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Food Research International 38 (2005) 867–871

Impact of various factors on the composition and stability ofblack currant anthocyanins

M. Rubinskiene a, P. Viskelis a,*, I. Jasutiene b, R. Viskeliene b, C. Bobinas a

a Lithuanian Institute of Horticulture, Kauno 30, LT-4335 Babtai, Lithuaniab 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 freshberries from cv Minaj Smyriov and Kupoliniai: these varieties contained 220.5 and 186.7 mg 100 g�1 of ascorbic acid in berries. Thehighest amount of anthocyanins was found in cake produced from berries cv Kupoliniai and Kriviai: 14.65 and 15.42 mg g�1, respec-tively. The major pigment determined in Kupoliniai variety was delphinidin-3-rutinoside; in Ben Lomond,Minaj Smyriov, Kriviai andGagatai 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 treat-ment at 95 �C, while cyanidin and delphinidin rutinosides were the most stable during storage for 12 months at 8 �C. Fructose has agreater 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 de-pends on the variety and agroclimatic conditions of theircultivation. The composition and properties of blackcurrant berry pigments grown in Lithuania have beenlittle investigated. The information on such pigments isimportant for the production of food colorants, berryprocessing and for expanding our scientific knowledgeon this topic.

Since natural pigments are susceptible to the impactof such technological factors as pH, temperature, oxy-gen and enzymes (Jackman, Yada, Tung, & Speers,1987; Wrolstad, 2000), it is important to study the effect

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* Corresponding author. Tel.: +370 37 555 439; fax: +370 37 555176.

E-mail address: biochem@lsdi.lt (P. Viskelis).

of these factors on the properties and structural changesof colorants. These changes may be monitored by mea-suring pigment concentration and colour analysis.Regression analyses did not show significant variationin the loss of total pigment concentration, however thechanges of individual major pigments were significant(Taylor, 1989). This study also reported that cyanidinwas the major pigment in the all examined British culti-vars, while delphinidin was dominating pigment in Scan-dinavian cultivars. Skrede, Wrolstad, Lea, and Enersen(1992) studied the colour and pigment stability of straw-berry and black currant syrups, Iversen, Kidmose, andPoulsen (1996) measured the breakdown of total antho-cyanins in black currant juice during storage, Goiffon,Mouly, and Gaydou (1999) studied stability of anthocy-anins in black currant juice and concentrates. Thedegradation of anthocyanins and ascorbic acid in blackcurrant 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% ofthe original content of monomeric anthocyanins re-mained after 6 months storage at 20 �C. The stabilitiesof delphinidins and cyanidins during storage were simi-lar. Torskangerpoll and Andersen (2005) focused studyon the impact of anthocyanin structures such as 5-gluco-sidic substitution and aromatic acylation on anthocya-nin, colour and stability at various pH values.Mikkelsen and Poll (2002) studied decomposition andtransformation of anthocyanins during black currantjuice processing. It was found that between 25% and30% of the anthocyanins were lost during juice process-ing; the heating treatments seem to be the most destruc-tive process steps. The aim of such studies is to predictthe impact of factors mentioned above on the qualityof final product and consequently to avoid negative ef-fects by applying the achievements in modern theoryof molecular chemistry and physics.

The objective of this study was to investigate chemi-cal and technological properties of black currant berriesfrom different plant varieties, to evaluate the amount ofanthocyanins and their composition, and to obtain somenew scientific data on suitability of black currant antho-cyanins for production of red food colorants.

2. Materials and methods

2.1. Berries

Black currant berries were grown in the experimentalfield of the Lithuanian Institute of Horticulture (LIH).The berries from the following varieties were used: reg-istered variety Minaj Smyriov, European commercialvarieties Ben Lomond, Ben Alder; and the varieties devel-oped at the LIH Kriviai, Kupoliniai, Gagatai, Joniniai,Almiai andVakariai. Berries were picked at ripenessphase, frozen and stored (at �28 �C) in polyethylenebags.

2.2. Chemical composition

Chemical composition of berries was determined bystandard methods. The following analyses were per-formed each year: the content of soluble solids by refrac-tometer, titratable acidity expressed as citric acid bytitration with 0.1 M NaOH solution, ascorbic acid con-tent by titration with 2,6-dichlorophenolindophenolsodium salt solution, inverse sugar and sucrose by Ber-trand method.

