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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Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Index

vii Blueberries: Market, Cultivars, Chemical Composition and Antioxidant Capacity Paula Becker Pertuzatti, Isidro Hermosín-Gutiérrez and Helena Teixeira Godoy Bioactive Compounds, Color and Physicochemical Parameters of Blueberries Paula Becker Pertuzatti, Milene Teixeira Barcia, Andressa Carolina Jacques and Rui Carlos Zambiazi Blueberries: Antioxidant Properties, Health and Innovative Technologies Guillermo Petzold, Jorge Moreno, Pamela Zúñiga, Karla Mella and Patricio Orellana 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 Blueberry Extracts Protect against Gross Mouse Fetal Defects Induced by Alcohol Toxicity Zach S. Gish, Sharang Penumetsa, Diana J. Valle and Roman J. Miller

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31

55

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89

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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

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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 obesityassociated inflammation is known to be a key feature of the etiology of noncommunicable 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

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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

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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 Editor: Malcolm Marsh

ISBN: 978-1-63484-885-5 © 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 University Castilla -La Mancha (UCLM), Regional Institute for Applied Scientific Research (IRICA), Ciudad Real, Spain 3 State University of Campinas (UNICAMP), Campinas, SP, Brazil

2

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 * [email protected].

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

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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

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P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy

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 –

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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

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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 Bluecrop

Category Highbush

Darrow

Highbush

O’Neal

Highbush

Georgiagem

Highbush

Misty

Highbush

Sharpblue

Highbush

Millenia

Highbush

Star

Highbush

Elliot

Highbush

Coville

Highbush

Brigitta Blue Aliceblue

Highbush

Bluebelle

Rabbiteye

Bluegem Briteblue Delite Climax Powderblue Woodard Tifblue Beckyblue

Rabbiteye Rabbiteye Rabbiteye Rabbiteye Rabbiteye Rabbiteye Rabbiteye Rabbiteye

Brightwell Bonita

Rabbiteye Rabbiteye

Windy

Rabbiteye

Rabbiteye

Characteristic Created by USDA – New Jersey, medium to large size, light blue, acid flavor, firm pulp, requirement more than 600 h of cold Large to very large size, light blue, medium firm pulp, slightly tart From North Carolina, requirement 200 – 600 h of cold, large size, light blue From Georgia, requirement 200 – 600 h of cold, medium size From Florida, requirement 150 – 200 h of cold, large size, light blue, firm pulp From Florida, medium size, dark blue, medium firm pulp From Florida, medium size, light blue, firm pulp, requirement of 300 h of cold From Florida, large size, dark blue, firm pulp, requirement of 400 h of cold Medium size, light blue, requirement more than 800 h of cold Large size, firm pulp, requirement more than 800 h of cold Large size, light blue, firm pulp, requirement more than 700 h of cold From Florida, low requirement of cold, sweetsour taste, light blue From Georgia, firm pulp, small to medium size, light blue From Florida, dark blue From Georgia, large size, light blue, firm pulp From Georgia, small size, sweet-sour taste From Georgia, medium size, dark blue From Maryland, small to medium size, light blue From Georgia, light blue, soft pulp From Georgia, small size, light blue, firm pulp Firm pulp, requirement 300-400 h of cold, medium blue Medium to large size, firm pulp From Florida, medium to large size, light blue, astringent flavor From Florida, medium to large size, firm pulp

References 29

29 31 31 28, 31 28 28 31 29 32 29, 32 28 28 28 31 28, 31 28 28, 29 28 29, 31 30 32 28 28

Market, Cultivars, Chemical Composition and Antioxidant Capacity

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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

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P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy

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

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

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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

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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.

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P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy 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-3glucoside 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.

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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, mcoumaric, 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 Gentisic Gallic Ellagic p-Hydroxybenzoic o-Pyrocatechuic Protocatechuic Salicylic Syringic Vanillic Veratric Chlorogenic Caffeic m-coumaric o-coumaric p-coumaric 3,4-dimethoxycinnamic Ferulic 2-Hydroxycaffeic p-hydroxyphenyl acetic

Content 28.6 17.6-294.1 14.0-44.0 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

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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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[74] Jaakola, L., Maatta-Riihinen, K., Karenlampi, S., Hohtola, A., 2004. Activation of flavonoid biosynthesis by solar radiation in bilberry (Vaccinium myrtillus L.) leaves. Planta, 218, 721–728. [75] Wang, C.Y., Chen, C., Wang, S.Y., 2009. Changes of flavonoid content and antioxidant capacity in blueberries after illumination with UV-C. Food Chemistry, 117, 426–431. [76] Borges, G., Degeneve, A., Mullen, W., Crozier, A., 2010. Identification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cranberries. Journal of Agricultural and Food Chemistry, 58, 3901-3909. [77] Gao, L., Mazza, G., 1994. Quantitation and distribution of simple and acylated anthocyanins and other phenolics in blueberries. Journal of Food Science, 59, 1057–1059. [78] Giusti, M.M., Jing, P., Natural pigments of berries: Functionality and application. In: Zhao, Y., 2007. Berry Fruit: Value-Added Products for Health Promotion. CRC PRESS. [79] Ma, C., Dastmalchi, K., Flores, G., Wu, S., Pedraza-Peñalosa, P., Long, C., Kennelly, E.L., 2013. Antioxidant and metabolite profiling of north american and neotropical blueberries using LC-TOF-MS and multivariate analyses. Journal of Agricultural and Food Chemistry, 61, 3548-3559. [80] Sun, L., Ding, X., Qi, J., Yu, H., He, S., Zhang, J., Ge, H., Yu, B., 2012. Antioxidant anthocyanins screening through spectrum–effect relationships and DPPH-HPLC-DAD analysis on nine cultivars of introduced rabbiteye blueberry in China. Food Chemistry, 132, 759-765. [81] Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Gebhardt, S.E., Prior, R.L., 2006. Concentrations of anthocyanins in common foods in the united states and estimation of normal consumption. Journal of Agricultural and Food Chemistry, 54, 4069-4075. [82] Krikorian, R., Shidler, M.D., Nash, T.A., Kalt, W., Vinqvist-Tymchuk, M.R., Shukitt-Hale, B., Joseph, J.A., 2010. Blueberry supplementation improves memory in older adults. Journal of Agricultural and Food Chemistry, 58, 3996–4000. [83] Seeram, N.P., Adams, L.S., Zhang, Y., Lee, R., Sand, D., Scheuller, H.S., Heber, D., 2006. Blackberry, Black Raspberry, Blueberry, Cranberry, Red Raspberry, and Strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. Journal of Agricultural and Food Chemistry, 54, 9329-9339.

