Vittorio Maria Moretti
email: vittorio.moretti@unimi.it

Tiziana Mentasti

Federica Bellagamba

Umberto Luzzana

Fabio ; Caprino

Giovanni Mario Turchini

Ivan Giani

Franco Valfrè

Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchuss mykiss

Keywords: Chromatography -HPLC, Traceability Additives/Contaminants: Additives general, Colours, Feeding, Nutrition Food Types: Animal, Animal feed, Feeding, Fish

variable distribution of the isomers deriving from the different biological organism that synthesised it. The high performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin was performed in different pigment sources, such as red yeast Phaffia rhodozyma, alga meal Haematococcus pluvialis, krill meal and oil, and shrimp meal. With the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 60 days diets containing astaxanthin from synthetic source, Haematococcus pluvialis algae meal and Phaffia rhodozyma red yeast. Moreover, the distribution of optical isomers of astaxanthin in trout purchased on the Italian market was investigated. A characteristic distribution of astaxanthin stereoisomers was detected for each pigment sources and such distribution was reproduced in the flesh of trout fed with that source.

Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources showed no significant differences. Similarly, different sources of pigment (natural or synthetic) produced colour values of fresh fillet with no relevant or significant differences. The coefficient of distance computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers was a useful tool to identify the origin of the pigment used on farm.

Introduction

Carotenoids are widely distributed in nature and present in many plants, algae, micro-organisms and animals. Although carotenoids are largely diffused in the plant and animal kingdoms, they are synthesized de novo only in higher plants and protists [START_REF] Goodwin | Distribution of carotenoids. Methods in Enzymology, Carotenoids, Part A[END_REF]. Consequently, the traceability of carotenoids in animal reflects the presence of their sources along with the food chain. Animal carotenoids mainly result from metabolic transformation (oxidation and/or reduction) of those present in the food chain. Aquatic animals constitute a considerable rich source of carotenoids, which are in many cases responsible of organoleptic characteristics considered by the consumer (bright colours in lobster, shrimp, salmon, fish eggs) [START_REF] Shahidi | Carotenoid pigments in seafoods and aquaculture[END_REF][START_REF] Matsumo | Aquatic animal carotenoids[END_REF]. Moreover, among the large number of pigments present in living organisms (i.e. chlorophylls, anthocyanins and porphyrins), carotenoids play an important role in protecting cells against photosensitised oxidation [START_REF] Palozza | Antioxidant effects of carotenoids in vivo and in vitro: an overview[END_REF].

In farmed salmonid, astaxanthin is mainly used for pigmentation and it is usually included in the feed as a synthetic product. However, other natural dietary sources of astaxanthin such as shrimp or krill wastes, algae meal or yeasts are also available on the market. All-trans astaxanthin may exists as three stereoisomers: two enantiomers (3R,3'R and 3S,3'S) and a meso form (3R,3'S; 3S,3'R). The distribution of the all-trans stereoisomers in natural astaxanthin differs from that of the synthetic product. This latter is a racemic mixture of three isomers, with a typical ratio of 1:2:1 (3S,3'S : 3R,3'S : 3R,3'R), while astaxanthin from natural sources has a Crustaceans belong to the class of marine animals that can oxidise carotenoids at the 3,3 ' and 4,4' position. Particularly,shrimp (Penaeus spp.) are able to convert by oxidative transformation dietary carotenoids such as ß-carotene, zeaxanthin and canthaxanthin into astaxanthin and they are also able to absorb astaxanthin (Matsumo 1992[START_REF] Schiedt | Metabolism of carotenoids and in vivo racemization of (3S,3'S)-astaxanthin in the crustacean Penaeus[END_REF][START_REF] Sachindra | Carotenoids in different body components of Indian shrimps[END_REF].

Similarly, in krill (Euphausia spp.) astaxanthin and its esters represent the major carotenoid component. A metabolic transformation to astaxanthin stereoisomers from other carotenoid sources is hypothesized as well in zooplanktonic crustaceans [START_REF] Takaichi | Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry[END_REF].

The red yeast Phaffia rhodozyma produces and accumulates carotenoids in lipid droplets into the cytoplasmatic membranes. [START_REF] Andrewes | Carotenoids of Phaffia rhodozyma a red pigmented fermenting yeast[END_REF] reported that free astaxanthin was the most abundant carotenoid in Phaffia with 83-87% of the total pigment mixture. Other carotenoids such as ß-carotene (2-2.5% of total carotenoids), echinenone (2-4% of total carotenoids), 3hydroxyechinenone (3-4.5% of total carotenoids) and phoenicoxanthin (5-7% of total carotenoids) were also isolated red yeast culture. The predominant optical isomer of Phaffia rhodozyma unesterified astaxanthin is the 3R,3'R form.

The Haematococcus pluvialis microalgae mainly contains astaxanthin monoesters linked to short chain fatty acids. The astaxanthin pool of encysted Haematococcus is approximately 70% monoesters, 25% diesters and 5% free (Lorenz and Cysewski 2000). All of free astaxanthin and its mono and di-esters in Haematococcus pluvialis have predominantly the 3S,3'S form. Since commercially grown Haematococcus pluvialis can accumulate >30 g astaxanthin of dry matter, the alga meal represents a good source of natural astaxanthin that found an increasing use in nutrition of farmed fish (Olaizola and Huntly 2003).

The distribution of astaxanthin stereoisomers has been investigated in different tissues of crustaceans (Renstrøm et al 1981, Coral-Hinostroza and[START_REF] Coral-Hinostroza | Astaxanthin from the red crab langostilla (Pleuroncodes planipes): optical R/S isomers and fatty acid moieties of astaxanthin esters[END_REF], trout (Oncorhynchus mykiss) [START_REF] Storebakken | Pigmentation of rainbow trout[END_REF][START_REF] Bjerkeng | Bioavailability of all-E-astaxanthin and Z-isomers of astaxanthin in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Østerlie | Accumulation of astaxanthin all-E, 9Z and 13Z geometrical isomers and 3 and 3' RS optical isomers in rainbow trout (Oncorhynchus mykiss) is selective[END_REF] and salmon (Oncorhynchus spp and Salmo salar) [START_REF] Arai | Pigmentation of juvenile coho salmon with carotenoid oil extracted from Antarctic krill[END_REF][START_REF] Turujman | Rapid liquid chromatographic method to distinguish wild salmon from aquacultures salmon fed synthetic astaxanthin[END_REF]. Astaxanthin deposition in white muscle of salmonid reflects the optical isomers ratio of dietary astaxanthin [START_REF] Foss | Carotenoids in diets for salmonids. I. Pigmentation of rainbow trout with the individual optical isomers of astaxanthin in comparison with canthaxanthin[END_REF][START_REF] Storebakken | Pigmentation of rainbow trout[END_REF][START_REF] Storebakken | Utilization of astaxanthin from yeast, Xanthophyllomyces dendrorhous, in rainbow trout, Oncorhynchus mykiss: effect of enzymatic cell wall disruption and feed extrusion temperature[END_REF]). On the contrary, the distribution of astaxanthin stereoisomers in skin and posterior kidney of rainbow trout has been observed to be different from that of dietary source, indicating some selectivity in the tissue metabolism of the optical isomers of astaxanthin. In particular Østerlie et al. (1999) found in rainbow trout fed synthetic astaxanthin an higher percentage of 3S,3'S isomer and a lower percentage of 3R,3'R isomer in kidney and skin, if compared to the percentage of optical isomers in the diet, suggesting a degree of epimerization of (3S,3'S)-astaxanthin in these tissues.

High performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin has been described in recent years by several authors [START_REF] Vecchi | Separation of (3S,3'S)-, (3R,3'R)-and (3S,3'R)-astaxanthin via (-)-camphanic acid esters[END_REF][START_REF] Maoka | Direct diastereomeric resolution of carotenoids: I. 3-hydroxy-4-ocyβ-end group[END_REF][START_REF] Turujman | Rapid direct resolution of the stereoisomers of all-trans astaxanthin on a Pirkle covalent L-leucine column[END_REF][START_REF] Turujman | Rapid liquid chromatographic method to distinguish wild salmon from aquacultures salmon fed synthetic astaxanthin[END_REF][START_REF] Østerlie | Accumulation of astaxanthin all-E, 9Z and 13Z geometrical isomers and 3 and 3' RS optical isomers in rainbow trout (Oncorhynchus mykiss) is selective[END_REF]. The separation of optical isomers could be achieved on an achiral column after derivatization with a suitable derivatizing reagent in order to obtain the diasteroisomers, that could be easily resolved on an achiral phase column. The derivatizing reagent more used for this purpose in literature is camphenic acid chloride [START_REF] Vecchi | Separation of (3S,3'S)-, (3R,3'R)-and (3S,3'R)-astaxanthin via (-)-camphanic acid esters[END_REF]Müller 1979, Bowen et al. 2002). Another approach is to perform direct HPLC analysis of underivatized astaxanthin on a chiral column. This latter method permits to [START_REF] Turujman | Rapid direct resolution of the stereoisomers of all-trans astaxanthin on a Pirkle covalent L-leucine column[END_REF][START_REF] Turujman | Rapid liquid chromatographic method to distinguish wild salmon from aquacultures salmon fed synthetic astaxanthin[END_REF]).

