Processing and Impact on Active Components in Foodhttp://dx.doi.org/10.1016/B978-0-12-404699-3.00018-4 Copyright © 2015 Elsevier Inc. All rights reserved.

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C H A P T E R

18Freezing of Fruits and Impact on Anthocyanins

Shyam S. SablaniBiological Systems Engineering Department, Washington State University, Pullman, USA

INTRODUCTION

Anthocyanins are polyphenolic compounds pres-ent in a variety of fruits, responsible for their attrac-tive color. Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyry-lium salt. Concentrations and types of anthocyanins in fruits vary significantly (Table 18.1). Some factors, such as the types and cultivars of fruits (food matrix), as well as growth conditions, weather at the growing site,

maturity, material preparation, and analysis methods could create differences in total anthocyanins in fruits. Growth conditions and environmental stress, such as high exposure to UV light and temperature, are impor-tant factors influencing the levels of bioactive content as a result of plant defense response (Leong and Oey, 2012). Maturity level also significantly contributes to the total anthocyanin content of fruits.

The anthocyanin content in fruits is at its highest dur-ing the ripening stage, in which the biosynthesis rate is accelerated due to the action of the ripening hormone (ethylene), triggering the activation of many enzymes involved in anthocyanin biosynthesis, and eventually declining at the end of maturation stage. Since anthocya-nins are synthesized at an increasing rate during matura-tion, the total anthocyanin content quantified here may serve as the index of maturity and an important quality parameter. The level of total anthocyanins also depends upon the cultivar. De Ancos et al. (2000) found higher total anthocyanin contents in the late red raspberry cul-tivars, Zeva (116 mg/g of fruit) and Rubi (96.08 mg/g of fruit) compared to the early cultivars, Heritage and Autumn Bliss (31.13 and 37.04 mg/g of fruit, respec-tively), which showed less than half of the late cultivars’ concentration. The anthocyanins are significant because of their nutraceutical benefits, antioxidant, and anticar-cinogenic properties. Anthocyanins can also be used as natural food colorants in the food industry. However, anthocyanins are labile in nature, and therefore are sus-ceptible to deterioration during processing and storage (Syamaladevi et al., 2011).

Freezing is one of the most common methods of pres-ervation of fruits for long-term storage. Frozen fruits are used as ingredients in many food formulations such as jams, jellies, sauces, purees, toppings, syrups, juice concentrates, as well as bakery and dairy products. The freezing process and frozen storage may change the anthocyanin content of fruits, thereby affecting the anti-oxidant capacity and possible health benefits of the fruit.

CHAPTER POINTS

• The maturity level and cultivar of fruit significantly affect the total anthocyanin content and total antioxidant activity.

• In comparison to fresh fruit, the anthocyanin content either increased or did not change significantly after the freezing process.

• Different measurement methods yield slightly different results; there are 3–9% differences between total anthocyanin contents obtained with the pH-differential and HPLC methods.

• Freezing and frozen storage allow a better extraction of total anthocyanins.

• During the initial 4–6 months of frozen storage, there is no significant difference or a slight decrease in content of total anthocyanins, after which the rate of degradation increases.

• Long-term frozen storage has a significant impact on the total anthocyanins and total antioxidant capacity of fruits, depending on the fruit variety and storage time.

• Anthocyanins make a greater contribution to the antioxidant activity of fruits than other phenolic compounds.

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TABLE 18.1 Effect of Frozen Storage Temperature on Stability of Total Anthocyanins

Fruit Initial Value Storage Conditions Comments Reference

Cherry 46.1–547 mg/g DW Just after freezing −20°C 48% increase Leong and Oey (2012)

Nectar 10.9–17.1 mg/g DW 22% increase

Peach 7.7–16.2 mg/g DW 69% increase

Plum 108% increase

Pomegranate 334 days at −23°C Turfani et al. (2012)

Unclarified juice 1091* and 1005** mg/kg FW 7% decrease

Clarified juice 863* and 839** mg/kg FW No loss

Strawberry 24 h at −20°Cthawing at 20°C/8 h

Holzwarth et al. (2012)

Senga Sengana 559 mg/100 g DW 7% decrease

Candonga 333 mg/100 g DW 19% decrease

Sabrosa 325 mg/100 g DW 14% decrease

Black carrot juice 3747* and 4123** mg/kg juice 319 daysat −23°C

0* and 2%** decrease Turkyılmaz and Ozkan (2012)

Date 1 month at −20°C Allaith et al. (2012)

