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Food and Chemical Toxicology 46 (2008) 16351644 www.elsevier.com/locate/foodchemtox

Food safety evaluation of broccoli and radish sprouts

Cristina Martnez-Villaluenga a, Juana Fras a, Piotr Gulewicz b, Krzysztof Gulewicz b, Concepcion Vidal-Valverde a,*
b a Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, Madrid 28006, Spain Institute Bioorganic Chemistry, Polish Academy of Sciences, Zygmunta Noskowskiego 12/14, 60-704 Poznan, Poland

Received 1 August 2007; accepted 2 January 2008

Abstract Three cultivars of broccoli seeds (Brassica oleracea var. italica), cv. Tiburon, cv. Belstar and cv. Lucky, and two cultivars of radish seeds (Raphanus sativus), cv. Rebel and cv. Bolide, were germinated for three and ve days and safety aspects such as microbiological counts and biogenic amines were investigated. Cytotoxicity evaluation was also carried out. Broccoli and radish sprouts contained numbers of mesophilic, psychrotrophic, total and faecal coliform bacteria which are the usual counts for minimally processed germinated seeds. Putrescine, cadaverine, histamine, tyramine, spermidine and spermine increased during sprout production although these levels were below those permitted by legislation (5 mg/100 g of edible food). Broccoli and radish sprouts demonstrated no toxic eects on proliferation and viability of HL-60 cells and should be included in our diets as healthy and safe fresh foods. 2008 Elsevier Ltd. All rights reserved.
Keywords: Germination; Radish; Broccoli; Biogenic amines; Food safety; Sprouts; HL60 cytotoxicity

1. Introduction The use of seed sprouts as food has spread in the past few decades from Far Eastern countries to parts of the Western world. Consumers can nd on the market an extraordinary variety of dierent types of sprouts in which the Cruciferae family is well represented. Several studies have demonstrated that cruciferous sprouts such as broccoli (Brassica oleracea L. var. italica) and radish (Raphanus sativus) are very rich in health-promoting phytochemical constituents such as glucosinolates related to cancer prevention as well as having antioxidant properties (Fahey et al., 1997; Tian et al., 2005; Barillari et al., 2005), phenolic compounds and ascorbic acid in these vegetables (Zielinski et al., 2002, 2003; Takaya et al., 2003). Moreover, a recent study performed in vivo showed that Japanese radish sprouts have the potential to alleviate hyperglycemia in diabetes cases and are eective in the primary prevention
*

Corresponding author. Tel.: +34 915622900x241; fax: +34 915644873. E-mail address: icv12@i.csic.es (C. Vidal-Valverde).

of diabetes mellitus in animal models (Taniguchi et al., 2006). Eating the fresh sprouts is the best way of gaining all of the health benets claimed for cruciferous sprouts because only minor losses in health-promoting components are likely to occur. Most consumers and retailers do not cook sprouts, and since bacteria on the seed surface can become internalized during sprouting, washing sprouts is probably an ineective way to eliminate spoilage and pathogenic bacteria (Mohle-Boetani et al., 2001). The importance of this problem is reected by a number of specic recommendations that were developed in 1997 by US National Advisory Committee of Microbiological Criteria for Foods (NACMCF, 1999). Also the US Food and Drugs Administration (USFDA) issued guidance to enhance the safety of sprouts (1999). It is generally accepted that microbial populations exceeding 5 106 cfu/g may produce measurable metabolites, not only in terms of spoilage parameters but also in relation to toxic metabolites. Biogenic amines are usually generated by decarboxylation of amino acids or by amination and transamination

0278-6915/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.01.004

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C. Martnez-Villaluenga et al. / Food and Chemical Toxicology 46 (2008) 16351644 To determine total mesophilic and psychrotrophic aerobic bacteria counts, appropriate serial dilutions were surface-plated on Triptic Soy Agar (TSA, Scharlau Chemie, Spain). Plates were incubated at 32 C for mesophilic bacteria counts for 48 h and at 8 C for psychrotrophic bacteria counts for 10 days. Total and faecal coliforms were determined on Violet-Red Bile Agar (VRBA, pH 7.4, Scharlau Chemie, Spain) and incubated at 37 C (for total coliforms) or 44 C (faecal coliforms) for 24 h under anaerobic conditions. Faecal Streptococci were surface-plated on Slanetz Bartley Agar (Scharlau Chemie, Spain) and incubated at 37 C for 24 h.