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[84] Smith, S.H., Tate, P.L., Huang, G., Magee, J.B., Meepagala, K.M., Wedge, D.E., Larcom, L.L., 2004. Antimutagenic activity of berry extracts. Journal of Medicinal Food, 7(4), 450-455. [85] Yi, W., Fischer, J., Krewer, G., Akoh, C.C., 2005. Phenolic compounds from blueberries can inhibit colon cancer cell proliferation and induce apoptosis. Journal of Agricultural and Food Chemistry, 53, 7320-7329. [86] Katsube, N., Iwashita, K., Tsushida, T., Yamaki, K., Kobori, M., 2003. Induction of apoptosis in cancer cells by bilberry (Vaccinium myrtillus) and the anthocyanins. Journal of Agricultural and Food Chemistry,51, 68-75. [87] Heinonen, M.I., Ollilainen, V., Linkola, E.K., Varo, P.T., Koivistoinen, P.E., 1989. Carotenoids in finnish foods: vegetables, fruits, and berries. Journal of Agricultural and Food Chemistry, 37, 655-659. [88] Jimenez-Garcia, S.N., Guevara-Gonzalez, R.G., Miranda-Lopez, R., Feregrino-Perez, A.A., Torres-Pacheco, I., Vazquez-Cruz, M.A., 2013. Functional properties and quality characteristics of bioactive compounds in berries: Biochemistry, biotechnology, and genomics. Food Research International. 54, 1195-1207. [89] Marinova, D., Ribarova, F., 2007. HPLC determination of carotenoids in Bulgarian berries. Journal of Food Composition and Analysis, 20, 370–374. [90] Bunea, A., Rugina, D., Pintea, A., Andrei, S., Bunea, C., Pop, R., Bele, C., 2012. Carotenoid and fatty acid profiles of bilberries and cultivated blueberries from Romania. Chemical Papers, 66(10), 935-939. [91] Lashmanova, K.A., Kuzivanova, O.A., Dymova, O.V., 2012. Northern Berries as a source of carotenoids. Acta Biochimica Polonica, 59(1), 133-134. [92] Youdim, K.A., Shukitt-Hale, B., Mackinnon, S., Kalt, W., Joseph, J.A., 2000. Polyphenolics enhance red blood cell resistance to oxidative stress: in vitro and in vivo, Biochim. Biophys. Acta, 1523, 117–122. In: Bagchi, M., Zafra-Stone, S., Losso, J.N., Sen, C.K., Roy, S., Hazra, S., Bagchi, D., 2007. Role of Edible Berry Anthocyanins in Angiogenesis. CRC PRESS. [93] García-Alonso, M., Pascual-Teresa, S., Santos-Buelga, C., RivasGonzalo, J.C., 2004. Evaluation of the antioxidant properties of fruits. Food Chemistry, 84, 13–18. [94] Rice-Evans, C.A., Miller, N.J., Bolwell, P.G., Bramley, P.M., Pridham, J.B., 1995. The relative antioxidant activities of plant-derived polyphenolic flavonoids, Free Radical Research, 22(4), 375–383.

In: Blueberries Editor: Malcolm Marsh

ISBN: 978-1-63484-885-5 © 2016 Nova Science Publishers, Inc.

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: [email protected].

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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).

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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

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P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.

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

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35

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).

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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

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collection site. The tannins can be categorized into hydrolyzable, and nonhydrolyzable 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.

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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

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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 Cultivar

Parts of the fruit Peel Pulp Whole fruit 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/ 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). CAE = catechin equivalent; GAE = gallic acid equivalent.

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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 βcarotene, 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 g1 fresh fruit) and loquat (24 µg of β- carotene g-1 fresh Fruit) [8], in which the carotenoids were found as the major pigments.

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

Parts of the fruit Peel Pulp Whole fruit 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/ 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).

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].

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P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al. 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 Powderblue Bluebelle Briteblue Climax Delite

16,70 22,57 17,15 16,67 19,63 18,54

bB2/ aB bB bB abB abB

Woodard Powderblue Bluebelle Briteblue Climax Delite

10,12 8,51 9,40 10,71 4,54 8,46

aA aA aB aA aB aA

Woodard Powderblue Bluebelle Briteblue Climax Delite

- 8,62 - 7,10 - 8,02 - 8,38 - 5,41 - 8,45

bB abB bB bB aB bB

29,25 47,97 31,82 28,38 39,96 25,24

cdA aA cA cdA bA dA

25,49 4,07 23,40 14,32 13,89 27,46

aB cA aA bA bA aB

8,26 8,29 4,87 5,25 12,13 7,21

bA bA dA cdA aA bcA

a*

b*

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 row do not differ by Tukey test (p ≤ 0.05).

Bioactive Compounds, Color and Physicochemical Parameters …

43

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 0100. 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.

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P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.

a

b Figure 1. Graphic representation of the values of L, a* and b* obtained in Minolta CR300 colorimeter; (a) colored solid in three dimensions (b) location of the peel and pulp of Rabbiteye blueberry cultivars in colorimetric space.

Bioactive Compounds, Color and Physicochemical Parameters …

45

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

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P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.

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 between cultivars P-Climax

ΔE pulp 13.2

Classification pulp Very large

ΔE peel 5.2

P-Brite P-Blue

22.3 25.4

Very large Very large

6.4 5.6

P-Delite

32.6

Very large

4.3

P-Wood Climax-Brite Climax-Blue

28.4 13.5 14.5

Very large Very large Very large

6.3 7.5 6.0

Climax-Delite

20.6

Very large

5.1

Climax-Wood Brite-Blue

16.3 6.0

Classification peel Easily distinguishable Very large Easily distinguishable Easily distinguishable Very large Very large Easily distinguishable Easily distinguishable Very large Easily distinguishable Distinguishable Very small

Very large 7.1 Easily 3.1 distinguishable Brite-Delite 13.7 Very large 2.9 Brite-Wood 3.9 Easily 0.5 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.

