591 Kaohsiung J Med Sci December 2004 • Vol 20 • No 12
A
NTIMICROBIAL
E
FFECT
OF
E
XTRACTS
OF
C
RUCIFEROUS
V
EGETABLES
Shu-Hui Hu, Jinn-Chyi Wang,1 Hsien-Feng Kung,1 Jih-Terng Wang,1 Wei-Lun Lee,2 and Yi-Hsin Yang3
Departments of Technology for Medical Science and 3Dental Hygiene, Kaohsiung
Medical University, Kaohsiung, and Departments of 1Food Science and Technology and
2
Environmental Engineering and Sanitation, Tajen Institute of Technology, Pingtung, Taiwan.
The cruciferous vegetables cauliflower, broccoli, cabbage, Chinese radish, Chinese kale, and Chinese kitam were used in this study to prepare water-soluble and methanol-water extracts. Crude protein extracts were also obtained by diethylaminoethyl (DEAE) anion exchange chromatography. Water-soluble polysaccharides were prepared by ethanol precipitation followed by ultrafiltration. The antimicrobial effects of all these extracts were evaluated against Gram-positive bacteria, Gram-negative bacteria, and yeast. Crude protein extracts exhibited the greatest antimicrobial activity in monoculture experiments. The antimicrobial effects of cruciferous vegetables were also studied by steeping beef, carrot, and celery in chlorine (10 ppm) or citric acid solution (1%) containing the crude protein extract (500 ppm) for different time periods. Total aerobic plate counts and coliform counts on these foods decreased significantly after 10 minutes in all steeping solutions (p < 0.05).
Key Words: cruciferous, extracts, crude protein, water-soluble polysaccharide, antimicrobial effect
(Kaohsiung J Med Sci 2004;20:591–9)
Received: August 27, 2004 Accepted: December 2, 2004 Address correspondence and reprint requests to: Dr. Jinn-Chyi Wang, Department of Food Science and Technology, Tajen Institute of Technology, 20 Wei-Shin Road, Shin-Erh Village, Yen-Pu Shiang, Ping Tung 90703, Taiwan.
E-mail: [email protected]
Many plants and vegetables contain compounds with antimicrobial activity. For example, shepherin (a peptide) in the root of shepherd’s purse inhibits Gram-negative bacteria and fungi [1]. Steroid and non-methylated fatty acids extracted from Diplotaxis harra Forsle inhibit fungi, yeast, and Gram-negative and Gram-positive bacteria [2]. An isothiocyanate (6-methyl sulfinylhexyl isothiocyanate) in Japanese horseradish may inhibit aggregation of human platelets [3]. Some spices and herbs inhibit the growth of Shigella sonnei and Shigella flexneri [4].
Cruciferous vegetables such as cauliflower, cabbage, mustard, Chinese cabbage, carrot, Chinese kale, and turnip
are edible plants that are low in calories, high in fiber, rich in vitamins and minerals, and have physiologic effects in humans [5,6]. Some important enzymes such as chitin-ase, glutathione transferchitin-ase, and epoxide hydrolase are also found in cruciferous vegetables [7,8]. Indole and isothiocyanate, enzymatic products of myrosinase from glucosinolate found in cauliflower and cabbage, lower the incidence of tumor formation and have an antioxidative effect [9]. There are also antifungal components such as iprodione [10], chitosan [11–13], and a peptide [14] in cruciferous vegetables.
The aim of this research was to study the antimicrobial activities of cruciferous vegetables. Various components from crucifers, such as those in water extracts, methanol-water extracts, and crude protein extracts (CPEs) and methanol- water-soluble polysaccharides (WSPS), were prepared and tested for their ability to inhibit microorganisms associated with food spoilage and illness. The results of these analyses could be useful in selecting some of these extracts as natural
disinfectants in food preparation. To further understand the practical applications of these extracts, their antimicrobial effects on the surfaces of foods were also studied.
M
ATERIALSANDM
ETHODSMicroorganisms
The following microorganisms were used in this study: Bacillus cereus CCRC 14665 (Culture Collection and Research Center, Taiwan), Bacillus subtilis CCRC 14199, Enterobacter aerogenes CCRC 10370, Escherichia coli CCRC 11634, Listeria monocytogenes CCRC 14845, Proteus vulgaris CCRC 12153, Pseudomonas aeruginosa CCRC 10773, Pseudomonas fluorescens CCRC 13902, Salmonella enterica serotype Typhimurium CCRC 12497, S. sonnei CCRC 10773, Streptococcus faecalis CCRC 10066, Staphylococcus aureus CCRC 11863, and Candida albicans CCRC 21538. Bacteria were cultured in nutrient broth (Difco, Sparks, MD, USA) at 37°C for 18 hours. Yeasts were cultured in Sabouraud medium (Difco) at 28°C for 40 hours.
Vegetable preparation
Six different kinds of fresh cruciferous vegetables including cauliflower (Brassica oleracea var. italica), broccoli (B. oleracea var. botrytis), cabbage (B. oleracea var. capitata), Chinese radish (Raphanus sativus), Chinese kale (B. oleracea var. alboglabra), and Chinese kitam (Brassica rapal) were purchased from a local market in Ping Tung, Taiwan.