of aldehydes and ketones (Askar and Treptow, 1986). Biogenic amines can be naturally present in plant food since they are required in cellular metabolism and in growing tissues (Matilla, 1996; Santos, 1996). However, they are also a consequence of microbial activity (Gloria et al., 2005; Hal` asz et al., 1994). The presence of biogenic amines in foods and beverages is considered important because of their inuence on physiological activities, which may cause symptoms such as skin irritations, headache, dizziness, vomiting and diarrhoea (Pechanek et al., 1983). The presence of some mono-, di- and polyamines has been suggested as a supplementary criterion to indicate the freshness and quality of food. In particular, putrescine, cadaverine, spermidine, spermine, histamine and tyramine have been suggested as indicators of food deterioration (Ramantanis et al., 1985; Paulsen et al., 1997). On the other hand, amines are also investigated as a possible mutagenic precursor, since some of them may be nitrated or act as precursors for other compounds capable of forming nitrosamines, which are carcinogenic for various species of animals and a potential hazard to humans (Shalaby, 1996). Moreover, in order to ensure the food safety of these cruciferous sprouts it would be interesting to determine the eect of seed germination on human cell viability. Therefore, the aim of this work is to determine the hygienic status, biogenic amine content and cytotoxicity of three cultivars of broccoli (B. oleracea var. italica) and two cultivars of radish (R. sativus) after sprouting.
2. Materials and methods 2.1. Plant material
Broccoli seeds (B. oleracea L. var. italica cv. Tiburon, cv. Belstar and cv. Lucky) were purchased at Bejo Iberica (Spain). Radish seeds (R. sativus cv. Rebel and cv. Bolide) were purchased at Bejo Iberica and Man Fong Pacic Trading (Spain), respectively.

2.4. Biogenic amine determination

Determination of biogenic amines was carried out by acid extraction, derivatization with dansyl chloride and HPLC quantication according to Fras et al. (2007). Putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tyramine, spermidine trihydrochloride, spermine tetrahydrochloride were purchased from Fluka (Spain). A stock standard aqueous solution of amines was prepared by adding an accurately weighed amount of each standard (ca. 80 mg) to a 25 mL volumetric ask. Standards were derivatized as described for the samples. The chromatographic system consisted of an Alliance Separation Module 2695 (Waters, Milford, USA), a Photodiode Array detector 996 (Waters) and a personal computer running the Empower II for windows chromatographic software (Waters). The sample (20 lL) was injected onto a C18 Kromasil 250 4.6 mm i.d., 5 lm size (Symta) column equipped with a C18 guard column (Symta) both thermostatted at 30 C. The mobile phase for DCl-derivatives separation consisted of bidistilled water (solvent A) and HPLC-grade acetonitrile (solvent B). The elution programme was held at 65% of B for 1 min, ramped at 80% (10 min), 90% (12 min), 100% of B (16 min) and held until the end of the run (23 min) with a ow rate of 0.8 mL/min. Calibration curves were obtained for standard amines and r was always above 0.990.

2.5. Cytotoxicity evaluation

To obtain the extracts, 50 mg of the seed our were subjected to extraction with 2.5 mL of deionized water and sonicated for 30 min (Sonorex AK103H). Then, the extracts were centrifuged for 15 min at 12,000 rpm. The supernatant was ltered through 0.22 lm membranes into sterile test tubes. One mL of each ltered supernatant was evaporated in tarred vessels and the obtained dry masses were weighed after 24 h desiccation with P2O5. The residues were dissolved in sterilized water to a nal concentration of 1 mg/mL. The human leukemic cell line (HL-60), derived from a patient with acute promyelocytic leukemia, was obtained from American Type Culture Collection (ATCC). The culture was maintained on RPMI medium containing 10% fetal calf serum and 1% penicillin G (Sigma) and streptomycin (Sigma), at 37 C in a humidity-controlled incubator at 90% relative humidity and 5% CO2. After a few passages, cells were centrifuged, resuspended in fresh medium at the concentration of 0.30 106 cell per millilitre and transferred onto several plates in 2 mL volumes. The cells were exposed to 100 lg/mL of raw and germinated seed extracts and control (sterilized water) during 24, 48 and 72 h. Then, trypan blue dye (Sigma) was added to cell cultures in a ratio of 1:1 and left for 10 min. Cell suspension (20 lL) was then loaded by micropipette into Burker cham bers. The cells were counted under a microscope at 100 magnication. The number of viable cells was determined for cell proliferation approach. The tetrazolium reduction assay (MTT) was performed in duplicate following the method of Denizot and Lang (1986). Briey, 100 lL of the cultured medium was transferred into Eppendorf tubes and centrifuged for 5 min at 1.600 rpm. The supernatants were removed and cells resuspended in 100 ll of fresh medium. The cells were exposed to 100 lg/mL of raw and germinated seed extracts and control (sterilized water) for 24 h. After this time, 20 lL of MTT (Sigma) at a concentration of 5 mg/mL in PBS, were added to each sample. All plates were incubated for 4 h at 37 C