This study was designed in order to investigate the ratios of optical isomers of astaxanthin from different synthetic and natural sources used for rainbow trout pigmentation. Different ingredients such as yeasts of the genus Phaffia (Phaffia rhodozyma), Haematococcus pluvialis algae meal, Antarctic krill meal, krill oil and shrimp meal were analysed. Afterwards, with the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 60 days diets containing 60 mg kg -1 of astaxanthin from synthetic source, Haematococcus pluvialis algae meal and Phaffia rhodozyma yeast.

Moreover, in order to investigate the reliability of the optical isomers distribution to distinguish the type of pigmentation of commercially farmed trout available on the Italian market, nine specimens of pigmented rainbow trout farmed in different aquaculture plants of Northern Italy were also purchased in local retailers and analysed.

Materials and methods

Chemicals and reagents

Fish and experimental conditions

Three outdoor raceways (L=50 X W=4 X H=1.5) were stocked each with 1900 kg rainbow trout (average weight 350 g) and were reared under normal production scale for 60 days. During the trial, oxygen levels were always over 80% saturation and water temperature was 11±1 °C. Three pigmented diets were used in this study. Diet 1 contained Carophyll Pink (CP), diet 2 contained Haematococcus pluvialis algae meal (HP) and diet 3 contained Phaffia rhodozyma red yeast (PR). The level of inclusion in all three diets was 60 mg kg -1 astaxanthin (free or esterified). The three groups of fish were fed with commercial extruded feed (41% protein, 26% fat) coated with different synthetic source. The feed ratio was set at 1% of the biomass per day. No mortalities occurred during the experiment. At the end of the trial, ten fish per group were taken and transported to the laboratory for colour measurements and astaxanthin analysis. Parallelly, nine specimens of pigmented rainbow trout from unknown origin were purchased from local retailers and analysed.

Astaxanthin extraction from feed ingredients

Carotenoids from algae and yeast pigments were extracted by suspending 25 mg of sample in 5 mL of dimethyl sulfoxyde (DMSO) and incubating in a thermostatic shaking bath at 50°C for 30 minutes. At the end, the sample was centrifuged at 4000 rpm for 10 minutes and the supernatant collected. The obtained pellet was homogenized for 2 min using an UltraTurrax (Janke & Kunkel, IKA, Staufen, Germany) in 5 mL of acetone, then centrifuged at 4000 rpm for 15 minutes. Acetone extraction was repeated until the organic phase resulted colourless. Similarly, acetone extraction was used with Antarctic krill meal, krill oil, shrimp meal and with synthetic astaxanthin source. To avoid oxidation and isomerisation of astaxanthin, all manipulations were performed in diffuse light and conditioned room (20°C), under acid-free conditions and using pure peroxide-free solvents (Liaaen-Jensen 1971).

Enzymatic hydrolysis of Haematococcus pluvialis extract

ThreemL of Haematoccoccus acetone extract was added to 2 mL of Tris HCl (0.05 M, pH 7.00) were added and put in a thermostatic bath at 37°C for 2 minutes. Then, 100 µL of cholesterol esterase solution (50 unit/mL) were added and incubated in a water bath at 37°C for 30 minutes with gently shaking. After the incubation 1.0 g of sodium sulphate decahydrate and 2 mL of petroleum ether were added and mixed vigorously by vortex. The upper petroleum ether phase was collected in a clean tube and the procedure repeated two times to make the organic phase clear. Petroleum ether phase containing astaxanthin was added with sodium sulphate anhydrous mixing by vortex for 30 seconds and the ethereal phase transferred in a clean tube, then dried under a stream of nitrogen and reconstituted in 200 µL of hexane. . Two measurements were taken on each site and the measuring head was rotated 90° between each measurement. The left fillet was packed under vacuum and stored at -20°C until astaxanthin analysis, which was performed within a few days.

Astaxanthin extraction from fish fillets

Astaxanthin was extracted from flesh according to the method of [START_REF] Turujman | Rapid liquid chromatographic method to distinguish wild salmon from aquacultures salmon fed synthetic astaxanthin[END_REF].

Briefly, 10 g of tissue were sampled at the centre of the fillet and homogenized using an Ultra

Turrax for 2 min with 10x2 mL of hexane, to remove most of lipids. The homogenate was centrifuged at 4000 rpm for 10 minutes and hexane phase was discharged. Astaxanthin was extracted from the pellet resulted after hexane extraction with 10x2 mL of acetone, following the procedure mentioned above. The acetone phase was evaporated with a rotary evaporator (Rotavapor R 110, Büchi, Switzerland) and the aqueous residue (approximately 4 mL) reconstituted in 20 mL of methylene chloride and vigorous swirled to dissolve astaxanthin. The residual water was removed with a separatory funnel and the organic phase was dried over approximately 1 g of anhydrous sodium sulfate. The methylene chloride phase containing astaxanthin was transferred in a clean tube. The amount of astaxanthin extracted was determined

HPLC analysis of astaxanthin

The astaxanthin was purified by loading the methylene chloride phase onto a silica gel Sep-Pak cartridge (Waters, Milford, MA, USA) pretreated with hexane. Astaxanthin was eluted from the cartridge with 5 mL of chloroform and the eluate dried under a stream of nitrogen. Dry residue was dissolved in 200 µL of hexane and 20 µL were injected into the HPLC system.

The HPLC system consisted of a Jasco model 980-PU pump (Ishikawa-cho, Japan), a Jasco model LG-980-02 gradient unit, a Jasco model AS-950 autosampler and a Waters model 996

Photodiode Array Detector (Waters, Milford, MA, USA). Chromatograms were recorded and integrated on a Millennium 32 Chromatography Software (Waters, Milford, MA, USA).

Analyses were performed with a Pirkle L-leucine chiral column (Regis Technologies, Morton Grove, IL, USA). Mobile phase was hexane-tetrahydrofuran-triethylamine-acetonitrile (77:19:2:2 v/v) at a flow rate of 1.5 mL min -1 . Isocratic condition and ambient temperature were used, and the monitoring wavelength was 474 nm. In these conditions the retention times of the 3S,3'S, meso and 3R,3'R forms were approximately 10.5, 11.5 and 12.5 minutes, respectively.

Each astaxanthin extract was analyzed in duplicate.

Statistical analysis

Data are reported as mean value ± standard error of the mean. Homogeneity of variance was confirmed and comparison between means was by one-way ANOVA. Significance was accepted at probabilities of 0.05 or less. and "test" samples (pigment source and trout fillet, respectively) was computed.

This coefficient was originally developed to estimate the similitude of aquatic animals on the base of their fatty acids composition. Therefore, the smaller the value of the coefficient of distance D the higher the probability that trout have been fed with a diet containing that specific pigment source.

For comparison of the whole astaxanthin stereoisomers profile of "reference" (feed ingredient) and "test" (trout fillet), the coefficient of distance D [START_REF] Mcintire | Fatty acid in lotic periphyton: another measure of community structure[END_REF] was computed using the following equation:

( )       - ∑ = = n i ih ij P P Djh 1 2 2 / 1
where, D jh is the degree of difference (coefficient of distance) between samples j ("reference" diet) and h ("test" trout fillet), P ij and P ih are percentage of astaxanthin stereoisomers i in sample j and h, for each i stereoisomers. All the statistical analysis were performed by SPSS 12.0 (SPSS Inc. Chicago, Illinois).

Results and Discussion

In Figure 1 the chromatograms of a standard solution (32 µg mL -1 ) of syntetic astaxanthin and of the extracts obtained from different sources are presented. The three peaks corresponding to 3S,3'S isomer, meso form and 3R,3'R isomer were eluted at 10.5, 11.5 and 12.6 minutes, respectively. Haematococcus pluvialis algae meal contained 87.6% of the 3S,3'S enantiomer, 10.1% of the meso form and 2.3% of the 3R,3'R enantiomer, while the red yeast Phaffia rhodozyma contained 1.5% of the meso form and 98.5% of the 3R,3'R enantiomer. The values of optical isomers in krill meal and krill oil were similar. In fact, 27.4% of 3S,3'S enantiomer, 21.8% of the meso form and 50.8% of 3R,3'R enantiomer were found in the krill meal whilst 35.8% of 3S,3'S enantiomer, 13.6% of the meso form and 50.6% of 3R,3'R enantiomer were found in krill oil.

