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Impact of various factors on the composition and stability of black currant anthocyanins
Food Research International 38 (2005) 867–871 www.elsevier.com/locate/foodres
Impact of various factors on the composition and stability of black currant anthocyanins M. Rubinskiene a, P. Viskelis
a,*
, I. Jasutiene b, R. Viskeliene b, C. Bobinas
a
a
b
Lithuanian Institute of Horticulture, Kauno 30, LT-4335 Babtai, Lithuania Food Institute of Kaunas University of Technology, Taikos 92, LT-51180 Kaunas, Lithuania Received 27 May 2004; accepted 27 February 2005
Abstract Nine black currant varieties cultivated in Lithuania were studied. The highest amount of ascorbic acid was established in fresh berries from cv Minaj Smyriov and Kupoliniai: these varieties contained 220.5 and 186.7 mg 100 g 1 of ascorbic acid in berries. The highest amount of anthocyanins was found in cake produced from berries cv Kupoliniai and Kriviai: 14.65 and 15.42 mg g 1, respectively. The major pigment determined in Kupoliniai variety was delphinidin-3-rutinoside; in Ben Lomond, Minaj Smyriov, Kriviai and Gagatai cultivars, cyanidin-3-rutinoside. The composition of the identified pigments was the following: cyanidin-3-rutinoside (33– 38%), delphinidin-3-rutinoside (27–34%), cyanidin-3-glucoside (8–10%) and delphinidin-3-glucoside (8–10%). Impact of storage, thermal treatment and addition of sweeteners were studied. Cyanidin-3-rutinoside was the most stable to the effect of thermal treatment at 95 C, while cyanidin and delphinidin rutinosides were the most stable during storage for 12 months at 8 C. Fructose has a greater effect on anthocyanin degradation compared with glucose and aspartame. 2005 Elsevier Ltd. All rights reserved. Keywords: Anthocyanins; Black currant; Colorant; Variety
1. Introduction Chemical composition of black currant berries depends on the variety and agroclimatic conditions of their cultivation. The composition and properties of black currant berry pigments grown in Lithuania have been little investigated. The information on such pigments is important for the production of food colorants, berry processing and for expanding our scientific knowledge on this topic. Since natural pigments are susceptible to the impact of such technological factors as pH, temperature, oxygen and enzymes (Jackman, Yada, Tung, & Speers, 1987; Wrolstad, 2000), it is important to study the effect *
Corresponding author. Tel.: +370 37 555 439; fax: +370 37 555 176. E-mail address: [email protected] (P. Viskelis). 0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.02.027
of these factors on the properties and structural changes of colorants. These changes may be monitored by measuring pigment concentration and colour analysis. Regression analyses did not show significant variation in the loss of total pigment concentration, however the changes of individual major pigments were significant (Taylor, 1989). This study also reported that cyanidin was the major pigment in the all examined British cultivars, while delphinidin was dominating pigment in Scandinavian cultivars. Skrede, Wrolstad, Lea, and Enersen (1992) studied the colour and pigment stability of strawberry and black currant syrups, Iversen, Kidmose, and Poulsen (1996) measured the breakdown of total anthocyanins in black currant juice during storage, Goiffon, Mouly, and Gaydou (1999) studied stability of anthocyanins in black currant juice and concentrates. The degradation of anthocyanins and ascorbic acid in black currant nectar was studied during processing and
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M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
storage (Iversen, 1999). It was found that about 50% of the original content of monomeric anthocyanins remained after 6 months storage at 20 C. The stabilities of delphinidins and cyanidins during storage were similar. Torskangerpoll and Andersen (2005) focused study on the impact of anthocyanin structures such as 5-glucosidic substitution and aromatic acylation on anthocyanin, colour and stability at various pH values. Mikkelsen and Poll (2002) studied decomposition and transformation of anthocyanins during black currant juice processing. It was found that between 25% and 30% of the anthocyanins were lost during juice processing; the heating treatments seem to be the most destructive process steps. The aim of such studies is to predict the impact of factors mentioned above on the quality of final product and consequently to avoid negative effects by applying the achievements in modern theory of molecular chemistry and physics. The objective of this study was to investigate chemical and technological properties of black currant berries from different plant varieties, to evaluate the amount of anthocyanins and their composition, and to obtain some new scientific data on suitability of black currant anthocyanins for production of red food colorants.
2. Materials and methods 2.1. Berries Black currant berries were grown in the experimental field of the Lithuanian Institute of Horticulture (LIH). The berries from the following varieties were used: registered variety Minaj Smyriov, European commercial varieties Ben Lomond, Ben Alder; and the varieties developed at the LIH Kriviai, Kupoliniai, Gagatai, Joniniai, Almiai andVakariai. Berries were picked at ripeness phase, frozen and stored (at 28 C) in polyethylene bags. 2.2. Chemical composition Chemical composition of berries was determined by standard methods. The following analyses were performed each year: the content of soluble solids by refractometer, titratable acidity expressed as citric acid by titration with 0.1 M NaOH solution, ascorbic acid content by titration with 2,6-dichlorophenolindophenol sodium salt solution, inverse sugar and sucrose by Bertrand method.
