf-life.
Chapter 4 - Inflammation is a natural defense mechanism triggered as a
response to an alteration of the physiological functions in the organism. This
process is responsible for the secretion of mediators crucial for tissues repair,
integrating different signalling pathways between distinct cells and organs.
Likewise, it has been observed that in metabolic diseases some classic
mediators present during short-term inflammation are involved, although the
features of its actions differ from the classic pathways. Thus it is considered as
a subclass of inflammation often referred as meta-inflammation. In the case of
obesity for example, this response is exacerbated and, at the long term, a
chronic inflammatory state associated with cardiovascular diseases, insulin
resistance and type-2 diabetes development is established. Since obesity-
associated inflammation is known to be a key feature of the etiology of non-
communicable diseases, several efforts have been made for identifying novel
agents with anti-inflammatory properties capable of ameliorate its negative
long-term effects. In this regard, blueberry consumption has been described to
induce important health benefits through anti-inflammatory and antioxidant
features. Therefore, in the present chapter, the authors will discuss the impact
x Malcolm Marsh
on the low-grade inflammatory status associated to metabolic diseases

provided by a blueberry treatment or diet, previously described in the
literature. In this context, will be addressed: a) in vitro studies over
inflammation in macrophages and changes in adipogenesis; b) in vivo studies
over pro-oxidant and inflammatory status, related to amelioration of insulin
resistance, hyperglycemia, dyslipidemia, hyperphagia and weight gain induced
by a high fat feeding, and improvement of blood pressure, renal function and
beta cell function; and c) human clinical evidence, over antioxidant defense
mechanisms and inflammation, influencing blood pressure and insulin
sensitivity susceptible subjects. In this sense, recent findings supports that a
blueberries-rich diet has been able to modulate the inflammatory status in a
positive manner, likewise exerting its effects in different crucial stages of
metabolic alterations development and hence contributing to the prevention
and reduction of obesity-associated comorbidities. It is still pending to deepen
into the cellular and molecular mechanisms in order to take advantage from a
commercially-available fruit for improve human life quality.
Chapter 5 - Alcohol is a powerful teratogen, systematically affecting
prenatal development as well as postnatal functioning in humans and other
mammals. Using a mouse model, this study explored the potential effects of
anthocyanins from blueberry extracts in protecting against alcohol-induced
prenatal developmental deficiencies.
Swiss mice were assigned to three experimental groups: control (CO),
binge alcohol (BA) and alcohol-anthocyanin (AA). CO mice were
administered normal saline (0.03 ml/g maternal body weight), while BA and
AA mice received alcohol (25% v/v of absolute ethanol in normal saline at
0.03 ml/g maternal body weight), through intraperitoneal injections on days 5
and 7 following impregnation. Supplemental anthocyanins via blueberry
extracts (0.03 mg/g maternal body weight) were additionally administered to
the AA group, through subcutaneous-neck injections on days 0, 5, 7 and 12.
Maternal mice were necropsied and fetuses removed at day 15 of gestation.
Statistical analysis (p < 0.05) showed that 15 day old mouse fetuses with
prior exposure to binge alcohol with anthocyanin supplementation (AA) were
partially protected from some gross developmental deficiencies over the binge
alcohol fetuses (BA). Group comparisons (CO vs BA vs AA) showed
significant fetal gross body differences in regards to average body weight (197
vs 90 vs 162 mg, respectively), crown-rump length (11.2 vs 9.1 vs 10.7 mm,
respectively), liver surface areas (6.9 vs 2.5 vs 5.1 mm2 respectively) and
telencephalon (forebrain) surface areas (3.18 vs 1.47 vs 2.75 mm2
respectively).
Preface xi
Results support the hypothesis that properties found in blueberry extracts

serve to mitigate certain gross anatomical effects in mouse fetuses due to
maternal binge alcohol exposure during prenatal development.
In: Blueberries ISBN: 978-1-63484-885-5
Editor: Malcolm Marsh © 2016 Nova Science Publishers, Inc.
Chapter 1
BLUEBERRIES: MARKET, CULTIVARS,

CHEMICAL COMPOSITION AND
ANTIOXIDANT CAPACITY
Paula Becker Pertuzatti1,*, Isidro Hermosín-Gutiérrez2

and Helena Teixeira Godoy3
1
Federal University of Mato Grosso (UFMT) Barra do Garcas, MT, Brazil
2
University Castilla -La Mancha (UCLM), Regional Institute for Applied
Scientific Research (IRICA), Ciudad Real, Spain
3
State University of Campinas (UNICAMP), Campinas, SP, Brazil
ABSTRACT
The production of blueberry in Brazil began at the decade of 80’s
and its commercialization during the decade of 90’s of the XXth century.
Despite being a new crop in the country, it is observed that each day the
fruit has been gaining ground, which led to an increase in the number of
producers and cultivars marketed. The growing in blueberry cultivation in
Brazil is mainly attributable to the privileged climatic conditions with
temperate regions in four states, Rio Grande do Sul, Santa Catarina,
Paraná and São Paulo. Thus, the country has production potential over all
the year. In addition, another economically important aspect is that
Brazilian production of this fruit mainly occurs from December to
February, which happens between the harvest seasons in the United
* paulapertuzatti@yahoo.com.br.
2 P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy
States and that of the European Union that are the main consumer centers
[1, 2]. The health benefits of blueberries became widely accepted after
Prior [3] reported that blueberries had the highest antioxidant capacity of
more than forty fruits and vegetables evaluated. In addition, most
researches have correlated the high antioxidant capacity with the phenolic
content in the fruit, especially flavonoids, because these kinds of
compounds are the major pigments found in these fruits [4]. Among the
phenolic compounds, the blueberries are rich in flavonoids, namely
anthocyanins, and flavanols, and hidroxycinamic acids [5], which are
associated in the literature with beneficial health effects due to their
ability to act as antioxidants, helping to protect the body against free
radicals and thus to avoid various types of cancer [6]. Roopchand et al.
[7] observed that the polyphenols present in blueberry may be useful for
the dietary management of diabetes, because lowered fasting blood
glucose levels, lowered serum cholesterol and reduced weight gain in
mice. Studies dealing with blueberries highlighted that, besides the
presence of phenolic compounds, other interesting compounds such as
carotenoids, tocopherols and ascorbic acid, also are present in this fruit,
being sometimes found in high levels [2, 8]. These compounds may also
impact on the antioxidant capacity of this fruit, once the antioxidant
capacity of carotenoids has been reported in the literature [9, 10, 11], as
well as for tocopherols and ascorbic acid [12, 13]. However, the
composition in bioactive compounds of blueberries can be highly
variable, depending on cultivar, stage of maturation and harvesting and
storage conditions, usually because of its non climacteric nature with
regard to their production and responsiveness to ethylene [14]. Therefore,
this chapter aimed to discuss about the blueberry producer and consumer
market, the importance of blueberry production in Brazil and in the
world, which are the cultivars produced in Brazil, in addition to its
chemical and bioactive composition and its influence in antioxidant
capacity.
Keywords: blueberries, anthocyanins, phenolic compounds, carotenoids
INTRODUCTION
Berry fruits, cultivated in temperate zones have increasingly attracted the
interest not only of consumers, due to its taste and high content of bioactive
compounds, but also the interest of producers, due to its high productivity,
good profitability and high demand. Because of this, the cultivation of
strawberry, blackberry, raspberry, cranberry and blueberry has been gaining
ground in several countries. According to data from Food and Agriculture
Market, Cultivars, Chemical Composition and Antioxidant Capacity 3
Organization of the United Nations [15] in 2013, more than 9 million tons of
berries were produced, contributing significantly to the increase availability of
berries.
Brazil has a privileged climatic condition for such cultivation, with
temperate regions in four states, including Rio Grande do Sul, Santa Catarina,
Paraná and São Paulo. Thus, the country has the potential to produce
blueberries throughout the year. In addition, another economically important
aspect is that Brazilian production of this fruit mainly occurs from December
to February and can extend until April, depending on the cultivar produced,
thus including the harvest season in the United States and that of the European
Union that are the main consumer centers [1, 2].
Berries are widely recognized as having a basic chemical composition that
accentuates its sweet taste, fruity aroma and beneficial health properties, which
are appreciated worldwide. This can be highly variable, depending on cultivar,
stage of maturation and harvesting and storage conditions, usually because of
its non climacteric nature with regard to its production and responsiveness to
ethylene [14].
Regarding blueberry, a small fruit native from North America, its
cultivation began in Brazil at 80’s, since then, the Brazilian Agricultural
Research Corporation (Embrapa) began developing researches in order to
verify the blueberry adaptability under the climatic conditions of the country,
in addition to organize workshops and other events, focused on small farmers
and scientific community, disseminating and encouraging the production of
this crop in the country, which contributed to increase in production and
marketing of this fruit in Brazil.
Worldwide, special attention began to be given to blueberry after Prior [3],
reported that blueberries had the highest antioxidant capacity of more than
forty fruits and vegetables. This finding sparked the interest of consumers and
researchers. Thereafter, several studies about antioxidant capacity of
blueberries began to be taken correlating this high value with the phenolic
content in the fruit, because this kind of compounds are their major pigments
[4].
Among the phenolic compounds, blueberries are rich in flavonoids,
namely anthocyanins and flavanols, and hydroxycinnamic acids [5], which are
associated in the literature with beneficial health effects due to their ability to
act as antioxidants, helping to protect the body against free radicals and thus to
avoid various types of cancer [6]. Roopchand et al. [7] observed that the
polyphenols presents in blueberries may be useful for the dietary management
of diabetes, because lowered fasting blood glucose levels, lowered serum

cholesterol and reduced weight gain in mice.
Studies dealing with blueberries highlighted that, besides the presence of
phenolic compounds, other interesting compounds such as carotenoids,
tocopherols and ascorbic acid, also are presents in this fruit, being sometimes
found in high levels [2, 8]. These compounds may also impact on the
antioxidant capacity [9, 10, 11, 12, 13]. However, according to Pertuzatti et al.
[11], the antioxidant capacity of lipophilic compounds in blueberry was five to
500-fold lower than hydrophilic compounds. However, the authors attribute
these low values to the kind of the antioxidant mechanisms (transfer of
hydrogens or electrons, and free radicals scavenging) involved in the different
assays performed in their study, which are more favorable to quantify the
antioxidant capacity of hydrophilic compounds, such as phenolic compounds.
PRODUCER AND CONSUMER MARKET, IMPORTANCE OF

BLUEBERRY PRODUCTION IN BRAZIL AND IN THE WORLD
According to the Food and Agriculture Organization (FAO) statistics,
from 1970 to 2011 the worldwide production of blueberry increased
approximately 7-fold [16]. According to the National Agricultural Statistics
Service (NASS) belonging to the United States Department of Agriculture
(USDA), the United States leads the world production of blueberry which
represented approximately 454 tons of fruit in 2012, which makes blueberry as
the second most produced and marketed berry in the country, second only after
strawberries [17]. The second largest blueberry producer is Canada with
31.5% of world production and Europe (mainly Poland) with 10.4%, the rest
of the world account for 2.9% of world production [16].
Blueberries cultivation (Vaccinium spp.) has increased in countries of
South America, such as Chile which has 2550 ha planted, Argentina with 1500
ha and Uruguay with 200 ha, being these areas characterized by not having a
very intense cold winter and to have a hot summer [18, 19]. The increase in
blueberry production in the Southern hemisphere is largely due to the demand
from the countries of Northern hemisphere and by the parallel production of
fresh blueberries in the not harvest season of the latter countries, which creates
a very interesting business opportunity for the Brazilian productive sector [2,
20].
In Brazil, the cultivation of blueberries is not still well known. The first
plants were brought from the University of Florida in 1980 by EMBRAPA –
Temperate Climate (Pelotas-Brazil), to evaluate varieties, being introduced

collections of cultivars belonging to Rabbiteye group, because is the most
adaptive to climatic conditions of the South region of Brazil [21]. The first
commercial initiative in the country started from 1990, in Vacaria city, by
introducing varieties of Highbush [22, 23]. Currently, the predominant
cultivars belong to Rabbiteye group. The current productive picture in the
country is mainly concentrated in the cities of Vacaria in Rio Grande do Sul
and Campos do Jordão in São Paulo [24], covering an acreage of more than
150 hectares [19]. In contrast, the national consumer market is still very
limited, with São Paulo and Rio de Janeiro as the main consumers.
Regarding consumer market worldwide, the United States have the highest
rates of consumption, with a strong demand which made per capita
consumption increase approximately 50% in the past fifteen years [25].
According to Madail and Santos [24] US imports about 82% of the world’s
remaining production. Despite being the largest producer, the country is not
self-sufficient and, except in May, June and July (harvest season) depends
directly on the Canada, Chile and Argentina supplies.
CULTIVARS
Blueberries are from the family Ericaceae, subfamily Vaccinoideae and
genre Vaccinium, and their fruits may be classified in three main categories or
groups: Highbush, Lowbush and Rabbiteye [14, 26].
The Highbush group is originally from the west coast of North America
and within it the predominant specie is V. corymbosum L., although the
species V. australe and V. darrowi can be used for breeding [27], their plants
have a need for 650 to 850 h of cold (with temperatures lower or equal to
7.2°C) and the fruits have the best quality, in size and taste or with regard to
pruine content (waxy skin responsible for blue color of fruits) [28].
Blueberries of Highbush group, can be divided into Northern Highbush,
which is the most commonly planted in the world, with a cold requirement of
over 800 h and Southern Highbush, developed to allow blueberry production
in regions with mild winters or warmer (200 to 300 h of cold) [29]. According
to Galletta and Ballington [30], Southern Highbush blueberry prefers plateau
areas, soil rich in organic matter, is not bothered by many pest problems and
produce great fruits with excellent quality. These fruits have a very early
production compared to the other blueberry groups. They are grown in the less
cold regions of the United States, Chile and predominantly in Argentina and
Uruguay. In Brazil, is produced in Vacaria, Pelotas and Serra Gaúcha, in the

State of Rio Grande do Sul and in Campos do Jordão, State of São Paulo.
Table 1. Cultivars, groups and characteristics of blueberries cultivated in

South America
Cultivar Category Characteristic References

Bluecrop Highbush Created by USDA – New Jersey, medium to large 29
size, light blue, acid flavor, firm pulp,
requirement more than 600 h of cold
Darrow Highbush Large to very large size, light blue, medium firm 29
pulp, slightly tart
O’Neal Highbush From North Carolina, requirement 200 – 600 h of 31
cold, large size, light blue
Georgiagem Highbush From Georgia, requirement 200 – 600 h of cold, 31
medium size
Misty Highbush From Florida, requirement 150 – 200 h of cold, 28, 31
large size, light blue, firm pulp
Sharpblue Highbush From Florida, medium size, dark blue, medium 28
firm pulp
Millenia Highbush From Florida, medium size, light blue, firm pulp, 28
requirement of 300 h of cold
Star Highbush From Florida, large size, dark blue, firm pulp, 31
requirement of 400 h of cold
Elliot Highbush Medium size, light blue, requirement more than 29
800 h of cold
Coville Highbush Large size, firm pulp, requirement more than 800 32
h of cold
Brigitta Highbush Large size, light blue, firm pulp, requirement 29, 32
Blue more than 700 h of cold
Aliceblue Rabbiteye From Florida, low requirement of cold, sweet- 28
sour taste, light blue
Bluebelle Rabbiteye From Georgia, firm pulp, small to medium size, 28
light blue
Bluegem Rabbiteye From Florida, dark blue 28
Briteblue Rabbiteye From Georgia, large size, light blue, firm pulp 31
Delite Rabbiteye From Georgia, small size, sweet-sour taste 28, 31
Climax Rabbiteye From Georgia, medium size, dark blue 28
Powderblue Rabbiteye From Maryland, small to medium size, light blue 28, 29
Woodard Rabbiteye From Georgia, light blue, soft pulp 28
Tifblue Rabbiteye From Georgia, small size, light blue, firm pulp 29, 31
Beckyblue Rabbiteye Firm pulp, requirement 300-400 h of cold, 30
medium blue
Brightwell Rabbiteye Medium to large size, firm pulp 32
Bonita Rabbiteye From Florida, medium to large size, light blue, 28
astringent flavor
Windy Rabbiteye From Florida, medium to large size, firm pulp 28
Cultivars belonging to that group were developed from interspecific

hybridization between Highbush blueberry (Vaccinium corymbosum), the
evergreen blueberry (Vaccinium darrowi) and Rabbiteye blueberry (Vaccinium
ashei Reade) [30], and are presented in Table 1, along with other cultivars of
Highbush and Rabbiteye groups.
The main cultivars planted in Latin America are: ‘Bluecrop,’ one of the
most cultivated in Chile; ‘Duke’ and ‘Brigitta;’ ‘Coville’ with a vigorous and
productive bush, fruit with large size and good bitter-sweet taste; ‘Elliot’
which is the cultivar with the most demanding in cold cultivated areas in
Brazil and is characterized by its late production, harvested from January to
April; ‘Bluecrispy’ which has a very firm pulp and an almost crunchy texture
of ripe fruit, the fruit of this cultivar are very sweet, have good conservation
and resist very well to transport presenting quality for export even when the
weather becomes hot and rainy [32, 33]. ‘O’Neal’ is the cultivar predominant
in Argentina, Uruguay and Chile, with the beginning of the harvest under
conditions of Argentina and Uruguay in October, which provides excellent
values for export. In Brazil, needs frost control, due to precocity of its first
flowering, which happens between July and August [31] and ‘Misty’ is widely
planted in Uruguay and Argentina [28].
Rabbiteye group is from North America, belongs to V. ashei Reade, has a
high yield per plant and its fruits have a higher post-harvest conservation,
however, the fruit size is lower than cultivars of Highbush group. One of its
important characteristics is the low requirement of cold (300 to 650 h), which
makes this species to have commercial importance in regions with lower
availability of cold, adapting well to South and South-East of Brazil. About
cultivars produced in South America, Tifblue was for many years the
Rabbiteye cultivar most planted in the world [31].
Lowbush blueberries mostly belong to V. angustifolium, although
according to Raseira and Antunes [28] Canadian blueberry (V. myrtilloides e
V. boreale) also belongs to this group, which has a high requirement in hours
of cold (up until 1100 h), due to this, Lowbush is produced in few places in the
world. The fruits are small and its main destination is the processing industry
[27].
There is another species of blueberry, Vaccinium myrtillus L., native from
Northern Europe and also found in parts of North America and Asia, called
bilberry. This fruit has an intense blue color and its pulp is pigmented,
different from the American blueberry pulp that has no anthocyanins. Bilberry
has found applications as dietary supplements and pharmaceuticals. The
commercial drug Difrarel, contains 100 mg of bilberry anthocyanins plus

5 mg of β-carotene and is prescribed for diseases of circulatory system [34].
CHEMICAL COMPOSITION
According to Moraes et al. [23], blueberry has carbohydrate content about
15%. The main sugars found are sucrose, glucose and fructose, occurring in
blueberry in concentrations of 0.12-1.14%, 3.28-3.87% and 3.34-3.88%
respectively, the high amount of fructose, usually the major sugar in
blueberries, making this fruit a good option for diabetics [6, 35]. Wang et al.
[36] support this statement saying that the main sugars found in blueberries are
fructose and glucose, while sucrose is found in lower amounts, probably due to
high activity of invertase during the final stage of maturation [37]. The
concentration of these sugars is important to fruit quality because fructose is
1.8 times sweeter than sucrose, while glucose presents 60% of fructose
sweetness. The cultivation system also influences the sugar concentration,
because blueberries grown under organic cultivation system have higher
amounts of sugar (fructose and glucose) than blueberries grown under
conventional system [36].
Among polysaccharides found in ripe blueberries cellulose, hemicellulose,
pectin and lignin, which are mainly found in the cell wall, stand out. Besides
contributing to the nutritional value, sugars and organic acids are also
responsible for texture and flavor of fruits [38, 39].
Sugar content in blueberries is compensated by the presence of organic
acids and also phenolic acids, which can give an additional bitter or astringent
flavor. The completion of organic acids with phenolic acids is responsible for
titratable acidity that is commonly measured as a global index of fruit quality.
Organic acids also help to stabilize ascorbic acid and are fundamental in fruit
color, stabilizing anthocyanins and extending the shelf life of fresh and
processed fruits [35]. The organic acids found in blueberries are citric acid and
malic acid. Talcott [35] reports only the presence of malic acid in blueberry,
ranging from 0.06 to 1.10% while Milivojevic et al. [37] found both organic
acids, with citric acid, 0.1–0.23% being the major organic acid while malic
acid had amounts of 0.05 – 0.12%.
Volatile compounds are typically esters, alcohols, acids, aldehydes and
ketones [38]. In blueberries, Simon et al. [40] demonstrated that the emission
levels of volatile compounds are very low compared with other fruits such as
strawberry, only butyl-acetate could be observed. However, according to Su
and Chien [41] blueberry aroma is composed by linalool, 2-trans-hexenol, 2-

trans-hexenal, 3-cis-hexenol and 3-cis-hexenal. Terpenes, unsaturated C6
aldehydes and unsaturated alcohols have been reported as the predominant
compounds identified in volatile extracts of Rabbiteye blueberries. Du,
Olmstead and Rouseff [42], characterized volatile profile of four cultivars of
Southern Highbush blueberry, Snowchase, Primadonna, Jewel and Kestrel,
finding 14 peaks, 11 of which were identified with (E)-2-hexenal as the major
compound.
Most proteins present in fruits have enzymatic functions and are found
mainly in cytoplasmic layer of cells. The protein content of fruits ranges from
less than 1% to more than 1.8% [23, 39]. Enzymes, that catalyze metabolic
processes within fruits, are important proteins in the reactions involved in
ripening and senescence of fruits [38]. Polyphenol oxidase and peroxidase are
enzymes reported to increase the maturity and quality deterioration in
blueberries [35]. However, Kader et al. [43] found that peroxidase plays no
role in the degradation of chlorogenic acid, only polyphenoloxidase is
involved and presents an optimum activity at pH 4.0 [44, 45], which is close to
the blueberry pH, 2.6-3.6 [23].
Lipids constitute less than 1% in most fresh fruits. However, lipids are
very important because constitute the cell membrane, forming the wax surface
(pruine) which contributes to blue color of blueberry and are also present in
cuticle that protects fruits against pathogens and water loss [38].
Blueberries contain a range of nutrients with recognized biological activity
that promote or contribute to health, including vitamins. Among water soluble
vitamins present in blueberry vitamin C, thiamine, riboflavin, niacin,
pantothenic acid, vitamin B6, folate and vitamin B12 are included, while the
fat soluble vitamins blueberry present vitamin A, vitamin K and tocopherols
[35]. These vitamins help the immune system, reduce inflammation and act as
antioxidants [6].
The content of tocopherols and tocotrienols in plants is higher in leaves
and other tissues more exposed to solar light, such as the fruit peels, and lower
in the roots and tissues with limited exposure to light [46]. However, the γ-
tocopherol content found in the peels of blueberries (0.5 – 1.7 mg of γ -
tocopherol.100g-1 peel) [2] can be compared with rich sources of tocopherols,
such as oils, which according to Lampi et al. [46], shows contents of 1.3 –
5 mg of γ -tocopherol.100g-1 olive oil, or sunflower oils with 0.4 – 3.0 mg of γ
-tocopherol.100g-1 oil; However, the same author showed that the total
tocopherol contents of these oils could reach 203 mg of tocopherols. 100g-1 oil
for olive oil and 91 mg of tocopherols.100g-1oil for the sunflower oil.
Fat soluble vitamins like tocopherols are not as sensitive to post-harvest

losses as water soluble vitamins like ascorbic acid [38]. The stability of
ascorbic acid is known to be influenced by many factors including
temperature, exposure to light, damaged fruits, food processing and ascorbic
acid oxidase [35], but when compared to other small fruits blueberry has little
amounts of vitamin C [8].
Blueberries contain the following minerals: calcium, copper, iron,
magnesium, manganese, phosphorus, potassium, selenium, sodium and zinc
[47]. Potassium is the most abundant mineral in fruits and found in high
amounts in blueberry (77 mg/100g of fruit), it usually occurs in combination
with organic acids [6]. High contents are usually associated with an increase in
acidity and improved color of fruits. Phosphorus is the second mineral with
highest amount in blueberry (12mg/100g of fruit), it is a constituent of the
cytoplasmic and nuclear proteins and plays an important role in the
metabolism of carbohydrates and energy transfer. Calcium and magnesium are
found in the same quantity in this fruit (6 mg/100g of fruit). Calcium is one
mineral component associated mainly with cell wall and magnesium is a
component of chlorophyll molecules [35, 38].
Phenolic Compounds
Berries are rich in polyphenols, especially flavonoids (Figure 1) [48].