On the other hand, shrimps meal contained 65.4% of 3S,3'S enantiomer, 27.9% of the meso form and 6.7% of 3R,3'R enantiomer. These results were consistent with literature values. In particular, [START_REF] Foss | Natural occurence of enantiomeric and meso astaxanthin: 7. Crustaceans including zooplankton[END_REF] analysed the content of astaxanthin stereoisomers in several species of crustaceans and demonstated that in Euphausia superba (krill) the 3R,3'R isomer predominated, whereas in Thysanoessa inermis and in Calanun finmarchicus, the 3S,3'S isomer was dominant. These latter two small crustaceans are important ingredient of diets of wilds salmonids, and their isomeric composition has been demonstrated similar to the isomeric composition encountered in wild salmon, in which the S-form predominated [START_REF] Turujman | Rapid liquid chromatographic method to distinguish wild salmon from aquacultures salmon fed synthetic astaxanthin[END_REF]. Astaxanthin isomer ratios in the flesh of fish fed different carotenoid sources is reported in Table 1. Dietary treatment influenced significantly the distribution of the optical isomers in the fillets, and the average values of the optical isomers in the flesh reflected the isomeric ratio of the dietary astaxanthin source. The astaxanthin isomers distribution in microalgae and in the corresponding trout flesh were similar and ranged from 80.0% to 87.6%, from 11.7% to 10.1% and from 2.4% to 8.3% for the 3S,3'S isomer, meso form and 3R,3'R isomer, respectively. In the 13 red yeast and in the corresponding trout flesh the 3R,3'R isomer predominated, ranging from 98.5% to 74.9%, while the meso form and the 3S,3'S isomer ranged from 1.5% to 10.7% and from 0% to 14.4%, respectively. In the flesh of trout fed synthetic astaxanthin, the averages of the distribution of optical isomer were 29.6%, 48.4% and 22.0% for the 3S,3'S isomer, meso form and 3R,3'R isomer, respectively. Also in this case the distribution resembles that of the diet and these results were in ageement with those of Østerlie et al. (1999), who found in rainbow trout fed syntetic astaxanthin a distribution of isomers in muscle of 28.5%, 49.0% and 22.5% for 3S,3'S, meso and 3R,3'R isomers, respectively. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources are presented in table 2. No significant differences were registered in different parts of the flesh. Similarly, different sources of pigment (natural or synthetic), used with the three different diets, produced colour values of fresh fillet (table 3) with no significant differences, although there was a tendency for the CP diet to give an higher value of redness (a*) and brightness (C*) when compared to HP diet. This confirms the better pigmenting efficacy of free astaxanthin in comparison with esterified astaxanthin from microalgae. [START_REF] White | The degree of carotenoid esterification influences the absorption of astaxanthin in rainbow trout, Oncorhynchus mykiss (Walbaum)[END_REF].

Among the specimens purchased on the market (table 4), two samples showed the algal astaxhantin isomeric profile (trout 3 and 4), and three (trout 2, 5, 7) were found to contain a different pigment source that on the basis of its retention time and UV/VIS absorption spectra was reasonably recognized as canthaxanthin. Three fish samples showed to contain only synthetic astaxanthin (trout 1, 8, 9) and one (trout 6) both synthetic carotenoids: astaxanthin and canthaxantin.

The coefficient of distance (table 5) computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers is a simple geometrical tool which can be useful to tentatively identify the origin of the pigment used on farm. The smallest D values registered in trout fed the Carophyll pink and trout fed microalgae were 5.4 and 9.8 when computed using the synthetic astaxanthin and the microalgae as "reference", respectively. The other D values, computed using the astaxanthin stereoisomers composition of the other analysed ingredients, were considerably higher. In trout fed the read yeast, D value (29.1) was similar to those calculated for krill meal (29.5) and krill oil (32.5).

This information suggests that the coefficient of distance can be used as a general tool to explain pigment source only amongst some sources and it is particularly useful to distinguish amongst synthetic and natural sources whilst is not sensitive enough to distinguish amongst different natural astaxanthin sources, or amongst mixtures of synthetic and natural sources.

Using the coefficient of distance to estimate the potential origin of the astaxanthin found in the market purchased trout, it is possible to point out that samples trout 1, trout 8 and trout 9 were with high probability fed with a diet containing a synthetic source of astaxanthin.

At the best of our knowledge, this is the first time the coefficient of distance was used as a tool for the identification of the astaxanthin origin in salmonids and further information, particularly as regard the inter-and intra-population variability and the dose-response effects are requested.

A U 0 ,0 0 0 ,0 2 0 ,0 4 0 ,0 6 0 ,0 8 0 ,1 0 0 ,1 2 0 ,1 4 2 ,0 0 4 ,0 0 6 ,0 0 8 ,0 0 1 0 ,0 1 2 ,0 1 4 ,0 1 6 ,0 1 8 ,0 In bold font, the actual coefficient of distance amongst pigment source and trout fillet of the known samples; in italics the possible coefficient of distance amongst pigment source and trout fillet of the unknown samples.

  the isomers deriving from the different biological organism that synthesised it.

  encountered during the derivatization step and to reduce the analysis time to less then 15 minutes

  Standard astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'dione) was a gift from Hoffmann-La-Roche Inc. (Basel, Switzerland).Hexane, tetrahydrofuran (THF), methylene chloride, 2-propanol, acetonitrile, acetone, petroleum ether were LC grade and from Merck (Darmstadt, Germany); dimethyl sulfoxide (DMSO), triethylamine, sodium sulphate decahydrate and sodium sulphate anhydrous supplied by Sigma-Aldrich (St. Louis, MO, USA). Cholesterol esterase from Pseudomonas fluorescens of carotenoids esters was purchased from Sigma (St. Louis, MO, USA). Sep-Pak silica gel cartridges were from Waters (Milford, MA, USA).

  Feed ingredientsUnesterified astaxanthin (Carophyll ® pink) and canthaxanthin (Carophyll ® red) were from Hoffmann-La-Roche Inc. (Basel, Switzerland), microalgae meal from Haematococcus pluvialis (Naturose ® ) purchased from Cyanotech Corporation (Kailua-Koma, HI, USA) and red yeast meal (Astaxin ® ) (Phaffia rhodozyma) was from Igene Biotecnology Inc. (Columbia, MD, USA). Antarctic krill meal, krill oil and shrimp meal were supplied by Tepual SA (Santiago, Chile).

  Colour measurements in filletThe fish were decapitated, gutted, filleted and the right fillet used for colour analysis. Colour measurements of the flesh samples were performed in the following sites: posterior to the head (neck), at the center below the dorsal fin (center) and anterior to the caudal fin (tail). Colour was measured using a Minolta Chroma Meter II Reflectance instrument (Minolta Camera Co. Ltd., Osaka, Japan). Results were recorded as a*, b*, L* values where a* describes redness, b* yellowness and L* lightness. Chroma values (brightness, C*) were calculated according to the following equation: C*

  and measuring the absorbance of the methylene chloride extract a 474 nm using a Jasco model V-530 spectrophotometer (Ishikawa-cho, Japan).

  whole astaxanthin stereoisomers profile of trout fillet and the pigment ingredients of the diet, the coefficient of distance D[START_REF] Mcintire | Fatty acid in lotic periphyton: another measure of community structure[END_REF] between "reference"

  Figure 2. A characteristic distribution of astaxanthin optical isomers was detected for each

Figure 1 :

 1 Figure 1: Chromatograms of stereoisomers composition of all-trans astaxanthin of: (a) a standard solution of syntetic astaxanthin (32 µg mL -1 ); (b) an Haematococcus pluvialis extract; (c) a Phaffia rhodozyma extract. HPLC conditions: Pirkle L-leucine chiral column; mobile phase hexane-tetrahydrofuran-triethylamine-acetonitrile (77:19:2:2 v/v); flow rate of 1mL min -1 ; monitoring wavelength 474 nm.

Table 1 .

 1 Percentage distribution of astaxanthin stereoisomers in fillet of rainbow trout fed with different pigment sources (means±s.e.)

							12,503
							(3R ,3'R )
						11,450 (m eso)
	2,00	4,00	6,00	8,00	10,00	12,00	14,00	16,00	18,00

Table 2 .

 2 Colour values measured in three different part of fillet of rainbow trout fed with different pigment sources(means±s.d., n=10)

			Food Additives and Contaminants	Page 22 of 25
	Color value	Neck	Center	Tail	P 1
	a*	14.8±0.74	14.5±0.66	15.8±0.33	0.28
	b*	18.9±1.13	17.3±1.17	19.3±0.87	0.40
	L*	44.8±1.31	42.3±1.42	43.8±1.60	0.48
	C*	24.1±1.31	22.6±1.28	25.0±0.86	0.37
	1 Significance accepted at probabilities of 0.05 or less

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Table 3 .

 3 Colour values measured in fillet of rainbow trout fed with different pigment sources(means±s.d., n=10)

	Page 23 of 25		Food Additives and Contaminants
	Color value	Synthetic astaxanthin	Phaffia rhodozyma	Haematococcus pluvialis	P 1
	a*	15.3±0.58	15.7±0.56	14.2±0.64	0.19
	b*	18.7±1.06	18.6±1.04	18.2±1.20	0.95
	L*	42.9±1.40	43.4±1.50	44.7±1.50	0.64
	C*	24.1±1.18	24.4±1.14	23.1±1.28	0.74
	1 Significance accepted at probabilities of 0.05 or less

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Table 4 .

 4 Percentage distribution of astaxanthin stereoisomers in flesh of rainbow trout purchased on the market.

	Samples	3S,3'S enantiomer (%)	3R,3'S meso (%)	3R,3'R enantiomer (%)	Presence canthaxanthin	of
	Trout 1	30.7	48.0	21.3	no
	Trout 2	nd	nd	nd	yes
	Trout 3	71.2	19.9	8.9	no
	Trout 4	68.4	21.8	9.8	no
	Trout 5	nd	nd	nd	yes
	Trout 6	40.5	26.9	32.6	yes
	Trout 7	nd	nd	nd	yes
	Trout 8	28.2	49.1	22.7	no
	Trout 9	29.8	48.3	21.9	no
	nd = not detected

Table 5 .

