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Food and Chemical Toxicology 46 (2008) 1635–1644 www.elsevier.com/locate/foodchemtox
Food safety evaluation of broccoli and radish sprouts
´ ´ Cristina Martınez-Villaluenga a, Juana Frıas 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 ﬁve 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 eﬀects on proliferation and viability of HL-60 cells and should be included in our diets as healthy and safe fresh foods. Ó 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 ﬁnd on the market an extraordinary variety of diﬀerent 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 eﬀective in the primary prevention
Corresponding author. Tel.: +34 915622900x241; fax: +34 915644873. E-mail address: iﬁcv12@iﬁ.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 beneﬁts 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 ineﬀective way to eliminate spoilage and pathogenic bacteria (Mohle-Boetani et al., 2001). The importance of this problem is reﬂected by a number of speciﬁc 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 Â 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 Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.01.004
italica cv. Rebel and cv. The residues were dissolved in sterilized water to a ﬁnal concentration of 1 mg/mL.2. seeds were soaked in 50 mL distilled water for 5½ h with shaking every 30 min. Plates were incubated at 32 °C for mesophilic bacteria counts for 48 h and at 8 °C for psychrotrophic bacteria counts for 10 days. Spain) and incubated at 37 °C for 24 h.5 mL of deionized water and sonicated for 30 min (Sonorex AK103H). tyramine. pH 7. 100% of B (16 min) and held until the end of the run (23 min) with a ﬂow rate of 0.1636
´ C. Total and faecal coliforms were determined on Violet-Red Bile Agar (VRBA. Biogenic amine determination
Determination of biogenic amines was carried out by acid extraction. The imbibed seeds were germinated at pilot scale by layering seeds over moist ﬁlter paper in a germination tray. Afterwards. histamine and tyramine have been suggested as indicators of food deterioration (Ramantanis et al. Calibration curves were obtained for standard amines and ‘‘r” was always above 0.4. The number of viable cells was determined for cell proliferation approach. The chromatographic system consisted of an Alliance Separation Module 2695 (Waters. The presence of biogenic amines in foods and beverages is considered important because of their inﬂuence on physiological activities.3.
2. Scharlau Chemie. After a few passages. The supernatant was ﬁltered through 0. cadaverine dihydrochloride. sativus) after sprouting. 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. in order to ensure the food safety of these cruciferous sprouts it would be interesting to determine the eﬀect of seed germination on human cell viability. Spain) in tubes.07% sodium hypochlorite for 30 min.. Spain). Plant material
Broccoli seeds (B. Cytotoxicity evaluation
To obtain the extracts.990. All plates were incubated for 4 h at 37 °C
2. cells were centrifuged. histamine dihydrochloride. Five grams of fresh samples were aseptically placed into a ﬂask with 45 mL of peptone water (Scharlau Chemie.. Therefore. Milford. spermine. (2007). which are carcinogenic for various species of animals and a potential hazard to humans (Shalaby. Cell suspension (20 lL) was then loaded by micropipette into Burker cham¨ bers. However. 48 and 72 h. The culture was maintained on RPMI medium containing 10% fetal calf serum and 1% penicillin G (Sigma) and streptomycin (Sigma). spermidine. On the other hand. a Photodiode Array detector 996 (Waters) and a personal computer running the Empower II for windows chromatographic software (Waters). respectively.6 mm i. 20 lL of MTT (Sigma) at a concentration of 5 mg/mL in PBS. Biogenic amines can be naturally present in plant food since they are required in cellular metabolism and in growing tissues (Matilla.600 rpm. Standards were derivatized as described for the samples. Faecal Streptococci were surface-plated on Slanetz Bartley Agar (Scharlau Chemie.1% buﬀered peptone water (Scharlau Chemie. dizziness. headache. 1994). Moreover. Martınez-Villaluenga et al. The supernatants were removed and cells resuspended in 100 ll of fresh medium. USA). derivatization with dansyl chloride and HPLC quantiﬁcation according to ´ Frıas et al. since some of them may be nitrated or act as precursors for other compounds capable of forming nitrosamines. 1983). Then.. 90% (12 min). Serial dilutions were made in 0.4. amines are also investigated as a possible mutagenic precursor.