Blueberry is particularly rich in anthocyanins, flavonols and chlorogenic acid
[49]. Skrede et al. [50] found 27 mg of chlorogenic acid, 40 mg of flavonols
glucosides and 10 mg of procyanidins, per 100 g of fresh Highbush blueberry.
However, different groups of blueberries (Highbush, Rabbiteye, Lowbush) and
different cultivars of them present significant differences in total phenolic
compounds content [11, 51] and also in individual composition of them [2].
Other factor influencing phenolic compounds content of blueberry can be the
degree of maturity, according Prior et al. [52] total phenolic compounds
increases with the evolution of maturation of two cultivars belonging to the
Rabbiteye group, namely Brightwell and Tifblue, by 169 and 113%,
respectively. However, the content of flavonols and hydroxycinnamic acids
decreases as fruit ripens [53]. Location and weather conditions during
blueberry cultivation also exert great influence in phenolic compounds
content, as observed by Pertuzatti et al. [11], who analyzed blueberries
produced in different years and cities and observed that higher the incidence of
UV radiation and consequently less rain during the maturation period, higher
is the phenolic content in fruits.
As can be seen in Table 2 the reported levels of total phenolic compounds
in Highbush, Lowbush, Rabbiteye and Southern Highbush blueberries ranged
between 118 and 461 mg/100 g of fresh fruit, from 299 to 374 mg/100 g of
fresh fruit, from 175 to 592 mg/100 g of fresh fruit, and between 116 and
586 mg/100 g of fresh fruit, respectively. According to Prior et al. [52], the
medium content of total phenolic in blueberry, in dry weight, is about
2500 mg/100g being one of the highest among fruits and vegetables.
Figure 1. Structure of major flavonoids found in blueberry. (a) quercetin, (b) (+)-
catechin, (c) myricetin, (d) (‒)-epicatechin, (e) kaempferol, (f) anthocyanidin.
Table 2. Content (mg/100g fresh fruit) of phenolic compounds and

anthocyanins in blueberry
Cultivar TPHa ACYb References

Rabbiteye cv. Florida 89.9-138.3 7.5-9.6 11
Rabbiteye cv. Powderblue 92.3-816.9 8.8-128 54, 11
Rabbiteye cv. Delite 750.5 72 54
Rabbiteye cv. Woodard 74.6-87.0 5.6-6.5 11
Rabbiteye cv. Brightwell 271.4-457.5 61.8-161.7 52
Rabbiteye cv. Bluebelle 377.3 70.2 2
Rabbiteye cv. Tifblue 361.1-409.3 87.4-154.2 52
Rabbiteye cv. Briteblue 77.9-86 5.7-6.4 11
Rabbiteye cv. Climax 82.1-230.8 6.9-90.8 11,52
Rabbiteye cv. Bluegem 717 242 51
Bilberry 525.0 299.6 52
Highbush cv. Elliot 78.0-115.6 7.4-11.9 11
Highbush cv. Bluecrop (N) 118-461 74-123 37, 51, 52, 55, 56
Highbush cv. Brigitta Blue (N) 246 103 51
Highbush cv. Duke (N) 274-305.9 127.4-278 51, 52, 55
Highbush cv. Rubel (N) 390.5-435 235.4-269 51, 52
Highbush cv. Northsky (S) 175-471 89-164 55, 56
Highbush cv. Northcountry (S) 175-592 131-220 55, 56
Highbush cv. Summit (S) 211 73 51
Highbush cv. O’neal (S) 227.3 92.6 52
Lowbush 299-374 91-255 52, 57
PH, total phenolics; ACY, total anthocyanins; (N), Northern Highbush; (S), Southern
Highbush; a expressed as gallic acid equivalents; b expressed as cyanidin-3-
glucoside equivalents.
Blueberry polyphenols are susceptible to losses during processing and

storage. Significant losses of anthocyanins and procyanidins were observed
during the processing of juices [23, 50, 58, 59], purees [58] and blueberry jam
[60]. Moraes et al. [23] observed that the losses occurred during processing of
juices are usually more severe compared to other methods of preservation, due
to the removal of seeds and peel of the fruit. While jam and jelly processing
results in significant losses of chlorogenic acid, according to Howard et al.
[60] Blueberry jams with and without added sugar had a loss of 15 and 20% of
chlorogenic acid, respectively.
Phenolic Acids
The predominant phenolic acids in berries are hydroxybenzoic and
hydroxycinnamic acids. Among the phenolic compounds analyzed by
Castrejón et al. [52] the hydroxycinnamic acids were the major group found in
blueberries in all maturity stages. This class of phenolic acids is found in all
parts of blueberry and bilberry plants [61].
These acids occur rarely as free acids, they are commonly found in the
conjugated form as esters and glucosides. In blueberries, hydroxybenzoic
acids: gentisic, gallic, protocatechuic, salicylic, syringic and vanillic, are
present in free, ester and glucoside forms, with ester and glucoside form of
predominant salicylic acid. The hydroxycinnamic acids, namely caffeic, m-
coumaric, o-coumaric, p-coumaric and ferulic acids, are also present in the
free, ester and glucoside forms, with sinapic acid and 3-4-dimethoxycinnamic
(veratric) acid present in ester and glucoside forms and dihydroxycinnamic
acid in ester form [48, 62]. In Table 3, are shown phenolic acids found in
blueberries.
Table 3. Total of phenolic acids in blueberry (mg/kg)
Phenolic acids Content References

Gentisic 28.6 63
Gallic 17.6-294.1 2,63
Ellagic 14.0-44.0 64
p-Hydroxybenzoic <1.0 56
o-Pyrocatechuic 0.26 63
Protocatechuic 21.4 63
Salicylic 91.8 63
Syringic 7.82 63
Vanillic 21.0 63
Veratric 1.43 63
Chlorogenic 227-1580 56, 65
Caffeic 1.0-223.0 56, 63, 65, 66
m-coumaric 89.1 63
o-coumaric 40.0 63
p-coumaric 2.0-143.2 63, 65, 66
3,4-dimethoxycinnamic 136.3 63
Ferulic 0.48-53.5 2, 56, 65
2-Hydroxycaffeic 136.1 63
p-hydroxyphenyl acetic 3.08 63
The chlorogenic acid is the predominant hydroxycinnamic acid in

blueberries [4, 5], its content in Northern Highbush, Lowbush and Southern
Highbush groups ranging between 22.7 mg/100g of fresh fruit to 1.58 g/kg of
fresh fruit [56, 65], and may represent up to 96.5% of phenolic acids present in
blueberry [65]. However, because they are one of the least stable phenolic
compounds [67], some studies have reported that there is a great degradation
of this compound in pomace from juice elaboration [68].
In addition to chlorogenic acid, specific derivatives of hydroxybenzoic
and hydroxycinnamics acids identified in blueberry include neochlorogenic
acid, 5-p-coumaroilquinic acid, 5-feruoilquinic acid, malonyl-caffeoilquinic
acid and the β-D-glucosides of caffeic, p-coumaric, ferulic, p-hydroxybenzoic,
protocatechuic and gallic acids [48, 69, 70].
According to Zadernowski et al. [63] the p-coumaric acid is the major
hydroxycinnamic acid in ester form, excluding chlorogenic acid that was not
identified in their study because is unstable under alkaline conditions,
degrading rapidly to caffeic acid, while 3,4-dimethoxycinnamic and 2-
hydroxycaffeic acids are the predominant glucosides in this fruit. Phenolic
acids are mainly in glucosides and esters forms, 56.7% and 40.7%,
respectively, while free acids only represent 2.6% in fruit.
Flavonols
Flavonols most commonly found in blueberries are quercetin, myricetin
and kaempferol, usually these compounds are present in fruits in glucoside
forms, with a sugar linked in C3 position. Glucose and galactose are the most
commonly sugar bonded, even though rutinose, xylose, arabinose and
rhamnose are also found [48]. According to Howard and Hager [48] fourteen
quercetin derivatives, including acylated compounds (caffeic and acetic acid),
together with four myricetin derivatives and two kaempferol derivatives were
found in blueberries.
Blueberry contains higher levels of flavonols than strawberry and
raspberry, quercetin levels ranged between 1.7 and 4.7 mg/100g of fresh fruit,
while myricetin shows a variation between 0.8 and 1.8 mg/100g of fresh fruit
[48]. Quercetin derivatives are the predominant flavonols in blueberry, being
quercetin 3-galactoside the major compound, however, some genotypes
contain appreciable amounts of quercetin 3-rhamnoside [70, 71]. According to
Johnson and Arjmandi [72], the potential of blueberry and apple juice blends,
rich in quercetin, in protection against induction of oxidative DNA damage in
human volunteers have been examined, in one of these studies [73] during four
weeks, volunteers consumed one liter of juice per day, that provided 97 mg of
quercetin, and the results showed an increase in plasma concentration of

quercetin and 20% protection against ex vivo H2O2-provoked oxidative DNA
damage.
The flavonols, quercetin and myricetin, are found mainly in the peel [2,
61]. Blueberry and bilberry leaves are composed, mainly, of quercetin and
kaempferol in such high quantities that made, mainly bilberry leaves, an
excellent source for flavonols extraction and use in cosmetics [61]. The
presence of flavonols, mainly in peel and leaves of fruits, is due to the
activation of flavonoid biosynthesis by solar radiation, which suggests that
these compounds have photoprotective activity [74]. Table 4 shows the
flavonols found in blueberries.
According to Kader et al. [62], often kaempferol is not found in blueberry,
however, differences in varieties [2, 48], stage of maturation, and in climatic
and agronomic factors such as soil nutrition, are responsible for changes in the
composition of flavonols in fruits.
Table 4. Flavonols composition of blueberries
Flavonol References
Myricetin 3-arabinoside 36, 75
Myricetin 3-galactoside 71, 76
Myricetin 3-glucoside 71
Myricetin 3 rhamnoside 71
Quercetin 3-galactoside 36, 62, 71, 75, 76
Quercetin 3-glucoside 36, 62, 70, 71, 75, 76
Quercetin-diglucoside 76
Quercetin 3-rutinoside (Rutin) 70, 71, 76
Quercetin 3-rhamnoside 62
Quercetin 3-arabinoside 70, 76
Quercetin 3-acetylglucoside 76
Quercetin 3-acetylrhamnoside 71
Kaempferol 3-glucuronide 75
Kaempferol 3-glucoside 62, 75, 76
Laricitrin 3-glucoside 70
Syringetin 3-glucoside 70
Anthocyanins
Anthocyanins are the subgroup of flavonoids most widely distributed in
nature and especially abundant in red, purple and blue small fruits, among
them blueberry, which are the main phenolic compounds found [6]. These
pigments are present in both peel and pulp of bilberry and just in the peel of
other species of blueberry, because of this the anthocyanin content of bilberry
is superior to the others, which makes it to be considered one of the best
sources of anthocyanins [61]. Chromatographic profiles have shown that
anthocyanins represent 35–74% of phenolic compounds in blueberry [70].
Studies indicate that blueberries of Rabbiteye group have higher levels of
anthocyanins than Highbush, Southern Highbush and Lowbush blueberries
[48].
According to Table 2 the total anthocyanin content in Highbush,
Lowbush, Rabbiteye and Southern Highbush blueberries ranges between 74
and 278 mg/100 g of fresh fruit, 91 to 255 mg/100 g of fresh fruit, 62 to 242
mg/100 g of fresh fruit, and 73 to 220 mg/100 g of fresh fruit, respectively.
Size is another factor related to the amount of anthocyanins in fruit, several
studies reported that smaller the berry size higher the amount of anthocyanins
is [51, 77].
Anthocyanidins are very unstable and rarely found in free form among
plant tissues [48, 78]. Anthocyanidins occur mainly in glycosylated form
(anthocyanins), because the substitution of sugar increases its stability and
solubility [78]. Due to the diversity of monoglucosides and acylation with
aliphatic acids such as malonic and acetic acid, many anthocyanins have been
identified in different blueberry groups (Table 5). In Highbush blueberry
extracts, 15 non-acylated anthocyanins and 5 acylated anthocyanins were
identified [65, 79], however in Rabbiteye blueberries none acylated
anthocyanin was found and 14 non-acylated anthocyanins were found [80].
Regarding Lowbush blueberries cultivars, 25 anthocyanins (11 acylated) were
reported [77].
According to Cho et al. [71] the percentage of monomeric anthocyanin
distribution in five genotypes of blueberry belonging to Highbush, Southern
Highbush and Rabbiteye groups are based on the following anthocyanidin
structures: delphinidin (27% to 40%), malvidin (22% to 33%), petunidin (19%
to 26%), cyanidin (6% to 14%) and peonidin (1% to 5%); with regard to the
percentage of glycosylated anthocyanin distribution, predominate galactosides,
60 to 67%, followed by arabinosides (26 to 32%) and glucosides (2 to 29%).
Table 5. Anthocyanin composition of blueberries
Anthocyanin References
Delphinidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Delphinidin 3-glucoside 36, 50, 71, 76, 77, 79, 81
Delphinidin 3-arabinoside 36, 50, 71, 75, 76, 77, 79, 81
Cyanidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Cyanidin 3-glucoside 50, 71, 77, 79, 81
Cyanidin 3-arabinoside 71, 76, 77, 79, 81
Petunidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Petunidin 3-glucoside 36, 50, 71, 75, 77, 79, 81
Petunidin 3-arabinoside 36, 71, 75, 76, 77, 79, 81
Peonidin 3-galactoside 50, 71, 76, 77, 79, 81
Peonidin 3-glucoside 79
Peonidin 3-arabinoside 71, 76, 77, 79
Malvidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Malvidin 3-glucoside 36, 50, 71, 75, 76, 77, 79, 81
Malvidin 3-arabinoside 36, 50, 71, 75, 76, 77, 79, 81
Delphinidin 3-(acetyl)-galactoside 50, 77
Delphinidin 3-(6”-acetyl)-glucoside 50, 71, 76, 77
Delphinidin 3-(malonyl)-glucoside 81
Cyanidin 3-(6”-acetyl)-galactoside 77, 81
Cyanidin 3-(6”-acetyl)-glucoside 77, 81
Cyanidin 3-(acetyl)-arabinoside 77
Cyanidin 3-(malonyl)-glucoside 81
Petunidin 3-(acetyl)-galactoside 77
Petunidin 3-(6”-acetyl)-glucoside 71, 76, 77, 81
Petunidin + pentose 81
Peonidin 3-(6”-acetyl)-galactoside 77, 81
Peonidin 3-(6”-acetyl)-glucoside 77, 81
Malvidin 3-(6”-acetyl)-galactoside 50, 77, 81
Malvidin 3-(6”-acetyl)-glucoside 50, 71, 76, 77, 81
Malvidin 3-(malonyl)-glucoside 81
Malvidin + acetyl + hexose 81
In several studies, anthocyanins have shown potential benefits to human

health, due to its anti-inflammatory [82], anti-carcinogenic [78, 83] and anti-
mutagenic activities, the latter being able to block cellular metabolism of
cancer or kill cancer cells [6, 84]. Another benefit associated to anthocyanins
consumption, is related to anti-diabetic effect. Roopchand et al. [7] observed

that mices feed for 13 weeks with soybean flour enriched with anthocyanins
extracted from blueberry juice, upon receiving a diet which provided 4.4 mg of
anthocyanins per day, lowered fasting blood glucose levels, reduced serum
cholesterol and reduced weight gain, demonstrating beneficial effects in the
dietary management of diabetes.
Anthocyanin extracts obtained from three Rabbiteye blueberry cultivars
(Briteblue, Tifblue, Powderblue) showed the greatest anti-proliferation effect
using two colon cancer cell lines (HT-29 and Caco-2), as compared with three
other phenolic fractions used (phenolic acids, flavonols and tannins) obtained
from the same fruit [85]. However, bilberry extracts are more effective at
inhibiting the growth of human promyelocytic leukemia cells (HL60) and
human colon carcinoma cells (HCT116) in vitro [86].
The consumption of bilberry has also been associated, for many years,
with benefits for eyes for improving night vision and for the treatment of
glaucoma and retinopathy, since anthocyanins of this fruit are responsible for
regenerate retinal pigments, increase circulation within the capillaries of the
retina and protect eyes from ultraviolet light [34]. According to Giusti and Jing
[78] anthocyanin extracts of blueberry have been marketed as dietary
supplements. In Europe, bilberry extracts have been prescribed for eyesight,
particularly night vision, this benefit is the primary reason for the product’s
popularity in Korea and Japan, where it is used to relieve computer-induced
eyestrain [34].
Another use of bilberry anthocyanins as a supplement is related to benefits
to circulation, while supplements of blueberry are used to maintain healthy
urinary tract and improve brain functions [78]. Krikorian et al. [82]
investigated the relationship between blueberry supplementation and the
improvements in memory of older adults with mean age of 76 years, and
observed that daily consumption of blueberry juice can confer neurocognitive
benefit, because anthocyanins have been associated with increased neuronal
signaling in brain centers.
Carotenoids
Carotenoids are another class of pigments present in blueberry. According

to Jacques et al. [54] and Pertuzatti et al. [2], their concentrations ranging from
0.3 to 10.8 μg/g of fresh fruit, being found in higher levels in peel than in pulp
of fruit. However, in bilberries the content of carotenoids can reach 37 μg/g of
fresh fruit [87]. Although these concentrations are low compared with other
fruits rich in carotenoids, its presence indicates the diversity of phytochemicals
in this fruit as well as contributing as an aroma precursor, when converted in
norisoprenoids, compounds identified as responsible for grape aroma [35, 88].
In the literature data on carotenoid content in berries is limited. Heinonen
et al. [87] found lutein+zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene
in blueberry and Marinova and Ribarova [89] found lutein, zeaxanthin, β-
cryptoxanthin and β-carotene, and found that there was no presence of α-
carotene because during the fruit ripening occurs hydroxylation of this
carotenoid and then it is converted into lutein, which together with β-
cryptoxanthin and β-carotene are major carotenoids in blueberry [2, 89, 90].
Regarding to bilberry, six carotenoids were found, namely, neoxanthin,
violaxanthin, anteraxanthin, lutein, zeaxanthin and β-carotene [91].
Antioxidant Capacity
Due to the high phytochemical content present in blueberry, several

studies about antioxidant capacity of this fruit began to be performed, Prior et
al. [52] were one of the first authors in studying antioxidant capacity of
blueberry, they used Rabbiteye, Highbush, Southern Highbush and Lowbush
blueberries from different regions of the United States and bilberry, to verify
how much phenolic compounds, anthocyanins, vitamin C, maturity degree and
variety of species influencing the antioxidant capacity of this fruit, and among
their findings observed that blueberry, when compared with other fruits, had
the highest antioxidant capacity (when using the ORAC, oxygen radical
absorbance capacity, method). However, they also observed that there was
much variation in antioxidant capacity due to the difference between blueberry
species (13.9 to 45.9 µmol Trolox equivalents/g of fresh berry) and it is mainly
due to variations in anthocyanin content and other phenolic compounds in
fruit. Prior et al. [52] also reported that increased maturity at harvest increased
ORAC values because of an existing linear relationship between antioxidant
capacity and anthocyanins content (rxy = 0.77) or total phenolics content
(rxy = 0.92) since its total phenolic level and anthocyanin content increased
with increasing maturity.
Through a HPLC-PDA online antioxidant detection system applied to
blueberry by Borges et al. [76], these authors found that by presenting a low
vitamin C level, this compound does not contribute to the antioxidant capacity
of blueberry, which is primarily due to high amount of anthocyanins presents
in fruit, accounting for 84% of antioxidant capacity of blueberry. Among

blueberry anthocyanins, these authors found that the main responsible for
antioxidant capacity was delphinidin 3-galactoside (20.4% ± 0.8%), cyanidin
3-galactoside+delphinidin 3-arabinoside (15.4% ± 0.7%), petunidin 3-
galactoside (12.2% ± 0.7%) and malvidin 3-galactoside (11.7% ± 1.7%).
In another study, the antioxidant properties of blueberry anthocyanins
were analyzed, in vitro and in vivo using red blood cell resistant to reactive
oxygen species (ROS). In vitro incubation with anthocyanins and
hydroxycinnamic acids (0.5 and 0.05 mg/mL respectively) significantly
increased red blood cell resistance and induced production of ROS. A similar
protective effect was also observed in vivo by oral supplementation of mices
with 100 mg/mL. Only group fed with anthocyanins had a significant
protection at 6 and 24 h after supplementation, but this was not consistent with
the measurements of anthocyanin plasma levels. In fact, anthocyanin plasma
concentrations were higher after 1 h, decreasing considerably after 6 h and not
being detected after 24 h. The difference in absorption between anthocyanins
and hydroxycinnamic acids probably contributed to the differences observed
in protection capacity of red blood cells. This protection has a positive role,
after blueberry consumption, against ROS formation in red blood cells in vivo
[92].
Currently, there are several studies that evaluate antioxidant capacity of
blueberry and other fruits. However, in the last years, researches began to use
different assays to measure antioxidant capacity in fruits and extracts, once it
varies according to the analytical method used due to the different antioxidant
mechanisms presented by different compounds, or even when the same
mechanism is evaluated the antioxidant capacity may be different depending
on the free radical used.
García-Alonso et al. [93] when evaluating the antioxidant capacity of
twenty-eight fruits from Spain observed that blueberry has obtained the fifth
highest antioxidant capacity (strawberry > raspberry > cherry > blackberry >
blueberry) by TBARS assay, which determines the inhibition of lipidic
peroxidation, and the same fruit obtained the third highest antioxidant
capacity, values lower than persimmon and blackberry, when using the method
of free radical capture ABTS which according to Rice-Evans et al. [94] was
detected in blueberries as the main phenolic compounds in aqueous extract:
quercetin > cyanidin > catechin.
When used the FRAP (ferric reducing antioxidant power) assay, Borges et
al. [76] found that blueberry obtained the second highest antioxidant capacity
(black currant > blueberry > raspberry > red currant > cranberry), ranging
from 18.5 to 161.4 µmol of Fe2+/g of fresh fruit [51, 56], this wide range of
results occurs due to different factors such as, cultivar, maturity degree, season
and storage conditions [76].
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Chapter 2
BIOACTIVE COMPOUNDS, COLOR