Khalas 1.17 mg/100 g FW 69% decrease

Khunaizi 1.27 mg/100 g FW 47% decrease

Red raspberry 0.78 mg/g DW 378 days Syamaladevi et al. (2011)

at −20°C 21% increase

at −35°C 29% increase

at −80°C 16% decrease

Serviceberry 53.2 mg/100 g FW 10 months at −20°C 13% increase Michalczyk and Macura (2010)

BlackberryTupy

141 mg/100 g fruit 6 months at −10°C 57% decrease Jacques et al., (2010)

6 months at −18°C 50% decrease

6 months at −80°C 31% decrease

Strawberry 31.0 mg/100 g FW 10 months at −18°C 25% decrease Poiana et al. (2010a)

Sweet cherry 49.9 mg/100 g FW 15% decrease

Sour cherry 93.2 mg/100 g FW 12% decrease

Blueberry 206 mg/100 g FW 10 months at −18°C 13% decrease Poiana et al. (2010b)

Red raspberry 39.7 mg/100 g FW 15% decrease

Blackberry 194 mg/100 g FW 8% decrease

Blackberry 12 months at −18°C Kopjar et al. (2009)

Thornfree 1306 mg/l puree # 31% decrease

Cacanska bestrna 1201 mg/l puree # 30% decrease

Strawberry 6 months at −20°C Oszmianski et al. (2009)

Elkat 682 mg/kg FW 7% increase

Kent 299.4 mg/kg FW 10% decrease

Myrtle berries 2307 mg/l 12 months at −20°C 18% decrease Tuberoso et al. (2008)

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Therefore, it is vital to understand the stability of antho-cyanins during frozen storage. For frozen fruits, the retention of anthocyanins depends on the freezing rate, composition, pH, cultivar, temperature, and the pres-ence/absence of oxygen (Wrolstad et al., 1970; Mazza and Miniati, 1993).

The freezing of fruits involves the conversion of water into ice, which includes two successive processes: the formation of ice crystals (nucleation) and the subse-quent increase in crystal size (growth) (Zaritzky, 2012).

Figure 18.1 shows a typical plot of time–temperatures for the freezing of pure water (Figure 18.1A) and food (Figure 18.1B). The cooling of pure water involves the removal of sensible heat (point 1 to point 2), followed by the removal of latent heat at the freezing point the water is converted to ice (point 3). Nucleation is neces-sary to initiate freezing, and the temperature can fall below 0°C without the formation of ice crystals. Point 2 indicates the supercooling of water before crystalliza-tion begins. The heat of solidification is liberated after

Fruit Initial Value Storage Conditions Comments Reference

Blackberry 248 mg/100 g FW 6 months at −20°C No significant change Hager et al. (2008a)

Black raspberry 1113 mg/100 g FW 6 months at −20°C No significant change Hager et al. (2008b)

Blackcurrant 0.9 g/100 g FW Collected from supermarket

28% lower than fresh Hollands et al. (2008)

BlueberryTifblue (extract)

115 (33.6) mg/100 g FW 2 months at −20°C (26% decrease) Srivastava et al. (2007)

Powderblue (extract) 121 (36.1) mg/100 g FW (25% decrease)

Strawberry(six different genotypes)

37.1–122.3 mg/100 g FW 1 month at −23°C 8% decrease (Totem genotype)

Ngo et al. (2007)

Red RaspberryHeritage

33.0 mg/100 g FW 12 months at −18°C No significant change Sousa et al. (2005)

Blueberry 7.2 mg/g DW 3 months at −20°C No significant change Lohachoompol et al. (2004)

Red raspberry Ample 770 nmol/g FW After freezing at −80°C No significant change Mullen et al. (2002)

Red raspberry 12 monthsat −20°C

De Ancos et al. (2000)

Autumn Bliss 31.1 mg/100 g FW 5% increase

Heritage 37.0 mg/100 g FW 17% increase

Zeva 116.3 mg/100 g FW 18% decrease

Rubi 96.1 mg/100 g FW 4% decrease

Strawberry n.a. 12 months at −20°C 23% decrease Garcia-Viguera et al. (1999)

Strawberry 130 mg/g FW 2 months at −20°C 42% decrease Larsen and Poll (1995)

Strawberry n.a 6 months−20°C to −80°C

Deng and Ueda (1993)

Hoko-wase 57–67% decrease

Toyonaka 73–80% decrease

Nyoho 66–71% decrease

Sour cherry Season 1: 12 months at −20°C

150–350% increase depending upon variety30–45% decrease depending upon the variety

Urbanyi and Horti (1992)

Eardi 541, 1321 mg/1000 g dry matter

Pandi 1101, 1039 mg/1000 g dry matter

Ujfehertoi 793, 1413 mg/1000 g dry matter

* pH differential method.** HPLC, #5 weeks of storage.