2. Microbiological determinations
Microbial counts were carried out using the poured plate technique.
of aldehydes and ketones (Askar and Treptow. 1986). 5 lm size (Symta) column equipped with a C18 guard column (Symta) both thermostatted at 30 °C.1. Spain) to achieve a 10À1 dilution and shaken with vortex for 1 min. vomiting and diarrhoea (Pechanek et al. putrescine. Brieﬂy.d. The germination process was carried out in triplicate and the rate was higher than 90%. The sample (20 lL) was injected onto a C18 Kromasil 250 Â 4. The elution programme was held at 65% of B for 1 min.
2. di. L. they are also a ´ consequence of microbial activity (Gloria et al. oleracea L.000 rpm. Paulsen et al.. Bolide) were purchased at Bejo Iberica and Man Fong Paciﬁc Trading (Spain). 1997). sativus cv. These seeds were drained and washed with distilled water until they reached neutral pH. 1996. the extracts were centrifuged for 15 min at 12. trypan blue dye (Sigma) was added to cell cultures in a ratio of 1:1 and left for 10 min. The cells were exposed to 100 lg/mL of raw and germinated seed extracts and control (sterilized water) for 24 h. The tray was placed in a seed germinator G-120 model (ASL Snijders International S. ramped at 80% (10 min). Tiburon. oleracea var. 1985. Belstar and cv. cadaverine. Radish seeds (R. Lucky) were purchased at Bejo Iberica (Spain). The tetrazolium reduction assay (MTT) was performed in duplicate following the method of Denizot and Lang (1986). 2005. Putrescine dihydrochloride.30 Â 106 cell per millilitre and transferred onto several plates in 2 mL volumes. var.. Sprouted seeds were collected after three and ﬁve days. Holland) and seeds were continuously watered by capillary.22 lm membranes into sterile test tubes. / Food and Chemical Toxicology 46 (2008) 1635–1644 To determine total mesophilic and psychrotrophic aerobic bacteria counts. were added to each sample. 80 mg) to a 25 mL volumetric ﬂask. After this time. at 37 °C in a humidity-controlled incubator at 90% relative humidity and 5% CO2. Santos.8 mL/min. Scharlau Chemie.and polyamines has been suggested as a supplementary criterion to indicate the freshness and quality of food. The human leukemic cell line (HL-60). spermidine trihydrochloride.. Spain) and incubated at 37 °C (for total coliforms) or 44 °C (faecal coliforms) for 24 h under anaerobic conditions. One mL of each ﬁltered supernatant was evaporated in tarred vessels and the obtained dry masses were weighed after 24 h desiccation with P2O5. appropriate serial dilutions were surface-plated on Triptic Soy Agar (TSA.5. The presence of some mono-. 100 lL of the cultured medium was transferred into Eppendorf tubes and centrifuged for 5 min at 1. which may cause symptoms such as skin irritations. derived from a patient with acute promyelocytic leukemia. The mobile phase for DCl-derivatives separation consisted of bidistilled water (solvent A) and HPLC-grade acetonitrile (solvent B). was obtained from American Type Culture Collection (ATCC).
2. The cells were exposed to 100 lg/mL of raw and germinated seed extracts and control (sterilized water) during 24.
. italica) and two cultivars of radish (R.. Materials and methods 2. the aim of this work is to determine the hygienic status. Hal` asz et al. Then. 1996). resuspended in fresh medium at the concentration of 0. 50 mg of the seed ﬂour were subjected to extraction with 2. Seeds germination
Seeds (10 g) was soaked with 50 mL of 0. 1996). biogenic amine content and cytotoxicity of three cultivars of broccoli (B. In particular. A stock standard aqueous solution of amines was prepared by adding an accurately weighed amount of each standard (ca. The cells were counted under a microscope at 100Â magniﬁcation. spermine tetrahydrochloride were purchased from Fluka (Spain). cv.
1983) and up to 4 log cycles in kidney bean (Kimanya et al. Rebel. respectively. 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. 0.6.
