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Freezing of Fruits and Impact on Anthocyanins
C H A P T E R
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Freezing of Fruits and Impact on Anthocyanins Shyam S. Sablani Biological Systems Engineering Department, Washington State University, Pullman, USA
CHAPTER POINTS • T he 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.
INTRODUCTION Anthocyanins are polyphenolic compounds present in a variety of fruits, responsible for their attractive color. Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium 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,
Processing and Impact on Active Components in Food http://dx.doi.org/10.1016/B978-0-12-404699-3.00018-4
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 important 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 during 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 anthocyanins are synthesized at an increasing rate during maturation, 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 cultivars, 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, respectively), which showed less than half of the late cultivars’ concentration. The anthocyanins are significant because of their nutraceutical benefits, antioxidant, and anticarcinogenic properties. Anthocyanins can also be used as natural food colorants in the food industry. However, anthocyanins are labile in nature, and therefore are susceptible to deterioration during processing and storage (Syamaladevi et al., 2011). Freezing is one of the most common methods of preservation 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 antioxidant capacity and possible health benefits of the fruit.
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18. FREEZING IMPACTS ON ANTHOCYANINS
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
Unclarified juice
1091*
Clarified juice
863*
and
and
1005**
839**
mg/kg FW
7% decrease
mg/kg FW
Strawberry
Turfani et al. (2012)
No loss 24 h at −20°C thawing 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
Date
0* and 2%** decrease
319 days at −23°C 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
Turkyılmaz and Ozkan (2012)
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)
Blackberry Tupy
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
10 months at −18°C
25% decrease
Strawberry
31.0 mg/100 g FW
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
Red raspberry
39.7 mg/100 g FW
15% decrease
Blackberry
194 mg/100 g FW
8% decrease
10 months at −18°C
Blackberry Thornfree Cacanska bestrna
13% decrease
12 months at −18°C 1306 mg/l puree
31% decrease
1201 mg/l puree
#
30% decrease 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
2. FRUIT
Poiana et al. (2010b)
Kopjar et al. (2009)
#
Strawberry
Poiana et al. (2010a)
18% decrease
Tuberoso et al. (2008)
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Introduction
TABLE 18.1 Effect of Frozen Storage Temperature on Stability of Total Anthocyanins—cont’d 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)
Blueberry Tifblue (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 Raspberry Heritage
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 months at −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
Hoko-wase
Deng and Ueda (1993) 57–67% decrease
Toyonaka
73–80% decrease
Nyoho
66–71% decrease
Sour cherry Eardi
541, 1321 mg/1000 g dry matter
Pandi
1101, 1039 mg/1000 g dry matter
Ujfehertoi
793, 1413 mg/1000 g dry matter
Season 1: 12 months at −20°C
150–350% increase depending upon variety 30–45% decrease depending upon the variety
Urbanyi and Horti (1992)
* pH differential method. ** HPLC, #5 weeks of storage.
Therefore, it is vital to understand the stability of anthocyanins during frozen storage. For frozen fruits, the retention of anthocyanins depends on the freezing rate, composition, pH, cultivar, temperature, and the presence/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 subsequent 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 necessary 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 crystallization begins. The heat of solidification is liberated after
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18. FREEZING IMPACTS ON ANTHOCYANINS
FIGURE 18.1 Typical plot of time–temperatures for the freezing of (A) pure water and (B) food.
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 initial 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 anthocyanins 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 preserve important nutrients and aesthetic qualities, maximizing 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 flavylium ion form (Hollands et al., 2008). In most plant tissues, anthocyanins are in the intensely colored flavylium 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 process 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 formation 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 maximumfreeze-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 considerably 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 anthocyanins 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 transition 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 significant 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).