TABLE 18.1 Effect of Frozen Storage Temperature on Stability of Total Anthocyanins—cont’d

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initial supercooling, thus increasing the temperature from point 2 to point 2′ (Figure 18.1A), which represents the onset of ice crystallization. Further removal of heat reduces the temperature to point 4. The freezing of foods is more complex than the freezing of pure water. The ini-tial cooling behavior from point 1 to point 2 and point 2′ is similar to that of pure water. However, point 2′, which represents the initial freezing point of the solution, is lower than that of pure water. Further cooling from point 2′ to point 3 represents the growth of ice crystals and ice formation. As freezing continues from point 2′ to point 3, water is separated in the form of ice, which increases the solute concentration and further suppresses the freezing point (Zaritzky, 2012).

This chapter examines the influence of the freezing process and frozen storage on the stability of anthocya-nins and antioxidant capacity in fruits, and also explores other factors that affect the stability of anthocyanins in fruits. An understanding of these processes is integral to the development of food processing techniques that pre-serve important nutrients and aesthetic qualities, maxi-mizing benefits to the consumer.

MECHANISMS OF ANTHOCYANIN DEGRADATION

The mechanisms of anthocyanin degradation during processing and storage are well-known. Anthocyanins are unique among the flavonoids because they carry a positive charge associated with the C-ring in the fla-vylium ion form (Hollands et al., 2008). In most plant tissues, anthocyanins are in the intensely colored flavy-lium form, often due to copigmentation which enhances the anthocyanin color and stability. However, when the plant cells are ruptured and anthocyanins are exposed to a higher pH (near neutral), they can form a carbinol pseudo-base, quinoidal-base, or a chalcone; this pro-cess is associated with a loss of color. The rate of color is dependent on the pH and temperature, light conditions, the presence/concentrations of metal ions, oxygen,

ascorbic acid, enzymes, and anthocyanin concentration. In addition, anthocyanin instability can lead to the for-mation of polymeric forms, which is associated with a change in color to a browner and less desirable shade (Mazza and Miniati, 1993).

Attempts have been made to explain the stability of anthocyanins in frozen fruits using the molecular mobility concept. At sufficiently low temperatures, fruit products form a maximum-freeze-concentrated matrix which is described by two temperatures, i.e., the glass transition temperatures of the maximum-freeze-concentrated matrices (Tg″) and the onset of ice melting temperature (Tm″) (Syamaladevi et al., 2011). The transition from the reversible liquid/rubber state to the glassy state starts in the fruit matrix, below the temperature corresponding to the onset of ice melting temperature (Tm′). According to the glass transition concept, foods are most stable in the glassy state, i.e., at temperatures below their glass transition temperature. Below Tg′, viscosity becomes great enough to consid-erably slow down the rates of chemical reactions. The physical and chemical degradation reactions of frozen food systems may be related to molecular mobility, and thus the Tg′ (Torreggiani et al., 1999; Syamaladevi et al., 2011). However, a recent study by Syamaladevi et al. (2011) found that storage temperature does not significantly influence the degradation of anthocya-nins in red raspberries. Rizzolo et al (2003) report no significant difference in total anthocyanin content of blueberry juices after 6 months of storage when frozen at −10, −20 and −30°C. Others found that the glass tran-sition and storage temperatures had no effect on the degradation of anthocyanins during frozen storage of blueberry juice, with or without added sugars (Rizzolo et al., 2003). Torreggiani et al. (1999) reported a signifi-cant loss of strawberry anthocyanins at −10°C during 4 months of storage, but no direct relationship between anthocyanin loss and Tg′. Several studies also suggest no evidence that the degradation of anthocyanins in frozen raspberries is diffusion-limited or dependent on molecular mobility (Syamaladevi et al., 2011).

FIGURE 18.1 Typical plot of time–tempera-tures for the freezing of (A) pure water and (B) food.