. 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.8 log cfu/ g in raw radish seeds cv. 2001.46 ± 0.24 ± 0. which remained unchanged in three-day and ﬁve-day broccoli sprouts. Rises in bacterial number observed in broccoli and radish sprouts are similar to others described in the literature.44 ± 0. Martınez-Villaluenga et al. 5% CO2 and 120 lL of DMSO (Sigma) were added. Tiburon 0 3 5 cv.77 ± 0.41 ± 0.13c 5.12b 7. 2006).34ab 8.17c 6.
2..26 ± 0. Results and discussion 3. The amount of faecal streptococci in raw broccoli seeds showed levels <2 log cfu/g.´ C.19b Total coliforms 6.07b 8. microbial populations increased $2 log cycles in rice (Piernas and Guirand.17c Faecal Streptococci <2 <2 <2 <2 <2 <2 <2 <2 <2
* Results are expressed as mean value ± SD of three repetitions.05a 7.1.12a 8.44 ± 0.. ´ Piernas and Guirand.70 ± 0.0 Program (Statistical Graphics Corporation.12b 9. respectively. Lucky 0 3 5 cv.20c Psychrotrophic Bacteria 6. and germination led to gradual increases of about $3 log cycles for both types of populations after ﬁve days. Table 2 shows the microbial population of raw and germinated radish cultivars (cv.28 ± 0.41 ± 0. Fecal coliform numbers amounted to 4. and presented an upward trend during sprouting.00b 8.22 ± 0. 1997). Feng (1997) showed that alfalfa and mungbean seeds routinely contained high numbers of microbial ﬂora (102À106 cfu/g) including coliforms (104 cfu/g) and faecal coliforms (102À103 cfu/g)..31 ± 0.43 ± 0.29 ± 0. 1982.48 ± 0. After 3 h of incubation at 37 °C samples were centrifuged for 5 min at 1. Rockville. Psychrotrophic bacteria numbers in raw radish seeds were $7 log cfu/g which increased up to $2 log cycles after ﬁve days of germination.03b 9.07b 7.50 ± 0. Diﬀerent superscript in the same column for each cultivar means signiﬁcant diﬀerence (P 6 0.49 ± 0.06b 6. 1991.09a 8. 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 ﬁve days of germination. Belstar.06b 8.600 rpm and 25 lL of Sorensen buﬀer (0.24 ± 0..34 ± 0. reaching numbers up to $7 log cfu/g after ﬁve days.05) growing up to $2 log cycles after ﬁve days of germination in cv.82 ± 0.05).
3. Hygienic status of broccoli and radish sprouts Table 1 shows the microbial population of raw seeds and broccoli sprout cultivars Lucky. and
Table 1 Microbial population in raw and sprouted broccoli (Brassica oleracea var. Tiburon and cv.45 ± 0.44 ± 0. 2006).40 ± 0.69 ± 0.05a 8. Total coliform counts corresponded to 4.29b 9.44 ± 0. italica) cultivars* Germination time (days) Counts (log 10 cfu/g fresh sprout) Mesophilic bacteria cv.64 ± 0..4 log cfu/g for radish seeds cv.11a 7. Belstar 0 3 5 7. 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 signiﬁcantly (P 6 0.32a 7.12b 6. pH = 10. Total and faecal coliforms achieved $6 and 5 log cfu/g in raw broccoli seeds. Splittstoesser et al. The number of total aerobic mesophilic bacteria in raw radish seeds reached $8 log cfu/g. Bolide and cv. and that these microorganisms form part of the normal seed microﬂora. Absorbance of the formazan product was measured at 570 nm.22a 6.39 ± 0.48c 6.5) were added to supernatants. 2003).42 ± 0. / Food and Chemical Toxicology 46 (2008) 1635–1644 in a humidity-controlled incubator at 90% relative humidity. Martınez-Villaluenga et al.06c Faecal coliforms 5. Diﬀerences in the microbial population of diﬀerent seeds can occur due to diﬀerent composition of the seed coat and diﬀerent cultivation and storage conditions (Piernas and Guirand.44 ± 0.48 ± 0.1 M NaCl.and disaccharides) and amino acids providing available substances for microbial growth. lupin and fenugreek (Martınez-Villaluenga et al. Bolide and cv.08b 9. counts higher than those found in broccoli seeds. whilst it remained unmodiﬁed in cv. 3 log cycles in alfalfa and mungbean (Andrews et al. 1997. Statistical analyses