95% (v/v) food grade ethanol acidified with 0.1 M HCl. The cake was ground with quartz sand and the extraction was continued with 20 ml portions of solvent until the sample became colourless. The extract was diluted with acidified ethanol; the absorption was measured on a spectrophotometer Genesys-5 (Thermo Spectronic, Rochester, USA) at 544 nm. The concentration of anthocyanins was determined from the calibration curve, which was constructed by measuring the absorption of cyanidin-3-rutinoside (MW 595.2, e = 28,800) reference solutions. Purified cyd-3-rut was donated by the Danish Institute of Agricultural Sciences (Department of Fruit, Vegetable and Food Science). The concentration of anthocyanins was calculated and expressed in milligrams of cyd-3-rut in gram of cake. The concentration of the monomeric anthocyanins in juice was determined by the pH differential method as described by Giusti and Wrolstad (2001). Absorbance was measured on a spectrophotometer Genesys-5 (Thermo Spectronic, Rochester, USA) at 510 nm and at 700 nm in buffer at pH 1.0 and pH 4.5. Results were expressed as milligram per litre. 2.4. HPLC/UV analysis of anthocyanins Anthocyanin profiles of black currant ethanol extracts were characterized by HPLC using a reversed phase C18 LiChrospher 100 RP 18e (5 lm, 125 · 4 mm) column and guard column LiChrospher 100 RP 18 (5 lm) (Merck, Darmstad, Germany). The eluents were: (A) 4% H3PO4 in water, (B) 100% HPLC grade acetonitrile (Merck, Darmstad, Germany). Chromatographic conditions were as follows: 3% B in A at the time of injection (20 ll), 45 min 25% B in A, 46 min 30% B in A, 47 min initial conditions. Flow rate was 1 ml/min, 10 ll was injected. Samples were filtered through a 0.45 lm cellulose syringe filter before analysis. Detection was performed using a UV detection system L-7400 LaChrom Merck Hitachi (Merck, Darmstad, Germany) at 520 nm. 2.5. Extraction and drying of pigments The pigments were isolated from the frozen (at 28 C) black currant cake with hot water acidified with 0.2% of citric acid (G.R. 99.8%, Lach-Ner, Neratovice, Czech Republic) during 3 h at 60 C. The extracts were spray-dried in a Bu¨chi 190 Mini Spray Drier (Buchi, Flawil, Switzerland) by using potato starch derivative Paselli MD (Avebe, Vendam, Netherlands) as a carrier.
2.3. Total anthocyanin assay 2.6. Determination of stability Fruit juices were pressed out in a conventional juicer and the cake was stored in a freezer until extraction. The pigments were extracted from 5 g of frozen cake with
The water solutions of spray-dried colorant (0.1% w/v), freshly prepared water and ethanol extracts of
M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
colouring substances from black currant cake were used for anthocyanin stability studies. To determine the impact of temperature and thermal treatment time on the stability of colouring substances they were placed in closed test tubes and heated at 75, 85 and 95 C in a water thermostat. The absorption was measured every 30 min. The composition of individual anthocyanins was monitored by HPLC with UV detector L-7400 LaChrom (Merck, Darmstad, Germany) at 520 nm. To determine the impact of additives to the stability of water solutions of spray-dried colorant 10%, 20% and 40% of sucrose, fructose and aspartame, respectively, were added and the samples were heated at 70 C for 2 h. Thermostability of pigments was assessed by measuring absorption of the solutions after heating. Duration of experiment was five years. All assays were performed in triplicate and multiple comparisons between means were performed. The least significant difference (LSD) test was used to specify differences. Data were statistically assessed by one-way analysis of variance (ANOVA, vers. 3.43, 2002). Significance of difference was estimated at 5% level. The impact of meteorological factors on biochemical composition of black currant cultivars was assessed by using coefficient of variation (CV).