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Effect of Frozen Storage on Anthocyanins
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 attributed 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 technique 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 molecules 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 texture. 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 frozen, smaller ice crystals form, which reduce cell structure 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 anthocyanin retention in fruits found that the effects of differing freezing technologies is minor; however, different thawing 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 anthocyanin 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 cyanidin 3-glucoside concentrations, showed a more evident degradation of total anthocyanins caused by the
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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 activity 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 differences have also been found between early and late cultivars: 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 anthocyanins 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 anthocyanin 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 carrot 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; Srivastava 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 anthocyanins during frozen storage, including red raspberries (Syamaladevi et al., 2011; De Ancos et al., 2000); serviceberries (Michalczyk and Macura, 2010); strawberries (Oszmianski et al., 2009); sour cherries (Urbanyi and Horti, 1992). This has been attributed to a concentration 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., clarified 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 raspberries were found to increase slightly, at 17% and 5%, respectively, after frozen storage for 360 days. The relative 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 cultivars 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 storage 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 significant 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 buffer solutions (10mMNa/K phosphate buffer in 2% HCl) were spiked with 150 μM delphinidin-3-glucoside, cyanidin-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 temperature 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 cyanidins showed a significant loss following freeze–thaw cycling, while delphinidins remained stable. Pelargonidins showed a linear rate of degradation, with significant 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 anthocyanin content increased linearly during the first year of storage. In studies of shorter storage times in the second 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 maturity. 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 anthocyanins 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 clarified juice compared to unclarified juice. The apparent decrease in anthocyanin content is caused by the interaction 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 physiological processing, the most damaging of which are the reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radicals. Many studies of
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Effect of Frozen Storage on Antioxidant Capacity
anthocyanin content changes in fruits after freezing and during frozen storage have also evaluated the total antioxidant capacity of fruits. The presence of other phenolic compounds such as flavonoids, phenolic acids, and vitamins C and E also contribute to the total antioxidant 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 different 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
Date Khalas Khunaizi
mmol/100 g FW 2.69 3.48
1 month at −20°C
Pulp: Guava Grape Fig Strawberry Apple Pear
TEAC(μmol/g) 27.0 22.0 5.1 16.0 11.1 7.3
3 months at −15°C
Strawberry Sweet cherry Sour cherry
mM Fe2+/kg FW 24.4 13.5 43.1
10 months at −18°C
Blueberry Red raspberry Blackberry
mM Fe2−/kg FW 58.3 40.2 49.6
10 months at −18°C
Serviceberry
g FW/g DPPH 21.0
10 months at −20°C
Apple Orange
n.a n.a
Blackberry Thornfree Cacanska bestrna
Comments
Reference Allaith et al. (2012)
88% increase 24% decrease Bof et al. (2012) 26% reduction No significant change No significant change 18% reduction No significant change 45% reduction Poiana et al. (2010a) 42% decrease 39% decrease 35% decrease 23% decrease 38% decrease 35% decrease
Poiana et al. (2010b)
25% decrease
Michalczyk and Macura (2010)
10 days at −18°C at −70°C
No significant change
Polinati et al. (2010)
n.a.
12 months at −18°C
8% decrease 8% decrease
Kopjar et al. (2009)
Blackberry
(μmol of TE/g of FW) 97.2
6 months at −20°C
No significant change
Hager et al. (2008a)
Black raspberry
μmol of TE/g of FW) 192
6 months at −18°C
18% increase
Hager et al. (2008b)
Blueberry Tifblue (extract) Powder blue (extract)
(μm/g FW) 26.1 (17.0) 27.3 (17.5)
2 months at −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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18. FREEZING IMPACTS ON ANTHOCYANINS
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% depending 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, followed 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 activity 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); blueberries (Srivastava et al., 2007); and red raspberries (Mullen et al., 2002). However, a few studies reported an increasing 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/Spectrophotometric method and high pressure liquid chromatography (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 HPLCDAD-MS
Turkyılmaz and Ozkan (2012)
Date
TA: pH differential method TAC: 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 methods TAA: 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 method TAC: 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)
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155
References
TABLE 18.3 Analytical Techniques Used to Quantify TA and TAC—cont’d Fruit
TA and TAC Measurement Methods
Reference
Blackberry
TAA: ORAC
Hager et al. (2008a)
Black raspberry
TA: HPLC TAC: ORAC
Hager et al. (2008b)
Strawberry
TA: Spectrophotometric method
Ngo et al. (2007)
Blueberry
TA: Spectrophotometric method TAC: TEAC
Srivastava et al. (2007)
Blueberry
TA: Spectrophotometric method
Lohachoompol et al. (2004)
Red raspberry
TA: Spectrophotometric method TAC: 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)
the HPLC method is used to quantify individual anthocyanins. 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 compounds. Other fruit substances such as pectin, proteins, lipids, and polyphenol compounds are known to interfere with anthocyanin measurements (Leong and Oey, 2012). Turfani et al. (2012) determined the total anthocyanin content of pomegranate juice with both pH differential (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, respectively; 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 differential 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.