AND PHYSICOCHEMICAL PARAMETERS
OF BLUEBERRIES
Paula Becker Pertuzatti1,, Milene Teixeira Barcia2,

Andressa Carolina Jacques3 and Rui Carlos Zambiazi4
1
Federal University of Mato Grosso (UFMT), Barra do Garcas , MT, Brazil
2
Federal University of Santa Maria (UFSM), Santa Maria, RS, Brazil
3
Federal University of Pampa (Unipampa), Bagé, RS, Brazil
4
Federal University of Pelotas (UFPel), Pelotas, RS, Brazil
ABSTRACT
According to Food and Agriculture Organization (FAO) statistics,
from 1970 to 2011 the world production of blueberries increased
approximately 7 times (FAOSTAT, 2012). According to the National
Agricultural Statistics Service (NASS) of the United States Department
of Agriculture (USDA), the United States is the world leading producer
of blueberries, being the second most produced and commercialized small
fruit in the country after strawberry. There are three main groups of
blueberries commercialized and produced in the world: the lowbush, the
highbush and the Rabbiteye (RASEIRA e ANTUNES, 2004). The high

Corresponding author: Paula Becker Pertuzatti. Universidade Federal de Mato Grosso (UFMT),
Av.Senador Valdon Varjão 6390, Barra do Garças - MT, 78600-000, Brazil. E-mail:
paulapertuzatti@yahoo.com.br.
32 P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.
amount of bioactive compounds present in both the pulp and in the peel
of the blueberries, makes it a fresh fruit rich in natural antioxidants.
Therefore, the identification and quantification of the major bioactive
compounds and some physicochemical parameters in the peel, pulp and
entire fruit of six blueberry cultivars belonging to the Rabbiteye group are
discussed in this chapter. Phenolic compounds, anthocyanins, color,
hydrolyzed and condensed tannins, carotenoids and physicochemical
analyses were done. There were evaluated Powderblue, Briteblue,
Bluebelle, Climax, Delite and Woodard cultivars. The blueberry fruits
showed as rich sources of phenolic compounds and anthocyanins, besides
to have considerable amounts carotenoids and tannins. All
phytochemicals analyzed were found at the highest levels in the peels of
blueberry cultivars tested.
Keywords: anthocyanins, phenolic compounds, tannins, carotenoids, acidity,

soluble solids
INTRODUCTION
Statistics data from 1970 to 2011 relate that the world production of
blueberries increased approximately 7 times [1]. The United States follows as
the world leading producer of this fruit. The increase in blueberry production
can be related with the special attention given to blueberry after that Prior et
al. [2] found its high antioxidant capacity, related with the large amount of
bioactive constituents such as phenolic compounds, vitamins and carotenoids.
Some quality factors influence the fresh-market value and the suitability
of the berries for processing. Color, related to the anthocyanins content, and
taste, related to the physicochemical composition are some of these. Studies by
Bargmann, Wu and Powers [3] have established a significant relation between
low titratable acidity/high pH and the acceptability testing of a few varieties.
Furthermore, varieties that showed higher absorbance, due to the darker color,
were judged superior in appearance.
Therefore, this chapter aimed to discuss about the identification and
quantification of the major bioactive compounds in the peel, pulp and entire
fruit of six blueberry cultivars belonging to the group Rabbiteye (Powderblue,
Climax, Briteblue, Bluebelle, Delite and Woodard), from the 2007/2008
harvest, from the city of Pelotas, RS, Brazil. The material was donated by
Embrapa Temperate Climate (Brazil).
Bioactive Compounds, Color and Physicochemical Parameters … 33
BIOACTIVE COMPOUNDS
Bioactive compounds from plants may be nutritive or non-nutritive
substances which ones have important role when are ingested, due to the
biological activity such as antioxidant, anticarcinogenic, antimicrobial and
anti-inflammatory effects. Depending on the biological activity attributed to
the bioactive compounds, diets with high ingestion of fruits and vegetables has
been correlated with decrease of incidence of degenerative diseases.
Phenolic Compounds
Among the bioactive compounds found in blueberries, phenolic

compounds are the most abundant. These compounds are secondary
metabolites; therefore, they do not participate in metabolic pathways
responsible for growth and reproduction, and their nature and concentration
vary greatly [4]. These compounds have a number of beneficial health
properties related to their antioxidant capacity [5] which is based mainly in the
molecule resonance capacity as a function of this property phenolic
compounds are able to donating a hydrogen atom of a hydroxyl group (OH) of
its aromatic structure to a free radical and still maintain stability.
The determination of total phenolic content allows one to estimate the
content of all compounds belonging to the subclass of phenolic compounds
present in a sample, i.e., that have at least one aromatic ring attached to one or
more hydroxyl groups in their structure, called phenolic ring. For phenolic
compounds determination, the Folin-ciocalteau method [6] is used. In our
studies it was used extracts that were diluted 1:100, and absorption was
measured at 735 nm. TPC (Total phenolic content) was expressed as mg of
gallic acid.100 g-1 of fresh-frozen fruit (Table 1). The data were analyzed for
their homoscedasticity and subsequently submitted to an analysis of variance
(P ≤ 0.05). The effects of the cultivar and the different parts of the fruit were
evaluated by a comparison of the means using the Tukey test (P ≤ 0.05).
The results showed that the total phenolic compounds differ significantly
between different blueberry parts (peel, pulp and whole fruit) as well as
between cultivars. The pulp showed a phenolic compounds content 72% lower
than that found in the peel of the Delite cultivar and up to 90% lower for the
content of the Bluebelle cultivar peel, indicating that a high concentration of
phenolic compounds is present in the peel of the blueberries. The phenolic
compounds content of the whole fruit ranged from 612.61 to 876.53 mg
GAE.100 g-1, with “Powderblue” and “Bluebelle” cultivars showing the

highest content of these compounds. These results are similar to those reported
by Carlson [7], who when working with seven different blueberry cultivars,
found that higher content of phenolic compounds were found in Powderblue
cultivar. Differences in the concentration of phenolic compounds are normal
among cultivars.
By comparing the values of total phenolic compounds of blueberry
cultivars to the study described by Jacques et al. [8] and Moyer et al. [9], a
similarity is observed in both. Analyzing small fruits of 107 genotypes of
Vaccinium, Rubus and Ribes, Moyer et al. [9] reported phenolic compound
content of 870 ± 20 mg GAE.100 g-1 for native cultivars originating from
Florida and Georgia, both belonging to the Rabbiteye group. The result is very
similar to that found for the Bluebelle cultivar, which is also originally from
Georgia [10], and the Powderblue cultivar belonging to the same group.
Phenolic compounds content similar to the cultivar Climax (612.61 mg GAE.
100 g-1) was also found for the Highbush group cultivar. In the work of
Jacques et al. [8], who analyzed various fruits, including blueberry,
blackberry, Butia capitata, loquat and pitanga (varieties: orange, purple and
red), a content of 816.9 mg GAE.100 g-1 was found for blueberry Powderblue
cultivar and 750.5 mg GAE.100 g-1 for the blueberry Delite cultivar. The same
authors found that among the evaluated fruits, the highest phenolic compounds
content was observed in the blueberry, which also showed the highest
anthocyanin content.
When compared with the values of Souza [11], it is clear that the whole
fruits analyzed in this study had higher values, since the authors report that the
blueberry has 305.38 mg GAE.100 g-1 fresh fruit.
Also analyzing the Pearson correlation coefficient, total phenolic
compounds showed a positive correlation (0.7) with the value of titratable
acidity.
Anthocyanins
Among the different subclasses belonging to the phenolic compounds

group, anthocyanins deserve special attention because they are found in large
quantities in blueberries, being the main pigments responsible for the color of
this fruit. Its color can range from bright red and purple/blue depending on
which electron donor groups (methoxy or hydroxyl) are bonded to the
aglycones, also called anthocyanidins, and their composition and
concentration. Only five types of anthocyanins are found in blueberries:

delphinidin, malvidin, petunidin, peonidin and cyanidin. However, due to the
instability, these molecules are most commonly found in the form of
anthocyanins (glycosylated form) and also in acylated form.
The quantification of anthocyanins in this study was performed according
to the Lees and Francis method [12]. The samples were extracted with ethanol
solution of pH 1.0 and the absorbance was measured at 520 nm in an
Ultrospec 2000 UV/Visible (Pharmacia Biotech) spectrophotometer. The ACY
(Total anthocyanins) was based on a Cyanidin 3-glucoside molar extinction
coefficient of 26900 and a molecular weight of 449.2. The total content was
expressed in terms of mg of anthocyanin 100 g-1 of fresh-frozen fruit (Table
1).
The total anthocyanin content ranged from 70.2 mg CYD-3-G 100 g-1 in
the Bluebelle cultivar to 217.55 mg CYD-3-G 100 g-1 for Climax cultivars,
which was the only one to differ significantly from the other cultivars. The
content was lower than that found by Su and Chien [13], who, on evaluating
blueberry of the Rabbiteye group, found an anthocyanin content of 363 ± 6.7
mg CYD-3-G.100 g-1. However, when compared the anthocyanin content of
the Climax cultivar (217.55 mg CYD-3-G.100 g-1) with the content found in
the Bluegem cultivar (242 mg CYD-3-G.100 g-1) belonging to the same group
(Rabbiteye), by Moyer et al. [9], great similarity can be seen. This similarity
extends to the comparison of the ACY/TPC relationship, since the authors
report that the anthocyanin content of “Bluegem” cultivar represents 34% of
all phenolic compounds, whereas in the “Climax” cultivar it represents 36% of
the total phenolic compounds in the fruit.
However, in this study the anthocyanin content was higher than that
reported in the study by Pertuzatti et al. [14], who reported an average
anthocyanin content of 218 mg CYD-3-G.100 g-1 (dry weight) for different
blueberry cultivars (2010/2011 harvest).
The highest anthocyanin content was found in the fruit peel in all
cultivars, an expected result because the blueberry has a skin with coloring and
accented blue tones and clear pulp.
The anthocyanins content in blueberry pulps showed no significant
differences among cultivars. Riihinem et al. [15], evaluating the
phytochemical content in different parts of blueberry, found a content of 1.9
mg CYD-3-G.100 g-1 in the pulp of the fruit, similar to the values found for
the Powderblue and Climax cultivars. However, the level that the authors
found in the fruit peel (622.3 mg CYD-3-G.100 g-1) was almost double that
found in this study (315.35-496.56 mg CYD-3-G.100 g-1).
Table 1. Total content of anthocyanins and phenolic compounds in peel,

pulp and whole fruit of blueberry cultivars
Cultivar Parts of the fruit

Peel Pulp Whole fruit
Total Anthocyanins (mg GYD-3-G 100 g-1)
Woodard 343.88 cA1/ 6.72 aC 108.97 bB
Powderblue 426.37 bA 2.22 aC 108.07 bB
Bluebelle 460.70 abA 0.97 aC 70.20 bB
Briteblue 458.89 abA 3.06 aC 117.62 bB
Climax 496.56 aA 2.61 aC 217.55 aB
Delite 315.35 cA 12.41 aC 109.55 bB
Total Phenolic Content (mg GAE 100 g-1)
Woodard 1418.57 cA 333.65 abC 816.83 bB
Powderblue 1637.58 aA 215.40 dC 876.53 aB
Bluebelle 1531.59 bA 155.51 eC 858.52 aB
Briteblue 1544.25 bA 324.39 bC 814.20 bB
Climax 1005.17 eA 264.30 cC 612.61 cB
Delite 1282.63 dA 359.55 aC 791.36 bB
ACY/TPC relationship (%)
Woodard 24.24 bA 2.01 aC 13.34 bB
Powderblue 26.05 bA 1.03 aC 12.33 bB
Bluebelle 30.07 bA 0.62 aC 8.18 bB
Briteblue 29.71 bA 0.95 aC 14.45 bB
Climax 49.41 aA 0.99 aC 35.51 aB
Delite 24.58 bA 3.45 aC 13.84 bB
1/
Means followed by the same lower case letter in the column and upper case in the
row do not differ by Tukey test (p ≤ 0.05).
GYD-3-G = cyanidin-3-glucoside; GAE = gallic acid equivalent; ACY = total
anthocyanins; TPC = total phenolic compounds.
Tannins
Among the bioactive compounds, tannins are also highlighted, which, like
the other phenolic compounds, are derived from the secondary metabolism of
plants. They are present in most plants and can vary in concentration
depending on the age and size of the plant part collected, the time or even the
collection site. The tannins can be categorized into hydrolyzable, and non-
hydrolyzable or condensed tannins [16, 17].
The hydrolyzable tannins are esters of phenolic acids (gallic, caffeic,
ellagic acids) linked to simple sugars. These compounds have smaller
molecular chains than condensed tannins and can be hydrolyzed more easily,
just by the action of dilute acids [18, 19]. Typically, hydrolysable tannins are
classified in gallotannins which produce gallic acid after hydrolysis and
ellagitannins which produce ellagic acid [20]. However, unlike other berries
such as raspberry, strawberry and blackberries, blueberries do not contain
ellagitannins nor other derivatives of ellagic acid. The ellagic acid content in
blueberry is lower than 5 mg.100 g-1 fresh fruit [21] after acid hydrolysis.
For the determination of hydrolyzable tannins in blueberry, the method
adapted from Brune et al. [22], which consists of the extraction of tannin with
methyl alcohol, was used. The methanol extract was then mixed with a
reaction solution of ferric ammonium sulfate, consisting of 89% urea buffer:
acetate, 10% arabic gum solution 1% in deionized water and 1% ferric
ammonium sulfate solution 5% in hydrochloric acid 1 mol.L-1. The absorbance
was read at 578 nm in a spectrophotometer (Ultrospec 2000). The
determination of the content of hydrolysable tannins was performed through a
gallic acid standard curve and results were expressed in mg of gallic acid.100
g-1 sample.
The non-hydrolyzable or condensed tannins, also known as
proanthocyanidins, are compounds formed by the polymerization of flavonoid
units, predominantly catechin and are present in a wide variety of foods, and
can be divided into two main classes that include procyanidins, mixtures of
oligomers and polymers composed of units of (+) -catechin and/or (-)-
epicatechins, and propelargonidins composed exclusively of epiafzelechin
units [23]. However, blueberries exclusively have procyanidins, which are
considered one of the major phenolic compounds present in the fruit pulp [15,
19, 21, 24]. These compounds are ranked according to their degree of
polymerization (DP) where DP = 1 indicates a monomer, while DP = 2-10 and
DP > 10 refer to oligomers and polymers, respectively [25].
The monomer units of procyanidins are connected via a C4-C8 or C4-C6
(type B) bond, and can coexist with a C2-O-C7 bond or, less frequently, with
C2-O-C5 bond (type A) [23]. Hwang et al. [26] found 300 mg procyanidin
B1.100 g-1 fresh fruit in blueberry extracts, well below the contents found for
black chokeberries (Aronia melanocarpa) of 2.5 g.100 g-1 fresh fruit, which is
known for its high astringency.
Procyanidin content in blueberries of Highbush and Lowbush group

varieties are often considered alike, ranging between 33-180 mg.100-1 of fresh
fruit for the Highbush group and 57-332 mg.100-1 fresh fruit in the Lowbush
group [21, 27]. Flavonol dimers represent the majority of flavonols present in
blueberries, approximately 24% of flavonols, while hexamers, monomers and
heptamers, represent an average percentage of 12.6, 11.2 and 11% respectively
[27]. The polymeric procyanidins of Lowbush blueberries were characterized
by Gu et al. [25], who reported that the degree of polymerization ranges from
20-114, with epicatechin representing 100% of the extension units, and
catechin and epicatechin representing 67% and 33% of the terminal units,
respectively.
According to Eskin and Snait [28], a number of proanthocyanidin
fractions were separated from blueberry extracts. Of these, only
proanthocyanidin oligomers of high molecular weight exhibited
antiproliferative and anti-adhesion properties. It was found that two fractions
composed predominantly of four to eight proanthocyanidin oligomers bonded
with an average degree of polymerization of 3.25 and 5.65 prevented
adherence of the organism responsible for urinary tract infections, Escherichia
coli. However, only the fraction with 5.65 proliferation showed
antiproliferative activity against human prostate cancer and in cancer cells of
mice liver.
To perform the determination of tannins, the method adapted from Price et
al. [29] was used. It consists of the extraction step of tannins with methanol,
and to the methanolic extract, a solution of 1:1 vanillin 1% in methyl alcohol
and 4% hydrochloric acid in methyl alcohol were added. The reading of
absorbance was done at 500 nm. The determination of the content of tannins
was performed using a catechin standard curve and results were expressed in
mg catechin.100 g-1 sample.
The content of condensed, hydrolyzable and total tannins in different parts
of Rabbiteye group blueberry cultivars are shown in Table 2.
Among the analyzed cultivars, “Powderblue” had the highest tannin
content, differing significantly from the others. Tannin content in the whole
fruit represents 6-22% of the content of phenolic compounds. For all cultivars,
higher tannin content (condensed, hydrolyzable and total) are present in the
peel, with the condensed tannin content being 140 times higher than the pulp
content on average. For total tannins, the average is 120 times higher than the
content present in the peel. For hydrolysable tannins, this average is around 12
times higher than the content of tannins in the peel, when compared to the
tannin content in the pulp. Only the pulp of the fruits showed no significant
differences in tannin content (condensed, hydrolyzable and total), between

different cultivars.
The condensed tannin content found in this study are similar to the content
found by Yi et al. [30], who worked with the Powderblue and Briteblue
cultivars. These authors found condensed tannin contents of 86.9 and 87.9 mg
CAE 100 g-1 respectively for these cultivars.
The blueberry cultivars showed an average condensed tannin content 90
times the hydrolysable tannin content. Sensorially this is a positive factor
because the condensed tannins have a lower complexing capacity with proteins
than hydrolysable tannins, resulting in lower astringency.
Table 2. Content of condensed, hydrolyzable and total tannins in peel,

pulp and whole fruit of blueberry cultivars

Condensed tannins (mg CAE 100 g-1)
Woodard 623.94 aA1/ 2.97 aC 85.57 bB
Powderblue 523.38 bA 3.02 aC 194.37 aB
Bluebelle 182.10 eA 4.92 aC 70.71 bB
Briteblue 432.75 cA 4.29 aC 71.62 bB
Climax 262.16 dA 0.93 aB 37.53 bB
Delite 329.50 dA 7.44 aB 44.69 bB
Hydrolyzable tannins (mg GAE 100 g-1)
Woodard 6.16 aA 0.30 aC 1.47 abB
Powderblue 0.99 dA 0.12 aA 0.92 abA
Bluebelle 0.50 dA 0.10 aA 0.45 bA
Briteblue 3.80 bA 0.30 aB 1.19 abB
Climax 2.40 cA 0.26 aB 1.60 aA
Delite 3.04 bcA 0.20 aC 1.12 abB
Total tannins (mg 100 g-1)
Woodard 630.10 aA 3.27 aC 87.05 bB
Powderblue 524.37 bA 3.13 aC 195.30 aB
Bluebelle 182.60 eA 5.02 aC 71.16 bB
Briteblue 436.55 cA 4.59 aC 72.82 bB
Climax 264.56 dA 1.20 aB 39.12 bB
Delite 332.54 dA 7.64 aB 45.80 bB
1/
CAE = catechin equivalent; GAE = gallic acid equivalent.
Carotenoids
Another group of bioactive compounds in blueberries are the carotenoids.

These compounds have non-polar nature due to their tetraterpenic structure,
which may contain terminal cyclic groups or not have any cyclization. One of
its distinct characteristics is the extensive system of conjugated double bonds,
which act as light absorption chromophores, thus being responsible for the
colors yellow, orange and red that these compounds confer to many foods [31,
32, 33].
In the present study the carotenoids were extracted with cold acetone and
partitioned with petroleum ether according to Rodriguez-Amaya [32]. The
absorbance reading of the ether extract was performed in an Ultrospec 2000
UV/Visible (Pharmacia Biotech) spectrophotometer at a wavelength of 450
nm. The total carotenoid was based on molar extinction coefficient of the β-
carotene, 2500, and molecular weight of 536.9. The carotenoid content was
expressed in mg of β-caroteno.100 g-1 of fresh-fruit. From the carotenoid
evaluation of blueberry, considering its peel, pulp and whole fruit (Table 3), it
can be observed that there was no significant difference between the content of
the pulp and the whole fruit of the analyzed cultivars; however, the carotenoid
content in the peel in both cultivars showed significant difference, with
“Briteblue” cultivar having the highest content (28.38 µg of β-carotene.g-1)
and “Delite” cultivar which presented the lower content of these compounds
(3.8 µg of β-caroteno.g-1). The high content of carotenoids in the peel of the
fruit has been documented in several studies with caja, mandarin and melon
[32], since in the same manner as phenolics, carotenoids have phytoprotective
action.
For the carotenoid content present in blueberry, it is observed that this
fruit contains a small amount of this pigment (0.16-0.66 µg of β-carotene g-1),
which supports data shown by Jacques et al. [8] who on analyzing various
fruits, they found that the blueberry was the one with the lowest content of
carotenoids (1.4 µg of β-carotene g-1 fresh fruit). According to Lima et al. [34],
fruits whose main compounds belong to the class of anthocyanins, the
carotenoid content reduces during ripening, consequently, fruits like blueberry,
have small amounts of carotenoids. However, in the peel of cultivars such as
Briteblue and Bluebelle, existing contents are considerable and can be
compared with the content found in fruits such as butia (28 µg of β-carotene g-
1 fresh fruit) and loquat (24 µg of β- carotene g-1 fresh Fruit) [8], in which the
carotenoids were found as the major pigments.