Bioactive Compounds, Color and Physicochemical Parameters …

47

Table 6. Physicochemical parameters of peel, pulp and whole fruit of six blueberry cultivars Cultivar

Parts of the fruit Pulp Whole fruit 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 row do not differ by Tukey test (p ≤ 0.05). Peel

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).

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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

Bioactive Compounds, Color and Physicochemical Parameters …

49

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 PerkinsVeazie 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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medicinais arbóreas simpátricas da caatinga (tannin content in three medicinal tree species sympatric of savanna). Árvore, 29(6), 999-1005. Sgarbieri, V.C., 1996. Proteínas em alimentos protéicos: propriedades – degradação – modificações (Protein - protein foods: properties degradation – modifications). São Paulo: Varela, Cap. 5: Deterioração e modificações químicas, físicas e enzimáticas de proteínas (Degradation and chemical changes, physical and enzymatic proteins). Taiz, L., Zeiger, E., 2004. Fisiologia Vegetal (Vegetal physiology). 3a ed. Porto Alegre: Artmed, 719p. Agostine-Costa, T.S., Lima, A., Lima, M.V., 2003. Determinação de tanino em pedúnculo de caju: método da vanilina versus método do butanol ácido (tannin determination in cashew apple: method of vanillin versus butanol acid method). Química Nova, 26(5), 763-765. Howard, L.R., Hager, T.J., 2007. Berry fruit phytochemicals. In: Zhao, Y., Berry Fruit: Value-Added Products for Health Promotion; CRC PRESS. Brune, M., Hallberg, L., Skanberg, A., 1991. Determination of ironbinding phenolic groups in foods. Journal food science. 56(1), 128-132. Apeeldoorn, M.M., Sanders, M., Vincken, J., Cheynier, V., Le Guernevé, C., Hollman, P. C. H., Gruppen, H., 2009. Efficient isolation of major procyanidin A-type dimers from peanut skins and B-type dimers from grape seeds. Food Chemistry, 117, 713-720. Silva, M.R., Silva, M.A.A.P., Aspectos nutricionais de fitatos e taninos (nutritional aspects of phytates and tannins). Nutrição, 12(1), 5-19. Gu, L., Kelm, M., Hammerstone, J.F., Beecher, G., Cunningham, D., Vannozzi, S., Prior, R.L., 2002. Fractionation of Polymeric Procyanidins from Lowbush Blueberry and Quantification of Procyanidins in Selected Foods with an Optimized Normal-Phase HPLC-MS Fluorescent Detection Method. Journal of Agricultural and. Food Chemistry, 50, 4852-4860. Hwang, S.J., Yoon, W.B., Lee, O., Cha, S.J., Kim, J.D., 2014. Radicalscavenging-linked antioxidant activities of extracts from black chokeberry and blueberry cultivated in Korea. Food Chemistry, 146, 7177. Rodriguez-Mateos, A., Cifuentes-Gomez, T., Tabatabaee, S., Lecras, C., Spencer, J.P.E., 2012. Procyanidin, Anthocyanin, and Chlorogenic Acid Contents of Highbush and Lowbush Blueberries. Journal of Agricultural and Food Chemistry, 60, 5772-5778.

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[28] Eskin, N.A.M., Snait, T., 2006. Dictionary of nutraceuticals and functional foods, CRC Press, US. [29] Price, M.L., Scoyoc, S.V., Butler, L.G., 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J. Agric. Food Chem. 28(5), 1214-1218. [30] Yi, W., Akoh, C.C., Fischer, J., Krewer, G., 2006. Effects of phenolic compounds in blueberries and muscadine grapes on HepG2 cell viability and apoptosis. Food Reserch International, 39, 628-638. [31] Fennema, O.R., Damodaran, S., Parkin, K.L., 2010. Química de Alimentos de Fennema – 4ª ed. - Editora Artmed (Fennema’s Food Chemistry - 4th ed. - Publisher Artmed). [32] Rodriguez-Amaya, D.B., 2001. A guide to carotenoid analysis in foods. ILSI Press. US. [33] Bobbio, F.O., Bobbio, P.A., 2003. Introdução à química de alimentos (Introduction to food chemistry). 3ª Ed., São Paulo: Livraria Varela. [34] Lima, V.L.A.G., Melo, E.A., Lima, D.E.daS., 2002. Fenólicos e Carotenóides Totais em Pitanga (Phenolics and carotenoids Total in Pitanga). Scientia Agrícola, 59(3). [35] Ngo, T., Wrolstad, R.E., Zhao, Y., 2007. Color quality of Oregon strawberries-impact of genotype, composition, and processing. Journal of Food Science, 72(1), 25-32. [36] Zhang, Y., Hu, X.S., Chen, F., 2008. Stability and color characteristics of PEF-treated cyaniding-3-glicoside during storage. Food Chemistry, 106, 669-679. [37] Silva, R.A., Petter, C.O., Schneider, I.A.H., 2007. Avaliacao da perda da coloração artificial de agatas (Evaluation of loss of artificial coloring of agates). REM: R. Esc. Minas, 60(3), 477-482. [38] DIN 6174, 1979. Farbmetrische Bestimmung von Farbabständen bei Körperfarben nach der CIELAB-formel (Colorimetric evaluation of color differences of surface colors according to the CIELAB formula), DIN-Deutsches Institut für Normung e. V (DIN German Institute for Standardization e. V). [39] Sachi, A.D, Biasi, L.A., 2008. Fruit maturation in four muscadine grape cultivars in Pinhais, Scientia Agraria, 9(2), 255-260. [40] Moraes, J.O., Pertuzatti, P.B., Corrêa, F.V., Salas-Mellado, M.D.L.M., 2007. Estudo do mirtilo (Vaccinium ashei Reade) no processamento de produtos alimentícios (Study Blueberry (Vaccinium ashei Reade) in the processing of food products). Ciência e Tecnologia de Alimentos, 27, 18-22.