References Allaith, A.A., Ahmed, S.H., Jafer, F., 2012. Effect of different thermal treatments and freezing on the antioxidant constituents and activity 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 polyphenolic composition of fresh and processed cherries. J. Food Sci. 69 (1), 73–83. De Ancos, B., Ibanez, E., Reglero, G., Pilar Cano, M., 2000. Frozen storage effects on anthocyanins and volatile compounds of raspberry fruit. J. Agric. Food Chem. 48, 873–879. Deng, H., Ueda, Y., 1993. Effects of freezing methods and storage temperature on flavor stability and ester contents of frozen strawberries. J. Japanese Soc. Horticultural Sci. 62 (3), 633–639. Garcia-Viguera, C., Zafrilla, P., Tomas-Barberan, F.A., 1999. Influence of processing and storage conditions in strawberry jam color. Food Sci. Technol. Int. 5 (6), 487–492. Gössinger, M., Moritz, S., Hermes, M., Wendelin, S., Scherbichler, H., Halbwirth, H., Stich, K., Berghofer, E., 2009. Effects of processing parameters on color stability of strawberry nectar from puree. J. Food Eng. 90, 171–178. Hager, T.J., Howard, L.R., Prior, R.L., 2008a. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blackberry products. J. Agric. Food Chem. 56, 689–695.
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Hager, A., Howard, L.R., Prior, R.L., Brownmiller, C., 2008b. Anthocyanins, percent polymeric color, and antioxidant capacity of processed black raspberry products. J. Food Sci. 73 (6), H134–H140. Hollands, W., Brett a, G.M., Radreau, P., Saha, S., Teucher, B., Bennett, R.N., Kroon, P.A., 2008. Processing blackcurrants dramatically reduces the content and does not enhance the urinary yield of anthocyanins in human subjects. Food Chem. 108, 869–878. Holzwarth, M., Korhummel, S., Carle, R., Kammerer, D.R., 2012. Evaluation of the effects of different freezing and thawing methods on color, polyphenol and ascorbic acid retention in strawberries (Fragaria×ananassa Duch.). Food Res. Int. 48, 241–248. Jacques, A.C., Pertuzatti, P.B., Barcia, M.T., Zambiazi, R.C., Chim, J.F., 2010. Stability of bioactive compounds in frozen pulp of blackberry (Rubus fructicosus) cv. Tupy, Quim. Nova 33 (8), 1720–1725. Kopjar, M., Tiban, N.N., Pilizota, V., Babic, J., 2009. Stability of anthocyanins, phenols and free radical scavenging activity through sugar addition during frozen storage of blackberries. J. Food process. Preservation 33, 1–11. Larsen, M., Poll, L., 1995. Changes in the composition of aromatic compounds and other quality parameters of strawberries during freezing and thawing. Z. Lebensm. Unters. Forsch. 201, 275–277. 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, carotenoids 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 drying and freezing. J. Biomed. Biotechnol. 5, 248–252. Mazza, G., Miniati, E., 1993. Introduction. In: Mazza, G. (Ed.), Anthocyanins 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 serviceberry (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 raspberries. J. Agric. Food Chem. 50, 5197–5201. Ngo, T., Wrolstad, R.E., Zhao, Y., 2007. Color quality of Oregon strawberries— 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 compounds, antioxidant properties and color indices of different kinds of berries. J. Food, Agri. Environ. 8 (1), 54–58.