Table 3. Carotenoid content in peel, pulp and whole fruit of

blueberry cultivars

Total carotenoids (µg de β-caroteno.g-1)
Woodard 6.25 cA1/ 0.55 aB 0.66 aB
Powderblue 7.57 cA 0.47 aB 0.55 aB
Bluebelle 14.71 bA 0.41 aB 0.60 aB
Briteblue 28.38 aA 0.46 aB 0.34 aB
Climax 6.47 cA 0.65 aB 0.16 aB
Delite 3.80 dA 0.30 aB 0.30 aB
1/
COLOR
Color is a primary indicator of food quality, because it has great
importance in evaluating the degree of maturity and freshness of fruits, the
storage conditions, postharvest handling and transportation. Therefore, color is
characterized as a decisive factor utilized at the time of choice and acceptance
of a product, especially blueberry which has as its remarkable characteristic
blue color.
Since color is a parameter used to describe quality, its determination is
useful to correlate with the concentration of the pigments present in the fruit.
Anthocyanin quantification methods and color indices have been
established and used in industrial control applications [35]. The data from
instrumental color evaluation in this study were performed with a colorimeter
(Minolta CR-300) for six blueberry fruit cultivars and are shown in Table 4.
The samples (about 3 g, equivalent to the average weight of a fruit) were
placed in Petri dishes of 5 cm diameter and 2 cm in height. The measured
color parameters were: L*, a* and b*, where L* indicates brightness (0 =
black and 100 = white) and a* and b* represent chromaticity coordinates (+ a*
= red, - a* = green, + b* = yellow, - b* = blue). The color parameters were
converted to color angle H° = tan-1 b/a, indicating the angle Hue (H°) of the
sample (0° or 360° = red; 90° = yellow; 180° = green; 270° = blue) [36].
Table 4. Brightness values (L), chromaticity coordinates

a* and b* color values and angles of the peel and pulp of
Rabbiteye blueberry cultivars
Parts of the fruit

Cultivar Peel Pulp
*1/
L
Woodard 16,70 bB2/ 29,25 cdA
Powderblue 22,57 aB 47,97 aA
Bluebelle 17,15 bB 31,82 cA
Briteblue 16,67 bB 28,38 cdA
Climax 19,63 abB 39,96 bA
Delite 18,54 abB 25,24 dA
a*
Woodard 10,12 aA 25,49 aB
Powderblue 8,51 aA 4,07 cA
Bluebelle 9,40 aB 23,40 aA
Briteblue 10,71 aA 14,32 bA
Climax 4,54 aB 13,89 bA
Delite 8,46 aA 27,46 aB
b*
Woodard - 8,62 bB 8,26 bA
Powderblue - 7,10 abB 8,29 bA
Bluebelle - 8,02 bB 4,87 dA
Briteblue - 8,38 bB 5,25 cdA
Climax - 5,41 aB 12,13 aA
Delite - 8,45 bB 7,21 bcA
h°
Woodard 319,29 aA 18,47 cB
Powderblue 318,56 aA 63,77 aB
Bluebelle 319,08 aA 11,86 cB
Briteblue 321,53 aA 20,16 cB
Climax 309,97 aA 41,48 bB
Delite 313,13 aA 14,70 cB
1/
L* (0 = black, 100 = white); a* (+ a = red, -a = green); b* (+ b = yellow, - b = blue);
h° angle (0° = red, 90° = yellow, 180° = green, 360° = blue).
2/
Means followed by the same lower case letter in the column and uppercase in the
The hue is an attribute by which the colors are identified where positive
values of a* indicate red, while negative values represent green colors.
Similarly, positive b* values relate to yellow and negative values express the
blue colors. The luminosity or brightness (L*) is presented as an attribute that
describes a gray scale of the measure, characterizing the color as lighter or
darker (between black and white) in a range of measurement ranging from 0-
100. The meeting of the three values sets the color of the product [37]. Thus, it
can be seen by the data of Table 4, Climax cultivar showed lower values of b*
and L, meaning that the peel of this fruit has a darker shade of blue, which is
very favorable commercially.
With respect to the values of Hue angle, it can be seen that all the peels
had values near blue (h° = 360), which is also very favorable for blueberry,
considering that consumers prefer blue fruits with intense shades. The fruit
pulp presented values between red and yellow, where the values varied
between 11.86-63.77.
When color is compared with the determination of anthocyanins and
phenolic compounds, there was a strong negative correlation, according to the
Pearson correlation coefficient between the b* value and the determination of
phenolic compounds (- 0,8). This means that as the concentration of these
phytochemicals increases the b* values reduce, i.e., the fruit gets bluer. With
the values of h°, the opposite occurred where there was a Strong positive
correlation with the content of phenolic compounds (0,8); Therefore, as the
amount of these compounds increases, the Hue angle also increases. The h°
angle is also positively correlated with the pH, this is due to the presence of
anthocyanins in the fruit, because as the pH increases, anthocyanins become
blue due to the deprotonation of the structure resulting in an increase in the h°,
thus being closest to the value for the blue color. However, as the pH
decreases anthocyanins are mostly in the form of flavylium cation and
acquires a red color, and Hue angle decreases, getting closer to this color.
Figure 1 is a graphical representation of the values of L, a* and b* in the
CIELAB scale obtained in a Minolta CR-300 colorimeter, for the peel and
pulp of blueberry cultivars.
By observing Figure 1 (b) one realizes that all blueberry pulps were
arranged in the first quadrant of the circle, however the display of color in a
graphical representation of two dimensions for this part of the fruit is not
enough because it does not take into account the luminosity values. Thus, it is
noticed (Figure 1 (a)) that since the values of L* are higher in the pulp, they
characterize the pulp as whitish, while the peel, which has smaller values of
L*, has a blue tone, tending to purple, characteristic of anthocyanins.
Figure 1. Graphic representation of the values of L, a* and b* obtained in Minolta CR-

300 colorimeter; (a) colored solid in three dimensions (b) location of the peel and pulp
of Rabbiteye blueberry cultivars in colorimetric space.
From Figure 1(b), it can also be observed that only the climax cultivar
presents difference in coloration, which was also statistically observed (Table
4). However, the location of the colors of the samples in the colorimetric
space, or even the statistic results of the color information are not sufficient to
express whether the color differences are possible to be distinguished visually,
but these differences may be calculated by the distances between two points in
three dimensional space (ΔE) defined by the parameters a*, b* and L*, and
these values can be compared with the classification used by the paint industry
for the perception of the human eye. In general, color differences of two
overlapping samples can be distinguished in ΔE values above 0.2-0.5 [37].
Mathematically, the colorimetric parameter ΔE is described by Equation 1 and
the values for the analyzed blueberry cultivars are shown in Table 5.
ΔEab = √(ΔL)2 + (Δa)2 + (Δb)2 (1)
where: ΔE = Color difference, ΔL = Difference in values of L*, Δa =

Difference in a* values, Δb = Difference in b* values.
When comparing the values in Table 5 with NORMA DIN 6174 [38], it is
clear that the color difference between the pulps of blueberries of different
cultivars was higher than the color difference between the peels, because while
the pulps gave values of ΔE from “distinguishable,” the peels showed
differences considered “very small” as in the color relationship between the
peels of Briteblue and Woodard cultivars. These results can be compared to
statistical analysis where the data of the pulps showed significant differences
among all the parameters (L*, a*, b* and h°) and from the data of the peel, the
difference related to L* and b* parameters was observed.
With instrumental color analysis one can see the importance of the
pigments in the constitution of the fruit. Therefore, there was a strong negative
correlation between the value of b* and the determination of phenolic
compounds, and a strong positive correlation between the value of ho with the
content of phenolic compounds and anthocyanins.
PHYSICOCHEMICAL PARAMETERS: SOLUBLE SOLIDS,

PH AND TITRATABLE ACIDITY
Determination of pH and acidity in blueberry, as well as in other fruits, is

related to the parameters for assessing the fruit ripeness. This is because the
pH of the fruit increases and the acidity decreases as the fruit ripens due to
degradation of organic acids with the evolution of maturation becoming acidic
salts [39].
The soluble solids (SS) are also indicators of the degree of maturation of
the fruit, because as the fruit ripens the soluble solids content increases, by
increasing the content of sugars. However, this measure is related to all solids
dissolved in water, including salts, acids, proteins and other soluble
compounds in addition to sugars.
Table 5. ΔE values for pulp and peel of blueberry Rabbiteye cultivars
Relationship ΔE Classification ΔE Classification

between cultivars pulp pulp peel peel
P-Climax 13.2 Very large 5.2 Easily
distinguishable
P-Brite 22.3 Very large 6.4 Very large
P-Blue 25.4 Very large 5.6 Easily
distinguishable
P-Delite 32.6 Very large 4.3 Easily
distinguishable
P-Wood 28.4 Very large 6.3 Very large
Climax-Brite 13.5 Very large 7.5 Very large
Climax-Blue 14.5 Very large 6.0 Easily
distinguishable
Climax-Delite 20.6 Very large 5.1 Easily
distinguishable
Climax-Wood 16.3 Very large 7.1 Very large
Brite-Blue 6.0 Easily 3.1 Easily
distinguishable distinguishable
Brite-Delite 13.7 Very large 2.9 Distinguishable
Brite-Wood 3.9 Easily 0.5 Very small
distinguishable
Blue-Delite 5.1 Easily 1.0 Small
distinguishable
Blue-Wood 4.7 Easily 1.0 Small
distinguishable
Delite-Wood 2.2 Distinguishable 1.7 Distinguishable
P = Powderblue; Brite = Briteblue; Blue = Bluebelle; Wood = Woodard.
Table 6. Physicochemical parameters of peel, pulp and whole fruit of

six blueberry cultivars

pH
Woodard 3.25 aB 1/ 2.29 cdC 3.57 aA
Powderblue 3.18 abA 2.58 bB 3.21 bcA
Bluebelle 2.96 cA 2.12 dB 2.87 dA
Briteblue 3.07 bcA 2.83 aB 3.11 cA
Climax 3.25 aA 2.68 abB 3.35 bA
Delite 3.29 aA 2.38 cB 3.24 bcA
Titratable Acidity (% of malic acid)
Woodard 0.10 aA 0.06 aB 0.09 aA
Powderblue 0.08 abA 0.05 abB 0.07 abAB
Bluebelle 0.10 aA 0.06 aB 0.06 bcB
Briteblue 0.09 aA 0.05 abB 0.06 bcAB
Climax 0.06 bA 0.03 bB 0.05 cAB
Delite 0.06 bA 0.05 abB 0.06 bcAB
Soluble Solids (°Brix)
Woodard 16.13 aA 8.87 cdC 12.93 cB
Powderblue 14.80 bA 11.87 bB 14.40 bA
Bluebelle 14.87 bA 8.20 dC 14.07 bB
Briteblue 13.47 cA 9.13 cC 12.33 cdB
Climax 16.00 aB 13.97 aC 17.87 aA
Delite 14.87 bA 11.60 bB 11.87 dB
1/
Means followed by the same lower case letter in the column and uppercase in the
Determination of SS was carried out in a refractometer bench (Analytik

Jena). The pH was measured using a digital potentiometer (pHmeter Digimed
MD-20). The titratable acidity was performed by titration with 0.1N NaOH to
pH 8.1.
From in natura blueberries (Vaccinium ashei Reade) of six cultivars:
Powderblue, Climax, Briteblue, Bluebelle, Delite and Woodard, 2007/2008
harvest, grown in the region of Pelotas, Brazil, similar results can be observed
with other authors from other regions. three distinct Blueberry parts were
evaluated: peel, pulp and whole fruit (Table 6).
The pH values of the peel and the whole fruit of blueberry cultivar did not
differ significantly from each other, staying in the range of 2.87-3.57.
However, when the pH values of the different parts of the fruit were observed
between the different cultivars, it was observed that there were differences in
the comparisons. It is also observed that for all cultivars, except the Delite, the
pH value of the pulp (2.38) was lower than that of the other parts of the fruit.
Comparing the values of this study with pH values found by Moraes et al.
[40], who worked with of Delite, Bluebelle and Woodard blueberry cultivars
(pH 2.67; 2.59 and 2.62, respectively), the pH of the fruits of this study
showed higher values, however when compared with the data obtained by
Perkins-Veazie et al. [41] in a study of blueberry (Vaccinium corymbosum) of
Collins and Bluecrop cultivars, the results are similar (3.5 and 3.3
respectively). As well as those found by Souza et al. [11], who reported a
3.64 pH for blueberry fruit.
Observing the pH values found in the pulp, inferior and significantly
different values are noted for the peel, for all cultivars, showing that there is a
higher concentration of dissociable organic acids in this part of the fruit, since
this determination quantifies only the total of ionizable hydrogen atoms
present in the fruit. This same statistical difference occurs with acidity content
of the peel and pulp, for all cultivars, with higher acidity in the peel, showing
that unlike the pH, non-dissociated organic acids are present in greater
amounts in the peel. The blueberry cultivars showed significant differences in
the whole fruit acidity levels, with Woodard (0.09%) and Powderblue (0.07%)
being the cultivars with higher acidity values. The acid content in blueberry is
mainly represented by the presence of malic acid which is the major organic
acid in small fruits.
The soluble solids content in the peel of blueberry cultivars showed higher
content than the content in the pulp (Table 6). Similar results were also
observed by an Embrapa Grape and wine [42] research, where the authors also
claim that in years of higher solar radiation intensity, grapes are produced with
higher soluble solids.
Among the analyzed fruits, the Woodard and Delite cultivars were the
ones that showed soluble solids similar to those of Raseira and Antunes [10],
Woodard cultivar, 12-13.9 °Brix and Delite cultivar 10.8-12.5 °Brix. The other
cultivars analyzed showed high soluble solids content (11.87-17.87 °Brix)
both when compared to the cultivars analyzed by Moraes et al. [40] (12-13.2
° Brix) as well as those analyzed by Perkins-Veazie et al. [41] Collins cultivar,
10.9 °Brix and Bluecrop cultivar 12 °Brix. These data demonstrate that the
fruits analyzed in the present study may have been collected with a greater
degree of ripeness.
Several factors can influence the content of soluble solids. Junior et al.
[43], when working with maturation curves and soluble solids in grapes,
describe climate as one of the factors that influence the accumulation of sugars
most. Volpe et al. [44] state that the temperature and the rains of months prior
to harvesting decisively influence the concentration of soluble solids of orange
juice. The soluble solids content of the blueberries in the present study showed
a positive correlation with pH (r = 0.7), which is justified by the increase in
value as the fruit ripens. This relationship was also observed by Perkins-
Veazie et al. [41], who found a coefficient of moderate Pearson correlation
(0.52) between pH and content of soluble solids in Highbush blueberry.
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Chapter 3
BLUEBERRIES: ANTIOXIDANT PROPERTIES,

HEALTH AND INNOVATIVE TECHNOLOGIES
Guillermo Petzold*, Jorge Moreno, Pamela Zúñiga,

Karla Mella and Patricio Orellana
Emerging Technologies and Bioactive Compounds in Food (TECBAL)
Department of Food Engineering, University of Bio-Bio, Chillán, Chile
ABSTRACT
Blueberries are a soft and small fruit native to North America with an
attractive blue color. In addition, blueberries are very popular because
they have low calories, high nutritional value and important antioxidant
properties. Blueberries have an interesting content of phenolic
compounds with high antioxidant capacity against free radicals and
reactive species, such that blueberry consumption may have a potential
beneficial effect on human health. Innovative technologies in the food
industry are new technologies based to develop more efficient process or
products, reduction of energy and water. Innovative technologies, such as
freeze concentration, osmotic dehydration and vacuum impregnation at
mild temperatures, are considered minimal processing techniques because
they preserve the fresh characteristics of fruits such as blueberries. Freeze
concentration is an innovative technology for producing a blueberry
concentrate juice in a process at low temperatures where no vapor/liquid
*
Emerging technologies and Bioactive Compounds in Food (TECBAL). Department of Food
Engineering, Universidad del Bío-Bío, Chillán, Chile. Email:gpetzold@ubiobio.cl.
56 Guillermo Petzold, Jorge Moreno, Pamela Zúñiga et al.
interface exists. On the other hand, osmotic dehydration and vacuum

impregnation of blueberries preserves different valuable attributes of the
fruit, providing products with an extended shelf-life.
Keywords: blueberries, antioxidant properties, innovative technologies
ANTIOXIDANT AND HEALTH PROPERTIES

OF BLUEBERRIES
In the last years, there has been a growing interest in food that can provide
beneficial effects to human health. It is widely known that a diet rich in fruits
and vegetables has beneficial effects due to the high amounts of antioxidants
and bioactive compounds in these foods, which have an essential role in
prevent certain diseases and reduce the risk of some health problems including
cardiovascular disease, neurodegenerative diseases, stroke and cancer
(Giampieri et al., 2014, Seeram et al., 2006; Neto, 2007; Kalt et al., 2008).
Accordingly, many native fruits have been studied for their potential as a
functional food. Recently, investigations have focused to improve the
nutritional value of fruits with emphasis in bioactive compounds (Scalzo et al.,
2005). Studies have also reported that specific berries, i.e., blueberries, have
antidiabetic effects. For instance, a study performed in mice (DeFuria et al.,
2009) found that supplementation with whole blueberries reduced the blood
glucose (Martineau et al., 2006; Vuong et al., 2007).
Fresh fruits of blueberry typically contain 1-3% seeds by weight, the
highly unsaturated seed oils of V. myrtillus (21-37%) is an excellent source of
essential fatty acids and oleic acid (Johansson et al., 1997).
Compounds capable of protecting against the effects of reactive oxygen
and nitrogen species (ROS and RNS) are known as antioxidants (Karadag et
al., 2009), they interact with unstable molecules such as free radicals and may
prevent the oxidative damage caused by the free radicals (Wang et al., 2012).
The high antioxidant activities in fruits are attributed to phenolic compounds,
such as anthocyanins, and other flavonoid compounds. However, the
antioxidant activity depends on their structure and content in berries.
Blueberries are a good sources of phenolic compounds, including
anthocyanins, flavonols, chlorogenic acid and procyanidins, that have high
antioxidant activity (Cho et al., 2004; Howard et al., 2003; Wang et al., 2012).
Antioxidant Properties, Health and Innovative Technologies 57
Figure 1. Clasification of phenolic compounds.
Phenolic compounds are secondary plant metabolites and are widespread

in all vegetables (Panico et al., 2009). Depending on their structure, phenolic
compounds are divided into non- flavonoids and flavonoids, which give rise to
other compounds of interest through their antioxidant capacity (see Figure 1).
In blueberries, the flavonols are predominately quercetin derivatives.
Quercetin glycosides accounted for >75% of total flavonols in the blueberry
genotypes (Cho et al., 2005). Similarly, with the study of Sellappan et al.,
(2002) which reported quercetin as the predominant flavonol in southern
highbush blueberries, followed by myricetin and kaempferol.
The in vitro antioxidant capacity of blueberries has been attributed to their
high concentration of phenolic compounds, especially anthocyanins (Kalt et
al., 1999). The anthocyanins are best known for their ability to impart red, blue
and purple color, in adding to their role as free radical scavengers and their
potentially significant interactions with biological systems, such as enzyme-
inhibiting, antibacterial and antioxidant effects (Mazza et al., 2002).
Prior et al. (2003) reported that consumption of a single meal of
blueberries is responsible in both hydrophilic and lipophilic antioxidant
capacity, according to their results the lipophilic component increased in a
37% following consumption of 189 g of blueberries in human subjects.
INNOVATIVE TECHNOLOGIES
Freeze Concentration
Concentration of liquid foods (such as fruit juices) is an important unit

operation, because concentrated products occupy less space and weight less,
and food manufacturers can potentially save on transportation costs,
warehousing costs, as well as handling costs for materials required for its
products (Ramaswamy and Marcotte, 2006).
The concentration of liquid foods (like fruit juices) is a delicate process,
since they are sensitive to thermal treatments. Even at moderate temperatures,
many of their components are unstable. At temperature between 40º and 70ºC
enzyme catalyzed reactions can alter juice properties within a few minutes. In
order to inactivate the enzymes, juices must be heat treated. Moreover, the
quality is strongly dependent on the concentration and composition of odorous
compounds. Most flavor and aroma components are volatile and can be lost by
evaporation (Deshpande et al., 1982).
Freeze concentration has long been recognized as one of the best
concentration techniques. As compared to evaporation and membrane
technology, freeze concentration has some significant potential advantages for
producing a concentrate with high quality because the process occurs at low
temperatures where no vapor/liquid interface exists, resulting in minimal loss
of volatiles (Morison and Hartel, 2007).
Freeze concentration process is particularly suited for the concentration of
heat labile liquid foods. In evaporators volatile aromas in the feed are almost
quantitatively lost with the water vapor. Normally, the quality can partly be
restored by separating the aromas from the vapor leaving the evaporator in a
distillation column and by feeding them back to the concentrated liquid. Very
volatile aromas, however, are lost with the inert gases and aromas with a
volatile equal to -or less than- the volatile of water in the solution cannot be
recovered from the vapor (Thijssen, 1974).
The general claim of the freeze concentration process is essentially that it
is capable of removing water by freezing it out from a solution as ice crystals.
Ideally, the ice formed should be free of solutes. First, the solution is partially
frozen, the ice crystals are physically separated from the residual solution
(concentrated solution), and the ice is melted to form the product water. Ice
crystals formed under the appropriate conditions can be very pure (Rahman et
al., 2007).
The industrial future of freeze concentration has been associated more