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[41] Perkins-veazie, P., Collins, J.K., Howard, L., 2008. Blueberry fruit response to postharvest application of ultraviolet radiation. Postharvest biology and Technology. 47, 280-285. [42] Tonietto, J., Mandelli, F., 2003. Sistema de produção (Production System), v. 4. URL: http://sistemasdeproducao.cnptia.embrapa.br/Fontes HTML/Uva/UvasViniferasRegioesClimaTemperado/clima.htm#element os. [43] Junior, M.J.P., Pommer, C.V., Martins, F.P., 1997. Curvas de maturação e estimativa do teor de sólidos solúveis para a videira ‘Niagara rosada’ com base em dados meteorológicos (maturation curves and estimation of soluble solids content for the vine ‘pink Niagara’ based on meteorological data). Bragantia, 56(2). [44] Volpe, C.A., Schöffef, E.R., Barbosa, J.C., 2000. Influência de algumas variáveis meteorológicas sobre a qualidade dos frutos das laranjeiras Valência’ e ‘Natal.’ (Influence of some meteorological variables on the quality of the fruit of the orange ‘Valencia’ and ‘Christmas’) Revista brasileira de agrometeorologia, 8(1), 85-94.

In: Blueberries Editor: Malcolm Marsh

ISBN: 978-1-63484-885-5 © 2016 Nova Science Publishers, Inc.

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:[email protected].

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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 enzymeinhibiting, 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.

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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).

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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

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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).

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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,

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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.

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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).

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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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Cho, M., Howard, L. R., Prior, R. L., Clark, J. R. (2004). Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry, and red grape genotypes determined by high-performance liquid chromatography/mass spectrometry. Journal of Science and Food Agriculture, 84, 1771-1782. Cho, M., Howard, L., Prior, R., Clark, J. (2005). Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high-performance liquid chromatography/mass spectrometry. Journal of Science and Food Agriculture, 85, 2149-2158. Cortés, M., Osorio, A. and García, E. (2007). Manzana deshidratada fortificada con vitamina E utilizando la ingeniería de matrices. Vitae, 14, 17-26. DeFuria, J., Bennett, G., Strissel, KJ., Perfield, JW. II, Milbury, PE., Greenberg, AS., Obin, MS. (2009). Dietary blueberry attenuates wholebody insulin resistance in high fat-fed mice by reducing adipocyte death and its inflammatory sequelae. Journal of Nutrition, 139 (8), 1510–1516. Deshpande, S.S., Bolin, H.R. and Salunkhe, D.K. (1982). Freeze concentration of fruit juices. Food Technology, 36(5), 68-82. Falade, K. O. and Igbeka, J. C. (2007). Osmotic dehydration of tropical fruits and vegetables. Food Reviews International, 23(4), 373-405. Gao, L., Sun, J., Zhang, M., Majumdar, A. S. and An, J. (2011). Effect of predrying and vacuum impregnation with nano-calcium carbonate solution on stawberry, Carrot, corn, and blueberry. European Drying ConferenceEuroDrying' 2011, Palma. Balearic Isl, Spain, 26-28 October 2011. Giampieri, F., Alvarez-Suarez, JM., Battino, M. (2014). Strawberry and Human Health: Effects beyond Antioxidant Activity. Journal of Agricultural and Food Chemistry, 62 (18), 3867-3876. Giovanelli, G., Brambilla, A. and Sinelli, N. (2013). Effects of osmo-air dehydration treatments on chemical, antioxidant and morphological characteristics of blueberries. LWT-Food Science and Technology, 54(2), 577-584. Giovanelli, G., Brambilla, A., Rizzolo, A. and Sinelli, N. (2012). Effects of blanching pre-treatment and sugar composition of the osmotic solution on physico-chemical, morphological and antioxidant characteristics of osmodehydrated blueberries (Vaccinium corymbosum L.). Food Research International, 49(1), 263-271. Howard, L. R., Clark, J. R., Brownmiller, C. (2003). Antioxidant capacity and phenolic content in blueberries as affected by genotype and growing season. Journal of Science and Food Agriculture, 83, 1238-1247. Hsieh, H.-C. (2008). Desalinating process. US Patent 7,467,526.

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In: Blueberries Editor: Malcolm Marsh

ISBN: 978-1-63484-885-5 © 2016 Nova Science Publishers, Inc.

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 metainflammation. 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.

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J. Soto-Covasich, M. Reyes-Farias, A. Ovalle-Marin et al. several efforts have been made for identifying novel agents with antiinflammatory 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

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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 lowgrade 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 lowgrade chronic inflammation in white adipose tissue (WAT). When adipocyte hypertrophy occurs, endoplasmic reticulum stress, hypoxia, and oxidative

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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 NFκBindependent (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

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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 antiinflammatory 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 antiinflammatory 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

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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 dosedependent (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

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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 lowlevel chronic inflammation associated with gastrointestinal dysfunction. The research group found that all blueberry fractions (crude extract; polyphenolrich (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

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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?)

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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.

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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 SpragueDawley 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

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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 endsystolic 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

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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 15weeks 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

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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 nontransmissible 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