Polinati, R.M., Faller, A.L.K., Fialho, E., 2010. The effect of freezing at −18oC and −70oC with and without ascorbic acid on the stability of antioxidant in extracts of apple and orange fruits. Int. J. Food Sci. Technol. 45, 1814–1820. Rizzolo, A., Nani, R.C., Viscardi, D., Bertolo, G., Torreggiani, D., 2003. Modification of glass transition temperature through carbohydrates addition and anthocyanin and soluble phenol stability of frozen blueberry juices. J. Food Eng. 56 (2–3), 229–231. Rommel, A., Wrolstad, R.E., Heatherbell, D.A., 1992. Blackberry juice and wine: Processing and storage effects on anthocyanin composition, color and appearance. J. Food Sci. 57, 385–391. Sahari, M.A., Mohsen Boostani, F., Zohreh, H.E., 2004. Effect of low temperature on the ascorbic acid content and quality characteristics of frozen strawberry. Food Chem. 86, 357–363. Sousa, M.B., Canet, W., Alvarez, M.D., Tortosa, M.E., 2005. The effect of the pre-treatments and the long and short-term frozen storage on the quality of raspberry (cv. Heritage). Eur. Food Res. Technol. 221, 132–144. Srivastava, A., Akoh, C.C., Yi, W., Fischer, J., Krewer, G., 2007. Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles. J. Agric. Food Chem. 55, 2705–2713. Syamaladevi, R.M., Sablani, S.S., Tang, J., Powers, J., Swanson, B.G., 2011. Stability of anthocyanins in frozen and freeze-dried raspberries during long-term storage—In relation to glass transition. J. Food Sci. 76, E414–E421. Tuberoso, C.I.G., Barra, A., Cabras, P., 2008. Effect of different technological processes on the chemical composition of myrtle (Myrtus communis L.) alcoholic extracts. Eur. Food Res. Technol. 226, 801–808. Torreggiani, D., Forni, E., Guercilena, I., Maestrelli, A., Bertolo, G., Archer, G.P., Kennedy, C.J., Bone, S., Blond, G., Contreras-Lopez, E., Champion, D., 1999. Modification of glass transition temperature through carbohydrates additions: effect upon color and anthocyanin pigment stability in frozen strawberry juices. Food Res. Int. 32, 441–446. Turfani, O., Turkyilmaz, M., Yemis, O., Özkan, M., 2012. Effects of clarification and storage on anthocyanins and color of pomegranate juice concentrates. J. Food Qual. 35, 272–282. Turkyılmaz, M., Ozkan, M., 2012. Kinetics of anthocyanin degradation and polymeric color formation in black carrot juice concentrates during storage. Int. J. Food Sci. Technol. 47, 2273–2281. Urbanyi, G., Horti, K., 1992. Changes of surface color of the fruit and of the anthocyanin content of sour cherries during frozen storage. Acta Alimentaria 21 (3–4), 307–323. Woodward, G., Kroon, P., Cassidy, A., Kay, C., 2009. Anthocyanin stability and recovery: implications for the analysis of clinical and experimental samples. J. Agric. Food Chem. 57, 5271–5278. Wrolstad, R.E., Skrede, G., Lea, P., Enersen, G., 1990. Influence of sugar on anthocyanin pigment stability in frozen strawberries. J. Food Sci. 55 (4), 1064–1072. Wrolstad, R.E., Putman, T.P., Varseveld, G.W., 1970. Color quality of frozen strawberries: effect of anthocyanin, pH, total acidity and ascorbic acid variability. J. Food Sci. 35, 448–452. Zaritzky, N., 2012. Physical-Chemical Principles in Freezing, In Handbook of Frozen Food Processing and Packaging. 2nd Edition CRC Press, Boca Raton, FL, p. 3–37.
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Freezing of Fruits and Impact on Anthocyanins Shyam S. Sablani Biological Systems Engineering Department, Washington State University, Pullman, USA
CHAPTER POINTS • T he 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.