 5 The coefficient of distance (D value) of the astaxanthin stereoisomers amongst known and unknown trout samples and the analysed astaxanthin sources

		Synthetic astaxanthin	Microalage (H.pluvialis)	Red yeast (X. dendrorhous)	Antartic Krill meal	Krill oil	Shrimps meal
	Trout fed Carophyll® pink	5.4	72.2	94.5	39.3	45.5	44.0
	Trout fed
	microalagae	69.1	9.8	121.0	68.4	61.2	21.9
	(H.plivualis)
	Trout fed red yeast (X. dendrorhous)	65.3	103.0	29.1	29.5	32.5	86.9
	Trout 1	6.8	70.9	95.2	39.6	45.5	42.7
	Trout 3	57.4	20.2	115.9	60.6	55.1	10.1
	Trout 4	53.9	23.7	113.8	58.0	52.9	7.5
	Trout 6	29.4	58.4	81.4	23.0	22.9	35.9
	Trout 8	3.7	73.9	93.8	39.2	45.8	45.7
	Trout 9	5.7	72.0	94.6	39.3	45.4	43.8

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Tomasz ; Borkowski

Keywords: broccoli, polyphenols, vitamin C, carotenoids, tocopherols, glucosinolates, 16 antioxidant activity, domestic processing 17 18

The effect of water-and steam-cooking on the content of vitamin C, polyphenols, carotenoids, 2 tocopherols and glucosinolates as well as on the antioxidant activity of broccoli are reported. 3 Flavonoids, phenolic acids, vitamin C and E, β-carotene, lutein, and glucosinolates in 4 domestically processed broccoli were quantified using HPLC methods; total polyphenols 5 were determined with Folin-Ciocalteu reagent. The antioxidant capacity of broccoli extracts 6 were evaluated using the Trolox Equivalent Antioxidant Capacity (TEAC) and 2,2-diphenyl-7 1-picrylhydrazyl (DPPH) methods. The results indicated that steam-cooking of broccoli 8 results in an increase in polyphenols, as well as the main glucosinolates and their total content 9 as compared to fresh broccoli, whereas cooking in water has opposite effect. Steam cooking 10 of broccoli has no influence on vitamin C, whereas cooking in water significantly lowers its 11 content. Both, water-and steam-cooking of broccoli results in an increase in β-carotene, 12 lutein, αand γ-tocopherols as compared to fresh broccoli. Similar effects of steaming and 13 water-cooking of broccoli on their antioxidant activity were observed. 14 15

PAWLAK-LEMAŃSKA 1 , JAROSŁAW CHMIELEWSKI 1 , TOMASZ BORKOWSKI 1

6

& BOśENA TYRAKOWSKA 1 Epidemiological data show that a diet rich in cruciferous vegetables such as broccoli, Brussels 3 sprouts, cabbage, cauliflower and kale can significantly reduce the risk of certain forms of 4 cancer [START_REF] Kohlmeier | Cruciferous vegetable consumption and colorectal cancer risk: 31 meta-analysis of the epidemiological evidence[END_REF]). The mechanism underlying the reduction of cancer by 5 cruciferous vegetables is not clear, however, it is well known that these vegetables are rich in 6 different health-promoting compounds including the antioxidant vitamin C and E, 7 polyphenols, glucosinolates, carotenoids and minerals. 8 9 Polyphenols are a large group of compounds, which includes flavonoids and phenolic acids. 10

Recently, polyphenols are of increased scientific interest because of their protective effects 11 against cardiovascular, photosensitivity-related diseases, aging, and various forms of cancer. 12 They may act as antioxidants or as agents in other mechanisms contributing to 13 cardioprotective or anticarcinogenic effects (Middleton et al. 1994[START_REF] Samman | Flavonoids and coronary heart 18 disease:dietary perspectives. Flavonoids in health and disease[END_REF]. 14 15 Another health-promoting compound with antioxidant activity is ascorbic acid. Vitamin C is 16 considered a most important water-soluble antioxidant present in the extracellular and 17 intracellular spaces in most biological systems where it can participate in redox reactions. It 18 can also directly scavenge superoxide radical, singlet oxygen, hydrogen peroxide and 19 hydroxyl radical. It is considered that the main contribution of vitamin C as a lipid 20 peroxidation chain-breaking agent is its ability to regenerate membrane-bound oxidised 21 vitamin E [START_REF] Kaur | Antioxidants in fruits and vegetables -the millennium's 22 health[END_REF]. [START_REF] Staack | A comparison of the individual 23 and collective effects of four glucosinolate breakdown products from Brussels sprouts on 24 induction of detoxification enzymes[END_REF] 23 Also carotenoids posses a range of important biological activities. Some carotenoids e.g. α-24 carotene, β-carotene and β-cryptoxanthine posses pro-vitamin A activity and they are 25 converted to retinal by mammals. Lutein and zeaxanthin are known to provide protection 26 against age-related macular degeneration, mediated by their ability to quench single oxygen 27 and blue light in the retina [START_REF] Landrum | Lutein, zeaxanthin and the macular pigment[END_REF]. Carotenoids are also potent antioxidants 28 and free radical scavengers, playing a role in the prevention of coronary heart disease 29 [START_REF] Kritchevsky | β-Carotene, carotenoids and the prevention of coronary heart 33 disease[END_REF]) and in the reduction of risk of developing lung cancer [START_REF] Block | Fruit, vegetables and cancer prevention: a review 10 of epidemiological evidence[END_REF]. 30 31 Carotenoids are biosynthetically related to other secondary metabolites such as tocopherols. 32 Vitamin E is well accepted as the most effective natural lipid-soluble antioxidant protecting 33 biological membranes, lipoproteins and fat deposit from peroxidative damage. It has been 34 suggested that it helps to protect against cancer induced by free-radical-generating 1 contaminants such as ozone or nitrogen dioxide. Reduction in the risk of coronary heart 2 diseases associated with a high intake of vitamin E has also been indicated [START_REF] Janero | Ischemic heart disease and antioxidants: mechanistic aspects of oxidative 20 injury and its prevention[END_REF], 3 Duell 1996). 4 5 Glucosinolates are a group of natural compounds present in especially cruciferous vegetables. 6

They are responsible for the pungent odour and biting taste of these vegetables. Recently, 7 glucosinolates are of great scientific interest because of their potential protection against the 8 lung, stomach and colon cancers [START_REF] Murillo | Cruciferous vegetables and cancer prevention[END_REF]. Glucosinolates themselves exhibit 9 minimal anticancer activity, however on chopping, crushing, or chewing the enzyme 10 myrosinase is released and it converts glucosinolates to isothiocyanates, thiocyanates, nitriles, 11 and the number of indolic compounds, including: indole-3-carbinol and products of its 12 oligomerisation, ascorbigens, as well as indole-3-acetonitrile and indole-3-acetic acid 13 [START_REF] Preobrazhenskaya | Ascorbigen 33 and other indole-derived compounds from Brassica vegetables and their analogs as 34 Page 18 of 30[END_REF][START_REF] Bjergegaard | 2 Glucosinolates and their transformation products -compounds with a broad biological 3 activity[END_REF]). Some of these products have been 14

reported to protect cells against cancer [START_REF] Faulkner | Selective increase of the potential 7 anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli[END_REF]). Several mechanisms have been 15

proposed for cancer prevention by hydrolysis products of glucosinolates, however the most 16 frequently indicated is induction of phase II enzymes, including quinone reductase and 17 glutathione-S-transferase, which protect against carcinogens and other toxic electrophiles 18 (Zhang et al. 1992, Williamson et al. 1997) 19 20 It is known that processing may affect, to a significant extent, the concentration and biological 21 activities of different compounds present in plants. This aspect seems to be very important 22 taking into account that only some vegetables are consumed in a raw state and most of them 23 are processed before consumption. Broccoli belongs to vegetables that are usually heat-treated 24 before eating thus it is important to know, which type of domestic processing is the best for 25 preserving health-promoting compounds present in this vegetable. To our knowledge, there is 26 no report discussing the changes in the contents of various groups of health-promoting 27 compounds in broccoli as an effect of different cooking methods. The literature data on the 28 changes in the content of different compounds in broccoli upon domestic processing concern 29 mostly one (e.g. carotenoids) or two (e.g. glucosinolates and vitamin C), rarely three different 30 groups of compounds [START_REF] Lessin | Quantification of cis-trans isomers of 12 provitamin A carotenoids in fresh and processed fruits and vegetables[END_REF][START_REF] Price | Composition and content 3 of flavonol glycosides in broccoli florets (Brassica oleracea) and their fate during cooking[END_REF], Vallejo et al. 2002, Zhang et al. 31 2004[START_REF] Turkmen | The effect of cooking methods on total phenolics 29 and antioxidant activity of selected green vegetables[END_REF]. Thus, the present study was undertaken to evaluate the effect of 32 domestic processing such as water-and steam-cooking on the contents of polyphenols, 33 vitamin C and E, carotenoids and glucosinolates in broccoli as well as on its antioxidant 34 isorhamnetin from Extrasynthese, (Genay, France) were used for the study. HPLC-grade 13 solvents and analytical grade reagents (or better) were used for all purposes. 14 15

Sample preparation 16

Broccoli of the Lord cultivar planted in April and harvested at optimal maturity in June 2004 17 was purchased from local farm less than 24 hours before sample preparation. The florets were 18 separated from the main stem and cut into pieces. Two 300-g portions of the florets of 19 broccoli were cooked in a food steamer (model PokyStreamer) for 10 min under atmospheric 20 pressure and in 1000 ml of boiling water for 5 min and then appropriate samples were 21 combined. Samples were allowed to stand at room temperature, under open air, for 15 min. 22 Subsamples of fresh, steamed and water-cooked broccoli were taken and immediately 23 analysed for vitamin C content. The rest of plant material was immersed in liquid nitrogen, 24 freeze-dried and dry weight was determined. The dry weight of fresh and processed broccoli 25 was found to be not significantly different. Water after broccoli cooking was also collected. 26 All samples were stored at -20 o C under nitrogen generally no longer than two weeks until 27 analysis. Before analyses, lyophilized samples were grounded into a powder. 28 29