Data were subjected to multi-factor analysis of variance by applying the least signiﬁcance diﬀerence test with the Statgraphic 4.06a 7.8 and 5.40 ± 0.77 ± 0.25 ± 0. Lucky.05a 7.08b 9.02c 7. The viable numbers of faecal streptococci present in radish seeds were < 2 log cfu/g and remained constant until the end of germination.46 ± 0. Soylemez et al. Thus.15c 7.69 ± 0. respectively. Rebel.. Md) for Windows.1 M glycine. Tiburon and Belstar after three and ﬁve days of germination. and germination brought about a rise in total coliform counts of $8 log cfu/g.33a 7. within two days of germination.67 ± 0.02b 9. Bolide and cv.03a 6.2 and 5.08c 5. Rebel).06a 6. Total aerobic plate counts increased gradually up to 1 log cycle after ﬁve days of germination
for the two radish cultivar sprouts. ´ 1997).
39a 8. tyramine.12b 5.07a 7.16 ± 0. Skowronek et al.78 ± 0. Moreover. 2003) and in some cases the germination capability of the treated seeds was aﬀected.m.78 ± 0. freshness and organoleptic properties of sprouts.11 ± 0.47b 8. On the other hand. 2007).01a 9. 2003.82 ± 0.02b 9.10c 5. Penas et al.m.86 ± 0. cv. studies have demonstrated that although seed decontamination can reduce the populations of human pathogens present.13 ± 0.6–3.3–11... Broccoli and radish sprouts contained numbers of mesophilic. several methods such as heat treatment (Jaquette et al. Sharma et al. 2006. (1990) demonstrated that Enterobacteria are the dominant microorganisms present in vegetable sprouts.18 ± 0.22 ± 0.1 mg/kg d. 2002) and high hydrostatic pressure (Wuytack et al.41 ± 0. showing that biogenic amine levels seem to depend on the seed type and variety ´ (Frıas et al.42 ± 0. Belstar) and radish (cv.
(b) the high moisture required and the warm temperatures during sprouting create a suitable environment for bacterial development (Feng.08 ± 0..12b 7. Total and individual amine content of diﬀerent legume and radish seeds reported in the literature were more than two-fold higher (Simon-Sarkadi and Holzapfel.07a 7. ´ 2005..2 mg/kg d. 1983). interventions should be carried out to prevent or minimize contamination of raw
sprouts and to remove pathogens prior to consumption.23 ± 0.59 ± 0.. cadaverine (10.) was the minor amine found.11b 6.5 mg/kg d. Shalaby. it cannot ensure the production of pathogen-free sprouts (Thomas et al. Proctor et al. 2003..34 ± 0. fenugreek or onion (Prokopowich and Blank. comprised up to 95–99% of the total microbial population of mungbean.7–12.62b 7. Bolide 0 3 5 cv. 2006).75 ± 0. Diﬀerences among cultivars in the individual and total amine content were more noticeable in broccoli than radish seeds. 2001. Rebel). 2000. Martınez-Villaluenga et al. 1991. Kimanya et al. chemical disinfectans (Beuchat et al. ´ Skowronek et al. 2007)..9–3. 2008) have ˜ been described for reducing the microbial ﬂora on seeds achieving P5 log reductions recommended by the National Advisory Committee on Microbial Criteria for Foods (NACMCF. The presence of biogenic amines has been described in diﬀerent legume and radish seeds (Simon-Sarkadi and Holzapfel. ´ ´ 1995. 2005).) and histamine (10.07a 8. Tiburon and cv.11b 7.02b 8. although no information has been found about these amines in broccoli seeds.. 1999).m.) while tyramine (1. spermidine and spermine in all cultivars. lupin.3 mg/kg d.1638