with developments in the configuration of one-step systems (block freeze
concentration or progressive freeze concentration) than conventional freeze
concentration systems (suspension crystallization), because of the simpler
separation step (Miyawaki et al., 2012; Petzold and Aguilera, 2009; Sánchez et
al., 2009, 2010). Other advantage of these one-step systems is their simplicity
in terms of both the construction and operation of the equipment (Sánchez et
al., 2009).
The basis of block freeze concentration (or freeze–thaw concentration) is
as follows: a food liquid solution is completely frozen, the whole frozen
solution is thawed and then the concentrated fraction is separated from the ice
fraction by gravitational thawing assisted or not by other techniques to
enhance the separation efficiency (Aider and de Halleux, 2008a,b). Under
these conditions, the ice block acts as a solid carcass through which the
concentrated fraction passes (Aider and de Halleux, 2009).
The alternatives of assisted techniques applied to block freeze
concentration are external forces such as vacuum or centrifugation. In this
way, vacuum (suction by a pump) has been proposed by Hsieh (2008) to get
drinkable water from sea water to separate salt, converting the ice of sea water
into fresh water, and Petzold et al. (2013) applying a vacuum improved the
efficiency in freeze concentration of sucrose solutions. Centrifugation has been
proposed by Bonilla-Zavaleta et al. (2006) in frozen pineapple juice to
separate ice from concentrated juice, while Luo et al. (2010) obtained ice
crystals of high purity during the freezing concentration of brackish water,
Virgen-Ortíz et al. (2012, 2013) proposed simple freeze centrifugation
methods to concentrate dilute protein solutions, and Petzold and Aguilera
(2013) presents an effective centrifugal freeze concentration method with
sucrose solutions.
On the other hand, the growing demand for fruit juices of high
organoleptic and nutritional quality has led to the search for new and improved
food processing technologies. Among the techniques for concentration of
liquid foodstuffs, freeze concentration is of particular interest due to the low
temperatures used in the process (Raventós et al., 2012).
These facts make it advisable to freeze concentrate blueberry juice,
especially for it’s interesting nutritional composition and high content of
bioactive compounds can be preserved through this technology (see first
section), together with an expected preserve that their fresh blueberry flavor.
In this way, Petzold et al. (2015) using a centrifuge operated for 10 min at
20°C and 4600 rpm, concentrate blueberry juice. This technique (block freeze
concentration assisted by centrifugation) has a good performance after the

third cryoconcentration cycle, reaching an increase of approximately 2.5 times
the initial concentrations of solids (see Figure 2), values close to 0.74 kg solute
per 1 kg initial solute, and approximately 60% of the percentage of
concentrate. Thus, using this technology was reaching approximately a
concentration of 33°Brix from a raw material near 12°Brix.
Figure 2. Performance of block freeze concentration assisted by centrifugation applied

to blueberries. Adapted by Petzold et al., 2015.
Osmotic Dehydration
Osmodehydration (OD) is considered as a minimal processing method that

preserves the fresh-like characteristics of fruits, such as color, firmness and
taste. This technology is a partial dehydration widely used to remove a portion
of water from plant material to obtain a product of intermediate moisture,
reducing undesirable effects (structural damage or loss in nutritional value)
which are characteristic of other processes of food preservation, such as
convective drying or freezing, due the water removal is conducted without
phase change (Kucner et al., 2013), and furthermore, the lack of oxygen during
the process can inhibit the oxidation of vegetable tissue and prevents
enzymatic browning (Sapers, 1992).
The OD process is carried out by immersion of the raw material in an

aqueous hypertonic solution, where there are two major simultaneous
countercurrent ﬂows: the natural flow of water and low molecular weight
components (sugars, vitamins, organic acids and mineral salts) from the raw
material into the solution; and the migration of solutes from the solution into
the food matrix (Falade and Igbeka, 2007). In multiphase food system, mass
transfer rates are attributed to the water and solute activity gradients across cell
membranes as solutes and water seek equilibrium.
Blueberries OD Application
Blueberries are fruits available in the growing season and show a limited
shelf-life due they are highly perishable commodities and sensitive to bacterial
and fungal contamination; hence, preservation and processing methods are
usually applied to extend berry commercial life and consumption occasions,
usually by either freezing or drying technologies.
Within existing methods available for drying blueberries, freeze-drying
and vacuum-drying obtain the highest-quality end product, considering the
losses in total anthocyanins and phenolics (Lohachoomplo et al., 2004).
However, these drying methods require a high input of energy and their cost
are elevated (Ratti, 2001).
On the other hand, osmotic dehydration is a simple and cheaper
dehydration method that has been proposed as an alternative for preserving
food products with high nutritional value, which has been successfully applied
to vegetables and fruits.
However, most berries (such as blueberries) have an impermeable
epidermis which behaves as a barrier that impedes mass transfer during
osmotic dehydration, slowing down the process (Ketata et al., 2013). This
epidermis is very thick and contains, among others, waxes and pectines which
are often necessary to pretreatment before processing the raw material.
To enhance skin permeability in blueberries, chemical, mechanical and
thermal pretreatments have been used to decrease the hydrophobicity of the
skin and promote moisture diffusion during drying of whole berries, which are
briefly described in Figure 3.
Nevertheless, the steam-blanching has been proving successful results
(using 85°C steam for 3 min) increasing mass transfer phenomena during the
osmodehydration treatments and reducing the loss of phenolic compounds,
improving the retention of antioxidant capacity in the final product. Blanched

berries had also deeper color and smoother surface (Giovanelli et al., 2012).
Actually, blanching and osmotic dehydration can be complemented with
subsequent air drying, denominated osmo-air dehydration, to further reduce
the final moisture content, increasing the stability and shelf-life, reducing
packaging and logistic costs and improving both sensory and nutritional end
products quality. Osmo-air dehydrated fruits have better texture, color and
flavor than conventionally air-dried fruits (Torreggiani and Bertolo, 2004).
However, osmotic treatment could cause significant losses in the antioxidant
activity and also, during air drying, losses in the bioactive compounds could
occur (Giovanelli et al., 2013), depending on the infusion process conditions
(sugar concentration, temperature, syrup to product ratio, infusion time and
agitation) and air-drying conditions (mainly air temperature and air flow-rate).
At 30–50°C, dehydration is not very effective, while the application of higher
temperatures leads to substantial losses of phenolic compounds in the
dehydrated material (Kucner et al., 2013).
On the other hand, cold pretreatments have been occasionally studied
regarding the acceleration of drying rates. Individual Quick Freezing (IQF) of
berries in a thin layer at -40°C for a specified time has been used in cycles
with slow thawing in the refrigerator at 4°C. This mild heat shocks (-40°C
to +4°C) together with the repetition in cycles lead to slight changes in the
permeability of the waxy cuticle, sufficient to increase the drying rate (Yang,
et al., 1987).
Figure 3. Pretreatment to decrease the hydrophobicity of the skin of blueberries and

promote moisture diffusion during drying.
The application of cryogenic pretreatments with liquid nitrogen to whole

berries can accelerate moisture loss and solids gain during osmotic
dehydration of blueberries. The epidermis weakens and the cuticular thickness
decrease, and decrease the amount of remaining epicuticular waxes after liquid
nitrogen pretreatments, facilitating the transfer of water and sugar during the
osmotic process (Ketata et al., 2013).
A promising method of pretreatment is immersing the fruit in lipolytic and
pectinolytic enzymes prior to dehydration, which leads to a greater increase of
dry matter content during osmotic dehydration, which increase dry matter
content with a low loss of phenolic compounds (Kucner et al., 2013).
Other pretreatment methods proposed in the literature include: ultrasound,
lower hydrostatic pressure, and exposure to a high intensity electric field
(Kucner et al., 2013), therefore, clearly there are various techniques for
enhance the mass transfer of blueberries in OD treatment. Nevertheless,
despite having increased the permeability of the skin of these fruits, there is a
disadvantage of the osmotic treatment per se: the long time required to reduce
the water activity. However, to improve the efficiency of the mass transfer and
the nutrient uptakes, vacuum impregnation process can be applied.
Vacuum Impregnation
Vacuum impregnation (VI) uses pressure gradients to accelerate the

incorporation of a solution into the structural matrix of high porosity food
samples. This implies that the gas is exchanged in the pores for the external
fluid due to the action of hydrodynamic mechanisms (HDM) promoted by
pressure changes. The process consists of two stages: in the first stage, the
system is vacuumed, and maintained at that lower pressure for a relatively
short period so that the gas in the fruit or vegetable can escape from the
interior of the porous solid. In the second stage, the system is restored to
normal pressure and maintained there for a certain length of times.
Interesting advantages of fruits and vegetables vacuum impregnation
(process at a relatively low temperature) are an evident reduction in the use of
energy, maintain the original color and the natural aroma, and protect some
heat sensitive nutrients (Gao et al., 2011).
The benefits of the combination of OD with VI is that it decrease time of
osmotic dehydration, the process occur in reduced oxygen environment and
also osmotic solution could be reused, making this process more practical from
nutritional and economical standpoints (Stojanovic and Silva, 2007).
Also, value-added products could be designed from berries by applying

these combined methods, where if the osmotic solution has physiologically
active compounds, they could be introduced into the solid food matrix to
enhance its nutritional or functional characteristics. VI has grown signiﬁcantly
in popularity because it has been successfully used to incorporate vitamins
(Cortés et al., 2007), minerals (Gao et al., 2011), probiotic microorganisms
(Betoret et al., 2003), into fruits and vegetables matrix structure without
substantially modifying their organoleptic properties, therefore this technology
has been recognized as a suitable technology for formulating new products.
ACKNOWLEDGMENTS
Author Guillermo Petzold is grateful for the financial support provided by
CONICYT through FONDECYT Project No. 11140747.
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Chapter 4
BLUEBERRY ANTI-INFLAMMATORY
EFFECTS OVER METABOLIC DISEASES
ASSOCIATED WITH OBESITY
J. Soto-Covasich, M. Reyes-Farias*, A. Ovalle-Marin,

C. Parra-Ruiz* and D. F. Garcia-Diaz
Department of Nutrition School of Medicine, University of Chile,
Santiago, Chile
ABSTRACT
Inflammation is a natural defense mechanism triggered as a response
to an alteration of the physiological functions of the organism. This
process is responsible for the secretion of mediators crucial for tissues
repair, integrating different signalling pathways between distinct cells and
organs. Likewise, it has been observed that in metabolic diseases some
classic mediators present during short-term inflammation are involved,
although the features of its actions differ from the classic pathways. Thus
it is considered as a subclass of inflammation often referred as meta-
inflammation. In the case of obesity for example, this response is
exacerbated and, at the long term, a chronic inflammatory state associated
with cardiovascular diseases, insulin resistance and type-2 diabetes
development is established. Since obesity-associated inflammation is
known to be a key feature of the etiology of non-communicable diseases,

Authors contribute equally.
72 J. Soto-Covasich, M. Reyes-Farias, A. Ovalle-Marin et al.
several efforts have been made for identifying novel agents with anti-
inflammatory properties capable of ameliorate its negative long-term
effects. In this regard, blueberry consumption has been described to
induce important health benefits through anti-inflammatory and
antioxidant features. Therefore, in the present chapter, we will discuss the
impact on the low-grade inflammatory status associated to metabolic
diseases provided by a blueberry treatment or diet, previously described
in the literature. In this context, will be addressed: a) in vitro studies over
inflammation in macrophages and changes in adipogenesis; b) in vivo
studies over pro-oxidant and inflammatory status, related to amelioration
of insulin resistance, hyperglycemia, dyslipidemia, hyperphagia and
weight gain induced by a high fat feeding, and improvement of blood
pressure, renal function and beta cell function; and c) human clinical
evidence, over antioxidant defense mechanisms and inflammation,
influencing blood pressure and insulin sensitivity in susceptible subjects.
In this sense, recent findings supports that a blueberries-rich diet has been
able to modulate the inflammatory status in a positive manner, likewise
exerting its effects in different crucial stages of metabolic alterations
development and hence contributing to the prevention and reduction of
obesity-associated comorbidities. It is still pending to deepen into the
cellular and molecular mechanisms in order to take advantage from a
commercially-available fruit for improve human life quality.
INTRODUCTION
Obesity is a Global Pandemic
One of the aspects that influence the most on each individual’s day-by-day
wellbeing is the body weight fluctuation. Despite the fact that humans require
the presence of adipose tissue in the organism, when this tissue develops
excessively several harmful consequences occur (Bray et al., 2004). Indeed, it
is well known that an excessive body fat accumulation is a masterpiece for
several associated clinical manifestations such as type 2 diabetes (T2D),
cardiovascular diseases (CVD), among others (Guh et al., 2009). Obesity
prevalence has been doubled from 6.4% in 1980 to 12.0% in 2008 in the entire
world. Half of this rise occurred from 2000 to 2008 (Stevens et al., 2012).
Obesity is defined as the pathogenic increment of the organism fat
content, accompanied by a total body weight augment, due to a positive
balance in the equation comprising both energy intake and energy expenditure
(Hartroft et al., 1960). This augmentation has been related specially with an
increase in white adipose tissue (WAT) content. WAT it is considered an
Blueberry Anti-Inflammatory Effects over Metabolic Diseases … 73
active endocrine organ that is able to secrete a large number of molecules

(adipokines, only secreted by it own, and other cytokines), which are involved
in a wide variety of physiological processes (Zulet et al., 2007). However, in
obesity, WAT overgrowth leads to a deregulated production of these
endogenous products, often presenting pro-inflammatory properties (Fantuzzi
et al., 2005). Therefore, it is known that increased body adiposity is habitually
accompanied by an increased systemic oxidative stress and by a chronic low-
grade inflammation condition in the adipose tissue (Furukawa et al., 2004;
Yudkin et al., 2007). In this regard, these adipokines, cytokines, and other
factors produced by this tissue, are possibly responsible for the induction and
maintenance of these oxidative and inflammatory processes (Fantuzzi et al.,
2005; Ferrante et al., 2007).
Inflammation as a Chronic Disease Inductor-Masterpiece
The inflammatory process are a group of biological responses that

involves a complex biological cascade of molecular and cellular signals that
alter physiology, resulting in clinical symptoms such as: pain, swelling, heat,
and redness (Libby, 2007). At the site of the injury, cells release molecular
signals that cause: recruitment and activation of immune cells (leukocytes,
monocytes, lymphocytes and dendritic cells), stimulation of the production of
different chemical mediators (such as cytokines, chemokines) and regulation
of signaling pathways such as: insulin, leptin, glucose, NFkB, among others
(González-Muniesa et al., 2015). The acute inflammation is a normal process
that protects and help to heal the body following physical injury or infection.
However, if the agent causing the inflammation persists for a prolonged period
of time, this inflammation becomes chronic. It is known that obesity is
associated with chronic low-grade inflammation (Feghali et al., 1997). In
relation to the above, epidemiological and clinical studies have described a
relation between the development of low-grade inflammatory responses and
metabolic diseases, specifically between obesity and type 2 diabetes
(Hotamisligil, 2006).
Obesity-Linked Inflammation and Its Outcomes: Focusing on Type 2

Diabetes and Cardiovascular Diseases
As mentioned before, obesity is often associated with the presence of low-
grade chronic inflammation in white adipose tissue (WAT). When adipocyte
hypertrophy occurs, endoplasmic reticulum stress, hypoxia, and oxidative
stress arise in WAT (Furukawa et al., 2004; Hosogai et al., 2007; Ozcan et al.,
2004). These stresses induce the release of inflammatory signals, which
initiate the infiltration of monocyte into this tissue (Suganami et al., 2010).
Adipose tissue inflammation is triggered by this macrophages infiltration; the
subsequent crosstalk between these cells and resident adipocytes highlights as
a key factor for the development of associated co-morbidities (Weisberg et al.,
2003; Xu et al., 2003), especially insulin-resistance (IR) (Weisberg et al.,
2003). In this sense, several inflammatory products produced by this
interaction, such as TNF-α, MCP-1 and NO, correlates with increased body
adiposity (Ferrante et al., 2007) and appear to participate in the induction and
maintenance of the chronic inflammatory state associated with obesity
(Shoelson et al., 2000). In addition, WAT overgrowth leads to downregulation
of several anti-inflammatory products, e.g., adiponectin (Maeda et al., 2002).
In this context, it has been observed that macrophages contribute to the
development of IR in obese patients, while weight loss reduces macrophage
infiltration and the expression of inflammation-related factors in adipose
tissue, being related with insulin-sensitivity improvement (Cancello et al.,
2005; Clement et al., 2004).
At the molecular level, the adipose tissue at enlargement presents
activation of some mitogen-activated protein kinases (MAPKs), such as ERK,
p38 MAPK, and JNK (Johnson et al., 2002; Bost et al., 2005; Hirosumi et al.,
2002). Once activated by upstream kinases, these enzymes are rapidly
inactivated by MKP-1 (Farooq et al., 2004). During the course of adipocyte
hypertrophy the expression of this protein is downregulated, which leads to an
increased secretion of MCP-1 (Ito et al., 2007). MCP-1 plays a crucial role in
the inflammatory response in obesity by enhancing monocyte migration and
activation of macrophages (Yu et al., 2006). Once inside the adipose tissue,
macrophages participate in the activation of inflammatory pathways mainly
through TNF-α secretion. TNF-α activates the hypertrophied adipocytes
through their TNF-α receptor, inducing pro-inflammatory cytokines
production by NFκB-dependent mechanisms and lipolysis by NFκB-
independent (MAPK-dependent) mechanisms (Suganami et al., 2005). On the
other hand, free fatty acids (FFA), which are consequently liberated by
adipocytes, bind TLR4 complex, which is essential for the activation of NFkB
signaling by lipopolysaccharides (LPS) stimulation in macrophages (Suganami
et al., 2007), establishing a vicious cycle (Suganami et al., 2010). TNF-α and
FFA both can induce disruption of the molecular signaling pathway of insulin
action mainly by aberrant key-proteins phosphorylation (e.g., on IRS1, Akt),
declining GLUT4 translocation to membrane (Hotamisligil et al., 1999; Boden
et al., 1999). This phenomenon is first observed locally (in WAT), and then at
the systemic level, the latter when advanced stages of obesity development are
reached.
Regarding other diseases, e.g., cardiovasculares diseases (CVD), which
includes heart and vascular disease, and atherosclerosis, are characterized also
as a chronic inflammatory condition. This condition relies majorly over the
secreted factors from the inflamed adipose tissue, that is, adipokines. These
molecules present contrasting actions for the cardiovascular system. In this
sense: adiponectin, apelin and omentin preserve normal cardiovascular
function, whereas leptin, resistin and visfatin contribute to inflammation and
endothelial dysfunction (Mattu and Randeva, 2013). It has been described that
an increase in proinflammatory state cause: alterations of vascular tone and
flow, increased expression of adhesion molecules (VCAM-1, ICAM-1),
increased vascular permeability (increase of VEGF), less fibrinolysis (increase
of PAI-1), increased cytokines and C-reactive protein (PCR) (Rajendran et al.,
2012). On the other hand, insulin-resistance itself (as a results of adipose tissue
local inflammation) it is known to contribute to CVD development (Li et al.,
2011). Therefore, adipose tissue inflammation, directly and indirectly, is
recognized as a key ignitor factor for cardiac tissue injury.
As it mentioned previously, obesity co-morbilities such as, insulin
resistance, T2D and cardiovascular disease have been reconsidered as
inflammatory diseases. In this regard, the treatment with anti-inflammatory
agents, such as polyphenolic compounds, could be an effective therapeutic
strategy. In this sense, blueberry (Vaccinium corymbosum, L.) is a fruit
worldwide known as a rich source of anthocyanins, one of the main
polyphenolic compounds. In this chapter, we will explore the anti-
inflammatory effect of blueberry over metabolic diseases associated with
obesity and its preventive effects of in reducing theses co-morbilities.
IN VITRO EVIDENCE
Effects of Blueberries Extracts on Inflammation Induced
In Vitro
It has benn described previously that a blueberry powder present anti-

inflammatory effects. Caco-2 intestinal cells were treated with IL-1β and three
different bioactive fractions (anthocyanin, phenolic, and water-soluble
fractions), obtained from an anthocyanin-rich wild blueberry (Vaccinium
angustifolium). These fractions reduced the activation of NFkB, induced by

this cytokine (Taverniti et al., 2014). In addition, it was reported that blueberry
extracts has anti-inflammatory properties on the chronic inflammatory process
sustained by microglia cells, whose play a crucial role in the progression of
neurodegenerative diseases. Lau et al. (2009) studied the effect of a
polyphenolic-enriched fraction of blueberry (PC18) on a cell line of microglia
(BV2) stimulated with LPS. They observed a reduction of NO secretion and a
reduced cyclooxygenase-2 (COX2) protein expression. In addition, blueberry
polyphenols inhibited NFkB nuclear translocation in LPS-activated BV2 cells.
This evidence support the role of bioactive compounds of blueberry as a
potential dietary agents with anti-inflammatory properties.
On the other hand, the majority of the anti-inflammatory and antioxidant
findings relative to the polyphenols present in fruits and vegetable extracts
came from studies that carried out with fractions composed of extractable
polyphenols (aqueous organic extracts), however, the total polyphenol content
in foods is composed of the extractable and non-extractable polyphenols; the
latter consist of high molecular weight proanthocyanidins and phenolics
associated with dietary fibre and indigestible compounds (Saura-Calixto et al.
2007). In this context, Cheng et al. (2015) evaluated and compared the effect
of two different fractions of extractable (EPP) and non-extractable
polyphenols (NEPP) from blueberries on the inflammatory response generated
by LPS-stimulated RAW 264.7 macrophages. They observed a dose-
dependent (ranged from 10 to 400 ug/ml) decreased NO secretion when EPP
and NEPP were added to the LPS-induced cells. Moreover, they reported an
inhibition of inducible NO synthase (iNOS) and COX-2 mRNA expression
after treatment with EPP or NEPP, and observed that this occurs through the
suppression of the main cellular effector of inflammation, NFkB, since the
treatment results in the inhibition at the level of phosphorylated p65 (P-p65).
In this study, the inhibitory effects of EPP was more significant than NEPP,
and the authors supposed that it may be a consequence of a higher
concentration of active compounds in EPP fraction. However, given these
results it becomes important to consider the NEPP fractions on further
investigations as a way to take advantage of the whole fruit (Cheng et al.
2015).
Obesity activates inflammatory pathways that contribute to the
pathogenesis of obesity-associated diseases, such as T2D and aterosclerosis
and so on. Particularly, the macrophage infiltration of WAT has been related
with an increase in inflammatory cytokine production. MCP-1, as described
above, is known to be responsible for recruiting macrophages to sites of
infection or inflammation (Salcedo et al., 2000). In this context, Esposito et al.

