Original Paper Ellagitannin and Anthocyanin Retention In

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Original paper

Ellagitannin and Anthocyanin Retention in Osmotically Dehydrated Blackberries

Agnieszka Sójka, Elżbieta Karlińska and Robert Klewicki

Institute of Food Technology and Analysis, Lodz University of Technology, Łódź, Poland

Received April 14, 2017 ; Accepted June 28, 2017

The objective of the work was to examine changes in the content of ellagitannins and anthocyanins, which are the main polyphenolic fractions of blackberries, during osmotic dehydration of the fruit in 50 – 65°Bx sucrose solutions. Frozen blackberries were found to be readily dehydrated under mild temperature conditions (30℃), with dry matter content more than doubling after 1 h, and reaching 48% after 3 h in 65°Bx solution. Total ellagitannin retention after 1 h of processing amounted to at least 80%, while after 3 h it ranged from 63.6% to 82.4%. The concentrations of the two main ellagitannins, lambertianin C (a trimer) and sanguiin H-6 (a dimer), revealed similar patterns of variation. The greatest losses, reaching up to 50% after 1 h of dehydration, were recorded for ellagic acid. After the same treatment time, the decrease in anthocyanin concentration was approx. 30 – 40%. The loss of polyphenolic compounds from fruit was attributable to their migration to the syrup. After 3 h of osmotic dehydration, ellagitannin concentration in the syrup amounted to 13.6 – 22.1 mg/100 mL, and that of anthocyanins was 6.7 – 11.2 mg/100 mL.

Keywords: osmotic dehydration, blackberries, ellagitannins, ellagic acid, anthocyanins

Introduction 2012), but data concerning blackberries are limited and mostly Osmotic dehydration is a food processing technology concern dehydration kinetics and sensory properties (Bedoya et al., consisting of partial water removal as a result of immersing plant 2004; Rodriguez-Barona S. et al., 2014; Giraldo G.A.G., 2005), tissue in concentrated saccharide or salt solutions. The method is while very little is known about polyphenolic retention. Osorio et particularly attractive in that it does not involve water phase al. (2007) provided such information only for monomeric change, which makes it possible to largely preserve the initial anthocyanins. properties of the raw material (Garcia-Martinez et al., 2002). Blackberry (Rubus fruticosus) is a shrub characterized by Moreover, any bioactive components of the hypertonic solution (if considerable intraspecific variability, belonging to the family present) may diffuse to the dehydrated material, enhancing its Rosaceae. The world production of blackberries is estimated at health benefits (Nambiar et al., 2016). A drawback of the method approx. 155,000 tons annually, with fruit harvested both from is the loss of valuable components from the dehydrated fruit or commodity plantations and wild-growing plants, especially in vegetables as a result of migration into the hypertonic solution North and South Americas, Europe, Asia, and Oceania. The short (syrup) or due to degradation. Therefore, the dehydration fruit-bearing period limits the possibility of consuming fresh conditions should be carefully selected to minimize the losses. blackberries, which leads to increased processing and a growing High concentrations of health-promoting polyphenols are range of products made from them, including frozen foods, jellies, found in berries, such as strawberries, blueberries, raspberries, jams, juices, as well as fruit stuffing for dairy products and gooseberries, and mulberries. The literature provides examples of confectionery (Kaume et al., 2012). their osmotic dehydration (Moreno et al., 2000; Stojanovic and In addition to their palatability and nutritional qualities, Silva, 2007; Bórquez, 2010; Kucner et al., 2014; Chottamom et al., blackberries also exhibit therapeutic properties, such as anti- 802 A. Sójka et al. inflammatory, antiviral, and antibacterial activity. Due to the high HPLC analysis at a wavelength of 210 nm, as described in the content of polyphenolic compounds, and especially anthocyanins section devoted to polyphenolic determination in fruit) were and ellagitannins, blackberries are also strong antioxidants (Cho et extracted with acetone from raspberry pomace and isolated by al, 2004; Hassimotto et al., 2008). preparative chromatography pursuant to Sójka et al., (2016). The Blackberry anthocyanins are a well-characterized polyphenolic molar masses of lambertianin C and sanguiin H-6 were confirmed group, predominantly consisting of cyanidin glucosides, using a Q Exactive Orbitrap mass detector (Thermo Fisher rutinosides, xylosides, and arabinosides (Kaume et al., 2012; Mertz Scientific, Walthman, MA) according to the procedure of Sójka et et al., 2007). The most abundant blackberry anthocyanins are al. (2016). monoglycosides, and, to a lesser extent, diglycosides, in which Osmotic dehydration Dehydration was conducted in tightly sugar residues are attached to the flavone carbon backbone at sealed plastic containers placed in a water bath shaken at a rate of position C3 (Wu et al., 2006). Anthocyanin content in blackberries 150 cycles/min. Frozen blackberries, stored at ‒18℃, were divided is variable and depends on such factors as cultivar, environmental into 13.5 g lots, which were placed in the containers, to which a conditions, geographic region, ripeness, and, in the case of sucrose solution (50, 57.5 or 65°Bx) was added after approx. processed fruit, technological conditions (Kaume et al., 2012). 20 min. The amount of the solution was four times the weight of Anthocyanin content typically ranges from 70 to 240 mg/100 g the fruit. Dehydration was conducted at 30℃. Samples were taken fresh weight (FW) (Cho et al., 2004; Fan-Chiang and Wrolstad after 1 h, 3 h, and 5 h. Each time, three containers were taken from 2005). the bath. The fruit was removed from the solution using a sieve Ellagitannins, which are hydrolyzable tannins (the other main (separately from each container). While still in the sieve, the group in this class of compounds being condensed tannins) are blackberries were immersed in distilled water three times to esters of 3,4,5,3’,4’,5’-hexahydroxydiphenic acid (HHDP) and a remove any remaining sucrose solution from the surface of the monosaccharide, usually β-D-glucose, or its oligomers. A fruit, which was subsequently dried using filter paper. fundamental property of ellagitannins is that they release HHDP, Dry matter determination First, 3 g samples of fruit ground in which spontaneously lactonizes to ellagic acid. This molecule, liquid nitrogen in a laboratory mill (IKA A11B) were weighed into which is further metabolized in humans and animals, is thought to weighing dishes containing dried sand (105℃, 1 h). The dishes be largely responsible for the health-promoting properties of were then placed in a vacuum dryer set to 70℃ and 60 mbar for ellagitannins. A diet rich in ellagitannins has a beneficial effect on 20 h. Subsequently, the dishes were cooled in a desiccator for the fermentation processes occurring in the gastrointestinal system 30 min and weighed. The measurements were made in triplicate. of humans (Battino et al., 2009; Jarosławska et al., 2011; Williams Determination of polyphenols in fruit Blackberry samples et al., 2004) and animals (Kosmala et al., 2014; Kosmala et al., were extracted three times with 70% acetone solution. Approx. 1 g 2015), and prevents cardiovascular diseases (Mullen et al., 2002) of fruit ground in liquid nitrogen was placed in 7 mL test tubes and cancer (Casto et al., 2002; Gonzalez-Sarrias et al., 2010; with stoppers. Then, the samples were immersed in 4 mL 70% Seeram et al., 2006; Umesalma and Sudhandiran, 2011). The most acetone, shaken on a vortex mixer, placed in an IS-4 ultrasonic abundant blackberry ellagitannins are sanguiin H-6 and cleaner (Intersonic, Olsztyn, Poland) for 15 min, and finally lambertianin C (Hager et al., 2010; Mertz et al., 2007). centrifuged in a MPW-260R laboratory centrifuge (Med The objective of the work was to determine the effect of Instruments, Warszawa, Poland) at 14,000 rpm for 5 min. Clear selected osmotic dehydration parameters (time, hypertonic solution extract was decanted into 10 mL volumetric flasks and the sediment concentration) on anthocyanin and ellagitannin retention in remaining in the test tubes was extracted in the same way two blackberries. times using 3 mL and 2 mL of the extractant. Following the extraction step, the solution in the volumetric flask was made up Materials and Methods with 70% acetone. The samples were diluted with methanol as a Materials Frozen blackberries (Rubus fruticosus) were ratio of 1:1, centrifuged (14,000 rpm), transferred to vials, and purchased from Cajdex PPHU (Łódź, Poland). analyzed chromatographically using a Knauer chromatograph The following reagents were used: sucrose (Diamant, Pfeifer (Berlin, Germany) equipped with a degasser (Manager 500), two and Langen Polska S.A., Polska), acetone (Poch S.A., Gliwice, pumps (P1000), stirrer, autosampler (3950), thermostat, and a PDA Polska), methanol (J.T.Baker, Deventer, Holland), acetonitrile (2800). Separation was conducted in a 5μ C18 110A 250×4.60 mm (LiChrosolv, Darmstadt, Germany), phosphoric acid (purity 85%, Gemini column (Phenomenex, Torrance, CA). The column was J.T. Baker, Deventer, Holland), formic acid (Sigma-Aldrich thermostated at 35℃. The mobile phase was: A – 0.05% (v/v) Chemie, Steinheim, Germany), cyanidin-3-O-glucoside phosphoric acid in water; B – 0.05% phosphoric acid in (Extrasynthèse S.A., Genay, France), ellagic acid (Extrasynthèse acetonitrile/methanol/water 63/20/17 (v/v/v) mixture. The flow rate S.A., Genay, France). Sanguiin H-6 with a purity of 90% and was 1.25 mL/min. The gradient program was: 0 – 5 min 5% B; lambertianin C with a purity of 95% (purity was determined by 5 – 30 min 5 – 28% B; 30 – 40 min 28 – 73% B; 40 – 45 min 73% Polyphenols in Osmo-dehydrated Blackberries 803