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EFFECT OF FREEZING ON ANTHOCYANINS

Studies show either no major difference or an increase in the concentration of anthocyanins immediately after freezing compared to fresh fruits (Table 18.1: Leong and Oey, 2012; Syamaladevi et al., 2011; De Ancos et al., 2000). Leong and Oey (2012) found a 22–108% increase in total anthocyanins after cherries, nectarines, peaches, and plums were frozen. This increase was dependent on the fruit matrix, with a 22% increase for nectarines and a 108% increase for plums. For other fruits, this change is much smaller. For example, Poiana et al. (2010a) found a slight increase in total anthocyanins in sweet cherries and strawberries immediately after freezing (1.6–2.9%), while Syamaladevi et al. (2011) and De Ancos et al. (2000) observed no significant change in total anthocyanins of red raspberries after freezing. The greater quantity of anthocyanins in fruits just after freezing has been attrib-uted to a higher extraction efficiency of anthocyanins from frozen fruits compared to that of fresh fruits, due to cellular disruption during freezing and thawing.

The rate of freezing and the type of freezing tech-nique may also influence the stability of anthocyanins in fruits. Freezing induces the formation of ice crystals, which favors localized concentrations of solutes such as anthocyanins, as well as the reallocation of water mol-ecules in the cell structure. However, cell damage from the growth of ice crystals due to temperature fluctuation and turgor loss often leads to the softening of fruit tex-ture. The rate of freezing has been found to influence ice crystal formation that expands the separation between cells in the fruit structure. When fruits are rapidly fro-zen, smaller ice crystals form, which reduce cell struc-ture disruption, while fruits that are frozen slowly form large intercellular ice crystals that cause more damage (Poina et al., 2010a). Studies examining the influence of different freezing and thawing procedures on anthocy-anin retention in fruits found that the effects of differing freezing technologies is minor; however, different thaw-ing regimes significantly affect anthocyanin retention after thawing. Holzwarth et al. (2012) observed higher retention of anthocyanins in strawberries when they were thawed at 20°C in a microwave oven compared to thawing at 4°C and 37°C.

De Ancos et al. (2000) observed that the two early cultivars of red raspberries, Heritage and Autumn Bliss, which have low total anthocyanin and cyanidin 3-glucoside concentrations, show no degradation after freezing and, indeed, a better extraction of total antho-cyanin content due to cellular disruption caused by the freezing process. Rubi and Zeva, the two late cultivars of red raspberry with high total anthocyanin and cyani-din 3-glucoside concentrations, showed a more evi-dent degradation of total anthocyanins caused by the

freezing process. This degradative effect could be due to the high content of the more reactive anthocyanin compound cyanidin 3-glucoside, or perhaps cellular disruption caused by the freezing process, producing a release of the oxidoreductase enzymatic system (PPO). De Ancos et al. (2000) report that their previous work showed more polyphenol oxidase (PPO) enzyme activ-ity in Rubi and Autumn Bliss raspberry tissues than in Zeva and Heritage tissues. Therefore, the degradative enzymatic reactions could be one of the main reasons for the total anthocyanin concentration losses in Rubi but not in Zeva. Physicochemical characteristic differ-ences have also been found between early and late cul-tivars: Rubi and Zeva show lower pH and higher °Brix values than the early cultivars, Heritage and Autumn Bliss. These results suggest that the stability of antho-cyanins during freezing mainly depends on the pH value, organic acid content, sugar concentration, initial concentration, and initial cyanidin 3-glucoside content.

EFFECT OF FROZEN STORAGE ON ANTHOCYANINS

Frozen storage affects the stability of total antho-cyanin content of fruits in different ways (Table 18.1). Several studies show a decreasing trend in total fruit anthocyanins during frozen storage, e.g., in unclarified pomegranate juice (Turfani et al., 2012); strawberries (Holzwarth et al., 2012, Poiana et al., 2010a; Oszmianski et al., 2009; Ngo et al., 2007; Garcia-Viguera et al., 1999; Larsen and Poll, 1995; Deng and Ueda, 1993); black car-rot juice (Turkyimaz and Ozkan, 2012); dates (Allaith et al., 2012); blackberries (Jacques et al., 2010; Poiana et al., 2010b; Kopjar et al., 2009); sweet cherries (Poiana et al., 2010a); blueberries (Poiana et al., 2010b; Srivas-tava et al., 2007); red raspberries (Poiana et al., 2010b; De Ancos et al., 2000); Myrtle berries (Tuberosco et al., 2008); blackcurrants (Hollands et al., 2008); and sour cherries (Urbanyi and Horti, 1992).