(2014) examined the capacity of polyphenol-rich wild blueberry extracts to
modulate the gene expression profiles of different biomarkers of acute and
chronic inflammation in LPS-activated macrophages. They used LPS as an
inducer of inflammatory response since is warranted its dual role in inducing
both acute inflammatory response and endothelial cell injury as well as low-
level chronic inflammation associated with gastrointestinal dysfunction. The
research group found that all blueberry fractions (crude extract; polyphenol-
rich (PPR); anthocyanin-rich (ANC); proanthocyanidin-rich (PAC) and ethyl
acetate fractions) suppressed at least one biomarker of inflammation (MCP-1,
IL-6, IL-1b, COX-2 and iNOS) at 50 μg/mL, a concentration easily to achieve
in the gastrointestinal tract after consumption of berries or juices. PPR, ANC
and PAC fractions suppressed effectively mRNA levels of COX-2, iNOS and
IL-1β. Besides, the researchers demonstrated that malvidin-3-glucoside is
more effective in reducing the expression of proinflammatory genes in vitro
compared with epicatechin or chlorogenic acid (Esposito et al., 2014).
Effects of Blueberries Extracts on Obesity-Related Diseases: In

Vitro Evidence
Obesity is the main risk factor for the development of other diseases, such
as insulin resistance, T2D, CVD, fatty liver disease, cancer and other
pathologies. The main works that describe beneficial effects of blueberry
treatment in this regards are described as follows.
Adipogenesis and Obesity

Moghe et al. (2012) determined that blueberry polyphenols may play an
effective role in inhibiting adipogenesis and cell proliferation. They assayed
the effects of three doses (150, 200, and 250 ug/mL) of blueberry polyphenols
on murine 3T3-F442A preadipocyte, and they determined intracellular lipid
content, cell proliferation, and lipolysis. The authors found that blueberry
inhibits adipocyte differentiation, evidenced in a reduced cellular lipid content
compared with the control group. Besides, cell proliferation was observed to
be significantly higher in controls compared with all the blueberry groups,
although there was no significant difference on cell proliferation among the
three doses of blueberry polyphenols. Nevertheless, when they tested lipolysis,
there was no significant difference observed among groups. These results
suggest that blueberry polyphenols may play an effective role in inhibiting
adipogenesis and cell proliferation. Other study has been demonstrated that the
treatment of 3T3-L1 preadipocytes with blueberry peel extract (BPE) results in
an important adipogenesis reduction. This finding is supported by the gradual
reduction in the number of lipid droplets in a dose-dependent manner (ranged
from 50 to 300 ug/ml of BP extract) and the diminished level of triglyceride
content (by 37, 7%) after seven days of treatment of differentiating cells with
BPE (200 ug/ml). Furthermore, the mRNA and protein levels of C/EBPb,
C/EBPa and PPAR also exhibit a reduction after concomitant treatment of
differentiating 3T3-L1 cells with increasing concentrations of BP extracts
(Song et al., 2013). This same study revealed that the phosphorylation of Akt
and its downstream substrate, phospho-GSK3 was also reduced by the
treatment of BPE in 3T3-L1 cells. Hence, the evidence shows that BP extracts
has the potential to modulate the adipogenic activity via PI3K/Akt/GSK3
pathway. However, further studies are required to assure the therapeutic
potential of blueberry to adipogenesis and obesity.
Insulin Resistance and T2D

Martineau et al. (2006) evaluated anti-diabetic properties of the Canadian
lowbush blueberry, using four extracts. Their study determined a 15-25%
increase in glucose uptake in C2C12 skeletal muscle cells incubated with 12.5
μg/mL of root, stem or leaf extracts during 18-21 h in presence and absence of
insulin (1nM and 100 nM), compared to cells incubated with vehicle only
(0.1% DMSO). An increased uptake (>25%) in 3T3-L1 adipocytes incubated
with root and stem extracts for 18-21 h in presence of 1nM insulin was
reported. Also, there was an increased uptake of these cells by incubating for 1
h with the same two extracts in presence and absence of insulin, reaching the
highest percentages in the latter condition (>65%). Moreover, an increase of
glucose-stimulated insulin secretion in growth-arrested β TC-tet cells
incubated for 18 h with 12.5 μg/mL leaf extract and a proliferative effect on
replicating β TC-tet cells treated with 12.5 μg/mL fruit extract for 24 h, were
observed. In addition to these insulin-sensitizing effect; root, stem and leaf
extracts were able to stimulate lipid accumulation in differentiating 3T3-L1
cells, and stem, leaf and fruit extracts promoted cytoprotection in PC12 cells
exposed to chronically elevated glucose (Wu et al., 2015). Another research
group determined that an ethanol extract of blueberry exerts a strong inhibitory
effect on the α-glycosidase enzyme, showing one of the lowest IC50 values
among the twenty extracts tested (IC50 = 13.0 mg/mL) (REF?)
Cardiovascular Disease
Sakaida et al. (2007) observed a potent inhibitory effect of blueberry leaf
extract on angiotensin converting enzyme activity, which was stronger
compared to other leaves of Ericaceae plants and Camellia sinensis L. (green
tea). Additionally, spontaneously hypertensive rats were fed a diet
supplemented with 3% of freeze-dried blueberry leaf and showed a decrease in
systolic blood pressure from the second week as compared to rats without
supplementation. These in vitro and in vivo results demonstrated the
antihypertensive property of blueberry leaf. Along with this, Louis et al.,
(2014) investigated the cardioprotective action of an aqueous blueberry extract
and five polyphenolic fractions (Phe: phenolic fraction; Flv: flavonoid
fraction; Acn: anthocyanins-enriched fraction; Hep: Heteropolymers-enriched
fraction; and Pac: proanthocyanidins-enriched fraction) on an in vitro model of
heart disease. Adult Sprague Dawley rats cardiomyocytes were isolated and
cultured, performing a pretreatment with extract and fractions and a further
treatment with norepinephrine (NE). Regarding hypertrophy and cell death of
cardiomyocyte, the Phe, Flv, Acn and Hep fractions presented a preventing
effect on cardiomyocyte injury by NE. Pretreatment of cardiomyocytes upon
Phe fraction prevented an increase of apoptosis, increase of oxidative stress,
decrease of superoxide dismutase and catalase activities, increase of calpain
activity and decrease of contractile function induced by NE treatment
compared to condition without pretreatment. As a first approach, these results
of in vitro model suggest a protective effect of blueberry over cardiovascular
diseases.
Non-Alcoholic Fatty Liver

Nonalcoholic fatty liver (NAFL) is one of the most common chronic liver
diseases worldwide. This disease is associated with metabolic syndromes, such
as obesity. It has been reported an inhibitory effect of polyphenols of Chinese
blueberries on oleic acid-induced hepatic steatosis in HepG2 cells (Liu et al.,
2011). The authors observed that a polyphenol-rich extract caused significant
inhibition in the cellular accumulation of triglycerides. The polyphenol-rich
extracts were separated previously into three fractions: anthocyanin-rich
fraction, phenolic acid-rich fraction, and ethyl acetate extract; being the second
one the most effective in the model.
IN VIVO EVIDENCE
Anorexic Effect of Blueberry Supplementation
It has been demonstrated that blueberry supplementation is an efective

strategy against hyperphagia. In fact, biotransformed blueberry juice
administration in a mice model of leptin resistance during 3-weeks had a
protective effect on hyperphagia and, hence, reducing weight gain. In fact,
anorexic effect was comparable with metformin administration (Vuong et al.,
2009). In addition, 3% unextruded or extruded pomace was able to reduce
leptin levels (Khanal et al., 2012).
Blueberry as Weight Loss Treatment
Numerous studies had been reported the slimming effect of blueberry

supplementation. In this sense, DeFuria et al. (2009) reported that
supplementation with blueberry juice or purified anthocyanins from blueberry
(ANCs) in high fat diet (HFD) showed lower weight and body fat than HFD
group, as also lower epididymal and retroperitoneal fat. Futhermore,
gastrointestinal administration of blueberry peel extract (BPE) in Sprague-
Dawley rats feed with high fat diet prevents weight increases and, once again,
epididymal and retroperitoneal fat gain (Song et al., 2013). Another study
shown that both inclusion of unextruded or extruded blueberry pomace at
concentration 1.5% and 3% in a model of metabolic syndrome of high fructose
diet on Sprague-Dawley rats diminish abdominal fat accumulation (Khanal et
al., 2012).
Blueberry Effects on Insulin Resistance Development
Some studies suggest that blueberry administration might acts as PPARs

agonist as well as thiazolidinediones drugs (Vuong et al., 2009, Seymour et al.,
2011), thus performing insulin sensitizer effects. Vuong et al. (2009) indicates
that blueberry juice administration improves glucose tolerance and reduces
glycemia in diabetic mice. Moreover, addition of 2% freeze-dried whole
blueberry powder in obesity-prone Zucker rats feeding with high fat diet
improves fasting insulin, HOMA-IR and glucose tolerance test compared with
group without blueberry addition. A similar study shows that C57BL/6J male
mice fed with high fat diet plus 4% freeze-dried blueberry powder show
improved glucose tolerance, similar to low-fat-diet group (DeFuria et al.,
2009). Moreover, fasting glucose values and HOMA-BCF score (β cell
function) were diminishes to normal levels in animals supplemented with
blueberry anthocyanins (Prior et al., 2010).
Improvement on Lipidic Parameters
Other biomedical feature ejerted by blueberry is the lipid profile

improvement. In this sense, supplementation with blueberry anthocyanins in
the drinking water in mice fed with high fat diet with 60% kcal from fat
restores to control levels serum triglycerides and cholesterol (Prior et al.,
2009). Song et al. (2013) demonstrated that administration of blueberry extract
in Sprague-Dawley rats fed with high fat diet increased HDL levels and
reduced total cholesterol and triglycerides levels. In addition, 3% unextruded
or extruded pomace was able to minimize plasma cholesterol levels (Khanal et
al., 2012). Moreover blueberry intake increased adipose and skeletal muscle
PPAR-α activity (Seymour et al., 2011). In this sense, it has been described
that PPAR-α is involved in lipid oxidation and fatty acid transport and its
agonist are used to reduce VLDL and LDL, improve HDL and fatty acid
oxidation/synthesis ratio (Kersten, 2014).
Blueberry Supplementation on Cardiovascular Disease
On the other hand, blueberry supplementation was tested on a myocardial

infarct (MI) induction. For this purpose, the left descending coronary artery
was ligated and animals were monitored to measure ejection fraction,
myocardial infarct and mortality after surgery. 2% blueberry-enriched diet
(BD) improves survival after artery ligation compared with control diet group.
When MI expansion was studied by echocardiography and histology, it was
observed that after 12 months BD significantly attenuates MI. In left
ventricular remodeling, BD attenuates end-diastolic volume (EDV) and end-
systolic volume (ESV) expansion, slightly improve eject fraction, and also
increases posterior wall thickness as compared to controls (Ahmet et al.,
2009a). Then cardioprotective effects of BD were studied in isolated
cardiomyocytes. Mitochondrial permeability transition (MPT) induction was
increased in BD animals, effects comparable with insulin, increasing
cardiomyocyte survival. In summary, this diet reduced the size of myocardial

infarction by attenuating cardiac necro-apoptosis. Additionally, Lopera et al.,
(2013) identified cardioprotective effects of a fermented nonalcoholic extract,
prepared from Colombian blueberries (Vaccinium meridionale Swartz), against
ischemia-reperfusion injury in isolated Wistar rat hearts. The isolated hearts
were treated 10 min before ischemia and the initial 10 min of reperfusion with
50 μg/mL of the blueberry extract (BE) and were evaluated versus an ischemic
control (IC) hearts without the extract treatment. The authors described that
hearts treated with BE improved the postischemic recovery of systolic and
diastolic myocardial function due to exhibited higher percentage of the left
ventricular developed pressure (87 ± 8% versus 42 ± 3% at the end of
reperfusion period) and of the maximal rise velocity of the left ventricular
pressure, and lower values of left ventricular and-diastolic pressure (18 ±
6mmHg versus 49 ± 6 mmHg at the end of reperfusion period) than IC hearts.
Likewise, treated hearts presented higher levels of reduced glutathione and
reduced levels of lipid peroxidation than IC hearts, which could be understood
as a reduction of oxidative stress in cardiac tissue. Additionally, was reported
by western blots that treatment with BE increased the eNOS and phospo-Akt
protein expression in isolated hearts. However, all the protective effects
detailed of the BE were reversed in presence of NOS inhibitor L-NAME. This
results suggest that BE could modulates an activation of eNOS via Akt in the
isolated heart, which would confer an important role to NO on the regulation
of oxidative stress and myocardial dysfunction (Lopera et al., 2013).
Blueberry and Renal Failure
Finally, it has been described that blueberry protects renal system. Obese
male Zucker rats treated with 2% lyophilized blueberry in water (BB) for 15-
weeks presented an improvement in glucose tolerance compared with
untreated group. When the kidney function was assessed, BB supplementation
decreased mean arterial pressure and renal vascular resistance, nevertheless it
improved glomerular filtration rate and renal blood flow. In addition BB
enriched diet attenuates the expression ACE and AT1 and improved the
expression levels of ACE2 and AT2 genes in renal tissue, exerting a protective
role. Kidneys examination showed severe glomerular adhesions, cortical and
medullary tubular lesions and interstitial nephritis in obese animal, however
BB supplementation exhibit a reduction in those pathological changes. At the
end, BB enriched diet increases SOD and catalase levels in kidneys and
promotes increased Nrf2 levels and reduced Keap-1, reestablishing the

antioxidant defense (Nair et al., 2013).
BLUEBERRY AND CLINICAL TRIALS

Summaryzing, it has been widely described the beneficial effects of berry
consumption, especially blueberries (Manganaris et al.?, 2014). Indeed,
blueberry treatment have been related to improvement in several chronic non-
transmissible diseases and conditions, such as cancer (Voung et al.?, 2016),
lung injury (Liu et al.?, 2015), ageing (Shukitt-Hale et al.?, 2015), periodontal
disease (Ben Lagha et al.?, 2015), glucose metabolism (Vendrame et al.?,
2015), metabolic syndrome (Stull et al.?, 2015), among others. Regarding
inflammation, it has been reported widely the anti-inflammatory potential of
this fruit (as fruit, extracts or some of its bioactive components) in several
contexts, such as: in corneal epithelial cells (Liu et al.?, 2015), against UV
radiation in lung tissue (Liu et al.?, 2015), ageing (Shukitt-Hale et al.?, 2015),
in intestinal cells (Taverniti et al.?, 2014), against high-fat meal ingestion
(Miglio et al.?, 2014), and cancer (Kanaya et al.?, 2014). However, to date just
a few of clinical trials (Guh et al., 2009) has been conducted regarding
benefitial outcomes of blueberry treatment specially in obesity in humans,
with no concomitant effect over inflammatory features.
As mentioned above, insulin resistance and T2D are closely related with
obesity. Stull et al. (2010) observed that blueberries improve insulin sensitivity
in obese subjects (man and woman). In this double-blinded study, participants
were randomized to consume either a smoothie containing 22.5 g blueberry
bioactives or a smoothie of equal nutritional value without added blueberry
bioactives, twice daily for 6 weeks. The group that consumed blueberry
showed an increase of insulin sensitivity without significant changes in
adiposity, energy intake, and inflammatory biomarkers. This results showed
that a daily dietary supplementation from blueberries improved insulin
sensitivity in obese, nondiabetic, and insulin-resistant participants.
Moreover, a study conducted in 2010 reported how a consumption of a
freeze dried beverage of 50 g freeze-dried blueberries (350 g fresh blueberries)
daily 8 weeks induced an improvement in variables related to CVD
progression, however no effects were observed in blood inflammatory
variables, such as PCR, vCAM, iCAM, and IL-6. Nevertheless, a significant
reduction in ox-LDL levels and stress oxidation markers were observed as
compared to controls (Basu et al.?, 2010). Riso et al. (2013) reported that
regular consumption of a wild blueberries drink for 6 weeks reduced the levels
of oxidized DNA bases and increased the resistance to oxidatively induced
DNA damage in subjects with risk factors for cardiovascular disease. In
addition, blueberries improve endothelial function in adults with metabolic
syndrome. Subjects received a blueberry or placebo smoothie twice daily for
six weeks. The group that consumed blueberry showed an increase in
endothelial function versus the placebo group (Stul et al., 2015). Moreover,
another study revealed that blueberry consumption improves blood pressure in
postmenopausal women with pre-and stage 1-hypertension (Johnson et al.,
2015). These studies reveal the protective effect of blueberry over
cardiovascular disease. However, further studies are required to assure the
therapeutic potential of blueberry.
CONCLUSION
All the listed evidence reveals important beneficial effects of the treatment
and/or consumption of blueberries. The low number of human trials that have
been performed in this sense, must lead future clinical trials in order to
confirm definitively the major potential of this fruit on human health, specially
on obesity and the metabolic diseases associated with.
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Chapter 5
BLUEBERRY EXTRACTS PROTECT AGAINST

GROSS MOUSE FETAL DEFECTS INDUCED
BY ALCOHOL TOXICITY
Zach S. Gish, Sharang Penumetsa, Diana J. Valle

and Roman J. Miller
Eastern Mennonite University,
Departments of Biology and Biomedicine,
Harrisonburg, Virginia, US
ABSTRACT
Alcohol is a powerful teratogen, systematically affecting prenatal
development as well as postnatal functioning in humans and other
mammals. Using a mouse model, this study explored the potential effects
of anthocyanins from blueberry extracts in protecting against alcohol-
induced prenatal developmental deficiencies.
Swiss mice were assigned to three experimental groups: control
(CO), binge alcohol (BA) and alcohol-anthocyanin (AA). CO mice were
administered normal saline (0.03 ml/g maternal body weight), while BA
and AA mice received alcohol (25% v/v of absolute ethanol in normal
saline at 0.03 ml/g maternal body weight), through intraperitoneal
injections on days 5 and 7 following impregnation. Supplemental
anthocyanins via blueberry extracts (0.03 mg/g maternal body weight)

Corresponding author: millerrj@emu.edu.
90 Zach S. Gish, Sharang Penumetsa, Diana J. Valle et al.
were additionally administered to the AA group, through subcutaneous-

neck injections on days 0, 5, 7 and 12. Maternal mice were necropsied
and fetuses removed at day 15 of gestation.
Statistical analysis (p < 0.05) showed that 15 day old mouse fetuses
with prior exposure to binge alcohol with anthocyanin supplementation
(AA) were partially protected from some gross developmental
deficiencies over the binge alcohol fetuses (BA). Group comparisons (CO
vs BA vs AA) showed significant fetal gross body differences in regards
to average body weight (197 vs 90 vs 162 mg, respectively), crown-rump
length (11.2 vs 9.1 vs 10.7 mm, respectively), liver surface areas (6.9 vs
2.5 vs 5.1 mm2 respectively) and telencephalon (forebrain) surface areas
(3.18 vs 1.47 vs 2.75 mm2 respectively).
Results support the hypothesis that properties found in blueberry
extracts serve to mitigate certain gross anatomical effects in mouse
fetuses due to maternal binge alcohol exposure during prenatal
development.
Keywords: anthocyanins, binge alcohol, fetus, mouse, telencephalon, liver
INTRODUCTION
Alcohol and Development
Pregnant mothers, who consume excessive alcohol, danger their

developing embryos/fetuses. As Goodlett and Horn report, alcohol exposure
during development increases oxidative stress, interferes with the activity of
growth factors, and changes the regulation of gene activity [1]. The
consequences of sustained maternal alcohol consumption can lead to
abnormalities in the mental and physical development of the offspring. The
abnormalities emerge from alcohol’s effects on migration and differentiation
of germ layers during the early embryonic period [2]. The sensitivity of early
developmental stages insures that teratogen introduction will affect multiple
natural developmental processes [3]. Abnormalities resulting from disturbed
cellular migrations and differentiation include, but are not limited to, stunted
growth, facial anomalies, and neurological damage in the central nervous
system (CNS).
Blueberry Extracts Protect against Gross Mouse Fetal Defects … 91
Alcohol Effects on Prenatal Mice
Alcohol administration in prenatal mice alters intracellular signaling

necessary for proper differentiation, leading to malformations, neurological
defects and, ultimately, cell death [4]. Additionally, alcohol lowers the overall
embryo size, suggesting that perigestational alcohol exposure induces
abnormal blastocyst growth, impairment of embryo-trophoblastic growth, and
expansion during implantation [5].
Alcohol Effects on Developing Nervous System
Alcohol is particularly dangerous to the developing nervous system, since

it has been shown to induce apoptosis in the cranial neural crest of embryos
[6]. Research on craniofacial malformation confirms that ethanol exposure
during gastrulation deforms the structure of the neural plate, a vital structure
needed in the development of the neural tube and the later spinal cord [7].
Alcohol exposure affects brain development through numerous pathways at all
stages from neurogenesis to myelination. Problems that occur during
neurogenesis ultimately lead to both behavioral and motor control
abnormalities [8]. Studies of cell migration and ethanol exposure show that
ethanol exposure to organisms will initially increase the number of cells in the
marginal zone and cortical plate, which will form the surface layers of the
neocortex. However, this proliferation of cells becomes prone to early cell
death, which will later lead to smaller numbers of cortical cells present in the
brain as well as to a thinner cortex at later stages of development [9].
Supportive studies [10] on ethanol exposure to developing cerebral cortex
demonstrate that brain volume, isocortical volume, isocortical thickness, and
isocortical surface area of rats exposed to ethanol were significantly smaller
than those regions in rats without ethanol exposure. Cerebral cortical cell
numbers and morphology in primary sensory areas exhibit high sensitivity to
ethanol exposure [11]. Embryonic brains of ethanol exposed mouse embryos
at day 10.5 of development (about the mid-point of the mouse gestation
period) show clear holoprosencephalic dysmorphic changes [12]. These
abnormal changes in the prosencephalon area of the brain include small and
incompletely divided telencephalic vesicles as well as anteriorly shifted and
connected nasal pits. Additionally again in the embryonic mouse model,
ethanol is responsible for irregular migration of pluripotent cells and, thus can
cause a variety of deformities, especially to the developing spinal cord [13].
In summary, research strongly indicates that ethanol exposure hinders