2.2. Seeds germination

Seeds (10 g) was soaked with 50 mL of 0.07% sodium hypochlorite for 30 min. These seeds were drained and washed with distilled water until they reached neutral pH. Afterwards, seeds were soaked in 50 mL distilled water for 5 h with shaking every 30 min. The imbibed seeds were germinated at pilot scale by layering seeds over moist lter paper in a germination tray. The tray was placed in a seed germinator G-120 model (ASL Snijders International S. L., Holland) and seeds were continuously watered by capillary. Germination of broccoli seeds was carried out at 25 C under photoperiod of 16 h light and 8 h darkness and germination of radish seeds was carried out at 25 C in darkness. Sprouted seeds were collected after three and ve days. The germination process was carried out in triplicate and the rate was higher than 90%.

2.3. Microbiological determinations

Microbial counts were carried out using the poured plate technique. Five grams of fresh samples were aseptically placed into a ask with 45 mL of peptone water (Scharlau Chemie, Spain) to achieve a 101 dilution and shaken with vortex for 1 min. Serial dilutions were made in 0.1% buered peptone water (Scharlau Chemie, Spain) in tubes.

 C. Martnez-Villaluenga et al. / Food and Chemical Toxicology 46 (2008) 16351644 in a humidity-controlled incubator at 90% relative humidity, 5% CO2 and 120 lL of DMSO (Sigma) were added. After 3 h of incubation at 37 C samples were centrifuged for 5 min at 1.600 rpm and 25 lL of Sorensen buer (0.1 M glycine, 0.1 M NaCl, pH = 10.5) were added to supernatants. Absorbance of the formazan product was measured at 570 nm.

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2.6. Statistical analyses

Data were subjected to multi-factor analysis of variance by applying the least signicance dierence test with the Statgraphic 4.0 Program (Statistical Graphics Corporation, Rockville, Md) for Windows.

3. Results and discussion 3.1. Hygienic status of broccoli and radish sprouts Table 1 shows the microbial population of raw seeds and broccoli sprout cultivars Lucky, Tiburon and Belstar after three and ve days of germination. The numbers of total aerobic mesophilic bacteria in broccoli seeds were $7 log cfu/g and this microbial population showed a rise of $9 log cfu/g after ve days of germination. The number of psychrotophic bacteria in broccoli seeds was $6 log cfu/ g and this microbial population increased $1 log cycle in three-day sprouts and continued signicantly (P 6 0.05) growing up to $2 log cycles after ve days of germination in cv. Tiburon and cv. Belstar, whilst it remained unmodied in cv. Lucky. Total and faecal coliforms achieved $6 and 5 log cfu/g in raw broccoli seeds, respectively, and germination led to gradual increases of about $3 log cycles for both types of populations after ve days. The amount of faecal streptococci in raw broccoli seeds showed levels <2 log cfu/g, which remained unchanged in three-day and ve-day broccoli sprouts. Table 2 shows the microbial population of raw and germinated radish cultivars (cv. Bolide and cv. Rebel). The number of total aerobic mesophilic bacteria in raw radish seeds reached $8 log cfu/g, counts higher than those found in broccoli seeds. Total aerobic plate counts increased gradually up to 1 log cycle after ve days of germination