3. Results and discussion New black currant varieties, namely Kupoliniai, Kriviai, Gagatai, Joniniai, Almiai andVakariai suitable for mechanic harvesting were produced at LIH. The Kupoliniai is an early season variety. Berries ripen in 8 days; which is the shortest period among the varieties cultivated in Lithuania. The varieties Gagatai and Kriviai are mid-season, berry-ripening time is short and coextensive. All new varieties are more productive compared to the currently grown in Lithuania variety Minaj Smyriov. The productivity is higher due to bigger berries and longer trusses containing 1 or 2 trusses per
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bud. New varieties were more resistant to diseases and pests comparing to currently grown varieties. Chemical composition of different black currant varieties varied, as well as sucrose, ascorbic acid and anthocyanins content of the same variety in different year (Table 1). The amounts of ascorbic acid and sucrose were most variable. The average amount of ascorbic acid varied from 112.3 to 220.5 mg 100 g 1. The content of anthocyanins in cake is the main index in terms of production of food colorants. The highest and statistically reliable pigment concentration (LSD0.05 1.046) was found in berries of varieties Kriviai and Kupoliniai. The concentration of anthocyanins was from 14.54 to 11.19 mg g 1 cakes in different harvesting years. The same tendency was observed in juice: the highest concentration was found in juice of Kupoliniai berries, 1956 mg l 1, the lowest one in Ben Lomond berries, 1199 mg l 1. Meteorological condition (amount of precipitation and air temperature) has significant impact on berry chemical composition. For dry food colorants production, an important index is the amount of inverse sugar because it increases sticking of food pigment particles to dryerÕs walls during spray-drying and consequently decreases the yield of dry colorant. Differences between concentrations of inverse sugar in berries of different varieties determined in this study were not significant. In order to establish the quantity of black currant anthocyanins were analyzed the ethanol extracts of berry cake, which had been frozen and stored at a temperature of 28 C. HPLC analysis showed that the main pigment in all investigated varieties was cyd-3-rut, except for Kupoliniai containing dpd-3-rut as the major component. The following pigments were identified and quantified in the black currant varieties: cyd-3-rut (33–38%), dpd-3-rut (27–34%), cyd-3-glu (8–10%) and dpd-3-glu (8–10%). The quantitative composition of anthocyanins is in agreement with previously reported Le Lous, Majoie, and Moriniere (1975), Hong and Wrolstad (1990), and Cacace and Mazza (2003).
Table 1 Chemical composition of black currant berriesa Variety
Dry solubles (Brix)
Inverse sugar content (mg 100 g 1)
Sucrose content (mg 100 g 1)
Ascorbic acid content (mg 100 g 1)
Titratable acidity express as citric acid (mg 100 g 1)
Total anthocyanin content express as cyd-3-rut (mg g 1 cake)
Ben Lomond Ben Alder Minaj Smyriov Almiai Joniniai Kupoliniai Kriviai Gagatai Vakariai
15.96 ± 9.06 15.37 ± 10.34 14.45 ± 4.30 14.01 ± 5.30 14.74 ± 15.51 15.90 ± 5.50 15.03 ± 8.60 14.50 ± 1.99 16.14 ± 14.52
6.01 ± 3.60 5.63 ± 9.34 6.63 ± 10.52 5.79 ± 7.32 6.36 ± 8.21 7.35 ± 9.80 5.82 ± 2.83 5.86 ± 2.10 4.75 ± 8.13
2.91 ± 4.20 1.22 ± 6.98 1.75 ± 9.56 1.65 ± 11.02 1.21 ± 10.85 2.08 ± 1.75 1.83 ± 7.94 1.94 ± 1.49 1.04 ± 5.32
156.6 ± 15.3 112.3 ± 15.6 220.5 ± 8.8 178.4 ± 12.9 154.4 ± 6.2 186.7 ± 8.6 164.4 ± 14.5 172.2 ± 6.9 129.9 ± 16.5
3.15 ± 16.71 3.11 ± 12.27 2.33 ± 10.16 2.63 ± 16.40 2.75 ± 1.83 2.57 ± 4.28 2.43 ± 17.77 2.30 ± 19.50 3.06 ± 9.45
11.19 ± 17.90 13.67 ± 10.78 11.79 ± 11.15 7.45 ± 19.12 10.39 ± 6.81 15.42 ± 17.33 14.65 ± 18.01 11.29 ± 10.01 13.46 ± 17.00
a
Data are expressed as mean ± variance.
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M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
The effect of thermal treatment and storage on the stability of main black currant pigments was also investigated. To determine the effect of temperature and time of thermal treatment on aqueous solution of dry colorant and water extract of pigments samples were heated at 75, 85 and 95 C for 150 min. It was found that proceeding at 75 C did not influence the stability of the aqueous solution and insignificantly decreased the stability of the water extract. At the temperature 85 C the colour intensity of both samples decreased by 20%. Heating at 95 C decreased colour intensity of the water extracts and aqueous solutions by 53% and 45%, accordingly. HPLC analysis shows that cyd-3-rut is most stable against thermal treatment. The average reduction of peak area of this pigment after 150 min of heating at 95 C was 35%; the peak of cyd-3-glu reduced by 53%, dpd-3-rut by 52% and dpd-3-glu by 63% (Fig. 1). The same tendency was observed analyzing the ethanol and water extracts of colouring substances obtained from thermally processed and freshly pressed cake. The concentration of cyd-3-rut in ethanol extract remained at 96.2%, and the concentration of other anthocyanins has amounted to 75.4–87.5% of the initial content. In water extract cyd-3-rut concentration made up 59.1% of the initial amount, other pigments from 41.5% to 53.1%. It can be concluded that in terms of thermal processing cyanidin-3-rutinoside is the most stable anthocyanin in ethanol (LSD0.05 8.33) and water (LSD0.05 9.10) extract of colouring substances. To establish the impact of storage, ethanol extracts of pigments were stored in closed test tubes at 8 C temperature in the dark. HPLC analysis showed that after 12 months the content of dpd-3-glu decreased by 48.3% and cyd-3-glu by 47.0%. The main black currant pigments cyanidin and delphinidin rutinosides were more stable and their concentrations on average decreased by 25.8% and 28.1%, respectively. Water-soluble anthocyanin dyes are often used in production of various drinks, jellies and puree. These
Peak area, arbitary units
120 100
Dpd-3-glu
80
Dpd-3-rut Cyd-3-glu
60
Fig. 2. Effect of concentration of additives on thermostability of dye solution.