A few studies found an increase in total anthocya-nins during frozen storage, including red raspberries (Syamaladevi et al., 2011; De Ancos et al., 2000); service-berries (Michalczyk and Macura, 2010); strawberries (Oszmianski et al., 2009); sour cherries (Urbanyi and Horti, 1992). This has been attributed to a concentra-tion effect from moisture loss or enhanced extraction of anthocyanins due to tissue softening (Hager et al., 2008b). Some studies show no significant change in total anthocyanins during frozen storage of fruits, e.g., clari-fied pomegranate juice (Turfani et al., 2012), black carrot juice (Turkyimaz and Ozkan, 2012), blackberries (Hager et al., 2008a), black raspberries (Hager et al., 2008b), red raspberries (Sousa et al., 2005; Mullen et al., 2002), and blueberries (Lohachoompol et al., 2004).

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It is not feasible to conduct a complete comparison between studies, since the fruit variety, maturity level, initial pH, storage temperature, and time differ. De Ancos et al. (2000) observed that frozen storage affects total anthocyanin content and individual anthocyanin distribution in different ways, depending on the cultivar. Total anthocyanins in Heritage and Autumn Bliss rasp-berries were found to increase slightly, at 17% and 5%, respectively, after frozen storage for 360 days. The rela-tive percentage of individual anthocyanins in the early raspberry cultivars, Heritage and Autumn Bliss, showed small changes during frozen storage, apparently due to increased extraction of the main pigments, cyanidin 3-sophoroside and cyanidin 3-rutinoside, during frozen storage. The total value of anthocyanins in late cultivars was significantly decreased (4–17%) in both Rubi and Zeva cultivars during frozen storage. These results could be explained by the different chemical compositions found between early cultivars (Heritage and Autumn Bliss) and late cultivars (Rubi and Zeva). Raspberry cul-tivars with low pH, high soluble solids content (°Brix), and high total anthocyanin content retained the initial anthocyanin concentration better during processing.

Many studies have shown that long-term frozen stor-age has a significant impact on anthocyanins depending on the type of fruit. However, during the initial period of frozen storage (4 to 6 months), there is little or no sig-nificant decrease in total anthocyanins, with the rate of degradation increasing after this period (Poiana et al., 2010a,b; Chaovanalikit and Wrolstad, 2004; Sahari et al., 2004). Woodward et al. (2009) studied the effect of freeze–thaw cycles on anthocyanin stability. To determine the storage stability of various anthocyanins, acidified buf-fer solutions (10mMNa/K phosphate buffer in 2% HCl) were spiked with 150 μM delphinidin-3-glucoside, cyan-idin-3-glucoside, pelargonidin-3-glucoside, delphinidin, cyanidin, or pelargonidin (individually). Samples were reanalyzed sequentially for six freeze–thaw cycles. One freeze–thaw cycle consisted of 24-h storage at −80°C, followed by a 20-min defrosting at ambient room tem-perature prior to HPLC analysis. Results showed that anthocyanin glucosides were stable during storage and the freeze–thaw treatment, with no significant losses. Of the three aglycone species, both pelargonidins and cyani-dins showed a significant loss following freeze–thaw cycling, while delphinidins remained stable. Pelargo-nidins showed a linear rate of degradation, with sig-nificant losses demonstrated at four freeze–thaw cycles and a total reduction of 11% at six freeze–thaw cycles. Cyanidins also showed a linear rate of degradation, with a maximum reduction of 6% at six freeze–thaw cycles.

Urbanyi and Horti (1992) found that total anthocy-anin content increased linearly during the first year of storage. In studies of shorter storage times in the sec-ond year, this change was reversed. They attributed this

behavior to various phases of storage time and different processes taking place at different levels of fruit matu-rity. In the less ripe fruits/1st year, the biosynthesis of anthocyanins continue, according to the results found by several researchers. In the case of riper fruits, this goes much slower or does not occur at all.

OTHER FACTORS AFFECTING ANTHOCYANIN DEGRADATION

Home-scale freezers take a longer time to freeze fruits compared to commercial freezers hence anthocyanins susceptible to oxidation suffer damages (Poiana et al., 2010a). This explains the higher loss of anthocyanins in domestic freezing of strawberries and cherries. Hollands et al. (2008) found that anthocyanin levels in thermally processed blackcurrant are extremely low compared to the high levels in frozen blackcurrant fruits. Adding sucrose to fruits during frozen storage has been shown to protect anthocyanins and also to retard browning and polymeric color formation (Wrolstad et al., 1990). Kopjar et al. (2009) also observed a high retention of anthocyanin content in two varieties of blackberries, Thornfree and Cacanska bestrna, after 12 months of storage at −18°C when samples were coated with glucose. This effect was explained by the fact that the addition of sugar reduces water activity.