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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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Prior, R. L., S, E. W., T, R. R., Khanal, R. C., Wu, X. & Howard, L. R. (2010). Purified blueberry anthocyanins and blueberry juice alter development of obesity in mice fed an obesogenic high-fat diet. J Agric Food Chem, 58, 3970-3976. Prior, R. L., Wu, X., Gu, L., Hager, T., Hager, A., Wilkes, S. & Howard, L. (2009). Purified berry anthocyanins but not whole berries normalize lipid parameters in mice fed an obesogenic high fat diet. Mol Nutr Food Res, 53, 1406-1418. Rajendran, K., Devarajan, N., Ganesan, M. & Ragunathan, M. (2012). Obesity, Inflammation and Acute Myocardial Infarction - Expression of leptin, IL-6 and high sensitivity-CRP in Chennai based population. Thromb J., 10(1), 13. Sakaida, H., et al., (2007). Effect of Vaccinium ashei reade leaves on angiotensin converting enzyme activity in vitro and on systolic blood pressure of spontaneously hypertensive rats in vivo. Biosci Biotechnol Biochem, 71(9), p. 2335-7. Salcedo, R., Ponce, M. L., Young, H. A., Wasserman, K., Ward, J. M., Kleinman, H. K., Oppenheim, J. J. & Murphy, W. J. (2000). Blood, 96, 34-40. Saura-Calixto, F., et al., (2007). Intake and bioaccessibility of total polyphenols in a whole diet. Food Chemistry, 101, p. 492–501. Suganami, T., et al. (2010). J Leukoc Biol, 88(1), 33-9. Seymour, E. M., Tanone, II., Urcuyo-Llanes, D. E., Lewis, S. K., Kirakosyan, A., Kondoleon, M. G., Kaufman, P. B. & Bolling, S. F. (2011). Blueberry intake alters skeletal muscle and adipose tissue peroxisome proliferatoractivated receptor activity and reduces insulin resistance in obese rats. J Med Food, 14, 1511-1518. Seeram, N. P. (2008). Berry fruits: compositional elements, biochemical activities, and the impact of their intake on human health, performance, and disease. J. Agric. Food Chem., vol. 56(3) pp. 627-9. Shoelson, S. E., et al. (2006). J Clin Invest, 116(7), 1793-801. Stevens, G. A., Singh, G. M., Lu, Y., et al., (2012). “National, regional, and global trends in adult overweight and obesity prevalences,” Population Health Metrics, vol. 10, no. 1, article 22. Stull, A. J., Cash, K. C., Johnson, W. D., Champagne, C. M. & Cefalu, W. T. (2010). Bioactives in blueberries improve insulin sensitivity in obese, insulin-resistant men and women. J Nutr., 140(10), 1764-8. Stull, A. J., Cash, K. C., Champagne, C. M., Gupta, A. K., Boston, R., Beyl, R. A., Johnson, W. D. & Cefalu, W. (2015). Blueberries improve endothelial

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function, but not blood pressure, in adults with metabolic syndrome: a randomized, double-blind, placebo-controlled clinical trial. Nutrients., 7(6), 4107-23. Song, Y., Park, H. J., Kang, S. N., Jang, S. H., Lee, S. J., Ko, Y. G., Kim, G. S. & Cho, J. H. (2013). Blueberry peel extracts inhibit adipogenesis in 3T3-L1 cells and reduce high-fat diet-induced obesity. PLoS One, 8, e69925. Suganami, T., et al. (2005). Arterioscler Thromb Vasc Biol, 25(10), 2062-8. Suganami, T., et al. (2007). Arterioscler Thromb Vasc Biol, 27(1), 84-91. Vuong, T., Benhaddou-Andaloussi, A., Brault, A., Harbilas, D., Martineau, L. C., Vallerand, D., Ramassamy, C., Matar, C. & Haddad, P. S. (2009). Antiobesity and antidiabetic effects of biotransformed blueberry juice in KKA(y) mice. Int J Obes (Lond), 33, 1166-1173. Weisberg, SP., et al. (2003). J Clin Invest, 112(12), 1796-808. Wu, T., Luo, J. & Xu, B. (2015). In vitro antidiabetic effects of selected fruits and vegetables against glycosidase and aldose reductase. Food Sci Nutr, 3(6), p. 495-505. Xu, H., et al. (2013). J Clin Invest, 112(12), 1821-30. Yu, R., et al. (2006). Obesity (Silver Spring), 14(8), 1353-62. Yudkin, J. S. (2007). Horm Metab Res, 39(10), 707-9. Zulet, M. A., et al. (2007). Nutricion Hospitalaria, 22(5), 511-27.

In: Blueberries Editor: Malcolm Marsh

ISBN: 978-1-63484-885-5 © 2016 Nova Science Publishers, Inc.

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 alcoholinduced 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: [email protected].

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Zach S. Gish, Sharang Penumetsa, Diana J. Valle et al. were additionally administered to the AA group, through subcutaneousneck 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).

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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].

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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 down- regulation 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.

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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

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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.

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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.

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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

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between groups were determined using One-Way ANOVA and StudentNewman-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).

Fertility Percent

Body Weight Day 1 (g)

Body Weight Day 8 (g)

Body Weight Day 15 (g)

Percent Increase in Body Weight

12 7 9 9.3

80% 58% 75% 71%

29.5 29.6 28.4 29.2

31.4 30.5 30.6 30.8

38.4 36.6 37.2 37.4

27% 21% 28% 25%

117 58 79

Average Number Fetuses / Mother

Number of Pregnant Mothers

CO BA AA Average

Total Fetuses

Experiment Groups

Table 1. Maternal Data: Average Body Weights and Pregnancy Results

9.75 8.29 8.78 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.

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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.

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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.

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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.

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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

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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.

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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

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(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

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histological and functional parameters of the liver and telencephalon to further elucidate this phenomenon.

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INDEX A adipose tissue, 72, 73, 74, 75, 87, 92, 107 alcohol consumption, 90, 92 alcohol exposure, 90, 91, 92, 105, 107, 109 alcohol toxicity, v, 89 alcohol-induced prenatal developmental deficiencies, x, 89 angiotensin converting enzyme, 79, 87 anthocyanins, viii, x, 2, 3, 7, 8, 10, 12, 16, 17, 18, 19, 20, 24, 25, 26, 27, 28, 29, 32, 34, 35, 36, 40, 43, 45, 50, 56, 57, 61, 66, 75, 79, 80, 81, 85, 87, 89, 90, 93, 94, 99, 102, 103, 104, 108 anti-cancer, 27 anti-inflammatory agents, 75 antioxidant, vii, viii, ix, 2, 3, 4, 9, 19, 20, 21, 24, 25, 26, 27, 28, 29, 32, 33, 50, 51, 55, 56, 57, 62, 65, 66, 67, 68, 69, 72, 76, 83, 93, 94, 107, 108 apoptosis, 28, 29, 52, 68, 79, 82, 91, 92, 105 arabinoside, 15, 17, 20 Argentina, 4, 5, 6, 7 ascorbic acid, viii, 2, 4, 8, 10, 21 atherosclerosis, 75