B; 45 – 47 min 73 – 5% B; 47 – 56 min 5% B. The injection volume was 20 μL. The detection conditions were 250 nm for ellagitannins and ellagic acid, and 520 nm for anthocyanins. Quantification of lambertianin C and sanguiin H-6 was conducted based on standard curves in the concentration ranges of 0.5 – 300 and 0.5 – 225 mg/L, respectively. Quantification of anthocyanins was done based on a standard curve for cyanidin-3-O-glucoside in the concentration range of 1.5 – 156 mg/L. Data were recorded using specialized software for chromatographic applications ClarityChrom v. 3.0.5.505 (Knauer, Berlin, Germany). Determinations were made in triplicate. Fig. 1. Dry matter content in blackberries osmotically dehydrated Determination of polyphenols in hypertonic solutions at 30℃ in sucrose solutions of various concentrations over different Following fruit removal, sucrose solutions were diluted: 3-fold periods of time. with methanol, and then 2-fold with 0.05% (v/v) phosphoric acid for 50°Bx, 4-fold with methanol and 2-fold with 0.05% (v/v) concentration in the sample prior to dehydration [mg/100 g] phosphoric acid for 57.5°Bx, or 5-fold with methanol and 2-fold CD = sample weight after dehydration [g] × polyphenolic with 0.05% (v/v) phosphoric acid for 65°Bx. The diluted solutions concentration in the sample after dehydration [mg/100 g] were centrifuged for 5 min in a MPW-260R laboratory centrifuge (Med Instruments, Warsaw, Poland) at 14,000 rpm, transferred to Results and Discussion vials, and analyzed chromatographically. Chromatographic Due to the fact that the studied material (frozen berries separation conditions were identical to those used for fruit analysis. consisting of drupes) was sensitive to processing conditions, a mild Determinations were made in triplicate. dehydration temperature (30℃) was used to minimize adverse Identification of ellagitannins and anthocyanins Ellagitannins effects (Moreno et al., 2000). Analysis of variation in dry matter and anthocyanins were identified using a Dionex 3000 Ultimate content (Fig. 1) shows that even at such a low temperature frozen high-performance liquid chromatograph (HPLC) coupled to a blackberries readily undergo osmotic concentration. The average diode array detector (DAD) and a Q Exacutive Orbitrap mass dry matter content in the starting material was 15.7%, while in detector (MS) (Thermo Fisher Scientific, Walthman, MA). dehydrated blackberries it ranged from 33.2% (for 57.5°Bx syrup, Separation was conducted in a Luna C18(2) 100A 250 mm × 1 h) to 51.3% (for 65°Bx syrup, 5 h). Thus, dry matter content 4.60 mm; 5 μm column (Phenomenex, Torrance, CA) with a 4 mm more than doubled after only 1 h. Moreover, the higher the × 3 mm guard column (Phenomenex, Torrance, CA). The column concentration of the sucrose solution, the higher the final dry was thermostated at 35℃. The mobile phase was: A – 1% (v/v) matter content. For instance, 5 h of blackberry dehydration in a formic acid in water, B – acetonitrile/methanol/water 63/20/17 (v/ 50°Bx solution led to a dry matter content of 40.8%, while at v/v) mixture. The flow rate was 1.0 mL/min. The gradient program 57.5°Bx and 65°Bx the dry matter content amounted to 46.5% and was: 0 – 6 min 5% B; 6 – 36 min 5 – 28% B; 36 – 48 min 28 – 73% 51.3%, respectively. The osmotic pressure gradient increases with B; 48 – 54 min 73% B; 54 – 60 min 73 – 5% B; 60 – 70 min 5% B. solution concentration, which translates into a more rapid tissue The injection volume was 20 μL. The detection conditions were dehydration and more intensive dry matter increments (Ahmed et 250 nm for ellagitannins and ellagic acid, and 520 nm for al., 2016). anthocyanins. Data were recorded using specialized software for Figs. 2, 3, and 4 show changes in ellagitannin content in chromatographic applications Xcalibur (Thermo). The MS system dehydrated fruit. The first two charts present data for the main was equipped with a H-ESI probe used in the negative mode. The blackberry ellagitannins: lambertianin C (standard, E3; source parameters were as follows: vaporizer temperature – 500℃, identification data are given in Table 1) and sanguiin H-6 (E4; ion spray voltage – 4 kV, capillary temperature – 400℃; sheath Table 1). Lambertianin C was the predominant ellagitannin in the gas and auxiliary gas flow rates – 75 and 20 units, respectively. fresh material (223.2 mg/100 g). Depending on the process The full MS (m/z: 200 – 2000) and full MS/dd-MS2 (normalized conditions, its content in the dehydrated fruit ranged from 171.2 to collision energy – 20) scan modes were used. 243.9 mg/100 g. Generally speaking, an increase in the Determination of polyphenolic retention in fruit Retention concentration of a chemical compound occurs when its was calculated according to the following formula: concentration increment resulting from loss of water outweighs the compound losses in the course of processing. In the case of R = (CD / C0) x 100% ······Eq. 1 blackberries, lambertianin C levels in most samples were lower where: than in the starting material, decreasing with treatment duration