´ C. without losses in the nutritional.m. exposure to ionizing radiation (Thayer et al.01c
Total coliforms 4.37 ± 0. / Food and Chemical Toxicology 46 (2008) 1635–1644
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. 2000. 2003). with the exception of soybean seeds which presented ´ lower levels (Gloria et al.. In this sense. 3.. Therefore. 1998).6 mg/kg d. 2003. Raw seeds showed the presence of putrescine. 2001. Weiss and Hammes. 1998.05 ± 0. histamine. Moreover.m while in radish seeds we found a narrow range from 47.7– 14.16a 5.) was the minor contributor to total amine levels. However...0 mg/kg d.22c
Faecal coliforms 4. Martınez -Villaluenga et al. it is noteworthy that putrescine and spermidine levels were higher in radish than broccoli seeds..) were the major amines found in radish seeds contributing about equally to the total amine content while tyramine (2. Nevertheless.2. 1997).03a 8. 2006) and even for commercial mungbean sprouts (Skowronek et al. Ghandi ´ and Matthews. Frıas et al.12a 9.79 ± 0. Geiges et al..33b 8. lentil and radish sprouts after two days of germination.05).01c
Faecal Streptococci <2 <2 <2 <2 <2 <2
Results are expressed as mean value ± SD of three repetitions.08a 6.21 ± 0. 1995. it is necessary to develop hygienic methods to improve the shelf-life and safety of sprouts. Fett and Cooke.. respectively.. Rebel 0 3 5
Psychrotrophic Bacteria 6.8 to 48.78 ± 0. Diﬀerent superscript in the same column for each cultivar means signiﬁcant diﬀerence (P 6 0. Bolide and cv. Total biogenic amine content in broccoli seeds ranged widely from 24–45 mg/kg d. cadaverine. Martınez-Villaluenga et al. Gloria et al.m. Cadaverine was predominant in broccoli seeds (ranging from 10.89 ± 0.. 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. total and faecal coliform bacteria which are the usual counts for minimally processed germinated seeds such as alfalfa. Inﬂuence of germination on proﬁle and levels of biogenic amines Table 3 and Table 4 show the individual and total biogenic amine content in seeds of diﬀerent cultivars of broccoli (cv. (2003) reported that the absence or low levels of pathogens in sprouts are not suﬃciently competitive with the background ﬂora during germination to increase to levels which lead to toxin production. (1998) reported that not only Enterobacteria but also Pseudomonas spp.18 ± 0.. Lucky. 1996.21b 9. Lang et al.. 2007) than values reported for broccoli and in radish seeds in this work. psychrotrophic.98 ± 0. Frıas et al.m. bean. It has been reported that the proﬁle and levels of amines in plants can be aﬀected by dif-
69c 1.50 ± 0.08 ± 0.38 ± 0.76 ± 0.54a 69.60 ± 0.33 ± 0.11c 32.89 ± 0.17a 14.95 ± 0.66 ± 1.49 ± 0.05 ± 0.47 ± 0. spermine and putrescine in soy sprouts to the greatest plant cellular multiplication and growth which occurs during sprouting.97 ± 0.31 ± 0.71a 15.87 ± 0. Belstar 0 3 5 Putrescine 7. (2005) attributed the signiﬁcantly higher levels of polyamines spermidine. Spermidine content also rose noticeably in broccoli and radish sprouts and levels of around 20 mg/kg dw and 30 mg/kg dw.90 ± 0. The detected amounts of cadaverine in broccoli and radish cultivars increased more than twice compared with raw seeds ranging from 27–35 mg/kg dw at the end of germination.70 ± 0.61c 10.64c 4.14a 16.34b 19. 2007). Shalaby.20a 20.42c 8.33b 27.72 ± 0.26 ± 0.28a 87.72c Spermidine 4. 2006.57b 22.76c 4..65c 12.78c 8.26c Tyramine 2.61c 3.21a 69.