products usually contain sugar or sweeteners; therefore it was reasonable to study the impact of sucrose, fructose and aspartame on the stability of pigment solutions prepared from the spray-dried powder. The importance of water activity on pigments was also reported (Simon, Drdak, & Altamirano, 1993), therefore simultaneously were investigated the impact of water activity on pigments thermostability. It is known that high concentration of sugar in fruit preserves stabilizes anthocyanins (Wrolstad, Skrede, Lea, & Enersen, 1990). This effect could be explained by the fact that sugar addition reduces water activity aw. Even low changes of sugar concentration and water activity can have impact on pigment stability. Low water activity stabilizes pigments, e.g., aw of dry powder is less than 0.3 and in this state hermetically packed dye can be stable for several years. In our study were added 10–40% of sucrose, fructose and aspartame to water solutions of spray-dried dye and the samples were heated at 70 C for 2 h. The addition of sugars and sweetener reduced water activity from 1.0 to 0.9. The impact of aspartame and sucrose was similar; thermostability was reduced when their concentration increased from 0% to 20%, while further increase of the concentration to 40%, had positive effect on the stability of pigments (LSD0.05 = 2.44) (Fig. 2). With the increase of the concentration of fructose the thermostability of pigments decreased linearly (y = 26.183x + 90.782; R2 = 0.9937). This is in agreement with earlier reported results, showing that fructose, arabinose, lactose and sorbose have a greater effect on anthocyanin degradation compared with glucose, sucrose and maltose (Elbe & Schwartz, 1996).
Cyd-3-rut
40
4. Conclusions
20 0 0
25
50
75
100
125
150
Time, min Fig. 1. Effect of heating time at 95 C on the concentration of main black currant pigments.
1. Nine black currant varieties cultivated in Lithuania were studied. The highest amount of ascorbic acid was established in fresh berries from cv Minaj Smyriov and Kupoliniai: these varieties contained 220.5 and 186.7 mg 100 g 1 of ascorbic acid in berries. The
M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
highest amount of anthocyanins was found in cake produced from berries cv Kupoliniai and Kriviai: 14.65 and 15.42 mg g 1, respectively. 2. The major pigment determined in Kupoliniai variety was delphinidin-3-rutinoside; in Ben Lomond, Minaj Smyriov, Kriviai and Gagatai cultivars, cyanidin-3rutinoside. The composition of the identified pigments was the following: cyanidin-3-rutinoside (33–38%), delphinidin-3-rutinoside (27–34%), cyanidin-3-glucoside (8–10%) and delphinidin-3-glucoside (8–10%). 3. Cyanidin-3-rutinoside showed the highest stability to the effect of thermal treatment at 95 C temperature. Cyanidin and delphinidin rutinosides were the most stable anthocyanins during storage for 12 months at 8 C temperature. 4. Addition of 10% and 20% of aspartame and sucrose reduced thermostability of pigments, while further increase of the sweeteners content up to 40% had positive effect on the stability of pigments. Addition of fructose had negative effect – the thermostability of pigments decreased linearly.
References Cacace, J. E., & Mazza, G. (2003). Optimization of extraction of anthocyanins from black currants with aqueous ethanol. Journal of Food Science, 68(1), 240–248. Elbe, H., & Schwartz, E. J. (1996). Colorants. In O. R. Fennema (Ed.), Food chemistry (pp. 651–718). NY: Marcel Dekker, Inc. Giusti, M. M., & Wrolstad, R. E. (2001). Characterization and measurement of anthocyanins by UV–visible spectroscopy. In R. E. Wrolstad & S. J. Schwartz (Eds.), Current protocols in food analytical chemistry (pp. F1.2.1–F1.2.13). New York: John Wiley & Sons, Inc.