C0 = sample weight prior to dehydration [g] × polyphenolic (especially up to 3 h). Lambertianin C retention values (understood 804 A. Sójka et al. ., 2008) ., 2008) ., 2010; ., 2010; et al et al ., 2011) ., 2011) ., 2011) ., 2007; ., 2007; ., 2007; ., 2007) ., 2008) ., 2008) et al et al et al et al et al et al et al et al et al et al et al ., 2007; Gasperotti ., 2007; Gasperotti Reference et al et al Hager Hager (Mertz (Mertz (Mertz (Mertz Jardheim Jardheim Jardheim ., 2010; Hager ., 2010; Hager (Gasperotti (Gasperotti (Mertz (Mertz et al et al

Fig. 2. Lambertianin C content in blackberries osmotically dehydrated at 30℃ in sucrose solutions of various concentrations over different periods of time (1.6% of d.e.) (4.9% of d.e.) (3.6% of d.a.) (3.1% of d.a.) (7.3% of d.a.) (65.2% of d.e.) (28.3% of d.e.) (86.0% of d.a.) Cyanidin-3-xyloside (percentage fraction) Structural assignment Cyanidin-3-rutinoside Lambertianin C isomer Lambertianin C isomer Sanguiin H-6 (standard) Lambertianin C (standard) Cyanidin-3-glucoside (standard) Cyanidin-3-(6''-malonylglucoside) , , , , −1 −1 −1 −1 −1 −1 −1 , [935.08] , [935.08] , [935.08] , [897.05] −1 −1 −1 −1 −1 −1 −1 −1 , [301.00] , [301.00] , [301.00] −1 −1 −1 −1 Fig. 3. Sanguiin H-6 content in blackberries osmotically dehydrated , [935.08] , [1235.07] , [1235.07] , [1235.07] , [301.00] , [285.04] , [285.04] , [285.04]

℃ −1

at 30 in sucrose solutions of various concentrations over different −1 −1 −1 −1 −1 −1 −1 periods of time [285.04] MS/MS data , [633.07] , [633.07] , [633.07] −1 −1 −1 [633.07] [447.09] [593.15] [417.08] , [1235.07] , [1567.14] , [1567.15] , [1567.14] −1 −1 −1 −1 [783.01] [783.07] [897.05] LC-MS identification of ellagitannins and anthocyanins in blackberries [1567.14] [1869.15] [1869.16] [1869.14] Table 1.Table 934 447 593 417 533 1401 1401 1401 value MS/MS