´ ferent cultivars (Cirilo et al.47 ± 0. Lucky 0 3 5 cv.74b 37.70 ± 0.38c Cadaverine 13.42c Histamine 7.13a 10.14a 13.35b 146..63 ± 0.86 ± 0. 1998. Martınez-Vill´ aluenga et al.35 ± 1.60d
Histamine 10.57b 15.98 ± 0.48c 5.18 ± 0.15a 16. 2003.88d
Spermine 5.66 ± 0.25d
Spermidine 8. The eﬀect of germination on the individual and total biogenic amine content of diﬀerent cultivars of broccoli (cv.26 ± 0.63c 2.29 ± 0.80 ± 0.12 ± 0.13b 42. Martınez-Villaluenga et al.10a 6. Rebel 0 3 5
Putrescine 9. However.24b 19. mineral deﬁciency (Smith 1985).46b 39.17b 15.40 ± 0.83 ± 0..34 ± 0.26a 10.43 ± 0.32 ± 0.89 ± 0.22 ± 0.53c 10.and post-harvest factors that aﬀect amine levels.61 ± 0. Tiburon and cv.76 ± 0.18b 15.20a 14.56 ± 0.01 ± 0. The pattern observed for total biogenic amines is similar to that previously reported in diﬀerent seeds (Simon-Sarkadi and Holzapfel.56 ± 0.31 ± 0. (2007) in alfalfa sprouts. there can be other pre.06a 7. Spermine content showed an increasing trend from early stages to the end
of germination for both broccoli and radish and achieved values between 22–24 and 27–30 mg/kg dw in ﬁve-day sprouts of broccoli and radish.07a 8.48 ± 0.03 ± 0.34 ± 0.39a 8.89 ± 0.25a 11.33 ± 0.57b 17.77 ± 0.12a 8..75 ± 0.44 ± 0.61 ± 2. Frıas et al.14a 12.27a 12.02 ± 0.73 ± 0.
Table 4 Biogenic amine content of raw and germinated radish (Raphanus sativus) cultivars* Germination time (days) cv.05). Serrano et al.60d
Total biogenic amines 47.00 ± 0.46d
Mean of four determinations expressed in mg/kg dry matter ± standard deviation.34b 38. / Food and Chemical Toxicology 46 (2008) 1635–1644 Table 3 Biogenic amine content of raw and germinated (Brassica oleracea var.23 ± 0.07 ± 0.79b 41. 2001).51b 30.82 ± 0.97 ± 1.68 ± 0.99 ± 0. were found..15a 16. such as water availability.21 ± 0.61a 71.53 ± 0.66 ± 0. although these levels are lower than those found by Simon-Sarkadi and Holzapfel (1995) in mung bean.93 ± 0.37b 30. respectively. Glo et al.30b 30. osmotic shock. Diﬀerent superscript in the same column for each cultivar means signiﬁcant diﬀerence (P 6 0.76 ± 0.50 ± 0.48b 18.11 ± 0. Lucky.58 ± 0.19 ± 1.58b 164.16b 20.08b 155. 1996.33a 10.53 ± 0. 2000. cv.53 ± 0. and by ´ Frıas et al..96 ± 0.38a 15.41b 16.03b 28.05).42a 83. Belstar) and radish (cv. respectively.12a 5.´ C.16b 140. but similar to those ´ ´ recorded in fenugreek sprouts (Frıas et al. The total amines showed an increasing trend throughout the germination period in broccoli and radish cultivars. respectively.88 ± 0.14b 35.71 ± 0.60 ± 0.13 ± 0.60c 14.67 ± 0. Rebel) is shown in Tables 3 and 4.48 ± 0.30c 8. Gloria et al.88 ± 0.86 ± 0. respectively. italica) cultivars* Germination time (days) cv.11a 17.57c 6.35c 10. 2007). 2005..39a 14.16a 7. The putrescine amounts detected in ﬁve day sprouts were >4-fold higher than those found in raw seeds reaching values $40 mg/kg dw for broccoli and radish cultivars.15 ± 0.27a 5.21b 19.26a 20..10b 15.98 ± 1. 1995.59d
Cadaverine 10.27a 13.77c Spermine 7. Tiburon 0 3 5 cv. Bolide and cv. Inﬂuence of germination on total levels of biogenic amines in broccoli and radish seeds is also shown in Tables 3 and 4.74b 30.82b 170. Gloria et al. lentil and radish.60 ± 0. 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 ﬁve days of germination.55c
* Mean of four determinations expressed in mg/kg dry matter ± standard deviation. chemical-caused damage (Conca et al. and chilling injury (Valero et al.14b 27.83 ± 0.35a 17.31b 19.80c