INTRODUCTION Anthocyanins are polyphenolic compounds present in a variety of fruits, responsible for their attractive color. Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium 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,
Processing and Impact on Active Components in Food http://dx.doi.org/10.1016/B978-0-12-404699-3.00018-4
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 important 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 during 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 anthocyanins are synthesized at an increasing rate during maturation, 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 cultivars, 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, respectively), which showed less than half of the late cultivars’ concentration. The anthocyanins are significant because of their nutraceutical benefits, antioxidant, and anticarcinogenic properties. Anthocyanins can also be used as natural food colorants in the food industry. However, anthocyanins are labile in nature, and therefore are susceptible to deterioration during processing and storage (Syamaladevi et al., 2011). Freezing is one of the most common methods of preservation 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 antioxidant capacity and possible health benefits of the fruit.
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18. FREEZING IMPACTS ON ANTHOCYANINS
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
Unclarified juice
1091*
Clarified juice
863*
and
and
1005**
839**
mg/kg FW
7% decrease
mg/kg FW
Strawberry
Turfani et al. (2012)
No loss 24 h at −20°C thawing 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
Date
0* and 2%** decrease
319 days at −23°C 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
Turkyılmaz and Ozkan (2012)
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)
Blackberry Tupy
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
10 months at −18°C
25% decrease
Strawberry
31.0 mg/100 g FW
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
Red raspberry
39.7 mg/100 g FW
15% decrease
Blackberry
194 mg/100 g FW
8% decrease
10 months at −18°C
Blackberry Thornfree Cacanska bestrna
13% decrease
12 months at −18°C 1306 mg/l puree
31% decrease
1201 mg/l puree
#
30% decrease 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
2. FRUIT
Poiana et al. (2010b)
Kopjar et al. (2009)
#
Strawberry
Poiana et al. (2010a)
18% decrease
Tuberoso et al. (2008)
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Introduction
TABLE 18.1 Effect of Frozen Storage Temperature on Stability of Total Anthocyanins—cont’d 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)
Blueberry Tifblue (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 Raspberry Heritage
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 months at −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
Hoko-wase
Deng and Ueda (1993) 57–67% decrease
Toyonaka
73–80% decrease
Nyoho
66–71% decrease
Sour cherry Eardi
541, 1321 mg/1000 g dry matter
Pandi
1101, 1039 mg/1000 g dry matter
Ujfehertoi
793, 1413 mg/1000 g dry matter
Season 1: 12 months at −20°C
150–350% increase depending upon variety 30–45% decrease depending upon the variety
Urbanyi and Horti (1992)
* pH differential method. ** HPLC, #5 weeks of storage.
Therefore, it is vital to understand the stability of anthocyanins during frozen storage. For frozen fruits, the retention of anthocyanins depends on the freezing rate, composition, pH, cultivar, temperature, and the presence/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 subsequent 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 necessary 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 crystallization begins. The heat of solidification is liberated after
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18. FREEZING IMPACTS ON ANTHOCYANINS
FIGURE 18.1 Typical plot of time–temperatures for the freezing of (A) pure water and (B) food.
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 initial 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 anthocyanins 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 preserve important nutrients and aesthetic qualities, maximizing 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 flavylium ion form (Hollands et al., 2008). In most plant tissues, anthocyanins are in the intensely colored flavylium 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 process 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 formation 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 maximumfreeze-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 considerably 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 anthocyanins 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 transition 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 significant 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).