−2 −2 −2 −1 −3 −3 −3 −2 −1 −1 −1 −1 MS data [934.07] [934.07] [934.07] [934.07] [447.09] [593.15] [417.08] [533.08] [1401.61] [1401.61] [1401.61] [1869.16] Fig. 4. Total ellagitannin content in blackberries osmotically dehydrated at 30℃ in sucrose solutions of various concentrations over different periods of time [min] 28.67 29.57 30.42 31.98 24.88 27.04 30.06 32.55 R t

as the ratio of the amount of the compound remaining in a given fruit sample after treatment to the amount of that compound present E1 E2 E3 E4 A1 A2 A3 A4 in the sample prior to treatment) indicate losses of 6.4% to almost (standard) - identification based on the standard compound; d.e. determined ellagitannins; d.a. anthocyanins Compound Ellagitannins Ellagitannins Ellagitannins Ellagitannins 35% (Table 2). The lowest decline (less than 20%) occurred after Anthocyanins Anthocyanins Anthocyanins Anthocyanins 1 h. No relationship was observed between solution concentration and the retention of this compound. The content of sanguiin H-6 in decreased sanguiin H-6 concentration by less 20%. Interestingly, the starting material amounted to 96.7 mg/100 g, as compared to an increase in treatment time from 3 h to 5 h did not cause a further 71 – 107 mg/100 g in the dehydrated fruit. Retention results were drop in retention, which amounted to 67.8 – 73.2% after 5 h. similar to those for lambertianin C. One hour of dehydration Similar results were obtained for total ellagitannins (including a Polyphenols in Osmo-dehydrated Blackberries 805

Table 2. Polyphenols retention in the fruit dehydrated in sucrose solutions of different concentrations

Retention [%] Compound Dehydration time [h] 50°Bx 57.5°Bx 65°Bx Lambertianin C 1 79.9 ± 10.2 92.5 ± 12.3 77.3 ± 3.9 3 62.6 ± 9.5 81.7 ± 5.2 72.6 ± 11.4 5 63.1 ± 6.7 71.0 ± 24.7 73.3 ± 6.2

Sanguiin H-6 1 80.0 ± 9.9 93.6 ± 7.9 80.4 ± 3.6 3 65.1 ± 6.1 82.4 ± 4.9 66.4 ± 2.5 5 67.8 ± 3.7 73.2 ± 21.4 68.4 ± 6.4

Total ellagitannins 1 79.7 ± 11.2 93.8 ± 11.3 78.1 ± 2.0 3 63.6 ± 8.5 82.4 ± 4.3 71.0 ± 9.1 5 64.2 ± 6.8 72.2 ± 24.0 72.0 ± 6.3

Ellagic acid 1 57.1 ± 20.4 72.7 ± 17.1 50.7 ± 4.9 3 70.5 ± 29.7 67.3 ± 7.4 44.2 ± 9.3 5 54.1 ± 14.9 64.2 ± 9.3 48.2 ± 3.2

Anthocyanins 1 61.6 ± 3.0 60.8 ± 8.7 68.6 ± 6.5 3 56.1 ± 8.6 50.5 ± 4.4 55.9 ± 13.8 5 51.7 ± 6.7 46.8 ± 4.7 53.6 ± 6.7