2.3. Total anthocyanin assay

Fruit juices were pressed out in a conventional juicerand the cake was stored in a freezer until extraction. Thepigments were extracted from 5 g of frozen cake with

95% (v/v) food grade ethanol acidified with 0.1 MHCl. The cake was ground with quartz sand and theextraction was continued with 20 ml portions of solventuntil the sample became colourless. The extract was di-luted with acidified ethanol; the absorption was mea-sured on a spectrophotometer Genesys-5 (ThermoSpectronic, Rochester, USA) at 544 nm. The concentra-tion of anthocyanins was determined from the calibra-tion curve, which was constructed by measuring theabsorption of cyanidin-3-rutinoside (MW 595.2, e =28,800) reference solutions. Purified cyd-3-rut was do-nated by the Danish Institute of Agricultural Sciences(Department of Fruit, Vegetable and Food Science).The concentration of anthocyanins was calculated andexpressed in milligrams of cyd-3-rut in gram of cake.

The concentration of the monomeric anthocyanins injuice was determined by the pH differential method asdescribed by Giusti and Wrolstad (2001). Absorbancewas measured on a spectrophotometer Genesys-5 (Ther-mo Spectronic, Rochester, USA) at 510 nm and at700 nm in buffer at pH 1.0 and pH 4.5. Results were ex-pressed as milligram per litre.

2.4. HPLC/UV analysis of anthocyanins

Anthocyanin profiles of black currant ethanol ex-tracts were characterized by HPLC using a reversedphase C18 LiChrospher� 100 RP 18e (5 lm,125 · 4 mm) column and guard column LiChrospher�

100 RP 18 (5 lm) (Merck, Darmstad, Germany). Theeluents were: (A) 4% H3PO4 in water, (B) 100% HPLCgrade acetonitrile (Merck, Darmstad, Germany). Chro-matographic conditions were as follows: 3% B in A atthe time of injection (20 ll), 45 min 25% B in A,46 min 30% B in A, 47 min initial conditions. Flow ratewas 1 ml/min, 10 ll was injected. Samples were filteredthrough a 0.45 lm cellulose syringe filter before analysis.Detection was performed using a UV detection systemL-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 with0.2% of citric acid (G.R. 99.8%, Lach-Ner, Neratovice,Czech Republic) during 3 h at 60 �C. The extracts werespray-dried in a Buchi 190 Mini Spray Drier (Buchi,Flawil, Switzerland) by using potato starch derivativePaselli MD (Avebe, Vendam, Netherlands) as a carrier.

2.6. Determination of stability

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) 867–871 869

colouring substances from black currant cake were usedfor anthocyanin stability studies. To determine the im-pact of temperature and thermal treatment time on thestability of colouring substances they were placed inclosed test tubes and heated at 75, 85 and 95 �C in awater thermostat. The absorption was measured every30 min. The composition of individual anthocyaninswas monitored by HPLC with UV detector L-7400 La-Chrom (Merck, Darmstad, Germany) at 520 nm. Todetermine the impact of additives to the stability ofwater solutions of spray-dried colorant 10%, 20% and40% of sucrose, fructose and aspartame, respectively,were added and the samples were heated at 70 �C for2 h. Thermostability of pigments was assessed by mea-suring absorption of the solutions after heating.

Duration of experiment was five years. All assayswere performed in triplicate and multiple comparisonsbetween means were performed. The least significant dif-ference (LSD) test was used to specify differences. Datawere statistically assessed by one-way analysis of vari-ance (ANOVA, vers. 3.43, 2002). Significance of differ-ence was estimated at 5% level. The impact ofmeteorological factors on biochemical composition ofblack currant cultivars was assessed by using coefficientof variation (CV).

3. Results and discussion

New black currant varieties, namely Kupoliniai, Kri-viai, Gagatai, Joniniai, Almiai andVakariai suitable formechanic harvesting were produced at LIH. The Kupol-iniai is an early season variety. Berries ripen in 8 days;which is the shortest period among the varieties culti-vated in Lithuania. The varieties Gagatai and Kriviai

are mid-season, berry-ripening time is short and coex-tensive. All new varieties are more productive comparedto the currently grown in Lithuania variety MinajSmyriov. The productivity is higher due to bigger ber-ries and longer trusses containing 1 or 2 trusses per

Table 1Chemical composition of black currant berriesa

Variety Dry solubles(�Brix)

Inverse sugarcontent (mg 100 g�1)