for the two radish cultivar sprouts. Psychrotrophic bacteria numbers in raw radish seeds were $7 log cfu/g which increased up to $2 log cycles after ve days of germination. Total coliform counts corresponded to 4.8 and 5.8 log cfu/ g in raw radish seeds cv. Bolide and cv. Rebel, respectively, and germination brought about a rise in total coliform counts of $8 log cfu/g. Fecal coliform numbers amounted to 4.2 and 5.4 log cfu/g for radish seeds cv. Bolide and cv. Rebel, respectively, and presented an upward trend during sprouting, reaching numbers up to $7 log cfu/g after ve days. The viable numbers of faecal streptococci present in radish seeds were < 2 log cfu/g and remained constant until the end of germination. Results obtained for broccoli and radish seeds on microbial count agree with those found in the literature for different kinds of seeds (Prokopowich and Blank, 1991; Piernas and Guirand, 1997; Soylemez et al., 2001; Martnez-Villaluenga et al., 2006). Feng (1997) showed that alfalfa and mungbean seeds routinely contained high numbers of microbial ora (102106 cfu/g) including coliforms (104 cfu/g) and faecal coliforms (102103 cfu/g), and that these microorganisms form part of the normal seed microora. Dierences in the microbial population of dierent seeds can occur due to dierent composition of the seed coat and dierent cultivation and storage conditions (Piernas and Guirand, 1997). Rises in bacterial number observed in broccoli and radish sprouts are similar to others described in the literature. Thus, within two days of germination, microbial populations increased $2 log cycles in rice (Piernas and Guirand, 1997), lupin and fenugreek (Martnez-Villaluenga et al., 2006), 3 log cycles in alfalfa and mungbean (Andrews et al., 1982; Splittstoesser et al., 1983) and up to 4 log cycles in kidney bean (Kimanya et al., 2003). Rises in bacterial numbers during sprouting are explained through nutritional and environmental factors: (a) germination improves the bioavailability of nutrients such as low molecular carbohydrates (mono- and disaccharides) and amino acids providing available substances for microbial growth, and

Table 1 Microbial population in raw and sprouted broccoli (Brassica oleracea var. italica) cultivars* Germination time (days) Counts (log 10 cfu/g fresh sprout) Mesophilic bacteria cv. Lucky 0 3 5 cv. Tiburon 0 3 5 cv. Belstar 0 3 5 7.24 0.05a 8.50 0.12b 9.42 0.15c 7.69 0.09a 8.77 0.08b 9.70 0.02c 7.39 0.12a 8.29 0.29b 9.46 0.20c Psychrotrophic Bacteria 6.40 0.05a 7.48 0.12b 7.41 0.12b 6.40 0.32a 7.77 0.06b 8.64 0.06b 6.31 0.33a 7.24 0.34ab 8.48 0.19b Total coliforms 6.67 0.05a 7.69 0.03b 9.44 0.17c 6.22 0.06a 7.28 0.02b 9.26 0.48c 6.49 0.11a 7.45 0.08b 9.82 0.06c Faecal coliforms 5.44 0.06a 6.44 0.07b 7.46 0.13c 5.43 0.22a 6.44 0.07b 8.44 0.08c 5.34 0.03a 6.41 0.00b 8.25 0.17c Faecal Streptococci <2 <2 <2 <2 <2 <2 <2 <2 <2

* Results are expressed as mean value SD of three repetitions. Dierent superscript in the same column for each cultivar means signicant dierence (P 6 0.05).

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Table 2 Microbial population in raw and sprouted radish (Raphanus sativus) cultivars* Germination time (days) Counts (log 10 cfu/g fresh sprout) Mesophilic bacteria cv. Bolide 0 3 5 cv. Rebel 0 3 5
*

Psychrotrophic Bacteria 6.82 0.03a 8.23 0.39a 8.42 0.11b 6.78 0.07a 8.18 0.33b 8.79 0.01c

Total coliforms 4.78 0.07a 7.59 0.11b 7.86 0.10c 5.78 0.07a 7.75 0.02b 8.22 0.22c

Faecal coliforms 4.16 0.16a 5.98 0.62b 7.11 0.12b 5.41 0.08a 6.18 0.12b 7.89 0.01c

Faecal Streptococci <2 <2 <2 <2 <2 <2

8.05 0.01a 9.08 0.21b 9.37 0.47b 8.21 0.12a 9.13 0.02b 9.34 0.02c

Results are expressed as mean value SD of three repetitions. Dierent superscript in the same column for each cultivar means signicant dierence (P 6 0.05).