In the juice production process, compounds such as pectin, fibers, semi-fibers, starch, and proteins must be clarified, which may affect the stability of anthocya-nins in juice during storage. Turfani et al. (2012) found that total anthocyanin content of pomegranate juice decreased substantially after clarification. The loss of total anthocyanins was more than 20% higher in clari-fied juice compared to unclarified juice. The apparent decrease in anthocyanin content is caused by the inter-action between anthocyanin and gelatin-tannin flocks formed during clarification. Similarly, studies show an 18% decrease in anthocyanin contents of blackberry juices after clarification (Rommel et al., 1992).

EFFECT OF FROZEN STORAGE ON ANTIOXIDANT CAPACITY

The health benefits of anthocyanins come from their antioxidant activity. Antioxidants are substances that, if present in low concentrations, significantly prevent the oxidation of a substrate. The human body produces reactive carbon, sulfur, nitrogen, and oxygen species as a result of interaction with ionizing radiation and physi-ological processing, the most damaging of which are the reactive oxygen species, such as superoxide, hydro-gen peroxide, and hydroxyl radicals. Many studies of

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anthocyanin content changes in fruits after freezing and during frozen storage have also evaluated the total anti-oxidant capacity of fruits. The presence of other pheno-lic compounds such as flavonoids, phenolic acids, and vitamins C and E also contribute to the total antioxi-dant activity of fruits. However, Bof et al. (2012) found a higher correlation between total anthocyanins and antioxidant capacity in comparison to total phenolics and antioxidant capacity, concluding that anthocyanins make a greater contribution to antioxidant activity than other phenolic compounds. Michalczyk and Macura

(2010) also observed that the antioxidant properties of serviceberries are affected mainly by total anthocyanins.

The total antioxidant capacity (TAC) of fruits can be affected by the freezing process and during frozen storage. Similar to total anthocyanins, frozen storage affects the total antioxidant capacity of fruits in differ-ent ways (Table 18.2). Several studies report a decrease in total antioxidant capacity during frozen storage, e.g., Khunaizi dates (Allaith et al., 2012), guava (Bof et al., 2012), strawberries (Bof et al., 2012; Poiana et al., 2010a), pears (Bof et al., 2012), cherries (Poiana et al., 2010a),

TABLE 18.2 Effect of Freezing and Frozen Storage on Antioxidant Activity

Fruit Initial Value Storage Conditions Comments Reference

DateKhalasKhunaizi

mmol/100 g FW2.693.48

1 monthat −20°C 88% increase

24% decrease

Allaith et al. (2012)

Pulp:GuavaGrapeFigStrawberryApplePear

TEAC(μmol/g)27.022.05.116.011.17.3

3 monthsat −15°C 26% reduction

No significant changeNo significant change18% reductionNo significant change45% reduction

Bof et al. (2012)

StrawberrySweet cherrySour cherry

mM Fe2+/kg FW24.413.543.1

10 monthsat −18°C 42% decrease

39% decrease35% decrease

Poiana et al. (2010a)

BlueberryRed raspberryBlackberry

mM Fe2−/kg FW58.340.249.6

10 monthsat −18°C 23% decrease

38% decrease35% decrease

Poiana et al. (2010b)

Serviceberryg FW/g DPPH21.0

10 monthsat −20°C 25% decrease

Michalczyk and Macura (2010)

AppleOrange

n.an.a

10 daysat −18°Cat −70°C

No significant change Polinati et al. (2010)

BlackberryThornfreeCacanska bestrna

n.a. 12 monthsat −18°C

8% decrease8% decrease

Kopjar et al. (2009)

Blackberry (μmol of TE/g of FW)97.2

6 monthsat −20°C

No significant change Hager et al. (2008a)

Black raspberry μmol of TE/g of FW)192

6 monthsat −18°C

18% increase Hager et al. (2008b)

BlueberryTifblue(extract)Powder blue(extract)

(μm/g FW)26.1(17.0)27.3(17.5)

2 monthsat −20°C

(No significant change)(6% decrease)

Srivastava et al. (2007)

Red raspberry Ample (number of Fermy’s radical × 1016 reduced)406

After freezing at −80°C No significant change Mullen et al. (2002)

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blueberries (Poiana et al., 2010b; Srivastava et al., 2007), blackberries (Poiana et al., 2010b; Kopjar et al., 2009), red raspberries (Poiana et al., 2010b), and serviceberries (Michalczyk and Macura, 2010). This decrease in total antioxidant capacity ranged from 8% to 45% depend-ing upon the fruit, storage temperature, and time. For example, Poina et al. (2010a) found that total antioxidant capacity of cherries and strawberries decreased during frozen storage. In the first 4 months of storage, there was a relatively small decrease in antioxidant capacity, fol-lowed by a significant decline in following months. At 10 months, antioxidant capacity had decreased by up to 35% for sour cherries, up to 38% for sweet cherries, and up to about 42% for strawberries. Also, at the end of 4 months of frozen storage, the loss of antioxidant activ-ity of sour cherries ranged about 15% for sour cherries, about 19% for sweet cherries, and 23% for strawberries (Poina et al., 2010a).