Total biogenic amines 24.59 ± 0.94 ± 0.73 ± 0.80 ± 0.81 ± 0.49b 32.97 ± 0. Matil´ria ´ la.27c 4. temperature or variation in altitude.50c 6.25 ± 0.54 ± 0..67 ± 1. Bolide 0 3 5 cv.38 ± 0.42b 23.41c
Tyramine 3.96 ± 0.23 ± 0.48c 9.76 ± 0.67c 48.26a 17.21a 11.18 ± 0. Tyramine levels increased sharply in ﬁve-day sprouts reaching values from 16 to 20 mg/kg dw in broccoli and $16 mg/kg dw in radish. 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 ﬁve days.42 ± 0.58c 44. 2003).00 ± 0.90 ± 0. 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
. Diﬀerent superscript in the same column for each cultivar means signiﬁcant diﬀerence (P 6 0.43b 22.43 ± 0. 2005).69b 19.61 ± 0.
1989) and (b) the increase in bacterial numbers which are known to possess a high decarboxylase activity. Martınez-Villaluenga et al. var. italica). 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)
(nº cells x 10000)
Lucky 3 days
Lucky 5 days
140 120 100 80 60 40 20 0 24h 48h
Exposure time (h)
(nº cells x 10000)
Tiburon 3 days
Tiburon 5 days
140 120 100 80 60 40 20 0 24h 48h
Exposure time (h)
Belstar 3 days
Belstar 5 days
Fig. 2003). 2001) and similar levels exist in the legislation of diﬀerent countries. / Food and Chemical Toxicology 46 (2008) 1635–1644
increase in cell size (Flores et al.. some strains of lactic acid bacteria naturally present on the seed surface and which could develop
(nº cells x 10000)
further during sprouting are tyramine and histamine producers (Suzzi and Gardini. 1.
´ C. The allowable limit in foods for each individual amine established by the US Food and Drug Administration is 5 mg/100 g (USFDA. Proliferation of HL-60 cells exposed to raw and germinated extracts of three cultivars of broccoli (Brassica oleracea L. since this enzyme plays a vital role in the metabolism of biogenic amines. especially cadaverine and putrescine (Simon-Sarkadi and Holzapfel. On the other hand.
Martınez-Villaluenga et al.3. Germination for three and ﬁve days did not cause signiﬁcant (P 6 0. 1994). proliferation of HL60 cells was diﬀerent depending on the germination conditions (two and four days. 20 and 30 °C.000) after 24. in alfalfa sprouts. However. 20 and 30 °C.3.4. and reached levels of 107–117 (Â10. 2). 3. with and without light). In patients treated with some psychoactive drugs.000) cells after 72 h exposure. 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.000) cells after 24 h exposure and cell counts increased gradually after 72 h exposure. A similar proﬁle was found for two cultivars of radish (Fig. 1.
. 6 mg intake of tyramine within a 4 h period can be deleterious (Tailor et al.. and after 72 h exposure and HL60 proliferation was equal to the control assay with cells exposed to distilled ´ water (Frıas et al.000) cells after 48 h. Proliferation of HL60 cells exposed to extracts of the two cultivars of raw radish reached levels of 56–61 (Â10.) since 100 g of edible portion would provide 1. For the three cultivars. the number of cells increased to 91–95 (Â10.4. 2007). irrespective of germination conditions (two and four days.000) cells after 24 h exposure to raw broccoli extract. 2007). respectively.05) diﬀerences in HL60 cell proliferation. 2. which seems to be independent of the germination conditions Similar results have been found in sprouted fenugreek extracts where cell proliferation increased.