(b) the high moisture required and the warm temperatures during sprouting create a suitable environment for bacterial development (Feng, 1997). Geiges et al. (1990) demonstrated that Enterobacteria are the dominant microorganisms present in vegetable sprouts. Skowronek et al. (1998) reported that not only Enterobacteria but also Pseudomonas spp. comprised up to 9599% of the total microbial population of mungbean, lentil and radish sprouts after two days of germination. This prevailing bacterial species found on sprouts probably originated from the seeds rather than being due to the sanitary conditions of sprout production (Splittstoesser et al., 1983). Moreover, Kimanya et al. (2003) reported that the absence or low levels of pathogens in sprouts are not suciently competitive with the background ora during germination to increase to levels which lead to toxin production. Broccoli and radish sprouts contained numbers of mesophilic, psychrotrophic, total and faecal coliform bacteria which are the usual counts for minimally processed germinated seeds such as alfalfa, bean, lupin, fenugreek or onion (Prokopowich and Blank, 1991; Lang et al., 2000; Ghandi and Matthews, 2003; Martnez-Villaluenga et al., 2006) and even for commercial mungbean sprouts (Skowronek et al., 1998). Nevertheless, it is necessary to develop hygienic methods to improve the shelf-life and safety of sprouts. In this sense, several methods such as heat treatment (Jaquette et al., 1996; Weiss and Hammes, 2003), exposure to ionizing radiation (Thayer et al., 2006), chemical disinfectans (Beuchat et al., 2001; Proctor et al., 2001; Fett and Cooke, 2003; Sharma et al., 2002) and high hydrostatic pressure (Wuytack et al., 2003; Penas et al., 2008) have been described for reducing the microbial ora on seeds achieving P5 log reductions recommended by the National Advisory Committee on Microbial Criteria for Foods (NACMCF, 1999). However, studies have demonstrated that although seed decontamination can reduce the populations of human pathogens present, it cannot ensure the production of pathogen-free sprouts (Thomas et al., 2003) and in some cases the germination capability of the treated seeds was aected. Therefore, interventions should be carried out to prevent or minimize contamination of raw

sprouts and to remove pathogens prior to consumption, without losses in the nutritional, freshness and organoleptic properties of sprouts. 3.2. Inuence of germination on prole and levels of biogenic amines Table 3 and Table 4 show the individual and total biogenic amine content in seeds of dierent cultivars of broccoli (cv. Lucky, cv. Tiburon and cv. Belstar) and radish (cv. Bolide and cv. Rebel), respectively. Raw seeds showed the presence of putrescine, cadaverine, histamine, tyramine, spermidine and spermine in all cultivars. The presence of biogenic amines has been described in dierent legume and radish seeds (Simon-Sarkadi and Holzapfel, 1995; Skowronek et al., 1998; Shalaby, 2000; Gloria et al., 2005; Fras et al., 2007), although no information has been found about these amines in broccoli seeds. Cadaverine was predominant in broccoli seeds (ranging from 10.7 14.5 mg/kg d.m.) while tyramine (1.93.1 mg/kg d.m.) was the minor contributor to total amine levels. On the other hand, cadaverine (10.712.3 mg/kg d.m.) and histamine (10.311.0 mg/kg d.m.) were the major amines found in radish seeds contributing about equally to the total amine content while tyramine (2.63.2 mg/kg d.m.) was the minor amine found. Moreover, it is noteworthy that putrescine and spermidine levels were higher in radish than broccoli seeds. Total biogenic amine content in broccoli seeds ranged widely from 2445 mg/kg d.m while in radish seeds we found a narrow range from 47.8 to 48.6 mg/kg d.m. Total and individual amine content of dierent legume and radish seeds reported in the literature were more than two-fold higher (Simon-Sarkadi and Holzapfel, 1995; Martnez -Villaluenga et al., 2006; Fras et al., 2007) than values reported for broccoli and in radish seeds in this work, with the exception of soybean seeds which presented lower levels (Gloria et al., 2005), showing that biogenic amine levels seem to depend on the seed type and variety (Fras et al., 2007). Dierences among cultivars in the individual and total amine content were more noticeable in broccoli than radish seeds. It has been reported that the prole and levels of amines in plants can be aected by dif-