B beneficial effect, ix, 18, 55, 56, 77, 83, 84, 104

benefits, vii, ix, 2, 18, 21, 63, 72, 84, 93 beta cell function, x, 72 binge alcohol, x, xi, 89, 90, 94, 97, 98, 104 binge drinking, 92, 106 biological activity, 9, 33 biological responses, 73 biological systems, 57, 93 biomarkers, 77, 83, 85 biosynthesis, 15, 28 biotechnology, 29 Bluebelle, ix, 6, 12, 32, 33, 34, 35, 36, 39, 40, 41, 42, 46, 47, 48 blueberries, vii, viii, ix, x, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 37, 38, 40, 45, 47, 49, 50, 52, 55, 56, 57, 60, 61, 62, 63, 65, 66, 68, 72, 76, 79, 82, 83, 84, 85, 87, 93 body composition, 101, 102 body fat, 72, 80 body size, 101, 102, 103 body weight, x, 21, 72, 89, 90, 95, 97, 99, 104 brain, 18, 91, 92, 101 brain functions, 18 Brazil, vii, 1, 3, 4, 5, 6, 7, 31, 32, 47 breeding, 5, 27 Briteblue, ix, 6, 12, 18, 32, 36, 39, 40, 41, 42, 45, 46, 47

112

Index

C Ca2+, 64 CAE, 39 calcium carbonate, 65 cancer, viii, 2, 3, 17, 28, 29, 38, 56, 67, 68, 77, 83, 93, 108 cancer cells, 17, 28, 29, 38, 68 carbohydrates, 8, 10 cardiovascular diseases (CVD), ix, 56, 71, 72, 75, 79, 84, 86 cardiovascular function, 75 cardiovascular risk, 84 cardiovascular system, 75 carotene, 8, 19, 21, 22, 40 carotenoids, viii, ix, 2, 4, 18, 19, 29, 32, 40, 41, 52 cell death, 79, 91, 102, 105, 109 cell lines, 18, 76 cell membranes, 61 cellulose, 8 central nervous system (CNS), 90, 92, 101 cerebral cortex, 91, 105 chemical, viii, 2, 3, 50, 51, 61, 65, 68, 73 chemical properties, 68 chemokines, 73 Chile, 4, 5, 6, 7, 55, 71 Chitosan, 68 chlorophyll, 10 cholesterol, viii, 2, 4, 18, 21, 81 Climax, ix, 6, 12, 32, 34, 35, 36, 39, 41, 42, 43, 46, 47 clinical symptoms, 73 clinical trials, 83, 84 coenzyme, 92 colon, 18, 29 colon cancer, 18, 29 consumption, ix, 5, 18, 20, 28, 55, 57, 61, 72, 77, 83, 84, 85, 93, 95, 98, 99, 105 contamination, 61 control group, 77, 93, 104 copper, 10 c-reactive protein, 75 crop, vii, 1, 3, 22 crystallization, 59

crystals, 58, 59 cultivars, vii, viii, 1, 5, 7, 9, 10, 16, 18, 21, 23, 24, 26, 27, 28, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 52 cultivation, vii, 1, 2, 3, 4, 5, 8, 10, 27 CVD, 75, 77, 83 cyclooxygenase, 76 cytokines, 73, 74, 75

D dehydration, ix, 55, 60, 61, 62, 63, 64, 65, 66, 68, 69 Delite, ix, 6, 12, 32, 33, 34, 36, 39, 40, 41, 42, 46, 47, 48 dendritic cell, 73 Department of Agriculture, vii, viii, 4, 31 derivatives, 14, 22, 37, 57 detection system, 19 developing brain, 108, 109 developmental change, 106, 109 developmental process, 90, 101 diabetes, viii, ix, 2, 4, 18, 71, 73, 85, 93, 108 dialysis, 68 diastolic pressure, 82 diet, x, 18, 56, 72, 79, 80, 81, 82, 84, 87, 88, 95, 107 dietary management, viii, 2, 4, 18 dietary supplementation, 83 diffusion, 61, 62 digestive enzymes, 107 dilated cardiomyopathy, 84 diseases, ix, 8, 33, 56, 71, 73, 75, 76, 77, 83, 84 distillation, 58 distribution, 16, 26, 28, 50 diversity, 16, 19 DNA, 14, 27, 84, 93, 108 DNA damage, 14, 27, 84, 93, 108 double bonds, 40 drinking water, 81 dry matter, 63, 66 dyslipidemia, x, 72

113

Index

E

F

elaboration, 14 Elam, 85 electric field, 63 electron, 34 electrons, 4 electrophoresis, 68 embryology, 105, 108 emission, 8, 24 endocrine, 72 endothelial dysfunction, 75 energy, ix, 10, 55, 61, 63, 72, 83 energy expenditure, 72 energy transfer, 10 engineering, 67 enlargement, 74 environment, 63 enzyme, 57, 58, 68, 78 enzymes, 9, 58, 63, 74 epidermis, 61, 63 epithelial cells, 83 equilibrium, 61 equipment, 59 ESI, 27 essential fatty acids, 56 ester, 13, 14 ethanol, x, 35, 78, 89, 91, 92, 93, 95, 98, 99, 101, 102, 103, 105, 106, 107, 108, 109 ethyl acetate, 77, 79 ethylene, viii, 2, 3 etiology, ix, 71 Europe, 4, 7, 18 European Union, vii, 2, 3 evaporation, 58 evidence, x, 67, 72, 76, 78, 84, 105 evolution, 10, 46 exercise, 22 exposure, x, xi, 9, 10, 63, 90, 91, 92, 93, 94, 99, 101, 103, 104, 105, 107, 109 extinction, 35, 40 extraction, 15, 37, 38 extracts, x, xi, 9, 16, 18, 20, 22, 27, 28, 29, 33, 37, 38, 50, 51, 68, 76, 77, 78, 79, 83, 88, 89, 90, 103, 104