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Goiffon, J. P., Mouly, P. P., & Gaydou, E. M. (1999). Anthocyanic pigment determination in red fruit juices, concentrated juices and syrups using liquid chromatography. Analytica Chimica Acta, 382(1–2), 39–50. Hong, V., & Wrolstad, R. E. (1990). Characterization of anthocyanincontaining colorants and fruit juice by HPLC/photodiode array detection. Journal of Agricultural and Food Chemistry, 38, 698–708. Iversen, C. K. (1999). Black currant nectar: effect of processing and storage on anthocyanin and ascorbic acid content. Journal of Food Science, 64(1), 37–41. Iversen, C.K., Kidmose, U., & Poulsen, N. (1996). Anthocyanins in fruit and juice. Deutsche Gesellschaft f u¨r Qualita¨tsforschung (pp. 109–112). E.V. XXXI. Vortragstagung, Kiel. Jackman, R. L., Yada, R. Y., Tung, M. A., & Speers, R. A. (1987). Anthocyanins as food colorants – a review. Journal of Food Biochemistry, 11(3), 201–247. Le Lous, J., Majoie, B., & Moriniere, J. (1975). Etude des flavonoides de Ribes nigrum. Annales Pharmaceutiques Francaises, 33(8–9), 33–93. Mikkelsen, B. B., & Poll, L. (2002). Decomposition and transformation of aroma compounds and anthocyanins during black currant (Ribes nigrum L.) juice processing. Journal of Food Science, 67(9), 3447–3455. Simon, P., Drdak, M., & Altamirano, R. C. (1993). Influence of water activity on the stability of betanin in various water/alcohol model systems. Food Chemistry, 46(2), 155–158. Skrede, G., Wrolstad, R. E., Lea, P., & Enersen, G. (1992). Color stability of strawberry and blackcurrant syrups. Journal of Food Science, 57(1), 172–177. Taylor, J. (1989). Colour stability of blackcurrant (Ribes nigrum) juice. Journal of the Science of Food and Agriculture, 49, 487–491. Torskangerpoll, K., & Andersen, ø. M. (2005). Colour stability of anthocyanins in aqueous solutions at various pH values. Food Chemistry, 89(3), 427–440. Wrolstad, R. E. (2000). Anthocyanins. In F. J. Francis & G. J. Lauro (Eds.), Natural food colorant (pp. 237–252). NY: Marcel Dekker, Inc. Wrolstad, R. E., Skrede, G., Lea, P., & Enersen, G. (1990). Influence of sugar on anthocyanin pigment stability in frozen strawberries. Journal of Food Science, 55(4), 1064–1065, 1072.
Impact of various factors on the composition and stability of black currant anthocyanins M. Rubinskiene a, P. Viskelis
a,*
, I. Jasutiene b, R. Viskeliene b, C. Bobinas
a
a
b
Lithuanian Institute of Horticulture, Kauno 30, LT-4335 Babtai, Lithuania Food Institute of Kaunas University of Technology, Taikos 92, LT-51180 Kaunas, Lithuania Received 27 May 2004; accepted 27 February 2005
Abstract Nine black currant varieties cultivated in Lithuania were studied. The highest amount of ascorbic acid was established in fresh berries from cv Minaj Smyriov and Kupoliniai: these varieties contained 220.5 and 186.7 mg 100 g 1 of ascorbic acid in berries. The highest amount of anthocyanins was found in cake produced from berries cv Kupoliniai and Kriviai: 14.65 and 15.42 mg g 1, respectively. The major pigment determined in Kupoliniai variety was delphinidin-3-rutinoside; in Ben Lomond, Minaj Smyriov, Kriviai and Gagatai cultivars, cyanidin-3-rutinoside. The composition of the identified pigments was the following: cyanidin-3-rutinoside (33– 38%), delphinidin-3-rutinoside (27–34%), cyanidin-3-glucoside (8–10%) and delphinidin-3-glucoside (8–10%). Impact of storage, thermal treatment and addition of sweeteners were studied. Cyanidin-3-rutinoside was the most stable to the effect of thermal treatment at 95 C, while cyanidin and delphinidin rutinosides were the most stable during storage for 12 months at 8 C. Fructose has a greater effect on anthocyanin degradation compared with glucose and aspartame. 2005 Elsevier Ltd. All rights reserved. Keywords: Anthocyanins; Black currant; Colorant; Variety
1. Introduction Chemical composition of black currant berries depends on the variety and agroclimatic conditions of their cultivation. The composition and properties of black currant berry pigments grown in Lithuania have been little investigated. The information on such pigments is important for the production of food colorants, berry processing and for expanding our scientific knowledge on this topic. Since natural pigments are susceptible to the impact of such technological factors as pH, temperature, oxygen and enzymes (Jackman, Yada, Tung, & Speers, 1987; Wrolstad, 2000), it is important to study the effect *
Corresponding author. Tel.: +370 37 555 439; fax: +370 37 555 176. E-mail address: [email protected] (P. Viskelis). 0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.02.027
of these factors on the properties and structural changes of colorants. These changes may be monitored by measuring pigment concentration and colour analysis. Regression analyses did not show significant variation in the loss of total pigment concentration, however the changes of individual major pigments were significant (Taylor, 1989). This study also reported that cyanidin was the major pigment in the all examined British cultivars, while delphinidin was dominating pigment in Scandinavian cultivars. Skrede, Wrolstad, Lea, and Enersen (1992) studied the colour and pigment stability of strawberry and black currant syrups, Iversen, Kidmose, and Poulsen (1996) measured the breakdown of total anthocyanins in black currant juice during storage, Goiffon, Mouly, and Gaydou (1999) studied stability of anthocyanins in black currant juice and concentrates. The degradation of anthocyanins and ascorbic acid in black currant nectar was studied during processing and
868
M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
storage (Iversen, 1999). It was found that about 50% of the original content of monomeric anthocyanins remained after 6 months storage at 20 C. The stabilities of delphinidins and cyanidins during storage were similar. Torskangerpoll and Andersen (2005) focused study on the impact of anthocyanin structures such as 5-glucosidic substitution and aromatic acylation on anthocyanin, colour and stability at various pH values. Mikkelsen and Poll (2002) studied decomposition and transformation of anthocyanins during black currant juice processing. It was found that between 25% and 30% of the anthocyanins were lost during juice processing; the heating treatments seem to be the most destructive process steps. The aim of such studies is to predict the impact of factors mentioned above on the quality of final product and consequently to avoid negative effects by applying the achievements in modern theory of molecular chemistry and physics. The objective of this study was to investigate chemical and technological properties of black currant berries from different plant varieties, to evaluate the amount of anthocyanins and their composition, and to obtain some new scientific data on suitability of black currant anthocyanins for production of red food colorants.