Extraction and determination of polyphenols 30

Polyphenols were extracted twice from 2 g of the dry powder (in triplicate) with 70% 31 methanol (50 ml) in ultrasonic bath for 15 min. The combined fractions were filtered, 32 evaporated in vacuum at 40 o C to approximately 20 ml and made up to 25 ml with water (Price 33 kaempferol and isorhamnetin O-glycosides were determined after their acid hydrolysis to 2 quercetin, kaempferol and isorhamnetin. Alkaline hydrolysis was done to determine 3 conjugated phenolic acids. For the determination of flavonols and phenolic acids after acid 4 and alkaline hydrolysis, polyphenols were extracted with 70% methanol containing TBHQ 5 (0.2%). Acid and alkaline hydrolysis of polyphenols was performed as described in literature 6 with minor modifications [START_REF] Vallejo | Characterisation of flavonols in 31 broccoli (Brassica oleracea L. var. italica) by liquid chromatography-UV diode-array 32 detection-electrospray ionisation mass spectrometry[END_REF]). Acid hydrolysis was performed by adding 1 7 ml 4N HCl to 1 ml of phenolic extract and this solution was incubated in a stoppered test tube 8 for 2 hours at 85 o C. Before HPLC analysis, all samples were centrifuged at 12.000 g for 5 9 min. Alkaline hydrolysis was carried out by adding 1 ml 4N NaOH to 1 ml of polyphenolic 10 extract and keeping the mixture for 20 h at room temperature in a stoppered test tube under N 2 11 atmosphere. After this time, the alkaline hydrolysis products were acidified with concentrated 12

HCl down to pH 1-2 and directly analysed using HPLC. 13 14 HPLC analyses were performed at room temperature on Waters 600 high performance liquid 15 chromatograph equipped with Symmetry C 18 column (3.9 x 150 mm, 5 µm, Waters, Millford, 16 Ma, USA) protected with a Symmetry C 18 guard column. Mobile phase, published by Crozier 17 et al. (1997), of acetonitrile (A) and water adjusted to pH 2.5 with trifluoroacetic acid (B) was 18 used according to the modified gradient: linear increment starting with 10% A to 26% A in 20 19 min, isocratic 26% A in 5 min, linear gradient from 26% A to 35% A in the next 5 min, and 20 the return to the initial conditions within the next 10 min with flow rate 1 ml/min. Flavonoids 21 and phenolic acids were detected using a Waters 996 photodiode-array detector set at 370 nm 22 and 320 nm, respectively. Quercetin, kaempferol, isorhamnetin, caffeic, p-coumaric, 23 ferulic/sinapic acids were identified on the basis of their absorption spectra and retention 24 times compared with those of standards. Quantification of flavonoids and phenolic acids was 25 done using calibration curve prepared for quercetin and sinapic acid, respectively. 26 27

Determination of total polyphenols 28

Total polyphenols were determined by the Folin-Ciocalteu method [START_REF] Singleton | Colorimetry of total phenolics with phosphomolybdic-21 phosphotungstic acid reagents[END_REF]) 29 using gallic acid as the standard. The results are expressed in mg of gallic acid equivalents per 30 100 g of dry and fresh weight of broccoli. 31

Extraction and determination of vitamin C 1

Extraction of vitamin C was performed essentially as described by [START_REF] Kurilich | Carotene. Tocopherol. And ascorbate contents in 2 subspecies of Brassica oleracea[END_REF]. 2 Briefly, 0.500 g of broccoli was extracted (in triplicate) with 2 ml of 1% meta-phosphoric 3 acid in ultrasonic bath for 15 min. Then, additional 1 ml of meta-phosphoric acid was added, 4 the mixture was sonicated for the next 15 min and made up to 5 ml with meta-phosphoric 5 acid. Samples were centrifuged at 600 g for 20 min. The supernatant (0.2 ml) and 5% DTT 6 (0.2 ml) were mixed and diluted to 2 ml with water. 7 8

The determination of vitamin C, using HPLC method, was performed as described previously 9

(Gliszczyńska-Świgło et al. 2003). Waters 600 high performance liquid chromatograph 10 (Waters, Millford, Ma, USA) equipped with LiChrospher C 18 (3.9 x 250 mm, 5µm, Merck, 11 Darmstadt, Germany) fitted with the same guard column was applied. A gradient of mobile 12 phase consisting of methanol (solvent A) and 5 mM KH 2 PO 4 pH 2.65 (solvent B) was used 13 according to the following gradient: linear increment starting with 5% A to 22% A in 6 min 14 and the return to the initial conditions within next 9 min with the flow rate 0.8 ml/min. The 15 eluate was detected using a Waters 996 photodiode-array detector set at 245 nm. Vitamin C 16 was identified by comparing its UV spectrum and retention time with that of standard. 17 Quantification of vitamin C was done using the external standard method. 18 19

Extraction and determination of carotenoids and tocopherols 20

Carotenoids and tocopherols were extracted according to the method described in Polish 21 Norm (PN-90/A-75101/12) with one minor modification: n-hexane was used instead of 22 acetone and n-hexane. Briefly, 1.000 g of dry material was extracted with 50 ml portion of n-23 hexane until the resulting extract was colourless. The fractions were combined and, if 24 necessary, dried using anhydrous Na 2 SO 4 . For simultaneous HPLC analysis of β-carotene, 25 lutein, αand γ-tocopherols, n-hexane extracts were evaporated in vacuum at 30 o C to the 26 dryness and dissolved in 2 ml of tetrahydrofuran. HPLC analyses were performed at room 27 temperature on Waters 600 high-performance liquid chromatograph (Waters, Millford, Ma, 28 USA) equipped with Lichrospher C 18 column (3.9 x 250 mm, 5µm, Merck, Darmstadt, 29 Germany) fitted with the same guard column. The mobile phase published by Kurilich et al. 30 (1999) was a combination of acetonitrile/methanol/tetrahydrofuran at 52:40:8 (v/v/v). Flow 31 rate was 1.5 ml/min to 7 min and then 2 ml/min to 20 min. β-Carotene and lutein were 32 detected using a Waters 996 photodiode-array detector set at 450 nm. For determination of α-33 325 nm with an excitation at 295 nm was used. Emission slit width was 10 nm, fluorometer 2 gain 100, and attenuation 1. Carotenoids and tocopherols were identified by comparing their 3 retention times with those of corresponding standards and by the spiking of samples with 4 appropriate standard. Additionally, photodiode-array detector was used to identify the 5 compounds on the basis of their absorption spectra. Quantification of carotenoids and 6 tocopherols was done using the external standard method. The content of carotenoids was 7 expressed as milligram β-carotene equivalents per 100 g of dry and fresh weight. 8 9

Extraction and determination of glucosinolates 10

Glucosinolates were analyzed by HPLC following enzymatic desulphatation according to the 11 Official Journal of European Communities (1990) as described earlier by [START_REF] Ciska | Content of glucosinolates in 24 cruciferous vegetables grown at the same site for two years under different climatic 25 conditions[END_REF]. 12 Individual glucosinolates were identified by comparing the retention times with those of 13 standards or on the basis of available literature data for glucoraphanin [START_REF] Kushad | Variation of glucosinolates in vegetable crops of Brassica oleracea[END_REF]). 14 Quantification of glucosinolates was based on the internal standard (glucotropaeolin) and 15 relevant relative response factors (Official Journal of European Communities 1990). [START_REF] Chandler | Isomerization and losses of trans-β-carotene in sweet 17 potatoes as affected by processing treatments[END_REF] 17

Determination of antioxidant capacity of broccoli extracts 18

The Trolox Equivalent Antioxidant Capacities (TEAC) of different broccoli extracts 19 (containing vitamin C, polyphenols and carotenoids together with tocopherols) to scavenge 20 ABTS •+ radical cation were determined by the method of Re et al. (1999). Briefly, ABTS was 21 dissolved in water. ABTS •+ was generated by the reaction of ABTS stock solution with 22 potassium persulfate (final concentration of ABTS and potassium persulfate was 7 mM and 23 2.45 mM, respectively). The mixture was allowed to stand in the dark at room temperature for 24 12-16 h before use. For the study of extracts, the ABTS •+ solution was diluted with methanol 25 to an absorbance of about 0.8 at 734 nm. Extracts and Trolox  were added as 1% (v/v) 26 solutions of 100 times concentrated stock solutions to give the final concentrations required. [START_REF] Kim | Vitamin C equivalent antioxidant 28 capacity (VCEAC) of phenolic phytochemicals[END_REF] For each experiment, solvent blank was run. The decrease in absorbance caused by extract, 28 measured at 6 min, reflects the ABTS •+ radical cation scavenging capacity and was plotted 29 against the concentration of the broccoli in extract. The TEAC value (in mmol Trolox/100 g 30 of dry and fresh weight) represents the ratio of the slope of the linear plot for scavenging of 31 ABTS •+ radical cation by the extract to the slope of the plot for ABTS

Statistical analysis of data 16

Data are presented as mean ± SD of at least triplicate experiments. Analysis of variance was 17 performed on the data obtained. Significance of differences between means was determined 18 by least significant differences (LSD) at P<0.05. 19