 C. Martnez-Villaluenga et al. / Food and Chemical Toxicology 46 (2008) 16351644 Table 3 Biogenic amine content of raw and germinated (Brassica oleracea var. italica) cultivars* Germination time (days) cv. Lucky 0 3 5 cv. Tiburon 0 3 5 cv. Belstar 0 3 5 Putrescine 7.88 0.17a 14.67 0.46b 39.83 0.50c 6.26 0.27a 12.63 0.74b 37.56 0.42c 8.71 0.20a 14.97 0.34b 38.76 0.38c Cadaverine 13.54 0.20a 20.81 0.14b 35.50 0.53c 10.72 0.15a 16.89 0.33b 27.80 0.60c 14.47 0.26a 20.11 0.74b 30.08 0.42c Histamine 7.07 0.21a 11.40 0.57b 17.94 0.35c 10.00 0.14a 13.76 0.41b 16.60 0.30c 8.86 0.14a 12.33 0.57b 15.89 0.26c Tyramine 2.02 0.10a 6.03 0.24b 19.90 0.69c 1.90 0.12a 5.50 0.10b 15.80 0.61c 3.05 0.27a 5.88 0.48b 18.61 0.72c Spermidine 4.23 0.12a 8.31 0.21b 19.59 0.27c 4.43 0.33a 10.76 0.34b 19.01 0.64c 4.34 0.06a 7.95 0.16b 20.73 0.77c Spermine 7.47 0.26a 10.43 0.57b 22.44 0.57c 6.12 0.13a 10.13 0.42b 23.58 0.48c 5.53 0.39a 8.42 0.43b 22.53 0.80c

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Total biogenic amines 24.21 0.61a 71.66 1.08b 155.19 1.11c 32.49 0.54a 69.66 0.16b 140.35 1.58c 44.96 0.21a 69.67 1.35b 146.61 2.55c

* Mean of four determinations expressed in mg/kg dry matter standard deviation. Dierent superscript in the same column for each cultivar means signicant dierence (P 6 0.05).

Table 4 Biogenic amine content of raw and germinated radish (Raphanus sativus) cultivars* Germination time (days) cv. Bolide 0 3 5 cv. Rebel 0 3 5
*

Putrescine 9.00 0.26a 17.22 0.79b 41.31 0.48c 9.29 0.39a 14.86 0.13b 42.32 0.59d

Cadaverine 10.70 0.38a 15.25 0.49b 32.99 0.65c 12.33 0.35a 17.48 0.03b 28.60 0.60d

Histamine 10.34 0.14a 16.68 0.69b 19.75 0.61c 10.98 0.71a 15.18 0.31b 19.96 0.41c

Tyramine 3.23 0.07a 8.76 0.18b 15.70 0.63c 2.61 0.16a 7.83 0.17b 15.89 0.25d

Spermidine 8.87 0.11a 17.48 0.30b 30.97 0.78c 8.53 0.15a 16.26 0.51b 30.38 0.88d

Spermine 5.73 0.27a 13.56 0.37b 30.18 0.76c 4.93 0.25a 11.77 0.14b 27.38 0.60d

Total biogenic amines 47.82 0.28a 87.98 1.82b 170.97 1.67c 48.66 0.42a 83.15 0.58b 164.60 0.46d

Mean of four determinations expressed in mg/kg dry matter standard deviation. Dierent superscript in the same column for each cultivar means signicant dierence (P 6 0.05).

ferent cultivars (Cirilo et al., 2003; Gloria et al., 2005). However, there can be other pre- and post-harvest factors that aect amine levels, such as water availability, mineral deciency (Smith 1985), chemical-caused damage (Conca et al., 2001), osmotic shock, temperature or variation in altitude, and chilling injury (Valero et al., 1998; Serrano et al., 2003). The eect of germination on the individual and total biogenic amine content of dierent cultivars of broccoli (cv. Lucky, cv. Tiburon and cv. Belstar) and radish (cv. Bolide and cv. Rebel) is shown in Tables 3 and 4, respectively. The putrescine amounts detected in ve day sprouts were >4-fold higher than those found in raw seeds reaching values $40 mg/kg dw for broccoli and radish cultivars. The detected amounts of cadaverine in broccoli and radish cultivars increased more than twice compared with raw seeds ranging from 2735 mg/kg dw at the end of germination. Histamine values showed a gradual increase up to 16 18 mg/kg dw in broccoli cultivars and up to $20 mg/kg dw in radish cultivars after ve days of germination. Tyramine levels increased sharply in ve-day sprouts reaching values from 16 to 20 mg/kg dw in broccoli and $16 mg/kg dw in radish. Spermidine content also rose noticeably in broccoli and radish sprouts and levels of around 20 mg/kg dw and 30 mg/kg dw, respectively, were found. Spermine content showed an increasing trend from early stages to the end