2. Materials and methods 2.1. Berries Black currant berries were grown in the experimental field of the Lithuanian Institute of Horticulture (LIH). The berries from the following varieties were used: registered variety Minaj Smyriov, European commercial varieties Ben Lomond, Ben Alder; and the varieties developed at the LIH Kriviai, Kupoliniai, Gagatai, Joniniai, Almiai andVakariai. Berries were picked at ripeness phase, frozen and stored (at 28 C) in polyethylene bags. 2.2. Chemical composition Chemical composition of berries was determined by standard methods. The following analyses were performed each year: the content of soluble solids by refractometer, titratable acidity expressed as citric acid by titration with 0.1 M NaOH solution, ascorbic acid content by titration with 2,6-dichlorophenolindophenol sodium salt solution, inverse sugar and sucrose by Bertrand method.
95% (v/v) food grade ethanol acidified with 0.1 M HCl. The cake was ground with quartz sand and the extraction was continued with 20 ml portions of solvent until the sample became colourless. The extract was diluted with acidified ethanol; the absorption was measured on a spectrophotometer Genesys-5 (Thermo Spectronic, Rochester, USA) at 544 nm. The concentration of anthocyanins was determined from the calibration curve, which was constructed by measuring the absorption of cyanidin-3-rutinoside (MW 595.2, e = 28,800) reference solutions. Purified cyd-3-rut was donated by the Danish Institute of Agricultural Sciences (Department of Fruit, Vegetable and Food Science). The concentration of anthocyanins was calculated and expressed in milligrams of cyd-3-rut in gram of cake. The concentration of the monomeric anthocyanins in juice was determined by the pH differential method as described by Giusti and Wrolstad (2001). Absorbance was measured on a spectrophotometer Genesys-5 (Thermo Spectronic, Rochester, USA) at 510 nm and at 700 nm in buffer at pH 1.0 and pH 4.5. Results were expressed as milligram per litre. 2.4. HPLC/UV analysis of anthocyanins Anthocyanin profiles of black currant ethanol extracts were characterized by HPLC using a reversed phase C18 LiChrospher 100 RP 18e (5 lm, 125 · 4 mm) column and guard column LiChrospher 100 RP 18 (5 lm) (Merck, Darmstad, Germany). The eluents were: (A) 4% H3PO4 in water, (B) 100% HPLC grade acetonitrile (Merck, Darmstad, Germany). Chromatographic conditions were as follows: 3% B in A at the time of injection (20 ll), 45 min 25% B in A, 46 min 30% B in A, 47 min initial conditions. Flow rate was 1 ml/min, 10 ll was injected. Samples were filtered through a 0.45 lm cellulose syringe filter before analysis. Detection was performed using a UV detection system L-7400 LaChrom Merck Hitachi (Merck, Darmstad, Germany) at 520 nm. 2.5. Extraction and drying of pigments The pigments were isolated from the frozen (at 28 C) black currant cake with hot water acidified with 0.2% of citric acid (G.R. 99.8%, Lach-Ner, Neratovice, Czech Republic) during 3 h at 60 C. The extracts were spray-dried in a Bu¨chi 190 Mini Spray Drier (Buchi, Flawil, Switzerland) by using potato starch derivative Paselli MD (Avebe, Vendam, Netherlands) as a carrier.
2.3. Total anthocyanin assay 2.6. Determination of stability Fruit juices were pressed out in a conventional juicer and the cake was stored in a freezer until extraction. The pigments were extracted from 5 g of frozen cake with
The water solutions of spray-dried colorant (0.1% w/v), freshly prepared water and ethanol extracts of
M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
colouring substances from black currant cake were used for anthocyanin stability studies. To determine the impact of temperature and thermal treatment time on the stability of colouring substances they were placed in closed test tubes and heated at 75, 85 and 95 C in a water thermostat. The absorption was measured every 30 min. The composition of individual anthocyanins was monitored by HPLC with UV detector L-7400 LaChrom (Merck, Darmstad, Germany) at 520 nm. To determine the impact of additives to the stability of water solutions of spray-dried colorant 10%, 20% and 40% of sucrose, fructose and aspartame, respectively, were added and the samples were heated at 70 C for 2 h. Thermostability of pigments was assessed by measuring absorption of the solutions after heating. Duration of experiment was five years. All assays were performed in triplicate and multiple comparisons between means were performed. The least significant difference (LSD) test was used to specify differences. Data were statistically assessed by one-way analysis of variance (ANOVA, vers. 3.43, 2002). Significance of difference was estimated at 5% level. The impact of meteorological factors on biochemical composition of black currant cultivars was assessed by using coefficient of variation (CV).