blackberry fruit is consistent with the data reported by Macierzyński et al. (2014) for 6 blackberry cultivars (32.1 – 163.1 mg/100 g FW for lambertianin C and 21.1 – 162.2 mg/100 g FW for sanguiin H-6). Gasperotti et al. (2010) found lower content of both ellagitannins in 5 blackberry cultivars from three harvest seasons: 306.3 – 665.2 mg/kg FW for lambertianin C and 188.9 – 418.3 mg/kg FW for sanguiin H-6. Also the free ellagic acid levels determined by those authors were lower and amounted to 45.6 – 84.6 mg/kg FW. Those results may be attributable to the application of mild extraction conditions (the fruits were extracted Fig. 5. Ellagic acid content in blackberries osmotically dehydrated twice for 1 min using a blender). at 30℃ in sucrose solutions of various concentrations over different The available literature does not provide any information on periods of time changes in ellagitannin content in osmotically dehydrated fruit. The available data concern other types of tannins and fruits other than small fraction of lambertianin C isomers: E1 and E2; Table 1) as blackberries. For instance, Kucner et al. (2013b) studied the can be seen from Fig. 4. Given that after 3 h further dehydration is content of procyanidins (condensed tannins) in blueberries unproductive due to very small growth in dry matter content, osmotically dehydrated (65°Bx sucrose, 40℃, 120 min) with a approx. 1/3 of ellagitannin content is lost during the treatment. hypertonic solution recycled 15 times; the reported mean retention Changes in the content of ellagic acid, a product of ellagitannin of those compounds was 77%. The retention of condensed tannins hydrolysis, were also analyzed. The concentration of this in dehydrated bananas was investigated by Almeida et al. (2014) compound in blackberries (Fig. 5) was much lower (19 mg/100 g) using 45 – 65% sucrose solutions at 30℃; the obtained results were than that of lambertianin C and sanguiin H-6. Osmotic dehydration 98% after 1 h and 79 – 94% after 3 h. Moreno et al. (2016) further reduced ellagic acid levels, especially in the first hour of reported data on catechins, which are components of condensed processing (11.1 – 12.7 mg/100 g). Ellagic acid retention after 1 h, tannins. Following dehydration in 65°Bx sucrose solution at 30℃ 3 h, and 5 h amounted to 50.7 – 72.7%, 44.1 – 70.4%, and 48.1 – for 4 h, catechin content in blueberry dry matter declined by more 64.2%, respectively. These results are lower than those for than 52%. An even greater decrease (over 71%) was found for lambertianin C and sanguiin H-6, which may be explained by epicatechin. On the other hand, lower losses were reported by facilitated migration of ellagic acid molecules, which are much Devic et al. (2010), who dehydrated apples (Gala and Marie smaller than those of the parent polymers. Menard) at a higher temperature (45℃), but for a shorter time (3 h), The content of lambertianin C and sanguiin H-6 in the studied using 60°Bx sucrose solution. In this case, an approx. 27% 806 A. Sójka et al. decrease in catechins and a 15% reduction in procyanidins (polymerized catechins) was found, which provides a good illustration of the relationship between the molecular mass and retention of a substance. Polymerized compounds diffuse into the hypertonic solution more slowly and, as noted by Devic et al. (2010), may more readily interact with cell wall lipopolysaccharides. More information is available on total or non-tannin polyphenolic compounds. In the study of Araya-Farias et al. (2014), the decrease in polyphenolics in osmotically dehydrated sea buckthorns was approx. 12% (60°Bx sucrose solution, 40℃, 6 h). Fig. 6. Anthocyanin content in blackberries osmotically dehydrated Kucner et al. (2013b) found that total polyphenolic retention in at 30℃ in sucrose solutions of various concentrations over different blueberries depended on the number of dehydration cycles and periods of time ranged from 2.4% to 29.4%. Hypertonic solution (65°Bx, 45℃, 4 h) was also recycled in experiments by Germer et al. (2016), who decline in anthocyanin content (by over 52%) in the dry mass of dehydrated guava. Over 15 treatment cycles, total polyphenolic mulberries was recorded in the work of Chottamom et al. (2012). retention varied from 87.5% to 98.5%. Ścibisz and Mitek (2006) A 10-day-long raspberry dehydration treatment conducted by Sette reported an 11% decline in polyphenolic content in blueberries (dry et al. (2015) had a very adverse effect on anthocyanins in the dry weight) after 2 h of dehydration at 20℃ and a 19% decrease after mass of fruit, with only 5% of their initial content retained (as 6 h. In turn, after dehydrating mulberries in 60% sucrose solution calculated on the basis of the reported data). for 6 h at 35℃, Chottamom et al. (2012) observed an approx. 58% The main cause of reduced polyphenolic content in osmotically reduction in polyphenolic content in dry matter. In the study of dehydrated fruit is the migration into the hypertonic solution Almeida et al. (2014), bananas dehydrated at 30℃ retained 89.8 – (Kucner et al., 2013a). Fig. 7 shows the concentration of 97% of polyphenols after 1 h and 77.7 – 98% after 3 h. Finally, ellagitannins (the main compounds and total) in hypertonic Sette et al. (2015) conducted raspberry dehydration in 61% sucrose solutions. Higher concentrations were recorded for lambertianin C, solution at ambient temperature for an extremely long period (10 as after 3 h of dehydration its levels in the solutions ranged from days). Calculations based on the data given in their paper show that 8.84 to 14.9 mg/100 mL, as compared to 3.97 – 6.20 mg/100 mL for polyphenolic content in dry matter decreased to approx. 18% of the sanguiin H-6. The sum of all ellagitannins determined in the syrup initial levels. ranged from 13.6 to 22.1 mg/100 g. Some small amounts of free As far as the other major group of polyphenols is concerned, ellagic acid were also recorded (1.40 – 1.90 mg/100 mL after 3 h, the most abundant anthocyanin was cyanidin-3-O-glucoside, which see Fig. 8). Finally, the content of anthocyanins ranged from 6.65 accounted for 86% of total anthocyanins determined (45.8 mg/100 g to 11.2 mg/100 mL (Fig. 9). Generally, within the studied range, of fruit). The other major compounds in this class included the concentration of the hypertonic solutions did not have a cyanidin-3-(6”-malonylglucoside) (3.94 mg/100 g), cyanidin-3-O- significant effect on their polyphenolic content. In most cases, the rutinoside (1.9 mg/100 g), and cyanidin-3-xyloside (1.64 mg/100 g). lowest content of the substances migrating from the fruit was found Fig. 6 shows changes in total anthocyanin content in the dehydrated for the lowest syrup concentration (50°Bx). However, it should be fruit, which substantially decreased after 1 h of dehydration (from noted that the final concentration of the compounds migrating from 53.3 to 38.3 – 42.0 mg/100 g). Further dehydration had a less the dehydrated material to the hypertonic solution depends not only dramatic effect on anthocyanin concentration (31.2 – 38.2 mg/100 g on the amount of the transported substance, but also on the volume after 3 h and 28.4 – 31.4 mg/100 g after another 2 h), which also of water transferred from the fruit to the solution, as well as on the translated into retention levels (60.8 – 68.6%, 50.5 – 55.9%, and amount of hypertonic solution absorbed by the fruit. This is a 46.8 – 53.6% after 1 h, 3 h, and 5 h, respectively). complicated system which may lead to major fluctuations in the In the study of Ścibisz and Mitek (2006), anthocyanin losses various substances. following 6 h of blueberry dehydration were lower (35%). In turn, The fact that migration (not degradation) played a major role in Moreno et al. (2016) found that the total content of the three most decreasing polyphenol content is confirmed by data presented in abundant classes of anthocyanins (delphinidins, cyanidins, and Table 3, which shows the balance of individual polyphenols in fruit malvidins) in the dry matter of blueberries declined by 48% and hypertonic solution before and after dehydration (in a 65°Bx following treatment in 65°Bx sucrose solution at 30℃ for 4 h. solution as an example). In the case of lambertianin C, 16.6, 23.8 Osorio et al. (2007) reported a 86% decrease in anthocyanins and 21.4% of the compound present in blackberries before (relative to sample weight) in Andes berries following dehydration dehydration migrated to syrups after 1, 3, and 5 h of processing, in 70% sucrose solution at 30℃ for 24 h. Moreover, a considerable respectively. In the case of sanguiin H-6, the value amounted to Polyphenols in Osmo-dehydrated Blackberries 807

Fig. 8. Ellagic acid content in hypertonic sucrose solutions of various concentrations used for blackberry dehydration over different periods of time