Sucrose content(mg 100 g�1)

Ben Lomond 15.96 ± 9.06 6.01 ± 3.60 2.91 ± 4.20Ben Alder 15.37 ± 10.34 5.63 ± 9.34 1.22 ± 6.98Minaj Smyriov 14.45 ± 4.30 6.63 ± 10.52 1.75 ± 9.56Almiai 14.01 ± 5.30 5.79 ± 7.32 1.65 ± 11.02Joniniai 14.74 ± 15.51 6.36 ± 8.21 1.21 ± 10.85Kupoliniai 15.90 ± 5.50 7.35 ± 9.80 2.08 ± 1.75Kriviai 15.03 ± 8.60 5.82 ± 2.83 1.83 ± 7.94Gagatai 14.50 ± 1.99 5.86 ± 2.10 1.94 ± 1.49Vakariai 16.14 ± 14.52 4.75 ± 8.13 1.04 ± 5.32

a Data are expressed as mean ± variance.

bud. New varieties were more resistant to diseasesand pests comparing to currently grown varieties.Chemical composition of different black currant varie-ties varied, as well as sucrose, ascorbic acid and antho-cyanins content of the same variety in different year(Table 1). The amounts of ascorbic acid and sucrosewere most variable. The average amount of ascorbicacid varied from 112.3 to 220.5 mg 100 g�1. The contentof anthocyanins in cake is the main index in terms ofproduction of food colorants. The highest and statisti-cally reliable pigment concentration (LSD0.05 1.046)was found in berries of varieties Kriviai and Kupoliniai.

The concentration of anthocyanins was from 14.54 to11.19 mg g�1 cakes in different harvesting years. Thesame tendency was observed in juice: the highest con-centration 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 pre-cipitation and air temperature) has significant impacton berry chemical composition.

For dry food colorants production, an important in-dex is the amount of inverse sugar because it increasessticking of food pigment particles to dryer�s walls duringspray-drying and consequently decreases the yield of drycolorant. Differences between concentrations of inversesugar in berries of different varieties determined in thisstudy were not significant.

In order to establish the quantity of black currantanthocyanins were analyzed the ethanol extracts of berrycake, which had been frozen and stored at a temperatureof �28 �C. HPLC analysis showed that the main pig-ment in all investigated varieties was cyd-3-rut, exceptfor Kupoliniai containing dpd-3-rut as the major compo-nent. The following pigments were identified and quan-tified 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 anthocyaninsis in agreement with previously reported Le Lous, Maj-oie, and Moriniere (1975), Hong and Wrolstad (1990),and Cacace and Mazza (2003).

Ascorbic acidcontent (mg 100 g�1)

Titratable acidityexpress as citricacid (mg 100 g�1)

Total anthocyanincontent express ascyd-3-rut (mg g�1 cake)

156.6 ± 15.3 3.15 ± 16.71 11.19 ± 17.90112.3 ± 15.6 3.11 ± 12.27 13.67 ± 10.78220.5 ± 8.8 2.33 ± 10.16 11.79 ± 11.15178.4 ± 12.9 2.63 ± 16.40 7.45 ± 19.12154.4 ± 6.2 2.75 ± 1.83 10.39 ± 6.81186.7 ± 8.6 2.57 ± 4.28 15.42 ± 17.33164.4 ± 14.5 2.43 ± 17.77 14.65 ± 18.01172.2 ± 6.9 2.30 ± 19.50 11.29 ± 10.01129.9 ± 16.5 3.06 ± 9.45 13.46 ± 17.00

Fig. 2. Effect of concentration of additives on thermostability of dyesolution.