2. FRUIT
Effect of Frozen Storage on Anthocyanins
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 attributed 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 technique 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 molecules 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 texture. 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 frozen, smaller ice crystals form, which reduce cell structure 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 anthocyanin retention in fruits found that the effects of differing freezing technologies is minor; however, different thawing 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 anthocyanin 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 cyanidin 3-glucoside concentrations, showed a more evident degradation of total anthocyanins caused by the
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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 activity 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 differences have also been found between early and late cultivars: 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 anthocyanins 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 anthocyanin 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 carrot 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; Srivastava 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 anthocyanins during frozen storage, including red raspberries (Syamaladevi et al., 2011; De Ancos et al., 2000); serviceberries (Michalczyk and Macura, 2010); strawberries (Oszmianski et al., 2009); sour cherries (Urbanyi and Horti, 1992). This has been attributed to a concentration 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., clarified 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 raspberries were found to increase slightly, at 17% and 5%, respectively, after frozen storage for 360 days. The relative 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 cultivars 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 storage 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 significant 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 buffer solutions (10mMNa/K phosphate buffer in 2% HCl) were spiked with 150 μM delphinidin-3-glucoside, cyanidin-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 temperature 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 cyanidins showed a significant loss following freeze–thaw cycling, while delphinidins remained stable. Pelargonidins showed a linear rate of degradation, with significant 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 anthocyanin content increased linearly during the first year of storage. In studies of shorter storage times in the second 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 maturity. 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 anthocyanins 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 clarified juice compared to unclarified juice. The apparent decrease in anthocyanin content is caused by the interaction 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 physiological processing, the most damaging of which are the reactive oxygen species, such as superoxide, hydrogen 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 antioxidant capacity of fruits. The presence of other phenolic compounds such as flavonoids, phenolic acids, and vitamins C and E also contribute to the total antioxidant 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 different 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
Date Khalas Khunaizi
mmol/100 g FW 2.69 3.48
1 month at −20°C
Pulp: Guava Grape Fig Strawberry Apple Pear
TEAC(μmol/g) 27.0 22.0 5.1 16.0 11.1 7.3
3 months at −15°C
Strawberry Sweet cherry Sour cherry
mM Fe2+/kg FW 24.4 13.5 43.1
10 months at −18°C
Blueberry Red raspberry Blackberry
mM Fe2−/kg FW 58.3 40.2 49.6
10 months at −18°C
Serviceberry
g FW/g DPPH 21.0
10 months at −20°C
Apple Orange
n.a n.a
Blackberry Thornfree Cacanska bestrna
Comments
Reference Allaith et al. (2012)
88% increase 24% decrease Bof et al. (2012) 26% reduction No significant change No significant change 18% reduction No significant change 45% reduction Poiana et al. (2010a) 42% decrease 39% decrease 35% decrease 23% decrease 38% decrease 35% decrease
Poiana et al. (2010b)
25% decrease
Michalczyk and Macura (2010)
10 days at −18°C at −70°C
No significant change
Polinati et al. (2010)
n.a.
12 months at −18°C
8% decrease 8% decrease
Kopjar et al. (2009)
Blackberry
(μmol of TE/g of FW) 97.2
6 months at −20°C
No significant change
Hager et al. (2008a)
Black raspberry
μmol of TE/g of FW) 192
6 months at −18°C
18% increase
Hager et al. (2008b)
Blueberry Tifblue (extract) Powder blue (extract)
(μm/g FW) 26.1 (17.0) 27.3 (17.5)
2 months at −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% depending 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, followed 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 activity 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); blueberries (Srivastava et al., 2007); and red raspberries (Mullen et al., 2002). However, a few studies reported an increasing 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/Spectrophotometric method and high pressure liquid chromatography (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 HPLCDAD-MS
Turkyılmaz and Ozkan (2012)
Date
TA: pH differential method TAC: 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 methods TAA: 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 method TAC: 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)
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References
TABLE 18.3 Analytical Techniques Used to Quantify TA and TAC—cont’d Fruit
TA and TAC Measurement Methods
Reference
Blackberry
TAA: ORAC
Hager et al. (2008a)
Black raspberry
TA: HPLC TAC: ORAC
Hager et al. (2008b)
Strawberry
TA: Spectrophotometric method
Ngo et al. (2007)
Blueberry
TA: Spectrophotometric method TAC: TEAC
Srivastava et al. (2007)
Blueberry
TA: Spectrophotometric method
Lohachoompol et al. (2004)
Red raspberry
TA: Spectrophotometric method TAC: 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)
the HPLC method is used to quantify individual anthocyanins. 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 compounds. Other fruit substances such as pectin, proteins, lipids, and polyphenol compounds are known to interfere with anthocyanin measurements (Leong and Oey, 2012). Turfani et al. (2012) determined the total anthocyanin content of pomegranate juice with both pH differential (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, respectively; 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 differential 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.