of germination for both broccoli and radish and achieved values between 2224 and 2730 mg/kg dw in ve-day sprouts of broccoli and radish, respectively. Inuence of germination on total levels of biogenic amines in broccoli and radish seeds is also shown in Tables 3 and 4. The total amines showed an increasing trend throughout the germination period in broccoli and radish cultivars, with levels of $70 mg/kg dw and 85 mg/kg dw after three days and $150 mg/kg dw and $170 mg/kg dw after ve days, respectively. The pattern observed for total biogenic amines is similar to that previously reported in dierent seeds (Simon-Sarkadi and Holzapfel, 1995; Matilria la, 1996; Shalaby, 2000; Glo et al., 2005; Martnez-Vill aluenga et al., 2006; Fras et al., 2007), although these levels are lower than those found by Simon-Sarkadi and Holzapfel (1995) in mung bean, lentil and radish, and by Fras et al. (2007) in alfalfa sprouts, but similar to those recorded in fenugreek sprouts (Fras et al., 2007). Gloria et al. (2005) attributed the signicantly higher levels of polyamines spermidine, spermine and putrescine in soy sprouts to the greatest plant cellular multiplication and growth which occurs during sprouting. Shalaby (2000) and Simon-Sarkadi and Holzapfel (1995) observed an increase in cadaverine levels during the germination of some legume and radish seeds that could be associated with two factors: (a) its role in elongation of the root and the

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increase in cell size (Flores et al., 1989) and (b) the increase in bacterial numbers which are known to possess a high decarboxylase activity, since this enzyme plays a vital role in the metabolism of biogenic amines, especially cadaverine and putrescine (Simon-Sarkadi and Holzapfel, 1995). On the other hand, some strains of lactic acid bacteria naturally present on the seed surface and which could develop
(n cells x 10000)

further during sprouting are tyramine and histamine producers (Suzzi and Gardini, 2003). The allowable limit in foods for each individual amine established by the US Food and Drug Administration is 5 mg/100 g (USFDA, 2001) and similar levels exist in the legislation of dierent countries. The results of the present work indicate that none of the biogenic amine levels in any

120 100 80 60 40 20 0 24h 48h

Exposure time (h)

72h

Control
(n cells x 10000)

Lucky raw

Lucky 3 days

Lucky 5 days

140 120 100 80 60 40 20 0 24h 48h

Exposure time (h)

72h

Control
(n cells x 10000)

Tiburon raw

Tiburon 3 days

Tiburon 5 days

140 120 100 80 60 40 20 0 24h 48h

Exposure time (h)

72h

Control

Belstar raw

Belstar 3 days

Belstar 5 days

Fig. 1. Proliferation of HL-60 cells exposed to raw and germinated extracts of three cultivars of broccoli (Brassica oleracea L. var. italica).

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of the broccoli and radish sprouts studied are of risk for healthy consumers and individuals with restricted activity of the detoxication enzyme monoamine oxidase (MAO EC. 1.4.3.4.) since 100 g of edible portion would provide 1.5 mg of total biogenic amines. In patients treated with some psychoactive drugs, 6 mg intake of tyramine within a 4 h period can be deleterious (Tailor et al., 1994). 3.3. Citotoxicity evaluation of broccoli and radish during sprout production In order to determine whether the germination process was responsible for the appearance of toxic compounds in broccoli and radish sprouts, studies of cell proliferation and citotoxicity (MTT assay) were carried out in vitro. Fig. 1 shows the proliferation of HL60 cells exposed to extracts of three cultivars of broccoli (raw seeds and sprouts) and control. For the three cultivars, cell proliferation reached levels of 5458 (10,000) cells after 24 h exposure to raw broccoli extract, the number of cells increased to 9195 (10,000) cells after 48 h, and reached levels of 107117 (10,000) cells after 72 h exposure. Germination for three and ve days did not cause signicant (P 6 0.05) dierences in HL60 cell proliferation, and the
(n cells x 10000)