central nervous system (CNS) formation. Overall, damage to the CNS can
result in learning difficultly, attention deficits and poor cause and effect
reasoning. Damage to the development of the frontal lobes, which form from
the telencephalon area of the brain, can be particularly harmful, since the
offspring will be more likely to engage in dangerous behaviors as a result of
prenatal alcohol exposure.
Alcohol Effects on Liver Development and Function
Adult human binge drinking is a concerning trend that is exhibited more in

liver pathologies than chronic alcohol consumption [14]. In the murine model,
binge drinking results in the downregulation of Hdac 1,7,9,10 and 11, while
up-regulating Hdac 3, leading to alcohol-induced microvesicular hepatic
steatosis and damage due to increased hepatic triglycerides [15]. In a
subsequent study, the up regulation of Hdac3 down regulates cpt1α
contributing to hepatic steatosis [16]. Additional research shows the
importance of tumor necrosis factor (TNF)-α in alcohol-induced liver injury
through the TNF-R1 pathway. Adult TNF-R1 knockout mice demonstrate no
hepatic pathology, detailing the importance of TNF-α in the onset of steatosis
and inflammation [17]. The transcription factor, early growth response (Egr)-
1, has also been shown to contribute to steatosis development following acute
ethanol exposure in mice [18]. Damage to the development of the liver may
affect gene expression and lipid metabolism in adult male mice which had
been injected with acute doses of ethanol [19].
Chronic alcohol exposure leads to hepatotoxicity through redox
manipulations, which alter reactive oxygen species and reactive nitrogen
species or glutathione concentrations inducing apoptosis and necrosis [20].
Chronic alcohol exposure in adult mice increases triacylglycerols in the liver
leading to a fatty liver, while decreasing their concentrations in epididymal
and subcutaneous white adipose tissues [21]. Liver steatosis also arises
partially from factors such as increased formation of NADH+ which allows for
reduced coenzyme for fatty acid synthesis [22]. Together these factors provide
detail as to the danger of both acute and chronic alcohol exposure on the liver.
Health Benefits of Blueberries
Many consumers are aware of the health benefits that fresh blueberries
provide [23]. These include basic nutrient properties, antioxidant activity, [24,
25] anti-aging properties, [26] cancer prevention, [27, 28] protection against
age-related neurological defects, [29] urinary tract health, protection against
diabetes, [30] and cardiovascular health [31]. The polyphenolics and
anthocyanins, found in ripened blueberries, are the primary health promoters
and protective antioxidant agents [32] In comparison to many other fruits,
blueberries contain higher levels of protective anthocyanins. These benefits are
based on various studies, many of them animal studies where the findings have
been superimposed on humans. This list has also been a clarion marketing call
and elicited many consumers to choose blueberries for consumption rather
than other fruits which have lower levels of antioxidants.
What if the health benefit list of blueberries or blueberry anthocyanins
could be expanded? This chapter, based on preliminary research in our
laboratory, documents that blueberry anthocyanins in the form of blueberry
extract can alleviate some of the teratogenic influences of maternally ingested
alcohol on embryonic/fetal development. That claim, if further verified, has
huge implications. The idea that anthocyanins protect against some of alcohol
teratogenic influences has been noted by several other investigators in other
biological systems; [33, 34] however to date, except for this model project,
that connection has not been demonstrated in an in vivo mammalian
developmental system.
Anthocyanin Interaction with Alcohol
Anthocyanins are natural pigments present in blueberries that belong to

the flavonoid class of compounds. A primary antioxidant compound found in
blueberries is cyanidin-3-glucoside (C3G). Research reveals that C3G is
capable of reducing neurodegenerative effects of ethanol exposure by
alleviating oxidative stress [35]. When bowel disease is induced in mice with
trinitrobenzene sulfonic acid (TNBS), the experimental groups that received
dietary blueberries along with TNBS had lower risk of induced bowel disease
than that of control groups [36]. Anthocyanin administration partially
eradicates free radicals from superoxide, peroxide, hydrogen peroxide, and
hydroxyl groups, which are responsible for the toxic responses to ethanol in
fetal tissues [37]. Anthocyanins also reduce DNA damage, which is a major
indicator in Fetal Alcohol Syndrome [38]. Similarly the antioxidant, vitamin

E, alleviates oxidative stress in ethanol exposed neonatal rats [39]. Since
identifying protective properties is important for creating therapeutic
treatments, these studies promote the role of antioxidants as preventive
measures against alcohol-induced damage.
Antioxidants have the power to shield against free radicals in the body that
can harm fetal cells. Lack of research, however, has slowed the transition of
antioxidants from research labs to the clinical field, as a medicinal treatment.
Mouse developmental studies can provide this transitional bridge. Mouse
gestational day 9 is comparable to human gestational day 20 in which the
neural plate begins to fold over the notochord. Mouse gestational day 11 is
comparable to human gestational day 30 in which the forebrain, somites and
1st, 2nd and 3rd pharyngeal arches are present. Mouse gestational day 15 is
comparable to human gestational day 55 in which the limbs, trunk, heart, liver
and even features of the face can be identified [40].
MODEL PROJECT
Experimental Objectives
The teratogenic effects that binge alcohol alone can have on a developing
embryo or fetus are well documented in various model systems. However, the
potential protection of anthocyanins against this alcohol toxicity has not been
examined in developmental model systems. Consequently our project
approach used an in vivo mouse development model to inspect the extent of
the protection that anthocyanins provide in combating the life-threatening
effects of oxidative stress on embryos and fetuses from alcohol induction
exposure during gestation. To investigate the extent of gross anatomical
malformations, three experimental groups of pregnant mice were used: control
(CO), binge alcohol (BA), and binge alcohol supplemented with anthocyanins
(AA). The goal for this experiment was to clearly demonstrate the protective
role of anthocyanins against the teratogenic influences of alcohol as shown in
gross anatomic parameters – whole body, forebrain, and liver in the mouse
fetuses.
Materials and Methods
Following approval from Eastern Mennonite University’s Animal Use and

Care Committee, Swiss outbred mice were obtained from a national supplier
[41], given free access to a diet of Purina rodent chow and water, and housed
in a separate room held at 24°C with a 12:12 light: dark cycle for the duration
of the experiment. Prior to the start of experimentation, male mice (average
24-26 g body weight) and female mice (average 20-22 g body weight) were
allowed to acclimate with their surroundings. A pre-trial group of three control
females was run to ensure proper experimental procedure. At the time of the
beginning of the experiment both male and female mice were young mature
adults averaging 50-60 days of age.
Experimental Groups and Design
Three experimentation groups were formed with female mice. Control

females (CO) received intraperitoneal (IP) saline injections (normal saline 0.03
ml/g per maternal body weight) on gestation days 5 and 7. Binge Alcohol
females (BA) received IP injections of alcohol (25% v/v of ethanol in normal
saline at 0.03 ml/g per maternal body weight) on gestation days 5 and 7.
Alcohol-Anthocyanin females (AA) received IP alcohol injections on days 5
and 7 of gestation (ethanol 0.03 ml/g 25% v/v of ethanol in normal saline per
maternal body weight) and subcutaneous-dorsal neck anthocyanin injections
on gestation days 0,5,7,12 (anthocyanin, 30 mg/kg per maternal body weight).
The anthocyanin injection solution was prepared at a concentration of 5 mg/ml
in normal saline from Life Extension Blueberry extract capsules [42]. The
concentrations of alcohol and anthocyanin were largely based on prior work in
other laboratories [43, 44].
On day 0, cohorts of female mice, representing the three experimental
groups, were mated with age-matched males. Gestation day one was
determined by the subsequent presence of a vaginal plug. Males were removed
3 days after vaginal plug appearance and rebred with the next cohort of
females. To maintain accuracy in food consumption measurements, males
were all removed on day four and feeding data measurements began.
Throughout the gestation period, food consumption, weight changes and
appearances of individual females were recorded.
DATA COLLECTION AND ANALYSIS

Necropsy
On gestation day 15, mothers were euthanized with an overdose of ether,

their uteruses were excised, and individual fetuses were isolated. After each
fetus was removed from its amniotic sac, the fetus was measured from the
cranial to caudal end (crown-rump length) using a calipers, weighed to the
nearest mg, and photographed, before being placed in either 10% buffered
formalin fixative for subsequent histological analysis or frozen for subsequent
biochemical analysis.
Measurements/Stereological Data
Random representative fetuses from each group (N = 12) were used for
morphometric and stereological data collection. Morphometric data consisted
of determining gross liver and telencephalon area with direct measurements
using a Nikon SMZ 74ST microscope and NIS Elements BR 3.2 software. In
each fetus the telencephalon, a part of the forebrain area (prosencephalon), and
the liver area were determined via specific somatic landmarks. These organ
areas were circumscribed and their surface areas estimated in mm2 using the
calibrated software program from the image camera. Subsequently, these areas
were compared with the total fetus body surface area.
To obtain primary stereological data, a coherent Weibel grid imprinted on
an acetate sheet was superimposed on photomicrographs of the fetuses.
Following an established protocol, [45, 46] simple point counts, based on the
number of Weibel grid points falling on the image of the parameter of interest,
e.g., liver vs total body area or telencephalon area vs. total body area, were
converted into volume density determinations. Each measured fetus was
contained within one counting field of view and represented an “n” of one.
Statistical Analysis
Means and standard errors were calculated as group statistics for measured
parameters: fetal weight, crown-rump length, and gross tissue measurements
(organ areas and organ volume density measures). Significant differences
between groups were determined using One-Way ANOVA and Student-

Newman-Keuls post-hoc statistical testing (p < 0.05) with SPSS 22 software.
RESULTS AND DISCUSSION

Maternal Responses
Throughout the experiment pregnant female health and weight were

ascertained daily. The maternal body weight of each trial group at the
beginning and at the end of the experiment revealed that the CO group
averaged 29.5 g at the beginning and finished with an average of 38.4 g. The
BA group began with an average of 29.6 g and finished with an average of
36.6 g. The AA group averaged 28.4 at the beginning of the experiment and
averaged 37.2 g at the end of the experiment. In summary the pregnancy
weight gains were similar for all three experimental groups with an average
increase during the first 15 days of 25% (See Table 1). While the CO and AA
group mothers trended toward higher body weight gains during their
pregnancies than the mothers in the BA group, these values were not
statistically different (27% and 28% versus 21% respectively).
Table 1. Maternal Data: Average Body Weights and Pregnancy Results

Body Weight Day 15
Number of Pregnant
Body Weight Day 1
Body Weight Day 8

Experiment Groups
Percent Increase in
Average Number
Fetuses / Mother
Fertility Percent
Total Fetuses
Body Weight
Mothers
(g)
(g)
(g)
CO 12 80% 29.5 31.4 38.4 27% 117 9.75

BA 7 58% 29.6 30.5 36.6 21% 58 8.29
AA 9 75% 28.4 30.6 37.2 28% 79 8.78
Average 9.3 71% 29.2 30.8 37.4 25% 8.94
Groups Definitions: CO = Control: pregnant mothers treated with two saline injections;
BA = Binge Alcohol: pregnant mothers treated with two binge alcohol injections;
AA = Alcohol-Anthocyanin: pregnant mothers treated with two binge alcohol
injections and supplemented with four anthocyanin injections.
Although the project was initiated with mating 15 female mice in the CO
group and 12 female mice in the BA and AA groups, the number of resultant
pregnant females in the BA and the AA groups was less than in the CO group:
CO = 12 pregnancies (80% fertility); BA = 7 pregnancies (58% fertility);
AA = 9 pregnancies (75% fertility). While the average number of fetuses per
pregnant mother varied slightly in the different groups with an average of 8.94
fetuses/mother, these differences were not statistically significant (See Table
1). Upon conclusion of the project, it was determined that one of the males
used was sterile, since each of the females this male mated throughout the
project did not produce offspring.
All females receiving binge alcohol injections exhibited similar patterns of
behavior following injections that included staggering and losing
consciousness within the span of a few minutes. But then later these females
revived and resumed normal activity after a period of time. Control females
with saline injections did not exhibit these behavioral patterns. Instead they
displayed mild agitation following injections.
The average food intake per day by each group generally reflected a
steady increase in consumption throughout the period of pregnancy. However
food intake did fluctuate as a consequence of alcohol injections (see Figure 1).
Figure 1. Daily food consumption by experimental group mothers over the course of
experiment. Control (CO) saline treated mice (N = 15), Binge Alcohol (BA) treated
mice (N = 12), Alcohol–Anthocyanin (AA) treated mice (N = 12). Feeding data began
after males were removed from female cages on day 4. Arrows denote days injections
were performed (large=ethanol) (narrow = anthocyanin). Values are expressed as
means ± standard error.
On day 6 following the day 5 alcohol injections, the BA and AA mothers

consumed less food than the CO mothers. More dramatically following the day
7 ethanol injections, the BA and AA mothers’ food consumption dropped by
almost 30%. In contrast the CO group food consumption remained stable.
Food consumption did rebound for both the BA and AA groups in the
following days and reached the level of the CO group by day 10 of gestation.
Fetal Whole Body Responses
Following necropsy, the average weight of the 117 CO fetuses was 196.7
mg (Figure 2) and the average crown to rump length was 11.19 mm (Figure 3).
The BA group of 58 fetuses averaged 90.4 mg for fetal weight at time of
collection and a length of 9.07 mm. The AA group of 79 fetuses averaged a
weight of 161.8 mg and a length of 10.71 mm. Both fetal body weight and
crown-rump length were not significantly different between the CO and AA
groups. However, significant differences were found between the CO and BA
groups as well as between AA and BA groups. Representative samples from
each experimental group demonstrating the developmental differences
pictorially are illustrated in Figure 4. Strong definition and detail differences in
the fetal mice can be more clearly observed in the CO (Figure 4A) and AA
(Figure 4C) mice while gross detail was far less distinct in the BA (Figure 4B)
mice. Especially in the BA group, the fetuses were frequently ill-formed, but
less so in the AA group when compared to the CO group.
In concordance with previous work, perigestational ethanol exposure does
retard gross fetal size, [47] however proactive administration of anthocyanins
appears to partially neutralize the deficits in fetal size. These results clearly
demonstrate that the anthocyanin dosages mitigate the detrimental effects of
the alcohol at the very least on a gross fetal body scale.
Fetal Telencephalon Response
In comparing telencephalon size with the rest of the fetal body (Figure 5),
BA fetuses demonstrated a trend of reduced telencephalon size (5%) compared
with those of CO (7%) and AA (7%). A similar trend was seen in another
study [48] that showed improper telencephalon division and development in
ethanol induced mouse embryos.
Figure 2. Fetal weights at gestation day 15 across experimental groups N = 254.

Control (CO) saline treated fetuses (N = 117), Binge Alcohol (BA) treated fetuses
(N = 58), Alcohol-Anthocyanin (AA) treated fetuses (N = 79). Black arrows
correspond with statistically significant differing values (p < 0.05). Values are
expressed as means ±standard error.
Figure 3. Fetal crown-rump length at gestation day 15 across experimental groups

N = 254. Control (CO) saline treated fetuses (N = 117), Binge Alcohol (BA) treated
fetuses (N = 58), Alcohol-Anthocyanin (AA) treated fetuses (N = 79). Black arrows
correspond with statistically significant different values (p < 0.05). Values are
expressed as means ± standard error.
Figure 4. Representative fetal photograph samples at 15 days gestation from each

experimental group demonstrating variations on gross body parameters. 4A: Control
(CO) saline treated mouse fetus; 4B: Binge Alcohol (BA) treated mouse fetus; 4C:
Alcohol–Anthocyanin (AA) treated mouse fetus.
Figure 5. Fetus body composition comparing telencephalon size to relative body size,
using a Weibel grid to determine volume density measures. Control (CO) saline treated
fetuses (N = 12), Binge Alcohol (BA) treated fetuses (N = 11), Alcohol-Anthocyanin
(AA) treated fetuses (N = 11). Values are expressed as means. Circle diameters reflect
relative differences in experimental group body sizes.
The reason behind this pattern can be explained by ethanol’s ability to

impede the regular migration of pluripotent cells, which plays a vital role in
the developing central nervous system [49]. In normal brain development, the
neural plate acts as a precursor to the developing spinal cord and brain. With
ethanol exposure however, the regular developmental processes in BA fetuses
may have been hindered, compromising the integrity of the overall size of the
brain [50]. The reduction in telencephalon size is not only due to reduced body
size but also reduced telencephalon size relative to body size.
Figure 6, which details exact measurements of telencephalon area,
demonstrates that AA fetuses have relatively similar telencephalon areas
compared with CO fetuses (2.75 versus 3.18 mm2) while BA fetuses (1.47
mm2) have significantly underdeveloped telencephalon areas. The sizeable
telencephalon difference between the experimental groups can be attributed to
alcohol-induced oxidative stress which is a major mechanism causing cell

death [51]. To explain why telencephalon measurements were similar between
AA and CO fetuses, oxidative stress has been shown to decrease upon the
administration of cyanidin-3-glucoside, the main anthocyanin compound [52].
Another study supports these protective properties of anthocyanins by showing
that anthocyanin administration prevented ethanol-induced neuronal cell death
in rat hippocampal cells [53]. The results from our experiment suggest that the
ability to maintain the size of the telencephalon in AA fetuses may be
attributed to anthocyanin buffering the rate of ethanol-induced neuronal cell
death.
Figure 6. Morphometric telencephalon analysis detailing average absolute

telencephalon area in each experimental groups. Control (CO) saline treated fetuses
(N = 12), Binge Alcohol (BA) treated fetuses (N = 11), Alcohol-Anthocyanin (AA)
treated fetuses (N = 11). Values are expressed as means ± standard errors.
Figure 7. Fetus body composition comparing liver size to relative body size, using a
Weibel grid to determine volume density measures. Control (CO) saline treated fetuses
(N = 12), Binge Alcohol (BA) treated fetuses (N = 11), Alcohol-Anthocyanin (AA)
treated fetuses (N = 11). Values are expressed as means. Circle diameters reflect
relative differences in experimental group body sizes.
Fetal Liver Response
Some of the current research is focused on postnatal liver functioning of

mice, which experienced perigestational ethanol exposure. These studies are
helpful in showing how binge ethanol exposure can increase hepatic
triglycerides and lead to hepatic steatosis through alterations in gene
expression [14, 15]. Additionally, in adult models, changes in gene expression
and lipid metabolism are central to the effects that acute doses of ethanol
prompt in the liver [30].
Figure 8. Morphometric liver analysis detailing average absolute liver area in each
experimental group. Control (CO) saline treated fetuses (N = 12), Binge Alcohol (BA)
treated fetuses (N = 11), Alcohol-Anthocyanin (AA) treated fetuses (N = 11). Values
are expressed as means ± standard errors.
In comparison, our results show prenatal fetal reductions of liver size in

relationship to relative body size as well as declining absolute liver area due to
perigestational ethanol exposure in the BA groups. The introduction of
anthocyanins (blueberry extracts) in combination with the ethanol however
effectively retains fetal liver size relative to body size as well as absolute liver
area. Figure 7 details the relationship between relative liver sizes to body sizes
among the experimental groups. Unsurprisingly, BA fetuses show reduced
liver size compared to body size (7%), while AA fetuses show retention of
liver sizes to body sizes in proportions similar to CO fetuses (11% versus 12%
respectively). When morphometric absolute liver area measurements among
experimental groups were compared (Figure 8), BA livers were much smaller
(2.54 mm2), while AA fetuses demonstrated retention of gross liver areas

similar to that of CO mice (5.05 vs 6.9 mm2). Together Figures 7 and 8 show
that BA fetuses not only exhibit reduced gross liver area compared with CO
and AA, they also exhibit reduced gross liver size even when their reduced
body size is accounted for. This trend is not observed in the AA group, but
rather retention of overall absolute liver area as well as liver size in proportion
to relative body size is demonstrated when compared with the CO group.
CONCLUSION
Our research demonstrates that anthocyanin supplementation (in the form
of blueberry extracts) given to developing embryos/fetuses in the mouse
development system mitigates some of the detrimental effects of concomitant
perigestational exposure of alcohol. This mitigating response is seen following
two binge alcohol exposures during the early period of gestation when
accompanied with four applications of anthocyanin supplementation given
before, during, and after the exposure to alcohol.
Initial gross fetal body assessments in comparison to control fetuses show
that binge alcohol exposure reduces average fetal body weight by 54% while
binge alcohol with anthocyanin supplementation reduces average fetal body
weight by 18%. When considering fetal size as determined by crown-rump
length, binge alcohol reduces size by 19% and binge alcohol supplemented
with anthocyanins reduces size by 4% when compared with control fetuses.
In looking at the size of two organs – telencephalon and liver – a similar
outcome is observed. Based on surface area, fetuses in the control group have
telencephalon and liver surface areas that represent 7% and 12% respectively
of their total body surface area. In contrast binge alcohol fetuses have
telencephalon and liver surface areas that represent 5% and 7% respectively of
their total body surface area. However, fetuses from the binge alcohol and
anthocyanin supplemented group have telencephalon and liver surfaces that
represent 7% and 11% respectively of their total body surface area paralleling
the control group data. Inspection of these data shows that anthocyanin
supplementation has a beneficial effect in reducing the influence of alcohol
toxicity.
The specific mechanism for the protective role of anthocyanins against
alcohol toxicity in the developing mouse system is not yet determined.
Subsequent studies extending this research are currently focusing on
histological and functional parameters of the liver and telencephalon to further

elucidate this phenomenon.
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INDEX
benefits, vii, ix, 2, 18, 21, 63, 72, 84, 93