3. Results and discussion New black currant varieties, namely Kupoliniai, Kriviai, Gagatai, Joniniai, Almiai andVakariai suitable for mechanic harvesting were produced at LIH. The Kupoliniai is an early season variety. Berries ripen in 8 days; which is the shortest period among the varieties cultivated in Lithuania. The varieties Gagatai and Kriviai are mid-season, berry-ripening time is short and coextensive. All new varieties are more productive compared to the currently grown in Lithuania variety Minaj Smyriov. The productivity is higher due to bigger berries and longer trusses containing 1 or 2 trusses per
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bud. New varieties were more resistant to diseases and pests comparing to currently grown varieties. Chemical composition of different black currant varieties varied, as well as sucrose, ascorbic acid and anthocyanins content of the same variety in different year (Table 1). The amounts of ascorbic acid and sucrose were most variable. The average amount of ascorbic acid varied from 112.3 to 220.5 mg 100 g 1. The content of anthocyanins in cake is the main index in terms of production of food colorants. The highest and statistically reliable pigment concentration (LSD0.05 1.046) was found in berries of varieties Kriviai and Kupoliniai. The concentration of anthocyanins was from 14.54 to 11.19 mg g 1 cakes in different harvesting years. The same tendency was observed in juice: the highest concentration was found in juice of Kupoliniai berries, 1956 mg l 1, the lowest one in Ben Lomond berries, 1199 mg l 1. Meteorological condition (amount of precipitation and air temperature) has significant impact on berry chemical composition. For dry food colorants production, an important index is the amount of inverse sugar because it increases sticking of food pigment particles to dryerÕs walls during spray-drying and consequently decreases the yield of dry colorant. Differences between concentrations of inverse sugar in berries of different varieties determined in this study were not significant. In order to establish the quantity of black currant anthocyanins were analyzed the ethanol extracts of berry cake, which had been frozen and stored at a temperature of 28 C. HPLC analysis showed that the main pigment in all investigated varieties was cyd-3-rut, except for Kupoliniai containing dpd-3-rut as the major component. The following pigments were identified and quantified in the black currant varieties: cyd-3-rut (33–38%), dpd-3-rut (27–34%), cyd-3-glu (8–10%) and dpd-3-glu (8–10%). The quantitative composition of anthocyanins is in agreement with previously reported Le Lous, Majoie, and Moriniere (1975), Hong and Wrolstad (1990), and Cacace and Mazza (2003).
Table 1 Chemical composition of black currant berriesa Variety
Dry solubles (Brix)
Inverse sugar content (mg 100 g 1)
Sucrose content (mg 100 g 1)
Ascorbic acid content (mg 100 g 1)
Titratable acidity express as citric acid (mg 100 g 1)
Total anthocyanin content express as cyd-3-rut (mg g 1 cake)
Ben Lomond Ben Alder Minaj Smyriov Almiai Joniniai Kupoliniai Kriviai Gagatai Vakariai
15.96 ± 9.06 15.37 ± 10.34 14.45 ± 4.30 14.01 ± 5.30 14.74 ± 15.51 15.90 ± 5.50 15.03 ± 8.60 14.50 ± 1.99 16.14 ± 14.52
6.01 ± 3.60 5.63 ± 9.34 6.63 ± 10.52 5.79 ± 7.32 6.36 ± 8.21 7.35 ± 9.80 5.82 ± 2.83 5.86 ± 2.10 4.75 ± 8.13
2.91 ± 4.20 1.22 ± 6.98 1.75 ± 9.56 1.65 ± 11.02 1.21 ± 10.85 2.08 ± 1.75 1.83 ± 7.94 1.94 ± 1.49 1.04 ± 5.32
156.6 ± 15.3 112.3 ± 15.6 220.5 ± 8.8 178.4 ± 12.9 154.4 ± 6.2 186.7 ± 8.6 164.4 ± 14.5 172.2 ± 6.9 129.9 ± 16.5
3.15 ± 16.71 3.11 ± 12.27 2.33 ± 10.16 2.63 ± 16.40 2.75 ± 1.83 2.57 ± 4.28 2.43 ± 17.77 2.30 ± 19.50 3.06 ± 9.45
11.19 ± 17.90 13.67 ± 10.78 11.79 ± 11.15 7.45 ± 19.12 10.39 ± 6.81 15.42 ± 17.33 14.65 ± 18.01 11.29 ± 10.01 13.46 ± 17.00
a
Data are expressed as mean ± variance.