870 M. Rubinskiene et al. / Food Research International 38 (2005) 867–871

The effect of thermal treatment and storage on thestability of main black currant pigments was also inves-tigated. To determine the effect of temperature and timeof thermal treatment on aqueous solution of dry color-ant and water extract of pigments samples were heatedat 75, 85 and 95 �C for 150 min. It was found that pro-ceeding at 75 �C did not influence the stability of theaqueous solution and insignificantly decreased the sta-bility of the water extract. At the temperature 85 �Cthe colour intensity of both samples decreased by 20%.Heating at 95 �C decreased colour intensity of the waterextracts and aqueous solutions by 53% and 45%, accord-ingly. HPLC analysis shows that cyd-3-rut is most stableagainst thermal treatment. The average reduction ofpeak area of this pigment after 150 min of heating at95 �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 etha-nol and water extracts of colouring substances obtainedfrom thermally processed and freshly pressed cake. Theconcentration of cyd-3-rut in ethanol extract remainedat 96.2%, and the concentration of other anthocyaninshas amounted to 75.4–87.5% of the initial content. Inwater extract cyd-3-rut concentration made up 59.1%of the initial amount, other pigments from 41.5% to53.1%. It can be concluded that in terms of thermal pro-cessing cyanidin-3-rutinoside is the most stable anthocy-anin 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 temper-ature in the dark. HPLC analysis showed that after12 months the content of dpd-3-glu decreased by48.3% and cyd-3-glu by 47.0%. The main black currantpigments cyanidin and delphinidin rutinosides weremore stable and their concentrations on average de-creased by 25.8% and 28.1%, respectively.

Water-soluble anthocyanin dyes are often used inproduction of various drinks, jellies and puree. These

0

20

40

60

80

100

120

0 25 50 75 100 125 150

Time, min

Peak

are

a, a

rbita

ry u

nits Dpd-3-glu

Dpd-3-rut

Cyd-3-glu

Cyd-3-rut

Fig. 1. Effect of heating time at 95 �C on the concentration of mainblack currant pigments.

products usually contain sugar or sweeteners; thereforeit was reasonable to study the impact of sucrose, fruc-tose and aspartame on the stability of pigment solutionsprepared from the spray-dried powder. The importanceof water activity on pigments was also reported (Simon,Drdak, & Altamirano, 1993), therefore simultaneouslywere investigated the impact of water activity on pig-ments thermostability. It is known that high concentra-tion of sugar in fruit preserves stabilizes anthocyanins(Wrolstad, Skrede, Lea, & Enersen, 1990). This effectcould be explained by the fact that sugar additionreduces water activity aw. Even low changes of sugarconcentration and water activity can have impact onpigment stability. Low water activity stabilizes pig-ments, e.g., aw of dry powder is less than 0.3 and in thisstate hermetically packed dye can be stable for severalyears. In our study were added 10–40% of sucrose, fruc-tose and aspartame to water solutions of spray-dried dyeand the samples were heated at 70 �C for 2 h. The addi-tion of sugars and sweetener reduced water activity from1.0 to 0.9. The impact of aspartame and sucrose wassimilar; thermostability was reduced when their concen-tration increased from 0% to 20%, while further increaseof the concentration to 40%, had positive effect on thestability of pigments (LSD0.05 = 2.44) (Fig. 2). Withthe increase of the concentration of fructose the thermo-stability of pigments decreased linearly (y = �26.183x +90.782; R2 = 0.9937). This is in agreement with earlierreported results, showing that fructose, arabinose, lac-tose and sorbose have a greater effect on anthocyanindegradation compared with glucose, sucrose and malt-ose (Elbe & Schwartz, 1996).

4. Conclusions

1. Nine black currant varieties cultivated in Lithuaniawere studied. The highest amount of ascorbic acidwas established in fresh berries from cv Minaj Smyr-

iov and Kupoliniai: these varieties contained 220.5 and186.7 mg 100 g�1 of ascorbic acid in berries. The

M. Rubinskiene et al. / Food Research International 38 (2005) 867–871 871

highest amount of anthocyanins was found in cakeproduced from berries cv Kupoliniai and Kriviai:14.65 and 15.42 mg g�1, respectively.

2. The major pigment determined in Kupoliniai varietywas delphinidin-3-rutinoside; in Ben Lomond, Minaj

Smyriov, Kriviai and Gagatai cultivars, cyanidin-3-rutinoside. The composition of the identified pigmentswas the following: cyanidin-3-rutinoside (33–38%),delphinidin-3-rutinoside (27–34%), cyanidin-3-gluco-side (8–10%) and delphinidin-3-glucoside (8–10%).

3. Cyanidin-3-rutinoside showed the highest stability tothe effect of thermal treatment at 95 �C temperature.Cyanidin and delphinidin rutinosides were the moststable anthocyanins during storage for 12 months at8 �C temperature.

4. Addition of 10% and 20% of aspartame and sucrosereduced thermostability of pigments, while furtherincrease of the sweeteners content up to 40% hadpositive effect on the stability of pigments. Additionof fructose had negative effect – the thermostabilityof pigments decreased linearly.

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