20

Results and discussion 21

Distribution of glucosinolates, polyphenols, vitamin C, carotenoids and tocopherols in fresh 22

and processed broccoli. 23 Table I lists 8 aliphatic, 4 indole and 1 aralkyl glucosinolates identified in fresh, water-cooked 24 and steamed broccoli The results of quantitative analysis show the differences in the content 25 of predominant and total glucosinolates present in fresh, steamed and water-cooked broccoli. 26 The predominant glucosinolates in fresh, water-cooked and steamed broccoli are 27 glucoraphanin, glucobrassicin, neoglucobrassicin and glucoiberin. Glucoraphanin and 28 glucobrassicin contents in fresh broccoli determined in this study are similar to those 29 previously reported by [START_REF] Kushad | Variation of glucosinolates in vegetable crops of Brassica oleracea[END_REF]. 30

Table I 31

The main glucosinolate in broccoli is glucoraphanin, frequently making up greater than 50% 32 of the total glucosinolates. In this study, glucoraphanin constitutes about 60% of total 33 glucosinolate content in fresh as well as in processed broccoli. Enzyme or acid hydrolysis of 1 glucoraphanin yields sulphoraphane (4-methylsulphinylbutyl isothiocyanate), which has been 2

shown to reduce the incidence of a number of tumors in various experimental models, both in 3 vivo in animals and in vitro in cell cultures due to induction of quinone reductase and 4 glutathione-S-transferase -phase II detoxification enzymes (Zhang et al. 1992). The 5 contributions of glucoiberin, glucobrassicin and neoglucobrassicin to the total content of 6 glucosinolates are 9%, 11% and 10%, respectively. Hydrolysis product of glucobrassicin 7 (indole-3-carbinol), similarly to sulphoraphane, have been associated with upregulation of 8 phase II detoxification enzymes and also with upregulation of cytochrome P-450 isoenzymes 9

(like for instance CYP 1A) [START_REF] Staack | A comparison of the individual 23 and collective effects of four glucosinolate breakdown products from Brussels sprouts on 24 induction of detoxification enzymes[END_REF]. 10 11The results of the present study indicate the increase in the main and total glucosinolate 12 contents in broccoli upon steam-cooking (Table I; Figure 1). The content of aliphatic 13 glucosinolates (glucoiberin and glucoraphanin) and indole glucosinolates (glucobrassicin and 14 neoglucobrassicin) increased 1.1-fold and 1.4-1.6-fold as compared to fresh broccoli, 15 respectively. Total glucosinolate content increased 1.2-fold. [START_REF] Conaway | Disposition of glucosinolates and sulforaphane in humans 30 after ingestion of steamed and fresh broccoli[END_REF] and 16 Vallejo et al. (2002) did not find any significant influence of steaming on glucosinolate 17 content in broccoli steamed for 15 and 3.5 minutes, respectively. Although, 10 min of 18 steaming, used in our study, is in the range of literature data, our results are different because 19 even small difference in cooking time may influence the results as it was shown in other 20 studies [START_REF] Ciska | Changes in glucosinolate content in selected 20 cruciferous vegetables during cooking[END_REF][START_REF] Ciska | The effect of cooking on the glucosinolates content in white 27 cabbage[END_REF]. It can not be excluded that just 10 minutes might be the 21 threshold time that causes the increase in the glucosinolate content upon steaming. The 22 steaming of broccoli for 3.5 minutes might be too short to observe significant increase of the 23 glucosinolates in the extracts from steamed broccoli. On the other hand, 15 minutes steaming 24 might be too long and therefore, after initial increase, some losses of glucosinolates may 25 occur. Moreover, the differences in our and literature data might be a result of difference in 26 broccoli cultivar. 27 [START_REF] Bjergegaard | Characterisation of proteins, including myrosinases, 7 associated to rapeseed dietary fibres, compared to proteins in pea dietary fibres[END_REF]. The presence of the enzyme-substrate system in the cell wall is also 1 a prerequisite for releasing of the degradation products only as a result of breaking the 2 structure of the cell wall. A hypothesis assuming the possibility of glucosinolates binding to 3 the cell walls, as in the case of other low-molecular compounds e.g. saccharides, glycosides, 4 and inositol phosphates, would be in line with a theory assuming the glucosinolates presence 5 in vacuole being their main reservoir in the cell [START_REF] Lüthy | The mustard oil bomb: rectified analysis of the subcellular 15 organization of the myrosinase system[END_REF]). 6 7 In contrast, cooking of broccoli in water for 5 min leads to considerable loss of glucosinolates 8 (Figure 1). The neoglucobrassicin is the most affected by the water-cooking; its concentration 9 decreased by 63%. The loss of other glucosinolates is 43% (glucoiberin), and 47% 10 (glucoraphanin and glucobrassicin); the total glucosinolate content decreased by 46%. The 11 observed losses of glucosinolates are most likely due to their leaching into the cooking-water. 12 13 Similar losses of glucosinolates and/or hydrolysis products upon various cooking methods 14 were reported in other studies. Vallejo et al. (2002) reported even higher loss of 15 glucosinolates in water-and microwave-cooked broccoli (74%) than that observed in our 16 study. Similar effect was reported by [START_REF] Rosa | The effect of cooking and processing on the 15 glukosinolate content: studies on four varieties of Portuguese cabbage and hybrid white 16 cabbage[END_REF] with cooked cabbage. According to 17 [START_REF] Howard | Retention of phytochemicals 18 in fresh and processed broccoli[END_REF], microwave cooking of broccoli at full power for 8 minutes, causes 18 considerable loss of sulforaphane. The decrease in the content of glucosinolates during 19 cooking of Brussels sprouts and white cabbage (up to 20 minutes), as a result of thermal and 20 initially also enzymatic degradation of glucosinolates, was reported by [START_REF] Ciska | Changes in glucosinolate content in selected 20 cruciferous vegetables during cooking[END_REF]Ciska et al. ( , 21 2001). The decrease in the content of glucosinolates in these vegetables was different for 22 individual compounds and it was dependent on the cooking time. Analysis of the cooking 23 water showed that the losses of glucosinolates were partly due to their leaching into the 24 cooking water [START_REF] Ciska | The effect of cooking on the glucosinolates content in white 27 cabbage[END_REF]. 25 26 The results presented in Table II show the influence of domestic processing on the content of 27 total polyphenols, flavonoids, phenolic acids, vitamin C and E, as well as carotenoids in 28

broccoli. 29

Table II 30 31 Fresh broccoli contains 681.2 mg/100 g d.w. (84.5 mg/100 g f.w.) of vitamin C. The cooking 32 of broccoli in water considerably affects the content of vitamin C, yielding losses of 23%. No 33 significant effect of steam-cooking on vitamin C content was observed (Figure 2). Similar 2004) the loss of vitamin C upon water-cooking for 5 min was 2 approximately 2-fold higher than observed in our study. According to literature data, the 3 content of vitamin C in edible parts of fresh broccoli, depending on variety, may vary from 4 43.2 to 146.4 mg/ 100 g of fresh weight (Vallejo et al. 2003). Based on available biochemical, 5 clinical, and epidemiological studies, the current recommended daily acceptance (RDA) for 6 ascorbic acid is suggested to be 100-120 mg/day to achieve cellular saturation and optimum 7 risk reduction of heart diseases, stroke and cancer in healthy individuals [START_REF] Naidu | Vitamin C in human health and disease is still a mystery? An overview[END_REF]). Thus, 8 steamed broccoli may be considered one of the most important sources of vitamin C in human 9 diet. 10

Figure 2 11

The content of total polyphenols in fresh broccoli 886.3 mg/100 g d.w. (109.9 mg/100 g f.w.), 12 determined in this study, is in agreement with the results previously reported (from 34.5 to 13 128 mg gallic acid/100 g f.w.) [START_REF] Leja | Antioxidant ability of broccoli 10 flower buds during short-term storage[END_REF][START_REF] Proteggente | The antioxidant activity of regularly 7 consumed fruit and vegetables reflects their phenolic and vitamin C composition[END_REF], Zhang et al. 2004). It 14 was found that cooking of broccoli in water affects the content of total polyphenols, yielding 15 loss of 13% (Figure 2). Zhang et al. ( 2004) reported that total polyphenols were retained in 16 28% in the cooked florets. 17 18 Acid hydrolysis of the broccoli phenolic extracts yields kaempferol as the main compound, 19 followed by quercetin and isorhamnetin. After alkaline hydrolysis caffeic, p-coumaric, sinapic 20 as well as ferulic acids were found to be the main acids present in the broccoli phenolic 21 extracts (results not shown). The losses of flavonoids and phenolic acids in water-cooked 22 broccoli were 72% and 52%, respectively as compared to fresh broccoli (Figure 2). These 23 results show that processing of broccoli by cooking in water has stronger effect on the content 24 of flavonoids than on phenolic acids. Similar losses, in case of flavonoids, were reported by 25 [START_REF] Price | Composition and content 3 of flavonol glycosides in broccoli florets (Brassica oleracea) and their fate during cooking[END_REF]. They found that during cooking of broccoli in water for 15 minutes, 14-26 28% of individual flavonol glycosides were retained in the cooked tissue, the remainder being 27 largely leached into the cooking water. 28