Fig. 9. Anthocyanin content in hypertonic sucrose solutions of various concentrations used for blackberry dehydration over different periods of time

concentration in the syrup amounting to approx. 1.3 mg/100 g. In their experiments, Kucner et al. (2013b) obtained much higher polyphenolic levels in 65°Bx syrup reused in 15 cycles of blueberry dehydration at 40℃. In that case, polyphenolic concentration Fig. 7. Lambertianin C (top), sanguiin (middle), and total ellagitannin (bottom) content in hypertonic sucrose solutions of various ranged from 4.75 to 34.3 mg/100 g of syrup. Following 10 h concentrations used for blackberry dehydration over different periods dehydration of raspberries, Sette et al. (2015) reported 45 mg of of time polyphenols (10% of which were anthocyanins) per 100 g of syrup. Similar anthocyanin content in sucrose solution (6.3 g/100 mL) was 16.3, 23,3 and 20.8%. 30.7, 36.0 and 35.9% of ellagic acid was found following dehydration of Andes berries (Osario et al., 2007). transferred to the hypertonic solutions. For anthocyanins, it was Polyphenolic migration into the hypertonic solution may be 51.6, 85.7 and 76.0%. A comparison of polyphenols in the whole considered from two points of view. On the one hand, limited system (fruit + syrup) before and after dehydration indicates that migration is desirable as a greater proportion of valuable blackberry ellagitannins were characterized by a relatively high polyphenols remains in the fruit, enabling it to retain more of its stability. The possible loss caused by degradation was 11% at the antioxidant properties. On the other hand, when polyphenolic most (sanguiin H-6, after 5 h). The highest loss was noted in the migration is significant, the hypertonic solution may be used as a case of ellagic acid (19.9%, after 3 h). valuable intermediate in the production of foodstuffs fortified with The available literature data on the migration of the substances polyphenols from the dehydrated fruit (Chwastek et al., 2016). found in fruits to the hypertonic solution mostly pertain to anthocyanins or total polyphenols. Considerable resistance to Conclusions anthocyanin loss was exhibited by blueberries dehydrated at 50℃ Frozen blackberries are readily dehydrated at 30℃. Dry matter in 70°Bx sucrose solution reused multiple times in a study by content can be more than doubled after 1 h of osmotic dehydration Grabowski et al. (2007), with the maximum anthocyanin conducted in 50 – 65°Bx sucrose solutions. The process should not 808 A. Sójka et al.

Table 3. Amount of individual polyphenols in the whole amount of fruit subjected to processing (before dehydration), in the whole amount of processed fruit (after dehydration), and in the whole amount of sucrose solution after dehydration (initial concentration 65°Bx)

Dehydration Before dehydration After dehydration Compound time [h] In fruit [mg] In fruit [mg] In solution [mg] Sum (fruit + solution) [mg] Lambertianin C 1 30.55 ± 0.17 23.62 ± 1.12 5.07 ± 0.66 28.69 3 30.85 ± 0.62 22.33 ± 3.03 7.35 ± 2.60 29.69 5 30.67 ± 0.39 22.48 ± 1.81 6.56 ± 2.45 29.04

Sanguiin H-6 1 13.24 ± 0.08 10.64 ± 0.53 2.21 ± 0.09 12.85 3 13.36 ± 0.27 8.87 ± 0.18 3.16 ± 0.95 12.03 5 13.29 ± 0.17 9.09 ± 0.82 2.74 ± 1.05 11.83 Total ellagitannins 1 46.82 ± 0.27 36.55 ± 0.85 7.65 ± 0.71 44.21 3 47.28 ± 0.95 33.50 ± 3.57 11.01 ± 3.67 44.51 5 47.00 ± 0.60 33.84 ± 2.85 9.76 ± 3.63 43.61 Ellagic acid 1 2.60 ± 0.01 1.32 ± 0.13 0.80 ± 0.09 2.12 3 2.63 ± 0.05 1.16 ± 0.24 0.95 ± 0.33 2.10 5 2.61 ± 0.03 1.26 ± 0.07 0.94 ± 0.34 2.19

Anthocyanins 1 7.29 ± 0.04 3.89 ± 0.84 3.76 ± 0.81 7.66 3 7.37 ± 0.15 3.47 ± 0.21 6.31 ± 1.43 9.78 5 7.32 ± 0.09 3.44 ± 0.26 5.57 ± 2.26 9.01 be carried out for more than three hours as dry matter gain after Araya-Farias, M., Macaigne, O., and Ratti, C. (2014). On the Development that time is minimal. In the presented experiments, the dry matter of Osmotically Dehydrated Seabuckthorn Fruits: Pretreatments, Osmotic content of blackberries was significantly affected by syrup Dehydration, Postdrying Techniques, and Nutritional Quality. Drying concentration (after 3 h, approx. 39% and 48% dry matter was Technol., 32, 813-819. obtained using 50°Bx and 65°Bx solutions, respectively). The Battino, M., Beekwilder, J., Denoyes-Rothan, B., Laimer, M., McDougall, retention of ellagitannins depended on dehydration time. The two G.J., and Mezzetti, B. (2009). Bioactive compounds in berries relevant to main compounds in this class, lambertianin C and sanguiin H-6, human health. Nutr. Rev., 67, 145-150. behaved similarly in this respect as approx. 80 – 90% of them Bórquez, R.M., Canales, E.R., and Redon, J.P. (2010). Osmotic dehydration remained the fruit after 1 h and approx. 60 – 80% after 3 h of raspberries with vacuum pretreatment followed by microwave- (depending on syrup concentration). A more dramatic decline was vacuum drying. J. Food Eng., 99, 121-127. noted for ellagic acid (up to 50% after 1 h). Furthermore, as much Casto, B.C., Kresty, L.A., Kraly, C.L., Pearl, D.K., Knobloch, T.J., Schut, as 30 – 40% of anthocyanins were lost after 1 h. The reduced H.A., Stoner, D.K., Mallery, S.R., and Weghorst, C.M. (2002). content of polyphenols in blackberries was caused by the migration Chemoprevention or oral cancer by black raspberries. Anticancer Res., of the compounds into the hypertonic solution, which contained 22, 4005-4015. 13.6 – 22.1 mg/100 mL ellagitannins and 6.7 – 11.2 mg/100 mL Cho, M. J., Howard, L. R., Prior, R. L., and Clark, J. R. (2004) Flavonoid anthocyanins after 3 h of osmotic processing. glycosides and antioxidant capacity of various blackberry and red grape genotypes determined by high-performance liquid chromatography/ mass Financing spectrometry. J. Sci. Food Agric., 84, 1771-1782. The study was financed by the Institute of Food Technology Chottamom, P., Kongmanee, R., Manklang, C., and Soponronnarit, S. and Analysis, Łódź University of Technology (2012). Effect of Osmotic Treatment on Drying Kinetics and Antioxidant Properties of Dried Mulberry. Drying Technol., 30, 80-87. References Chwastek, A., Klewicka, E., Klewicki, R., and Sójka, M. (2016). Lactic Ahmed, I., Quazi, I.M., and Jamal, S. (2016). Developments in osmotic acid fermentation of reed beet juice supplemented with waste highbush dehydration technique for the preservation of fruits and vegetables. blueberry-sucrose osmotic syrup as a method of probiotic beverage Innovative Food Sci. Emerging Technol., 34, 29-43. production. J. Food Process Preserv., 40, 780-789. Almeida, J.A.R., Mussi, L.P., Oliveira, D.B., and Pereira, N.R. (2015). Devic, E., Guyot, S., Daudin, J.-D., and Bonazzi, C. (2010). Effect of Effect of temperature and sucrose concentration on the retention of Temperature and Cultivar on Polyphenol Retention and Mass Transfer polyphenol compounds and antioxidant activity of osmotically during Osmotic Dehydration of Apples. J. Agric. Food Chem., 58, 606- dehydrated bananas. J. Food Process. Preserv., 39, 1061-1069. 614. Polyphenols in Osmo-dehydrated Blackberries 809