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Polinati, R.M., Faller, A.L.K., Fialho, E., 2010. The effect of freezing at −18oC and −70oC with and without ascorbic acid on the stability of antioxidant in extracts of apple and orange fruits. Int. J. Food Sci. Technol. 45, 1814–1820. Rizzolo, A., Nani, R.C., Viscardi, D., Bertolo, G., Torreggiani, D., 2003. Modification of glass transition temperature through carbohydrates addition and anthocyanin and soluble phenol stability of frozen blueberry juices. J. Food Eng. 56 (2–3), 229–231. Rommel, A., Wrolstad, R.E., Heatherbell, D.A., 1992. Blackberry juice and wine: Processing and storage effects on anthocyanin composition, color and appearance. J. Food Sci. 57, 385–391. Sahari, M.A., Mohsen Boostani, F., Zohreh, H.E., 2004. Effect of low temperature on the ascorbic acid content and quality characteristics of frozen strawberry. Food Chem. 86, 357–363. Sousa, M.B., Canet, W., Alvarez, M.D., Tortosa, M.E., 2005. The effect of the pre-treatments and the long and short-term frozen storage on the quality of raspberry (cv. Heritage). Eur. Food Res. Technol. 221, 132–144. Srivastava, A., Akoh, C.C., Yi, W., Fischer, J., Krewer, G., 2007. Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles. J. Agric. Food Chem. 55, 2705–2713. Syamaladevi, R.M., Sablani, S.S., Tang, J., Powers, J., Swanson, B.G., 2011. Stability of anthocyanins in frozen and freeze-dried raspberries during long-term storage—In relation to glass transition. J. Food Sci. 76, E414–E421. Tuberoso, C.I.G., Barra, A., Cabras, P., 2008. Effect of different technological processes on the chemical composition of myrtle (Myrtus communis L.) alcoholic extracts. Eur. Food Res. Technol. 226, 801–808. Torreggiani, D., Forni, E., Guercilena, I., Maestrelli, A., Bertolo, G., Archer, G.P., Kennedy, C.J., Bone, S., Blond, G., Contreras-Lopez, E., Champion, D., 1999. Modification of glass transition temperature through carbohydrates additions: effect upon color and anthocyanin pigment stability in frozen strawberry juices. Food Res. Int. 32, 441–446. Turfani, O., Turkyilmaz, M., Yemis, O., Özkan, M., 2012. Effects of clarification and storage on anthocyanins and color of pomegranate juice concentrates. J. Food Qual. 35, 272–282. Turkyılmaz, M., Ozkan, M., 2012. Kinetics of anthocyanin degradation and polymeric color formation in black carrot juice concentrates during storage. Int. J. Food Sci. Technol. 47, 2273–2281. Urbanyi, G., Horti, K., 1992. Changes of surface color of the fruit and of the anthocyanin content of sour cherries during frozen storage. Acta Alimentaria 21 (3–4), 307–323. Woodward, G., Kroon, P., Cassidy, A., Kay, C., 2009. Anthocyanin stability and recovery: implications for the analysis of clinical and experimental samples. J. Agric. Food Chem. 57, 5271–5278. Wrolstad, R.E., Skrede, G., Lea, P., Enersen, G., 1990. Influence of sugar on anthocyanin pigment stability in frozen strawberries. J. Food Sci. 55 (4), 1064–1072. Wrolstad, R.E., Putman, T.P., Varseveld, G.W., 1970. Color quality of frozen strawberries: effect of anthocyanin, pH, total acidity and ascorbic acid variability. J. Food Sci. 35, 448–452. Zaritzky, N., 2012. Physical-Chemical Principles in Freezing, In Handbook of Frozen Food Processing and Packaging. 2nd Edition CRC Press, Boca Raton, FL, p. 3–37.
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