29

The analysis of water, in which broccoli was cooked, confirmed that the losses of both 30 vitamin C and polyphenols are mainly due to their leaching into the cooking water. 31 32 In contrast, steaming of broccoli leads to the increase in the content of total polyphenols (1.6-33 fold), flavonoids (1.5-fold) and phenolic acids (1.3-fold) 2). Similar effect of steaming on the level of polyphenols in broccoli and green beans 1 was observed by [START_REF] Turkmen | The effect of cooking methods on total phenolics 29 and antioxidant activity of selected green vegetables[END_REF]. It was also reported that heat treatment causes the 2 increase of free flavonols in tomato-based products [START_REF] Stewart | 26 Occurrence of flavonols in tomatoes and tomatoe-based products[END_REF]. The apparent 3 increase in polyphenols is most likely due to disruption of complexes between polyphenols 4 and e.g. proteins resulting in better availability of these compounds to extraction from 5 steamed broccoli as compared to fresh one. 6 7 β-Carotene and lutein contents in fresh broccoli (Table I) are in the range of literature data 8 (from 0.37 to 2.42 mg/100 f.w. and from 0.80 to 2.83 mg/100 g f.w., respectively) (Heinonen 9 et al. 1989[START_REF] Khachik | Effect of food preparation on qualitative and quantitative distribution of 25 major carotenoids constituents of tomatoes and several green vegetables[END_REF][START_REF] Lessin | Quantification of cis-trans isomers of 12 provitamin A carotenoids in fresh and processed fruits and vegetables[END_REF][START_REF] Müller | Determination of the carotenoid content in selected vegetables and fruits by 24 HPLC and photodiode array detection[END_REF], Kurilich et a. 1999, Zhang et 10 al. 2004). The contents of tocopherols are similar to those previously published by Burns et al. 11 (2003) but they are much lower than those reported by [START_REF] Kurilich | Carotene. Tocopherol. And ascorbate contents in 2 subspecies of Brassica oleracea[END_REF] and Lessin et al. 12 (1997). In contrast to vitamin C and polyphenols, both cooking methods (boiling in water and 13 steaming) leads to the increase in β-carotene and lutein contents (Figure 3). The increase in β-14 carotene was 1.9-fold and 2.3-fold in steamed and water-cooked broccoli, respectively; for 15 lutein -4.1 and 6-fold, respectively. From the results presented (Figure 3) it follows that α-16 tocopherol content increased 1.2-fold and 1.7-fold upon steaming and cooking of broccoli in 17 water, respectively. Similar effect was found for γ-tocopherol (1.4-fold and 1.7-fold, 18 respectively). Carotenoids and tocopherols have not been detected in the cooking water. oxidizing lipids (Friedrich 1998), and it was also reported that little or no degradation of β-24 carotene occurs during thermal processing [START_REF] Chandler | Isomerization and losses of trans-β-carotene in sweet 17 potatoes as affected by processing treatments[END_REF][START_REF] Khachik | Effect of food preparation on qualitative and quantitative distribution of 25 major carotenoids constituents of tomatoes and several green vegetables[END_REF]), the 25 increase in the content of tocopherols and carotenoids observed in our study is most likely a 26 result of better availability of these compounds for extraction. For carotenoids, the increase in 27 their concentration in processed broccoli is most likely a result of improved extraction in part 28 due to disruption of carotenoprotein complexes and inactivation of carotene oxidizing 29 enzymes [START_REF] Lessin | Quantification of cis-trans isomers of 12 provitamin A carotenoids in fresh and processed fruits and vegetables[END_REF]). The higher content of carotenoids and tocopherols in water-30 cooked than in steamed broccoli seem to support the conclusion that the increase in the 31 content of these compounds is related to the improved extraction; availability of extracted 32 compounds from more disintegrated tissue of water-cooked broccoli is better than from 33 steamed broccoli. In the study of [START_REF] Lessin | Quantification of cis-trans isomers of 12 provitamin A carotenoids in fresh and processed fruits and vegetables[END_REF], where water-cooked broccoli was 1 compared with the fresh one, about 1.2-fold increase in the β-carotene content was observed. 2 Similar effect of heat-based processing on the content of lycopene in tomatoes was observed 3 by [START_REF] Re | Effects of food processing on flavonoids and 13 lycopene status in a mediterranean tomato variety[END_REF]. It was also reported that carotene content increases during blanching, lye 4 peeling and pureeing of sweet potatoes. It was also attributed to an enhanced extraction of 5 carotenoids from heat-treated samples [START_REF] Chandler | Isomerization and losses of trans-β-carotene in sweet 17 potatoes as affected by processing treatments[END_REF]). 6 7

Antioxidant capacities of broccoli extracts 8

The TEAC and DPPH antioxidant activities of fresh, steamed and water-cooked broccoli 9 (Table III) generally reflect the results of quantification of polyphenols, carotenoids, vitamin 10 C and E in broccoli. The antioxidant activities of water-cooked broccoli decreased by 6% for 11 polyphenol extract and 26% for vitamin C extract in the TEAC assay. In the DPPH assay, 12 antioxidant activities decreased by 29% and 16% for polyphenols and vitamin C extracts, 13 respectively. For steamed broccoli the increase in the antioxidant activities of polyphenol and 14 vitamin C extracts was observed as compared to fresh broccoli: 1.3-fold for both extracts in 15 the TEAC and DPPH assays (Figure 4). 16

Table III 17

Figure 4 18

The antioxidant activities of carotenoid and tocopherol extracts obtained from steamed and 19 water-cooked broccoli increased 2.2-fold and 2.9-fold, respectively as compared to that of 20 fresh broccoli (Figure 5). The effects of domestic processing on the TEAC values support the 21 conclusion on an increased availability of carotenoids and tocopherols for extraction upon 22 steam-and water-cooking. In DPPH assay, no significant activities of carotenoids and 23 tocopherol extracts were observed. Antioxidant activities of glucosinolates in broccoli 24 extracts were not determined because they are not effective free radical scavengers (Plumb et 25 al. 1996[START_REF] Faulkner | Selective increase of the potential 7 anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli[END_REF]. 26

Figure 5 27

Conclusions 28

The results indicate that steam-cooking of broccoli results in an increase in the content of 29 flavonoids and phenolic acids, as well as the main glucosinolates and their total content as 30 compared to fresh broccoli, whereas cooking in water has opposite effect. Steam-cooking of 31 broccoli has no influence on vitamin C content, whereas cooking in water significantly lowers 32 the content of this vitamin. Both water-and steam-cooking of broccoli results in an increase 33

2 3and methods 4 5

 245 because the data on changes of these compounds during cooking are still 1 limited and sometimes contradictory.MaterialsMaterialsLutein from Roth (Karlsruhe, Germany), 2,2-diphenyl-1-picrylhydrazyl (DPPH), γ-6 tocopherol, p-coumaric, ferulic and sinapic acids from Sigma (St. Louis, MO, USA), ABTS 7(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt from Roche 8 Diagnostics (Mannheim, Germany), quercetin dihydrate, kaempferol and tert-9 butylhydroquinone (TBHQ) from Fluka (Buchs, Switzerland), Trolox ® , α-tocopherol, all-10 trans-β-carotene, thero-1,4-dimercapto-2,3-butandiol (DTT) and caffeic acid from Aldrich 11 (Steinheim, Germany), ascorbic acid (vitamin C) from Merck (Darmstad, Germany), 12

  al. 1998). Concentration of flavonols present in broccoli, which occur as quercetin, 1

32

 32

  γ-tocopherols, a Waters 474 scanning fluorescence detector set at emission wavelength of 1

  scavenging activities of the same extracts were determined using a modified 2 method of Brand-Williams et al. (1995) as described by[START_REF] Kim | Vitamin C equivalent antioxidant 28 capacity (VCEAC) of phenolic phytochemicals[END_REF]. Briefly, 100 µM 3 DPPH • was dissolved in 80% aqueous methanol. The broccoli extracts (0.03 ml of different 4 concentrations) were added to 0.87 ml of DPPH • solution to give the final concentration 5 required. The reaction mixtures were incubated in the dark at room temperature for 30 min, 6 and the decrease in absorbance caused by the extract was measured at 517 nm. The 7 corresponding solvent blank readings were also taken and, from the decrease of absorbance, 8 the scavenged DPPH • was calculated. The percentage of the scavenged DPPH • was plotted 9 against the concentration of the broccoli extract. The DPPH radical scavenging activity of 10 broccoli extracts was expressed in Trolox equivalents (in mmol Trolox/100 g of dry and fresh 11 weight) calculated as the ratio of the slope of the linear plot for scavenging of DPPH • radical 12 by the extract tested to the slope of the plot for DPPH • radical scavenging by the water-13 soluble vitamin E analogue Trolox  , used as an antioxidant standard. 14 15

Figure 1 28 29

 28 Figure 1 28 29 According to Ciska et al. (2001) the increase in the content of glucosinolates may result from 30 a deep disintegration of plant tissue upon heat treatment because part of glucosinolates in 31 plant cells can be bound to the cell walls and released only after a deep disintegration of cell 32 structures. Such an assumption can be supported by the reports concerning the presence of 33 myrosinase, a native enzyme catalyzing the glucosinolates hydrolysis, in the cell wall 34

  on vitamin C content in broccoli was observed byVallejo et al. (2002). In 1 the study of Zhang et al. (