Fan-Chiang, H. J. and Wrolstad, R. E. (2005). Anthocyanin pigment and health benefits.J. Agric. Food Chem., 60, 5716-5727. composition of blackberries. J. Food Sci., 70, 198-202. Kosmala, M., Zduńczyk, Z., Juskiewicz, J., Jurgonski, A., Karlińska, E., García-Martínez, E., Martínez-Monzó, J., Camacho, M.M., and Martínez- Macierzyński, J., Jańczak, R., and Rój, E. (2015). Chemical composition Navarrete, N. (2002). Characterization of reused osmotic solution as of defatted strawberry and raspberry seeds and the effect of these dietary ingridient in new product formulation. Food Res. Int., 35, 307-313. ingredients on polyphenol metabolites, intestinal function, and selected Gasperotti, M., Masuero, D., Vrhovsek, U., Guella, G., and Mattivi, F. serum parameters in rats. J. Agric. Food Chem., 63, 2989-2996. (2010). Profiling and accurate quantification ai Rubus ellagitannins and Kosmala, M., Zduńczyk, Z., Karlińska, E., and Juśkiewicz, J. (2014). The ellagic acid conjugates using direct UPLC-Q-TOF HDMS and HPLC- effects of strawberry, black currant, and chokeberry extracts in a grain DAD analysis. J. Agric. Food Chem., 58, 4602-4616. dietary fiber matrix on intestinal fermentation in rats. Food Res. Int., 64, Germer, S.P.M., Morgano, M.A., da Silva, M.G., de Arruda, S.N.F., and de 752-761. Cássia, G.S.E. (2016). Effect of reconditioning and reuse of sucrose Kucner, A., Klewicki, R., Sójka, M., and Klewicka, E. (2014). Osmotic syrup in quality properties and retention of nutrients in osmotic concentration of gooseberry fruits – the influence of temperature, time dehydration of guava. Drying Technol., 34, 997-1008. and pretreatment methods on mass transfer and total polyphenol and Giraldo Bedoya, D.P., Arango Velez, L.M., and Marquez Cardozo, C.J. organic acid content. Food Technol. Biotechnol., 52, 411-419. (2004). Osmodehydration of blackberry (Rubus glaucusBenth) with three Kucner, A., Klewicki, R., and Sójka, M. (2013). The influence of selected sweetening. Rev. Fac. Nal. Agr., Medelin, 57, 2257-2274. osmotic dehydration and pretreatment parameters on dry matter and Giraldo, G., Duque, A., and Mejia, C. (2005). Pretreatment in the polyphenol content in highbush blueberry (Vaccinium corymbosum L) conservation of blackberry (Rubus glaucus) and uchuva (Physalis fruits. Food Bioprocess Technol., 6, 2031-2047. peruviana L.) with osmotic dehydration. Vitae, 12, 15-22. Kucner, A., Papiewska, A., Sójka, M., and Klewicki, R. (2013). Chemical Gonzalez-Sarrias, A., Gimez-Bastida, J.A., Garcia-Conesa, M.T., Gomez- and microbiological changes in blueberries and in hypertonic solution Sanchez, M.B., Garcia-Talavera, N.V., Gil-Izquierdo, A., Sanchez- during osmotic dehydration employing reused concentrate. J. Food Alvarez, C., Fontana-Compiano, L.O., Morga-Egea, J.P., Pastor- Process Eng., 36, 608-618. Quirante, F.A., Martinez-Diaz, F., and Tomas-Barberan, F.A. Espin, J.C. Macieżyński, J., Buczek, M., Zaweracz ,W., and Król, B. (2014). (2010). Occurrence of urolithins, gut microbiota ellagic acid metabolites Polyphenolic composition of Rubus fruticosus blackberry fruits. Food and proliferation markers expression response in the human prostate Sci. Technol. Qual., 5, 183-194. gland upon consumption of walnuts and pomegranate juice. Mol. Nutr. Mertz, C., Cheynier, V., Guanat, Z., and Brat, P. (2007). Analysis of Food Res., 54, 311-322. phenolic compounds in two blackberry species (Rubus glaucus and Grabowski, S., Marcotte, M., Quan, D., Tahérian, A.R., Zareifard, M.R., Rubus adenotrichus) by high-performance liquid chromatography with Poirier, M., and Kudra, T. (2007). Kinetics and Quality Aspects of diode array detection and electrospray ion trap mass spectrometry. J Canadian Blueberries and Cranberries Dried by Osmo-Convective Agric. Food Chem., 55, 8616-8624. Method. Drying Technol., 25, 367-374. Moreno, J., Chiralt, A., Escriche, I., and Serra, J.A. (2000). Effect of Hager, T. J., Howard, L. R., Liyanage, R., Lay, J. O., and Prior, R. L. blanching/osmotic dehydration combined methods on quality and (2008). Ellagitannin composition of blackberry as determined by HPLC- stability of minimally processed strawberries. Food Res. Int., 33, 609- ESIMS and MALDI-TOF-MS. J. Agric. Food Chem., 56, 661-669. 616. Hager, T.F., Howard, L.R., and Prior, R.L. (2010). Processing and storage Moreno, J., Gonzales, M., Zúñiga, P., Petzold, G., Mella, K., and Muñoz, O. effects on the ellagitannin composition of processed blackberry products. (2016). Ohmic heating and pulsed vacuum effect on dehydration J. Agric. Food Chem., 58, 11749-11754. processes and polyphenol component retention of osmodehydrated Hassimotto, N. M. A., Silva Pinto, M., and Lajolo, F. M. (2008). blueberries (cv. Tifblue). Innovative Food Sci. Emerging Technol., 36, Antioxidant status in humans after consumption of blackberry (Rubus 112-119. fruticosus L.) juices with and without defatted milk. J. Agric. Food Mullen, W., McGinn, J., Lean, M.E.J., MacLean, M.R., Gardner, P., Duthie Chem., 56, 11727-11733. G.G., Yokota, T., and Crozier, A. (2002). ellagitannins, flavonoids, and Jarosławska, J., Juśkiewicz, J., Wróblewska, M., Jurgoński, A., Król, B., other phenolics in red raspberries and their contribution to antioxidant and Zduńczyk, Z. (2011). Polyphenol-rich strawberry pomace reduces capacity and vasorelaxation properties. J. Agric. Food Chem., 50, 5191- serum and liver lipids and alters gastrointestinal metabolite formation in 5196. fructose-fed rats. J. Nutr., 141, 1777-1783. Nambiar, S.S., Basu, A., Shetty, N.P., Rastogi, N.K., and Prapulla, S.G. Jordheim, M., Enerstvedt, K.H., and Andersen, O.M. (2011). Identification (2016). Infusion of fructooligosaccharide in Indian gooseberry (Emblica of Cyanidin 3-O-β-(6’’-(3-Hydroxy3-methylglutaroyl)glucoside) and officinalis) fruit using osmotic treatment and its effect on the antioxidant other anthocyanins from wild and cultivated blackberries. J. Agric. Food activity of the fruit. J. Food Eng., 190, 139-146. Chem., 59, 7436-7400. Osorio, C., Franco, M.S., Castaño, M.P., González-Miret, M.L., Heredia, Kaume, L., Howard, L. R., and Devareddy, L. (2012). The blackberry fruit: a F.J., and Morales, A.L. (2007) Colour and flavour changes during review on its composition and chemistry, metabolism and bioavailability, osmotic dehydration of fruits. Innovative Food Sci. Emerging Technol., 8, 810 A. Sójka et al.