A few studies noticed no significant change in TAC during the frozen storage of fruits, e.g., grapes, figs, and apples (Bof et al., 2012); apples and oranges (Polinati et al., 2010); blackberries (Hager et al., 2008a); blueber-ries (Srivastava et al., 2007); and red raspberries (Mullen et al., 2002). However, a few studies reported an increas-ing trend in TAC during frozen storage, e.g., Khalas dates (Allaith et al., 2012) and black raspberries (Hager et al., 2008b).

ANALYTICAL TECHNIQUES

The most common methods to quantify anthocyanin content of fruits are pH differential method/Spectro-photometric method and high pressure liquid chro-matography (HPLC) (Table 18.3). The pH differential method is used to determine total anthocyanins, while

TABLE 18.3 Analytical Techniques used to Quantify TA and TAC

Fruit TA and TAC Measurement Methods Reference

Guava, grape, fig, strawberry, apple, pear

TAC: TEAC Bof et al. (2012)

Pomegranate TA: pH differential and HPLC Turfani et al. (2012)

Strawberry TA: HPLC-DAD-MS Holzwarth et al. (2012)

Black carrot juice TA: pH differential method and HPLC- DAD-MS

Turkyılmaz and Ozkan (2012)

Date TA: pH differential methodTAC: FRAP and DPPH assays

Allaith et al. (2012)

Cherry, nectar, peach, plum TA: pH differential method/ spectrophotometer (700 nm)

Leong and Oey (2012)

Red raspberry TA: Spectrophotometric method Syamaladevi et al. (2011)

Blackberry TA: Spectrophotometric method Jacques et al., (2010)

Serviceberry TA: pH differential methodsTAA: DPPH assay

Michalczyk and Macura (2010)

Strawberry, sweet cherry, sour cherry TA: Spectrophotometric methods (520 nm and 700 nm)TAC: FRAP

Poiana et al. (2010a)

Blueberry, Red raspberry, Blackberry TA: Spectrophotometric methods (520 nm and 700 nm)TAC: FRAP

Poiana et al. (2010b)

Apple and orange TAC: DPPH assay Polinati et al. (2010)

Blackberry TA: Spectrophotometric methodTAC: DPPH

Kopjar et al. (2009)

Strawberry TA: HPLC Oszmianski et al. (2009)

Strawberry TA: HPLC Gössinger et al. (2009)

Myrtle berries TA: Spectrophotometric method (520 nm)

Tuberoso et al. (2008)

Blackcurrant TA: HPLC Hollands et al. (2008)

REFEREnCES 155

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the HPLC method is used to quantify individual antho-cyanins. Although the pH differential method is fast and easy to perform, critics have noted that this method is not suitable for identifying the pattern of individual glycoside substitution of anthocyanins with sugar com-pounds. Other fruit substances such as pectin, proteins, lipids, and polyphenol compounds are known to inter-fere with anthocyanin measurements (Leong and Oey, 2012). Turfani et al. (2012) determined the total anthocy-anin content of pomegranate juice with both pH differ-ential (spectrophotometric) and HPLC methods. They found differences between total anthocyanin contents obtained by both methods; however, there was high correlation between the amounts of anthocyanins found in pomegranate juice with both methods. Similarly, Lee et al. (2002) found that total anthocyanin contents of blueberry juices obtained with the pH differential method differed from those obtained with HPLC. These differences are attributed to several factors: (i) different solvent systems utilized for HPLC and pH differential methods; (ii) different wavelengths, i.e., 520 and 512 nm utilized by HPLC and pH differential methods, respec-tively; and (iii) the interference of polymeric pigments during anthocyanin analyses. For example, polymeric pigments may be retained in the HPLC column and not included in HPLC measurements, whereas they may have contributed to the results from the pH differen-tial method (Lee et al., 2002). Turkyılmaz and Ozkan (2012) found that in comparison to the HPLC method, the spectrophotometric method determined a 9% lower

value of total anthocyanins in black carrot juice, while Turfani et al. (2012) observed an opposite trend, with a 3–8% lower value of anthocyanins in pomegranate juice using the HPLC method.