farmers, 3 fasting, viii, 2, 4, 18, 80 fasting glucose, 81 fat, 9, 72, 80, 81, 83, 87, 88 fat soluble, 9 fatty acids, 74 fermentation, 50 fertility, 98 fetal alcohol syndrome, 106, 109 fetal development, 93 fetus, 90, 94, 96, 101 filtration, 68, 82 flavanols, viii, 2, 3, 57 flavonoids, viii, 2, 3, 10, 11, 16, 29, 57, 85, 107 flavor, 6, 8, 58, 59, 62 folate, 9 food, ix, 10, 24, 41, 51, 52, 55, 56, 58, 59, 60, 61, 63, 64, 67, 95, 98, 99 Food and Agriculture Organization (FAO), vii, viii, 3, 4, 22, 31, 49 food industry, ix, 55, 64 food intake, 98 food products, 52, 61 forebrain, x, 90, 94, 96 formation, 20, 22, 92 free radicals, viii, ix, 2, 3, 4, 55, 56, 93, 94, 107 freeze concentration, ix, 55, 58, 59, 60, 65, 66, 67 freezing, 58, 59, 60, 61, 66, 68 fructose, 8, 80, 86 fruits, vii, ix, 2, 3, 5, 7, 8, 9, 10, 11, 14, 15, 16, 19, 20, 21, 24, 25, 29, 32, 33, 34, 38, 40, 41, 43, 45, 48, 50, 55, 56, 60, 61, 62, 63, 64, 65, 66, 76, 87, 88, 93, 107 functional food, 52, 56

G GABA, 109 gastrointestinal tract, 77

114

Index

gastrulation, 91, 106, 109 gene expression, 77, 92, 103, 106 genes, 77, 82 genomics, 29 genotype, 52, 65, 68 germ layer, 90 gestation, x, 90, 91, 94, 95, 96, 99, 100, 101, 104 glucose, viii, 2, 4, 8, 18, 56, 68, 73, 78, 80, 82, 83, 86 glucose tolerance, 80, 82 lucoside, 12, 13, 14, 15, 17, 35, 36, 77, 93, 102, 105, 108, 109 GLUT4, 74 glutathione, 82, 92, 107 glycogen, 105

H harvesting, vii, viii, 2, 3, 49 health, vii, ix, 2, 3, 9, 21, 22, 24, 25, 27, 33, 56, 72, 93, 97 health effects, vii, viii, 2, 3 health problems, 56 health promotion, 22, 25 hemicellulose, 8 hepatotoxicity, 92 herbal medicine, 25 hidroxycinamic acids, viii, 2 high fat, x, 65, 72, 80, 81, 85, 87 homeostasis, 86, 107 human health, ix, 17, 21, 55, 56, 84, 87 hybridization, 7 hydrogen, 33, 48, 93 hydrogen atoms, 48 hydrogen peroxide, 93 hydrolysis, 27, 37 hydrophobicity, 61, 62 hydroxyl groups, 33, 93 hyperglycemia, x, 21, 72 hyperphagia, x, 72, 80 hypertension, 84, 86 hypertrophy, 73, 74, 79 hypoxia, 73

I ICAM, 75 immune system, 9 impregnation, ix, x, 55, 63, 64, 65, 69, 89 in vitro, x, 18, 20, 21, 28, 29, 57, 68, 72, 77, 79, 85, 86, 87, 105, 109 in vivo, x, 20, 29, 72, 79, 87, 93, 94, 105, 107, 109 inflammation, ix, 9, 71, 73, 75, 76, 83, 85, 86, 92 inflammatory disease, 75 inflammatory responses, 73 ingestion, 33, 83 inhibition, 20, 76, 79, 105 inhibitor, 82 injections, x, 89, 95, 97, 98, 99 injury, 73, 75, 77, 79, 83, 92, 106, 107 insulin resistance, ix, 65, 71, 75, 77, 83, 85, 86, 87 insulin sensitivity, x, 72, 83, 87 interneurons, 105 interstitial nephritis, 82 iron, 10, 51 ischemia-reperfusion injury, 82

K kaempferol, 11, 14, 15, 57 ketones, 8 kidneys, 82 kinetics, 64

L LDL, 81, 83 lead, 62, 90, 91, 103 leptin, 73, 75, 80, 87 leukemia, 18 leukocytes, 73 lignin, 8 lipid metabolism, 92, 103, 106 lipid oxidation, 81 lipid peroxidation, 82

115

Index lipids, 9, 66, 107 lipolysis, 74, 77 liquid chromatography, 27, 65 listeria monocytogenes, 27 liver, x, 38, 77, 79, 90, 92, 94, 96, 102, 103, 104, 105, 106, 107 liver disease, 77, 79 low temperatures, ix, 56, 58, 59 low-grade inflammation, 73 lutein, 19 lymphocytes, 73

M macrophages, x, 72, 74, 76, 85 magnetic resonance imaging, 106 manganese, 10 mass spectrometry, 27, 65, 107 matrix, 61, 63, 64, 107 matrix metalloproteinase, 107 MCP-1, 74, 76 mean arterial pressure, 82 melon, 40 melting, 66 Mercosul, 23 metabolic, v, 71, 86 metabolic diseases, ix, 71, 73, 75, 84 metabolic disorders, 85 metabolic pathways, 33 metabolic syndrome, 79, 80, 83, 84, 88 metabolism, 10, 17, 83 metabolites, 33, 57 metformin, 80 methanol, 37, 38 microorganisms, 64 migration, 61, 74, 90, 91, 101, 106, 109 mitogen, 74 molecular weight, 35, 38, 40, 61, 76 molecules, 10, 35, 56, 72, 75 monomers, 38 morphology, 67, 91 morphometric, 96, 103 motor control, 91 mRNA, 76, 77, 78 myocardial infarction, 82, 84

N NADH, 92 NASS, 22 National Agricultural Statistics Service, vii, viii, 4, 31 National Agricultural Statistics Service (NASS), vii, viii, 4, 31 necrosis, 92 neocortex, 91 nervous system, 91, 105 neurodegenerative diseases, 56, 76 neurogenesis, 91 neuronal apoptosis, 109 neurons, 109 neurotoxicity, 108, 109 niacin, 9 Nile, 21 nitric oxide, 76 nitrogen, 56, 63, 66, 92 non-polar, 40 norepinephrine, 79 North America, ix, 3, 5, 7, 55 notochord, 94 Nrf2, 83 nutrient, 63, 93 nutrients, 9, 63, 108 nutrition, 15, 24 nutritional value, ix, 8, 55, 56, 60, 61, 83