19 20 Figure 3 21 22

 20321 Figure 3 21 22 Since tocopherols are largely resistant to heating up to 200 o C, in the absence of oxygen and 23

6 7

 6 carotene, lutein and vitamin E as compared to fresh one. The increase in the content of 1 polyphenols, carotenoids, glucosinolates and vitamin E is related to their enhanced 2 availability for the extraction, whereas the observed losses of the compounds are mainly due 3 to their leaching into the cooking-water. The changes in the content of health-promoting 4 compounds of broccoli upon domestic processing are generally reflected by the changes in the 5 antioxidant activities of broccoli extracts.Moreover, the results of the present study and the literature data show that the concentration 8 and availability of different health-promoting compounds present in broccoli are dependent 9 on the matrix, in which they are present. Furthermore, steam-cooking of broccoli may be 10 considered a "friendly" process, preserving health-promoting compounds that are important 11 for preventing adverse health effects and maintaining food quality. ., Tomas-Barberan, F. A.,Garcia-Viguera, C., 2003, Potential bioactive compounds 1 in health promotion from broccoli cultivars grown in Spain. Journal of the Science of 2 Food and Agriculture, 82, 1293-1297. 3 Vallejo, F., Tomas-Barberan, F. A., Garcia-Viguera, C., 2002, Glucosinolates and vitamin C 4 content in edible parts of broccoli florets after domestic cooking. European Food Research 5 and Technology, 215, 310-316. 6 Williamson, G., Dupont, M. S., Wanigatunga, S., Heaney, R. K., Musk, S. R. R., Fenwick, G. 7 R., Rhodes, M. J. C., 1997, Induction of glutathione-S-transferase activity in hepG2 cells 8 by extracts from fruits and vegetables. Food Chemistry, 60, 157-160. 9 Williamson, G., Faulkner, K, Plumb G. W., 1998, Glucosinolates and phenolics as 10 antioxidants from plant foods. European Journal of Cancer Prevention, 7, 17-21. 11 Zhang, D., Hamauzu, Y., 2004, Phenolics, ascorbic acid, carotenoids and antioxidant activity 12 of broccoli and their changes during conventional and microwave cooking. Food 13 Chemistry, 88, 503-509. 14 Zhang, Y., Talalay, P., Cho, C-G., Posner, G. H., 1992, A major inducer of anticarcinogenic 15 protective enzymes from broccoli: isolation and elucidation of structure; Proceedings of 16 the National Academy of Science USA, 89, 2399-2403. 17

1 Figure 1 . 1 Figure 2 . 1 Figure 3 . 4 PageFigure 4 . 2 Figure 5 .

 1112134425 g l u c o b r a s s i c i n G l u c o b r a s s i c i n G l u c o r a p h a n i n G l u c o i b e r i n T o t a l g l u c o s i n o l a t e s Concentration (% of fresh broccoli) The changes in the content of total and the main glucosinolates (referred to dry 2 weight) in broccoli upon water-and steam-cooking. n o l i c a c i d s F l a v o n o i d s T o t a l p o l y p h e n o l s Concentration (% of fresh broccoli) The changes in the content of vitamin C, total polyphenols, flavonoids and phenolic 2 acids (referred to dry weight) in broccoli upon water-and steam-cooking. a r o t e n e γ -T o c o p h e r o l α -T o c o p h e r o l L u t e i n Concentration (% of fresh broccoli) The changes in the content of β-carotene, lutein, αand γ-tocopherols (referred to 2 dry weight) in broccoli upon water-and steam-cooking. 3 The changes in the TEAC and the DPPH values of (A) polyphenol extracts and 4 (B) vitamin C extracts from domestically processed broccoli. 5 value [mmol Trolox/100 g d.w] The changes in the TEAC value of carotenoid and tocopherol extracts from 3

5 Page 1 Figure 1 . 3 Figure 2 . 5 Figure 3 . 7 Figure 4 . 9 Figure 5 .Food

 51132537495 Figure 1. The changes in the content of total and the main glucosinolates (referred to dry 2 weight) in broccoli upon water-and steam-cooking. 3

  in comparison to fresh broccoli

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

(Figure

Table I .

 I Glucosinolate content (µmol/g dry weight) in fresh, steamed and water-cooked

	1
	2	broccoli.
	3
						Glucosinolate content
					Fresh	Steamed	Water-
			Compound		broccoli	broccoli	cooked broccoli
		Aliphatics	Glucoiberin		1.43	1.58	0.81
			Progoitrin		0.18	0.19	0.14
		Glucoraphanin Napoleiferin Glucoalyssin Gluconapin F o r		9.60 0.31 0.07 Traces	10.19 0.26 0.15 Traces	5.09 0.14 0.10 Traces
		Aralkyl Indoles	Glucoibervirin Glucoerucin P Gluconasturtiin e 4-Hydroxyglucobrassicin Glucobrassicin e r	Traces Traces 0.10 0.63 1.76	0.05 Traces 0.14 0.76 2.78	Traces Traces 0.05 0.43 0.93
		Total	4-Metoxyglucobrassicin Neoglucobrassicin R e	0.36 1.60 16.04	0.48 2.21 18.79	0.30 0.59 8.58
	4 5	Traces = < 0.05 µmol/g dry weight	v i e
					w
					O n l
						y

Table II .

 II Distribution (mean ± SD) of compounds analysed in fresh and domestically-Due to the fact that literature data for polyphenol, flavonoid, vitamin C and E, and carotenoid 7 contents in broccoli are mostly referred to fresh weight , TableIIadditionally contains the results

	1
	2	processed broccoli (mg/100 g).
		Compounds		Fresh	Steamed	Water-cooked
		Total polyphenols 1	dry weight	886.3±104.3	1409.1±31.1	775.8±55.7
				fresh weight*	109.9±12.9	167.3±3.7	89.2±6.4
		Flavonoids 2		dry weight	25.4±1.4	38.7±0.8	7.0±0.8
				fresh weight	3.15±0.17	4.59±0.09	0.81±0.10
		Phenolic acids 3	dry weight	328.1±25.9	417.3±2.0	155.9±20.0
		Vitamin C β-Carotene	fresh weight dry weight fresh weight dry weight o F r	40.55±3.20 681.2±18.3 a 84.5±2.3 a 10.50±0.54	50.05±0.91 652.6±46.3 a 77.7±5.5 a 19.93±0.48	17.93±2.30 524.8±1.2 60.3±0.1 24.61±0.48
		Lutein		fresh weight dry weight P	1.30±0.07 6.47±0.33	2.37±0.04 26.60±1.60	2.83±0.06 38.8±1.85
				fresh weight e	0.80±0.04	3.16±0.19	4.46±0.21
	3 4 5 6 8	dry weight fresh weight e r dry weight fresh weight dry weight R 0.727±0.047 0.090±0.006 0.072±0.003 0.009±0.000 0.798±0.049 fresh weight 0.099±0.006 e 1 -measured using Folin-Ciocalteu method; 2 -determined by HPLC method after acid hydrolysis 0.895±0.012 1.272±0.044 α-Tocopherol 0.106±0.001 0.146±0.005 0.104±0.003 0.125±0.002 γ-Tocopherol 0.012±0.000 0.014±0.000 0.999±0.015 1.397±0.046 Vitamin E 4 0.119±0.002 0.161±0.005 and quantified as quercetin; 3 -determined by HPLC method after alkaline hydrolysis and quantified as sinapic acid; 4 -calculated as the sum of α-and γ-tocopherols; a -not significantly different at P<0.05. v i e w
	9	expressed in mg/100 g fresh weight.		O n l
						y

*

Table III .

 III Antioxidant activity (mmol Trolox/100 g) of polyphenol, vitamin C and 1 carotenoid/tocopherol extracts from fresh, water-cooked and steamed broccoli (mean ± SD) Due to the fact that literature data for the antioxidant activity of vegetables are mostly referred to 4 fresh weight, TableIIIadditionally contains the results expressed in mmol Trolox/100 g fresh weight.

	2
	3
			Fresh broccoli	Steamed broccoli	Water-cooked
					broccoli
		Antioxidant activity of polyphenol extracts
	TEAC value	dry weight	7.69±1.40	10.07±0.56	7.22±0.32
		fresh weight*	0.95±0.07	1.20±0.07	0.83±0.04
	DPPH value	dry weight	3.84±0.60	4.78±0.10	2.74±0.47
		fresh weight	0.47±0.07	0.57±0.01	0.31±0.05
		Antioxidant activity of vitamin C extracts
	TEAC value	dry weight	4.72±0.13	6.14±0.82	3.47±0.50
		fresh weight	0.58±0.02	0.73±0.10	0.40±0.05
	DPPH value	dry weight	3.03±0.04	3.84±0.46	2.56±0.38
		fresh weight	0.37±0.04	0.46±0.05	0.29±0.04
		Antioxidant activity of carotenoid and tocopherol extracts
	TEAC value	dry weight	0.138±0.016	0.299±0.011	0.403±0.007
		fresh weight	0.017±0.002	0.035±0.001	0.046±0.001
	DPPH value		inactive	inactive	inactive

*

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Acknowledgements 14

The grant from State Committee for Scientific Research (Poland), PBZ-KBN-

List of Tables 1

Table I. Glucosinolate content (µmol/g dry weight) in fresh, steamed and water-cooked 2 broccoli. 3

Table II. Distribution (mean ± SD) of compounds analysed in fresh and domestically-4 processed broccoli (mg/100 g). 5

Table III. Antioxidant activity (mmol Trolox/100 g) of polyphenol, vitamin C and 6 carotenoid/tocopherol extracts from fresh, water-cooked and steamed broccoli (mean ± SD). 7

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