140 120 100 80 60 40 20 0 24h 48h
Exposure time (h)
Control Bolide raw Bolide 3 days Bolide 5 days
(nº cells x 10000)
140 120 100 80 60 40 20 0 24h
Control Rebel raw
Rebel 3 days
Rebel 5 days
Fig.. 90 and 109 (Â10. and the
(nº cells x 10000)
levels were very close to those found for cells exposed to distilled water (control). / Food and Chemical Toxicology 46 (2008) 1635–1644
of the broccoli and radish sprouts studied are of risk for healthy consumers and individuals with restricted activity of the detoxiﬁcation enzyme monoamine oxidase (MAO EC. 48 and 72 h.. 1 shows the proliferation of HL60 cells exposed to extracts of three cultivars of broccoli (raw seeds and sprouts) and control. Proliferation of HL-60 cells exposed to raw and germinated extracts of three cultivars of radish (Raphanus sativus). cell proliferation reached levels of 54–58 (Â10. and levels were always ´ slightly lower than those found for the control (Frıas et al. studies of cell proliferation and citotoxicity (MTT assay) were carried out in vitro. Fig. where cell proliferation obtained was 52. These results suggest that germination promotes HL-60 proliferation. Germination for three and ﬁve days did not bring about signiﬁcant changes compared with raw seeds or with cells exposed to distilled water (control). with and without light).´ C.5 mg of total biogenic amines.
1997).0 0. 1992). Bolide and higher for cv. In radish extracts. Tiburon where a decrease was observed. all aspects linked to cancer preven-
tion.8 0. MTT colorimetric assay has been described to measure cytotoxicity and cell proliferation.. 3. This study.. 1996) since it was shown by substantial and extensively reviewed evidence to reduce cancer risk at several major sites (Block et al. Consumption of cruciferous vegetables has been included in dietary recommendations (ACS.
Fig.4 0.4 0. 3 shows the results of the MTT assay performed on HL60 cells exposed to extracts of raw and sprouted seeds of broccoli and radish. therefore. are low in energy and are sources of vitamins and minerals.1642
´ C. some of which might protect against carcinogenesis (Nestle. sulphoraphane present in broccoli sprouts induces phase 2 enzymes and inhibits carcinogenesis (Fahey et al.6 0. MTT assay results performed on HL-60 cells exposed to raw and germinated extracts of threee cultivars of broccoli and two cultivars of radish. At the same time. Rebel after three days where a decrease was observed (Fig. 1996). Gerlier and Thomasset. our ﬁndings showed that these fresh vegetables did not inhibit cell proliferation and did not show cytotoxicity. 2007).2 0. Furthermore. makes a scientiﬁc
. Steinmetz and Potter. / Food and Chemical Toxicology 46 (2008) 1635–1644
Brassica oleracea L . italica
1. 1992. ´ 1986. Frıas et al.8 0. In broccoli extracts.2 0. except in cv.
4... Ferrari et al. Extracts of broccoli sprouts contain 10– 100 times the phase 2 inducer activity of mature broccoli plants and are more eﬃcient inhibitors of rat tumorigenesis (Zhang et al. and the level of MTT is proportional to the viable cells (Mosmann.. MTT values for raw seeds were lower for cv. 1983. Martınez-Villaluenga et al.0 Lucky Tiburon Belstar
1. 1998). Rebel compared to control and germination brought about an increase in MTT values. and similar results have been ´ reported for alfalfa and fenugreek sprouts (Frıas et al. policies are needed to promote their consumption. MTT values for raw seeds were higher than control except for cv. but they also contain a large number of phytochemicals.0 0. Lucky and germination produced slight changes in MTT values.0 Bolide Rebel
Fig.6 0. Cruciferous vegetables contain little fat. 1990. 3). Conclusions Broccoli and radish sprouts are safe foods from the perspective of their microbiological and biogenic amine content. Since vegetable sprouts are considered as a healthy and fresh food. Var. The work carried out here on broccoli and radish sprouts shows no cytotoxicity for all tested samples. and its comparison with control (cell exposure to distilled water). except for cv.. 2007).
Ingham. Slutsker. ´ ´ ´ Martınez-Villaluenga.H. 119–125.. 239–247. C. ` ` Halasz.
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