353-359. ols, and Flavonols during the Processing of Red Raspberries (Rubus Rodriguez-Barona, S., Granada-Orozco, J., and Cruz-Rios, D. (2014). Ideaus L.) to Juice. J. Agric. Food Chem., 64, 5549-5563. Comparison of sucrose, inulin and fructooligosaccharides as osmotic Stojanovic, J. and Silva, J.L. (2007). Influence of osmotic concentration, agents in the Andean blackberry (Rubus glaucus Benth). Acta Hortic., continuous high frequency ultrasound and dehydration on antioxidants, 1016, 47-51. colour and chemical properties of rabbiteye blueberries. Food Chem., Ścibisz, I. and Mitek, M. (2006). Antioxidant activity and phenolics 101, 898-906. compound capacity in dried highbush blueberries (Vaccinium Umesalma, S. and Sudhandiran, G. (2011). Ellagic acid prevents rat colon corymbosum L). Food Sci. Technol. Qual., 4, 68-76. carcinogenesis induced by 1,2-dimethyl hydrazine through inhibition of Seeram, N.P., Adams, L.S., Zhang, Y., Lee, R., Sand, D., Schuller, H.S., AKT-phosphoinositide-3 kinase pathway. Eur. J. Pharmacol., 660, 249- and Heber, D. (2006). Blackberry, black raspberry, blueberry, cranberry, 258. red raspberry, and strawberry extracts inhibit growth and stimulate Williams, R.J., Spencer, J.P.E., and Rice-Evans, C. (2004). Flavonoids: apoptosis of human cancer cells in vitro. J. Agric. Food Chem., 54, 9329- antioxidants or signalling molecules? Free Radical Biol. Med., 36, 838- 9339. 849. Sette Paula, A., Franceschinis Lorena, E., and Schebor Carolina, Salvatori Wu, X., Beecher, G. R., Holden, J. M. Haytowitz, D. B., Gebhardt, S. E., Daniela. (2015). Osmotic Dehydrated Raspberries: Changes in Physical and Prior, R. L. (2006). Concentrations of anthocyanins in common Aspects and Bioactive Compounds. Drying Technol., 33, 659-670. foods in the United States and estimation of normal consumption. J. Sójka, M., Macierzyński, J., Zaweracz, W., and Buczek, M. (2016). Agric. Food Chem., 54, 4069-4075. Transfer and Mass Balance of Ellagitannins, Anthocyanins, Flavan-3-