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The effect of thermal treatment and storage on the stability of main black currant pigments was also investigated. To determine the effect of temperature and time of thermal treatment on aqueous solution of dry colorant and water extract of pigments samples were heated at 75, 85 and 95 C for 150 min. It was found that proceeding at 75 C did not influence the stability of the aqueous solution and insignificantly decreased the stability of the water extract. At the temperature 85 C the colour intensity of both samples decreased by 20%. Heating at 95 C decreased colour intensity of the water extracts and aqueous solutions by 53% and 45%, accordingly. HPLC analysis shows that cyd-3-rut is most stable against thermal treatment. The average reduction of peak area of this pigment after 150 min of heating at 95 C was 35%; the peak of cyd-3-glu reduced by 53%, dpd-3-rut by 52% and dpd-3-glu by 63% (Fig. 1). The same tendency was observed analyzing the ethanol and water extracts of colouring substances obtained from thermally processed and freshly pressed cake. The concentration of cyd-3-rut in ethanol extract remained at 96.2%, and the concentration of other anthocyanins has amounted to 75.4–87.5% of the initial content. In water extract cyd-3-rut concentration made up 59.1% of the initial amount, other pigments from 41.5% to 53.1%. It can be concluded that in terms of thermal processing cyanidin-3-rutinoside is the most stable anthocyanin in ethanol (LSD0.05 8.33) and water (LSD0.05 9.10) extract of colouring substances. To establish the impact of storage, ethanol extracts of pigments were stored in closed test tubes at 8 C temperature in the dark. HPLC analysis showed that after 12 months the content of dpd-3-glu decreased by 48.3% and cyd-3-glu by 47.0%. The main black currant pigments cyanidin and delphinidin rutinosides were more stable and their concentrations on average decreased by 25.8% and 28.1%, respectively. Water-soluble anthocyanin dyes are often used in production of various drinks, jellies and puree. These
Peak area, arbitary units
120 100
Dpd-3-glu
80
Dpd-3-rut Cyd-3-glu
60
Fig. 2. Effect of concentration of additives on thermostability of dye solution.
products usually contain sugar or sweeteners; therefore it was reasonable to study the impact of sucrose, fructose and aspartame on the stability of pigment solutions prepared from the spray-dried powder. The importance of water activity on pigments was also reported (Simon, Drdak, & Altamirano, 1993), therefore simultaneously were investigated the impact of water activity on pigments thermostability. It is known that high concentration of sugar in fruit preserves stabilizes anthocyanins (Wrolstad, Skrede, Lea, & Enersen, 1990). This effect could be explained by the fact that sugar addition reduces water activity aw. Even low changes of sugar concentration and water activity can have impact on pigment stability. Low water activity stabilizes pigments, e.g., aw of dry powder is less than 0.3 and in this state hermetically packed dye can be stable for several years. In our study were added 10–40% of sucrose, fructose and aspartame to water solutions of spray-dried dye and the samples were heated at 70 C for 2 h. The addition of sugars and sweetener reduced water activity from 1.0 to 0.9. The impact of aspartame and sucrose was similar; thermostability was reduced when their concentration increased from 0% to 20%, while further increase of the concentration to 40%, had positive effect on the stability of pigments (LSD0.05 = 2.44) (Fig. 2). With the increase of the concentration of fructose the thermostability of pigments decreased linearly (y = 26.183x + 90.782; R2 = 0.9937). This is in agreement with earlier reported results, showing that fructose, arabinose, lactose and sorbose have a greater effect on anthocyanin degradation compared with glucose, sucrose and maltose (Elbe & Schwartz, 1996).
Cyd-3-rut
40
4. Conclusions
20 0 0
25
50
75
100
125
150
Time, min Fig. 1. Effect of heating time at 95 C on the concentration of main black currant pigments.
1. Nine black currant varieties cultivated in Lithuania were studied. The highest amount of ascorbic acid was established in fresh berries from cv Minaj Smyriov and Kupoliniai: these varieties contained 220.5 and 186.7 mg 100 g 1 of ascorbic acid in berries. The
M. Rubinskiene et al. / Food Research International 38 (2005) 867–871
highest amount of anthocyanins was found in cake produced from berries cv Kupoliniai and Kriviai: 14.65 and 15.42 mg g 1, respectively. 2. The major pigment determined in Kupoliniai variety was delphinidin-3-rutinoside; in Ben Lomond, Minaj Smyriov, Kriviai and Gagatai cultivars, cyanidin-3rutinoside. The composition of the identified pigments was the following: cyanidin-3-rutinoside (33–38%), delphinidin-3-rutinoside (27–34%), cyanidin-3-glucoside (8–10%) and delphinidin-3-glucoside (8–10%). 3. Cyanidin-3-rutinoside showed the highest stability to the effect of thermal treatment at 95 C temperature. Cyanidin and delphinidin rutinosides were the most stable anthocyanins during storage for 12 months at 8 C temperature. 4. Addition of 10% and 20% of aspartame and sucrose reduced thermostability of pigments, while further increase of the sweeteners content up to 40% had positive effect on the stability of pigments. Addition of fructose had negative effect – the thermostability of pigments decreased linearly.
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