levels were very close to those found for cells exposed to distilled water (control), where cell proliferation obtained was 52, 90 and 109 (10,000) after 24, 48 and 72 h, respectively. A similar prole was found for two cultivars of radish (Fig. 2). Proliferation of HL60 cells exposed to extracts of the two cultivars of raw radish reached levels of 5661 (10,000) cells after 24 h exposure and cell counts increased gradually after 72 h exposure. Germination for three and ve days did not bring about signicant changes compared with raw seeds or with cells exposed to distilled water (control). These results suggest that germination promotes HL-60 proliferation, which seems to be independent of the germination conditions Similar results have been found in sprouted fenugreek extracts where cell proliferation increased, irrespective of germination conditions (two and four days, 20 and 30 C, with and without light), and after 72 h exposure and HL60 proliferation was equal to the control assay with cells exposed to distilled water (Fras et al., 2007). However, in alfalfa sprouts, proliferation of HL60 cells was dierent depending on the germination conditions (two and four days, 20 and 30 C, with and without light), and levels were always slightly lower than those found for the control (Fras et al., 2007).

140 120 100 80 60 40 20 0 24h 48h

Exposure time (h)
Control Bolide raw Bolide 3 days Bolide 5 days

72h

(n cells x 10000)

140 120 100 80 60 40 20 0 24h

Control Rebel raw

48h
Exposuretime (h)
Rebel 3 days

72h
Rebel 5 days

Fig. 2. Proliferation of HL-60 cells exposed to raw and germinated extracts of three cultivars of radish (Raphanus sativus).

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Brassica oleracea L . Var. italica

Absorbance

1.0 0.8 0.6 0.4 0.2 0.0 Lucky Tiburon Belstar

Raw

3 days

5 days

Control

Raphanus sativus
Absorbance

1.0 0.8 0.6 0.4 0.2 0.0 Bolide Rebel

Raw

3 days

5 days

Control

Fig. 3. MTT assay results performed on HL-60 cells exposed to raw and germinated extracts of threee cultivars of broccoli and two cultivars of radish.

Fig. 3 shows the results of the MTT assay performed on HL60 cells exposed to extracts of raw and sprouted seeds of broccoli and radish, and its comparison with control (cell exposure to distilled water). In broccoli extracts, MTT values for raw seeds were higher than control except for cv. Lucky and germination produced slight changes in MTT values, except for cv. Tiburon where a decrease was observed. In radish extracts, MTT values for raw seeds were lower for cv. Bolide and higher for cv. Rebel compared to control and germination brought about an increase in MTT values, except in cv. Rebel after three days where a decrease was observed (Fig. 3). MTT colorimetric assay has been described to measure cytotoxicity and cell proliferation, and the level of MTT is proportional to the viable cells (Mosmann, 1983; Gerlier and Thomasset, 1986; Ferrari et al., 1990; Fras et al., 2007). Consumption of cruciferous vegetables has been included in dietary recommendations (ACS, 1996) since it was shown by substantial and extensively reviewed evidence to reduce cancer risk at several major sites (Block et al., 1992; Steinmetz and Potter, 1996). Cruciferous vegetables contain little fat, are low in energy and are sources of vitamins and minerals, all aspects linked to cancer preven-

tion, but they also contain a large number of phytochemicals, some of which might protect against carcinogenesis (Nestle, 1998). Extracts of broccoli sprouts contain 10 100 times the phase 2 inducer activity of mature broccoli plants and are more ecient inhibitors of rat tumorigenesis (Zhang et al., 1992). At the same time, sulphoraphane present in broccoli sprouts induces phase 2 enzymes and inhibits carcinogenesis (Fahey et al., 1997). The work carried out here on broccoli and radish sprouts shows no cytotoxicity for all tested samples, and similar results have been reported for alfalfa and fenugreek sprouts (Fras et al., 2007). Since vegetable sprouts are considered as a healthy and fresh food, policies are needed to promote their consumption.

4. Conclusions Broccoli and radish sprouts are safe foods from the perspective of their microbiological and biogenic amine content. Furthermore, our ndings showed that these fresh vegetables did not inhibit cell proliferation and did not show cytotoxicity. This study, therefore, makes a scientic

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contribution to the eld of food safety of signicant interest for sprout consumers and producers. Conict of interest statement The authors declare that there are no conicts of interest. Acknowledgements We wish to thank Mr. Antonio Perez Romero for his skillful technical assistance. This research was funded by Project AGL-2004-00886/ALI from the Spanish CYCYT. Dr. Piotr Gulewicz thanks the Spanish Ministry of Education and Science for a postdoctoral fellowship. References
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