A beta cell function, x, 72
binge alcohol, x, xi, 89, 90, 94, 97, 98, 104
adipose tissue, 72, 73, 74, 75, 87, 92, 107
binge drinking, 92, 106
alcohol consumption, 90, 92
biological activity, 9, 33
alcohol exposure, 90, 91, 92, 105, 107, 109
biological responses, 73
alcohol toxicity, v, 89
biological systems, 57, 93
alcohol-induced prenatal developmental
biomarkers, 77, 83, 85
deficiencies, x, 89
biosynthesis, 15, 28
angiotensin converting enzyme, 79, 87
biotechnology, 29
anthocyanins, viii, x, 2, 3, 7, 8, 10, 12, 16,
Bluebelle, ix, 6, 12, 32, 33, 34, 35, 36, 39,
17, 18, 19, 20, 24, 25, 26, 27, 28, 29, 32,
40, 41, 42, 46, 47, 48
34, 35, 36, 40, 43, 45, 50, 56, 57, 61, 66,
blueberries, vii, viii, ix, x, 2, 3, 4, 5, 6, 7, 8,
75, 79, 80, 81, 85, 87, 89, 90, 93, 94, 99,
9, 10, 11, 13, 14, 15, 16, 17, 19, 20, 21,
102, 103, 104, 108
22, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34,
anti-cancer, 27
37, 38, 40, 45, 47, 49, 50, 52, 55, 56, 57,
anti-inflammatory agents, 75
60, 61, 62, 63, 65, 66, 68, 72, 76, 79, 82,
antioxidant, vii, viii, ix, 2, 3, 4, 9, 19, 20,
83, 84, 85, 87, 93
21, 24, 25, 26, 27, 28, 29, 32, 33, 50, 51,
body composition, 101, 102
55, 56, 57, 62, 65, 66, 67, 68, 69, 72, 76,
body fat, 72, 80
83, 93, 94, 107, 108
body size, 101, 102, 103
apoptosis, 28, 29, 52, 68, 79, 82, 91, 92, 105
body weight, x, 21, 72, 89, 90, 95, 97, 99,
arabinoside, 15, 17, 20
104
Argentina, 4, 5, 6, 7
brain, 18, 91, 92, 101
ascorbic acid, viii, 2, 4, 8, 10, 21
brain functions, 18
atherosclerosis, 75
Brazil, vii, 1, 3, 4, 5, 6, 7, 31, 32, 47
breeding, 5, 27
B Briteblue, ix, 6, 12, 18, 32, 36, 39, 40, 41,
42, 45, 46, 47
beneficial effect, ix, 18, 55, 56, 77, 83, 84,
104
112 Index
crystals, 58, 59
C cultivars, vii, viii, 1, 5, 7, 9, 10, 16, 18, 21,
23, 24, 26, 27, 28, 32, 33, 34, 35, 36, 38,
Ca2+, 64
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 52
CAE, 39
cultivation, vii, 1, 2, 3, 4, 5, 8, 10, 27
calcium carbonate, 65
CVD, 75, 77, 83
cancer, viii, 2, 3, 17, 28, 29, 38, 56, 67, 68,
cyclooxygenase, 76
77, 83, 93, 108
cytokines, 73, 74, 75
cancer cells, 17, 28, 29, 38, 68
carbohydrates, 8, 10
cardiovascular diseases (CVD), ix, 56, 71, D
72, 75, 79, 84, 86
cardiovascular function, 75 dehydration, ix, 55, 60, 61, 62, 63, 64, 65,
cardiovascular risk, 84 66, 68, 69
cardiovascular system, 75 Delite, ix, 6, 12, 32, 33, 34, 36, 39, 40, 41,
carotene, 8, 19, 21, 22, 40 42, 46, 47, 48
carotenoids, viii, ix, 2, 4, 18, 19, 29, 32, 40, dendritic cell, 73
41, 52 Department of Agriculture, vii, viii, 4, 31
cell death, 79, 91, 102, 105, 109 derivatives, 14, 22, 37, 57
cell lines, 18, 76 detection system, 19
cell membranes, 61 developing brain, 108, 109
cellulose, 8 developmental change, 106, 109
central nervous system (CNS), 90, 92, 101 developmental process, 90, 101
cerebral cortex, 91, 105 diabetes, viii, ix, 2, 4, 18, 71, 73, 85, 93,
chemical, viii, 2, 3, 50, 51, 61, 65, 68, 73 108
chemical properties, 68 dialysis, 68
chemokines, 73 diastolic pressure, 82
Chile, 4, 5, 6, 7, 55, 71 diet, x, 18, 56, 72, 79, 80, 81, 82, 84, 87, 88,
Chitosan, 68 95, 107
chlorophyll, 10 dietary management, viii, 2, 4, 18
cholesterol, viii, 2, 4, 18, 21, 81 dietary supplementation, 83
Climax, ix, 6, 12, 32, 34, 35, 36, 39, 41, 42, diffusion, 61, 62
43, 46, 47 digestive enzymes, 107
clinical symptoms, 73 dilated cardiomyopathy, 84
clinical trials, 83, 84 diseases, ix, 8, 33, 56, 71, 73, 75, 76, 77, 83,
coenzyme, 92 84
colon, 18, 29 distillation, 58
colon cancer, 18, 29 distribution, 16, 26, 28, 50
consumption, ix, 5, 18, 20, 28, 55, 57, 61, diversity, 16, 19
72, 77, 83, 84, 85, 93, 95, 98, 99, 105 DNA, 14, 27, 84, 93, 108
contamination, 61 DNA damage, 14, 27, 84, 93, 108
control group, 77, 93, 104 double bonds, 40
copper, 10 drinking water, 81
c-reactive protein, 75 dry matter, 63, 66
crop, vii, 1, 3, 22 dyslipidemia, x, 72
crystallization, 59
Index 113
E F
elaboration, 14 farmers, 3
Elam, 85 fasting, viii, 2, 4, 18, 80
electric field, 63 fasting glucose, 81
electron, 34 fat, 9, 72, 80, 81, 83, 87, 88
electrons, 4 fat soluble, 9
electrophoresis, 68 fatty acids, 74
embryology, 105, 108 fermentation, 50
emission, 8, 24 fertility, 98
endocrine, 72 fetal alcohol syndrome, 106, 109
endothelial dysfunction, 75 fetal development, 93
energy, ix, 10, 55, 61, 63, 72, 83 fetus, 90, 94, 96, 101
energy expenditure, 72 filtration, 68, 82
energy transfer, 10 flavanols, viii, 2, 3, 57
engineering, 67 flavonoids, viii, 2, 3, 10, 11, 16, 29, 57, 85,
enlargement, 74 107
environment, 63 flavor, 6, 8, 58, 59, 62
enzyme, 57, 58, 68, 78 folate, 9
enzymes, 9, 58, 63, 74 food, ix, 10, 24, 41, 51, 52, 55, 56, 58, 59,
epidermis, 61, 63 60, 61, 63, 64, 67, 95, 98, 99
epithelial cells, 83 Food and Agriculture Organization (FAO),
equilibrium, 61 vii, viii, 3, 4, 22, 31, 49
equipment, 59 food industry, ix, 55, 64
ESI, 27 food intake, 98
essential fatty acids, 56 food products, 52, 61
ester, 13, 14 forebrain, x, 90, 94, 96
ethanol, x, 35, 78, 89, 91, 92, 93, 95, 98, 99, formation, 20, 22, 92
101, 102, 103, 105, 106, 107, 108, 109 free radicals, viii, ix, 2, 3, 4, 55, 56, 93, 94,
ethyl acetate, 77, 79 107
ethylene, viii, 2, 3 freeze concentration, ix, 55, 58, 59, 60, 65,
etiology, ix, 71 66, 67
Europe, 4, 7, 18 freezing, 58, 59, 60, 61, 66, 68
European Union, vii, 2, 3 fructose, 8, 80, 86
evaporation, 58 fruits, vii, ix, 2, 3, 5, 7, 8, 9, 10, 11, 14, 15,
evidence, x, 67, 72, 76, 78, 84, 105 16, 19, 20, 21, 24, 25, 29, 32, 33, 34, 38,
evolution, 10, 46 40, 41, 43, 45, 48, 50, 55, 56, 60, 61, 62,
exercise, 22 63, 64, 65, 66, 76, 87, 88, 93, 107
exposure, x, xi, 9, 10, 63, 90, 91, 92, 93, 94, functional food, 52, 56
99, 101, 103, 104, 105, 107, 109
extinction, 35, 40
extraction, 15, 37, 38 G
extracts, x, xi, 9, 16, 18, 20, 22, 27, 28, 29,
GABA, 109
33, 37, 38, 50, 51, 68, 76, 77, 78, 79, 83,
gastrointestinal tract, 77
88, 89, 90, 103, 104
114 Index
gastrulation, 91, 106, 109

gene expression, 77, 92, 103, 106
I
genes, 77, 82
ICAM, 75
genomics, 29
immune system, 9
genotype, 52, 65, 68
impregnation, ix, x, 55, 63, 64, 65, 69, 89
germ layer, 90
in vitro, x, 18, 20, 21, 28, 29, 57, 68, 72, 77,
gestation, x, 90, 91, 94, 95, 96, 99, 100, 101,
79, 85, 86, 87, 105, 109
104
in vivo, x, 20, 29, 72, 79, 87, 93, 94, 105,
glucose, viii, 2, 4, 8, 18, 56, 68, 73, 78, 80,
107, 109
82, 83, 86
inflammation, ix, 9, 71, 73, 75, 76, 83, 85,
glucose tolerance, 80, 82
86, 92
lucoside, 12, 13, 14, 15, 17, 35, 36, 77, 93,
inflammatory disease, 75
102, 105, 108, 109
inflammatory responses, 73
GLUT4, 74
ingestion, 33, 83
glutathione, 82, 92, 107
inhibition, 20, 76, 79, 105
glycogen, 105
inhibitor, 82
injections, x, 89, 95, 97, 98, 99
H injury, 73, 75, 77, 79, 83, 92, 106, 107
insulin resistance, ix, 65, 71, 75, 77, 83, 85,
harvesting, vii, viii, 2, 3, 49 86, 87
health, vii, ix, 2, 3, 9, 21, 22, 24, 25, 27, 33, insulin sensitivity, x, 72, 83, 87
56, 72, 93, 97 interneurons, 105
health effects, vii, viii, 2, 3 interstitial nephritis, 82
health problems, 56 iron, 10, 51
health promotion, 22, 25 ischemia-reperfusion injury, 82
hemicellulose, 8
hepatotoxicity, 92
herbal medicine, 25
K
hidroxycinamic acids, viii, 2
kaempferol, 11, 14, 15, 57
high fat, x, 65, 72, 80, 81, 85, 87
ketones, 8
homeostasis, 86, 107
kidneys, 82
human health, ix, 17, 21, 55, 56, 84, 87
kinetics, 64
hybridization, 7
hydrogen, 33, 48, 93
hydrogen atoms, 48 L
hydrogen peroxide, 93
hydrolysis, 27, 37 LDL, 81, 83
hydrophobicity, 61, 62 lead, 62, 90, 91, 103
hydroxyl groups, 33, 93 leptin, 73, 75, 80, 87
hyperglycemia, x, 21, 72 leukemia, 18
hyperphagia, x, 72, 80 leukocytes, 73
hypertension, 84, 86 lignin, 8
hypertrophy, 73, 74, 79 lipid metabolism, 92, 103, 106
hypoxia, 73 lipid oxidation, 81
lipid peroxidation, 82
Index 115
lipids, 9, 66, 107

lipolysis, 74, 77
N
liquid chromatography, 27, 65
NADH, 92
listeria monocytogenes, 27
NASS, 22
liver, x, 38, 77, 79, 90, 92, 94, 96, 102, 103,
National Agricultural Statistics Service, vii,
104, 105, 106, 107
viii, 4, 31
liver disease, 77, 79
National Agricultural Statistics Service
low temperatures, ix, 56, 58, 59
(NASS), vii, viii, 4, 31
low-grade inflammation, 73
necrosis, 92
lutein, 19
neocortex, 91
lymphocytes, 73
nervous system, 91, 105
neurodegenerative diseases, 56, 76
M neurogenesis, 91
neuronal apoptosis, 109
macrophages, x, 72, 74, 76, 85 neurons, 109
magnetic resonance imaging, 106 neurotoxicity, 108, 109
manganese, 10 niacin, 9
mass spectrometry, 27, 65, 107 Nile, 21
matrix, 61, 63, 64, 107 nitric oxide, 76
matrix metalloproteinase, 107 nitrogen, 56, 63, 66, 92
MCP-1, 74, 76 non-polar, 40
mean arterial pressure, 82 norepinephrine, 79
melon, 40 North America, ix, 3, 5, 7, 55
melting, 66 notochord, 94
Mercosul, 23 Nrf2, 83
metabolic, v, 71, 86 nutrient, 63, 93
metabolic diseases, ix, 71, 73, 75, 84 nutrients, 9, 63, 108
metabolic disorders, 85 nutrition, 15, 24
metabolic pathways, 33 nutritional value, ix, 8, 55, 56, 60, 61, 83
metabolic syndrome, 79, 80, 83, 84, 88
metabolism, 10, 17, 83
metabolites, 33, 57
O
metformin, 80
obesity, ix, 71, 73, 74, 75, 76, 78, 79, 80,
methanol, 37, 38
83, 84, 87, 88
microorganisms, 64
obesity-associated inflammation, ix, 71
migration, 61, 74, 90, 91, 101, 106, 109
oleic acid, 56, 79, 86
mitogen, 74
oligomers, 37, 38
molecular weight, 35, 38, 40, 61, 76
olive oil, 9, 22
molecules, 10, 35, 56, 72, 75
organ, 72, 96
monomers, 38
organic matter, 5
morphology, 67, 91
organism, ix, 38, 71, 72
morphometric, 96, 103
organs, ix, 71, 104
motor control, 91
osmotic dehydration, ix, 55, 61, 62, 63, 64,
mRNA, 76, 77, 78
65, 66, 69
myocardial infarction, 82, 84
116 Index
oxidation, 60, 81, 83 pro-inflammatory, 73, 74

oxidative damage, 56 project, 93, 94, 98
oxidative stress, 22, 29, 73, 79, 82, 86, 90, proliferation, 18, 27, 29, 38, 77, 91
93, 94, 102, 109 prostate cancer, 38, 107
oxygen, 19, 60, 63, 67 protection, 14, 20, 93, 94, 107, 108
protective role, 82, 94, 104, 108
protein kinases, 74
P proteins, 9, 10, 39, 46, 51, 74
public health, 106
pantothenic acid, 9
pulp, vii, viii, 6, 7, 16, 18, 32, 33, 35, 36,
pathogenesis, 76
37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48
pathogens, 9
pathology, 92
pathways, ix, 71, 74, 76, 91 Q
PCR, 75, 83
periodontal disease, 83 quercetin, 11, 14, 15, 20, 57
permeability, 61, 62, 63, 75, 81
peroxidation, 20
peroxide, 93 R
pH, 9, 32, 35, 43, 45, 47, 48, 49
radiation, 11, 15, 28, 48, 53
phenolic compounds, viii, ix, 2, 3, 4, 10, 11,
radicals, 56
12, 13, 14, 16, 19, 20, 26, 27, 32, 33, 34,
reactive oxygen, 20, 56, 92
35, 36, 37, 38, 43, 45, 49, 50, 52, 55, 56,
receptor, 74, 86, 87, 109
57, 61, 62, 63
red blood cells, 20
phosphorus, 10
renal function, x, 72, 86
phosphorylation, 74, 78
resistance, x, 20, 29, 72, 74, 75, 80, 82, 84
photomicrographs, 96
response, ix, 53, 71, 74, 76, 77, 92, 104, 106
phytochemicals, ix, 19, 25, 32, 43, 51
reticulum, 73
PI3K, 78
retina, 18
placebo, 84, 86, 88
retinopathy, 18
plants, 5, 9, 13, 33, 36, 79
riboflavin, 9
plasma levels, 20
risk, 56, 77, 84, 93, 105
polyacrylamide, 68
risk factors, 84
polymerization, 37, 38
polymorphisms, 27
polyphenols, viii, 2, 4, 10, 12, 22, 26, 76, S
77, 79, 85, 86, 87, 107
polysaccharides, 8 Salmonella, 27
potassium, 10 salts, 46, 61
potential benefits, 17 seed, 56, 66
Powderblue, ix, 6, 12, 18, 32, 34, 35, 36, 38, selenium, 10
39, 41, 42, 46, 47, 48 senescence, 9, 107
preservation, 12, 60, 61 signaling pathway, 73, 74, 85
pressure gradient, 63 skeletal muscle, 78, 81, 87
prevention, x, 72, 93, 105 skin, 5, 35, 61, 62, 63
probiotic, 64 sodium, 10, 68
Index 117
sodium dodecyl sulfate, 68 urinary tract, 18, 38, 93

South America, 4, 6, 7, 22 urinary tract infection, 38
species, ix, 5, 7, 16, 19, 20, 27, 51, 55, 56, USDA, vii, viii, 4, 6, 22, 31
92, 107 UV radiation, 11, 83
spinal cord, 91, 101
Sprague-Dawley rats, 80, 81, 86
strawberry, vii, viii, 3, 8, 14, 20, 31, 37, 50, V
67, 68
vacuum impregnation, ix, 55, 63, 64, 65, 69
sucrose, 8, 59, 67
varieties, 5, 15, 23, 32, 34, 38
sulfate, 37
vascular diseases, 67
systolic blood pressure, 79, 87
VCAM, 75
vegetables, vii, 2, 3, 11, 21, 29, 33, 56, 57,
T 61, 63, 64, 65, 88
VEGF, 75
tannins, viii, 18, 32, 36, 37, 38, 39, 50, 51 vitamin A, 9
techniques, ix, 55, 58, 59, 63 vitamin B1, 9
technologies, ix, 55, 56, 59, 61 vitamin B12, 9
telencephalon, x, 90, 92, 96, 99, 101, 102, vitamin B6, 9
104, 105 vitamin C, 9, 10, 19, 22, 66
temperature, 10, 22, 49, 58, 62, 63 vitamin E, 94, 108
teratogen, x, 89, 90 vitamin K, 9
testing, 32, 97, 107 vitamins, 9, 10, 32, 61, 64, 108
thermal treatment, 58 VLDL, 81
thiazolidinediones, 80 volatile organic compounds, 24
tissue, 60, 72, 73, 74, 75, 82, 83, 96, 108
TLR4, 74, 86
TNF-α, 74, 92 W
tocopherols, viii, 2, 4, 9, 10, 21
water, ix, 9, 10, 37, 46, 55, 58, 59, 60, 61,
total cholesterol, 81
63, 66, 75, 82, 95
toxicity, 94, 104
weight changes, 95
translocation, 74, 76
weight gain, viii, x, 2, 4, 18, 72, 80, 97
treatment, x, 18, 62, 63, 65, 72, 75, 76, 77,
Woodard, ix, 6, 12, 32, 36, 39, 41, 42, 45,
78, 79, 82, 83, 84, 86, 94, 106
46, 47, 48
triglycerides, 79, 81, 92, 103
tumor necrosis factor, 92, 106
type 2 diabetes, 72, 73 Y
yield, 7
U
ultrasound, 63, 68 Z
United Nations, 3, 22, 49
United States, vii, viii, 2, 3, 4, 5, 6, 19, 31, zinc, 10
32

Blueberries Harvesting Methods, Antioxidant Properties and Health Effects

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Blueberries Harvesting Methods, Antioxidant Properties and Health Effects

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NUTRITION AND DIET RESEARCH PROGRESS

Additional books in this series can be found on Nova’s website

Additional e-books in this series can be found on Nova’s website

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. † New York

According to Food and Agriculture Organization (FAO) statistics, from

carotenoids and physicochemical analyses were done. There were evaluated

on the low-grade inflammatory status associated to metabolic diseases

Results support the hypothesis that properties found in blueberry extracts

BLUEBERRIES: MARKET, CULTIVARS,

Paula Becker Pertuzatti1,*, Isidro Hermosín-Gutiérrez2

Keywords: blueberries, anthocyanins, phenolic compounds, carotenoids

of diabetes, because lowered fasting blood glucose levels, lowered serum

PRODUCER AND CONSUMER MARKET, IMPORTANCE OF

Temperate Climate (Pelotas-Brazil), to evaluate varieties, being introduced

Uruguay. In Brazil, is produced in Vacaria, Pelotas and Serra Gaúcha, in the

Table 1. Cultivars, groups and characteristics of blueberries cultivated in

Cultivar Category Characteristic References

Cultivars belonging to that group were developed from interspecific

commercial drug Difrarel, contains 100 mg of bilberry anthocyanins plus

and Chien [41] blueberry aroma is composed by linalool, 2-trans-hexenol, 2-

Fat soluble vitamins like tocopherols are not as sensitive to post-harvest

Berries are rich in polyphenols, especially flavonoids (Figure 1) [48].

Table 2. Content (mg/100g fresh fruit) of phenolic compounds and

Cultivar TPHa ACYb References

Blueberry polyphenols are susceptible to losses during processing and

Table 3. Total of phenolic acids in blueberry (mg/kg)

Phenolic acids Content References

The chlorogenic acid is the predominant hydroxycinnamic acid in

quercetin, and the results showed an increase in plasma concentration of

Table 4. Flavonols composition of blueberries

Table 5. Anthocyanin composition of blueberries

In several studies, anthocyanins have shown potential benefits to human

consumption, is related to anti-diabetic effect. Roopchand et al. [7] observed

Carotenoids are another class of pigments present in blueberry. According

Due to the high phytochemical content present in blueberry, several

in fruit, accounting for 84% of antioxidant capacity of blueberry. Among

colour associated with the formation of b-carotene epoxides and

[23] Moraes, J.O., Pertuzatti, P.B., Corrêa, F.V., Salas-Mellado, M.D.L.M.,

[34] Camire, M.E., 2002. Phytochemicals in the Vaccinium Family:

corymbosum L.) extracts against the growth of Listeria monocytogenes

BIOACTIVE COMPOUNDS, COLOR

Paula Becker Pertuzatti1,, Milene Teixeira Barcia2,

Keywords: anthocyanins, phenolic compounds, tannins, carotenoids, acidity,

Among the bioactive compounds found in blueberries, phenolic

GAE.100 g-1, with “Powderblue” and “Bluebelle” cultivars showing the

Among the different subclasses belonging to the phenolic compounds

concentration. Only five types of anthocyanins are found in blueberries:

Table 1. Total content of anthocyanins and phenolic compounds in peel,

Cultivar Parts of the fruit

Procyanidin content in blueberries of Highbush and Lowbush group

differences in tannin content (condensed, hydrolyzable and total), between

Table 2. Content of condensed, hydrolyzable and total tannins in peel,

Cultivar Parts of the fruit

Another group of bioactive compounds in blueberries are the carotenoids.

carotenoids were found as the major pigments.

Table 3. Carotenoid content in peel, pulp and whole fruit of

Cultivar Parts of the fruit

Table 4. Brightness values (L), chromaticity coordinates

Parts of the fruit

Figure 1. Graphic representation of the values of L, a* and b* obtained in Minolta CR-

ΔEab = √(ΔL)2 + (Δa)2 + (Δb)2 (1)

where: ΔE = Color difference, ΔL = Difference in values of L*, Δa =