O obesity, ix, 71, 73, 74, 75, 76, 78, 79, 80, 83, 84, 87, 88 obesity-associated inflammation, ix, 71 oleic acid, 56, 79, 86 oligomers, 37, 38 olive oil, 9, 22 organ, 72, 96 organic matter, 5 organism, ix, 38, 71, 72 organs, ix, 71, 104 osmotic dehydration, ix, 55, 61, 62, 63, 64, 65, 66, 69

116

Index

oxidation, 60, 81, 83 oxidative damage, 56 oxidative stress, 22, 29, 73, 79, 82, 86, 90, 93, 94, 102, 109 oxygen, 19, 60, 63, 67

P pantothenic acid, 9 pathogenesis, 76 pathogens, 9 pathology, 92 pathways, ix, 71, 74, 76, 91 PCR, 75, 83 periodontal disease, 83 permeability, 61, 62, 63, 75, 81 peroxidation, 20 peroxide, 93 pH, 9, 32, 35, 43, 45, 47, 48, 49 phenolic compounds, viii, ix, 2, 3, 4, 10, 11, 12, 13, 14, 16, 19, 20, 26, 27, 32, 33, 34, 35, 36, 37, 38, 43, 45, 49, 50, 52, 55, 56, 57, 61, 62, 63 phosphorus, 10 phosphorylation, 74, 78 photomicrographs, 96 phytochemicals, ix, 19, 25, 32, 43, 51 PI3K, 78 placebo, 84, 86, 88 plants, 5, 9, 13, 33, 36, 79 plasma levels, 20 polyacrylamide, 68 polymerization, 37, 38 polymorphisms, 27 polyphenols, viii, 2, 4, 10, 12, 22, 26, 76, 77, 79, 85, 86, 87, 107 polysaccharides, 8 potassium, 10 potential benefits, 17 Powderblue, ix, 6, 12, 18, 32, 34, 35, 36, 38, 39, 41, 42, 46, 47, 48 preservation, 12, 60, 61 pressure gradient, 63 prevention, x, 72, 93, 105 probiotic, 64

pro-inflammatory, 73, 74 project, 93, 94, 98 proliferation, 18, 27, 29, 38, 77, 91 prostate cancer, 38, 107 protection, 14, 20, 93, 94, 107, 108 protective role, 82, 94, 104, 108 protein kinases, 74 proteins, 9, 10, 39, 46, 51, 74 public health, 106 pulp, vii, viii, 6, 7, 16, 18, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48

Q quercetin, 11, 14, 15, 20, 57

R radiation, 11, 15, 28, 48, 53 radicals, 56 reactive oxygen, 20, 56, 92 receptor, 74, 86, 87, 109 red blood cells, 20 renal function, x, 72, 86 resistance, x, 20, 29, 72, 74, 75, 80, 82, 84 response, ix, 53, 71, 74, 76, 77, 92, 104, 106 reticulum, 73 retina, 18 retinopathy, 18 riboflavin, 9 risk, 56, 77, 84, 93, 105 risk factors, 84

S Salmonella, 27 salts, 46, 61 seed, 56, 66 selenium, 10 senescence, 9, 107 signaling pathway, 73, 74, 85 skeletal muscle, 78, 81, 87 skin, 5, 35, 61, 62, 63 sodium, 10, 68

117

Index sodium dodecyl sulfate, 68 South America, 4, 6, 7, 22 species, ix, 5, 7, 16, 19, 20, 27, 51, 55, 56, 92, 107 spinal cord, 91, 101 Sprague-Dawley rats, 80, 81, 86 strawberry, vii, viii, 3, 8, 14, 20, 31, 37, 50, 67, 68 sucrose, 8, 59, 67 sulfate, 37 systolic blood pressure, 79, 87

T tannins, viii, 18, 32, 36, 37, 38, 39, 50, 51 techniques, ix, 55, 58, 59, 63 technologies, ix, 55, 56, 59, 61 telencephalon, x, 90, 92, 96, 99, 101, 102, 104, 105 temperature, 10, 22, 49, 58, 62, 63 teratogen, x, 89, 90 testing, 32, 97, 107 thermal treatment, 58 thiazolidinediones, 80 tissue, 60, 72, 73, 74, 75, 82, 83, 96, 108 TLR4, 74, 86 TNF-α, 74, 92 tocopherols, viii, 2, 4, 9, 10, 21 total cholesterol, 81 toxicity, 94, 104 translocation, 74, 76 treatment, x, 18, 62, 63, 65, 72, 75, 76, 77, 78, 79, 82, 83, 84, 86, 94, 106 triglycerides, 79, 81, 92, 103 tumor necrosis factor, 92, 106 type 2 diabetes, 72, 73

U ultrasound, 63, 68 United Nations, 3, 22, 49 United States, vii, viii, 2, 3, 4, 5, 6, 19, 31, 32

urinary tract, 18, 38, 93 urinary tract infection, 38 USDA, vii, viii, 4, 6, 22, 31 UV radiation, 11, 83

V vacuum impregnation, ix, 55, 63, 64, 65, 69 varieties, 5, 15, 23, 32, 34, 38 vascular diseases, 67 VCAM, 75 vegetables, vii, 2, 3, 11, 21, 29, 33, 56, 57, 61, 63, 64, 65, 88 VEGF, 75 vitamin A, 9 vitamin B1, 9 vitamin B12, 9 vitamin B6, 9 vitamin C, 9, 10, 19, 22, 66 vitamin E, 94, 108 vitamin K, 9 vitamins, 9, 10, 32, 61, 64, 108 VLDL, 81 volatile organic compounds, 24

W water, ix, 9, 10, 37, 46, 55, 58, 59, 60, 61, 63, 66, 75, 82, 95 weight changes, 95 weight gain, viii, x, 2, 4, 18, 72, 80, 97 Woodard, ix, 6, 12, 32, 36, 39, 41, 42, 45, 46, 47, 48

Y yield, 7

Z zinc, 10