ReferencesAllaith, A.A., Ahmed, S.H., Jafer, F., 2012. Effect of different thermal

treatments and freezing on the antioxidant constituents and activ-ity of two Bahraini date cultivars (Phoenix dactylifera L.). Int. J. Food Sci. Technol. 47, 783–792.

Bof, C.M.J., Fontana, R.C., Piemolini-Barreto, L.T., Sandri, I.G., 2012. Effect of freezing and processing technologies on the antioxidant capacity of fruit pulp and jelly. Braz. Arch. Biol. Technol. 55 (1), 107–114.

Chaovanalikit, A., Wrolstad, R.E., 2004. Anthocyanin and polypheno-lic composition of fresh and processed cherries. J. Food Sci. 69 (1), 73–83.

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Deng, H., Ueda, Y., 1993. Effects of freezing methods and storage tem-perature on flavor stability and ester contents of frozen strawber-ries. J. Japanese Soc. Horticultural Sci. 62 (3), 633–639.

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Fruit TA and TAC Measurement Methods Reference

Blackberry TAA: ORAC Hager et al. (2008a)

Black raspberry TA: HPLCTAC: ORAC

Hager et al. (2008b)

Strawberry TA: Spectrophotometric method Ngo et al. (2007)

Blueberry TA: Spectrophotometric methodTAC: TEAC

Srivastava et al. (2007)

Blueberry TA: Spectrophotometric method Lohachoompol et al. (2004)

Red raspberry TA: Spectrophotometric methodTAC: ESR spectra of Fermy’s radical

Mullen et al. (2002)

Red raspberry TA: Spectrophotometric method and HPLC

De Ancos et al. (2000)

Strawberry TA: HPLC Garcia-Viguera et al. (1999)

Strawberry TA: pH differential method/ spectrophotometer

Larsen and Poll (1995)

Strawberry TA: Spectrophotometric method Deng and Ueda (1993)

Sour cherries TA: Spectrophotometric method (530 nm) Urbanyi and Horti (1992)

Strawberry TA: Spectrophotometric method Wrolstad et al. (1990)

TABLE 18.3 Analytical Techniques used to Quantify TA and TAC—cont’d

18. FREEZING IMPACTS ON ANTHOCYANINS156

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Lee, J., Durst, R.W., Wrolstad, R.E., 2002. Impact of juice processing on blueberry anthocyanins and polyphenolics: Comparison of two pretreatments. J. Food Sci. 67, 1660–1667.

Leong, S.Y., Oey, I., 2012. Effects of processing on anthocyanins, carot-enoids and vitamin C in summer fruits and vegetables. Food Chem. 133, 1577–1587.

Lohachoompol, V., Srzednicki, G., Craske, J., 2004. The change of total anthocyanins in blueberries and their antioxidant effect after dry-ing and freezing. J. Biomed. Biotechnol. 5, 248–252.

Mazza, G., Miniati, E., 1993. Introduction. In: Mazza, G. (Ed.), Antho-cyanins in Fruits, Vegetables and Grains. CRC Press, Boca Raton, pp. 85–87.

Michalczyk, M., Macura, R., 2010. Effect of processing and storage on the antioxidant activity of frozen and pasteurized shadblow service-berry (Amelanchier canadensis). Int. J. Food Properties 13, 1225–1233.

Mullen, W., Stewart, A.J., Lean, M.E.J., Gardner, P., Duthie, G.G., Crozier, A., 2002. Effect of freezing and storage on the phenolics, ellagitannins, flavonoids, and antioxidant capacity of red rasp-berries. J. Agric. Food Chem. 50, 5197–5201.

Ngo, T., Wrolstad, R.E., Zhao, Y., 2007. Color quality of Oregon straw-berries— impact of genotype, composition, and processing. J. Food Sci. 72 (1), c25–c32.

Oszmianski, J., Wojdylo, A., Kolniak, J., 2009. Effect of L–ascorbic acid, sugar, pectin and freeze–thaw treatment on polyphenol content of frozen strawberries. LWT- Food Sci Technol. 42, 581–586.

Poiana, M.A., Moigradean, D., Alexa, E., 2010a. Influence of home-scale freezing and storage of antioxidant properties and color quality of different garden fruits. Bulgarian J. Agri. Sci. 16 (2), 163–171.

Poiana, M.A., Moigradean, D., Raba, D., Alda, L.-M., Popa, M., 2010b. The effect of long-term frozen storage on the nutraceutical com-pounds, antioxidant properties and color indices of different kinds of berries. J. Food, Agri. Environ. 8 (1), 54–58.

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