After rinsing in running tap water, the vegetables were freeze-dried (600 g, dry weight) and then macerated in methanol (Merck, Darmstadt, Germany) overnight. The methanol extracts were filtered and concentrated under vacuum. The residue was extracted with boiling distilled water (1:10 wt/vol) for 15 minutes and then centrifuged (4,000g) for 10 minutes. The supernatant obtained was lyophilized to provide a methanol-water extract.
Water extracts and CPEs were obtained by blending lyophilized vegetables in distilled water (1:10 wt/vol) in a blender (Osterizer, Sunbeam Products Inc, Boca Raton, FL, USA). The homogenate was maintained at 20°C for 10 hours and filtered through filter paper (Whatman No. 1, Whatman plc, Brentford, Middlesex, UK). Part of the fil-trate was lyophilized to give the water extract. The re-mainder was subjected to ammonium sulfate precipita-tion. Ammonium sulfate was added to the filtrate to a final concentration of 50% and the precipitate collected by centrifugation at 12,400g at 4°C for 15 minutes. The precipitate was dissolved in 0.02 M Tris-HCl (pH 8.2) and
applied to a diethylaminoethyl (DEAE)-Sepharose (Am-ersham Pharmacia Biotech AB, Uppsala, Sweden) anion-exchange column (40 cm × 3 cm). The column was washed stepwise with 0.02 M Tris-HCl (pH 8.2) solution containing 0.1, 0.3, and 0.8 M NaCl, respectively. The eluate with the highest protein content was collected, dialyzed overnight at 4°C and then lyophilized. The lyophilized sample was designated the CPE [15]. Protein content was determined by the Bradford assay [16].
WSPS were prepared by adding distilled water (1:10 wt/vol) to the vegetables and heating at 60°C for 20 minutes, then filtering through Whatman No. 1 filter pa-per. Ethyl alcohol (95%) was added to the filtrate, followed by 75% alcohol to precipitate polysaccharides [17]. The precipitate was collected by centrifugation and dissolved in distilled water and filtered (Militan system, ELO 04; Millipore Co, Billerica, MA, USA) to collect the components
with molecular weight below 105 Da. These components
were concentrated by lyophilization and then redissolved in deionized water (pH 7.0). The content of the WSPS was determined using the phenol-sulfuric acid method [18].
Antimicrobial activity of cruciferous vegetables
A preliminary disk diffusion test was used to investigate the antimicrobial activities of the vegetable preparations [19,20]. To determine antibacterial activity, melted medium (45°C, nutrient agar for bacteria, Sabouraud medium foryeast) containing 106 colony-forming units (CFU)/mL was
poured onto solidified medium on a plate. The solidified medium was plate count agar (Difco) for bacteria and YM agar (Difco) for yeast. A paper disk (Adventec, Toyo Roshi Kaisha Ltd, Tokyo, Japan), 8 mm in diameter and 0.85 mm thick, containing 30 µL of sample solution (0.5 µg/µL) was placed in the center of the plate after the top agar had solidified. Bacterial plates were incubated at 37°C for 14 hours while yeast plates were incubated at 28°C for 24 hours [21]. The antimicrobial activity was assessed by the diameter (mm) of the inhibition zones, measured using calipers.
Combined effect of CPE and other chemicals on
foods
Simulated foods including fresh beef (8 × 8 × 0.8 cm), celery and carrot (5 × 1.5 × 0.5 cm) dices were individually steeped in 100 mL solution at a final concentration of 500 ppm CPE from cabbage alone or with either 1% citric acid or 10 ppm chlorine. The steeped solutions were incubated at 20°C for 5, 10, or 30 minutes.
0.1 M potassium phosphate buffer (pH 7.0) and homogen-ized with a stomacher (Type BA 7021, Seward Medical, London, UK) for 1 minute. The homogenates were col-lected and total aerobic plate counts (TPC) and coliform counts were measured using Petrifilm [22,23]. The anti-microbial effect was expressed in terms of lethality, defined as the logarithmic ratio of microbial counts before and after treatment [24].
Statistical analysis
All treatments were conducted in three samples and duplicate tests were run for each treatment. Data from each treatment underwent analysis of variance. Duncan’s multiple range test was used to determine significant difference (p < 0.05) between treatments.
R
ESULTSAntimicrobial effect of extracts
The antimicrobial effects of the four extracts of cruciferous vegetables are shown in the Table. Water extracts from cabbage had the highest antimicrobial activity, especially against Gram-negative bacteria, and of all bacteria examined, E. aerogenes was the most inhibited by cabbage extracts. Water extracts from all six kinds of cruciferous vegetables also inhibited C. albicans, the only fungus used in this experiment. The inhibition of C. albicans by the six water extracts was greater than that of B. cereus and B. subtilis, but lower than that of other Gram-positive bacteria. None of the water extracts was able to inhibit P. vulgaris.
Methanol-water extracts of cabbage, Chinese kale, and Chinese kitam had antimicrobial effects against two species of Pseudomonas. Only the cabbage methanol-water extract inhibited the two Bacillus spp. Methanol-water extracts of broccoli, Chinese kale, and Chinese kitam exhibited greater inhibition of C. albicans than did their water-based counterparts.
WSPS from all six kinds of cruciferous vegetables had greater inhibitory effects against Gram-negative bacteria, especially E. coli and Salmonella enterica serotype Typhimurium, than against Gram-positive bacteria. Cabbage WSPS had greater effects against the two Gram-positive cocci than against Bacillus spp. (p < 0.05). The inhibition of S. sonnei by WSPS from Chinese radish was not significantly different from that by WSPS from Chi-nese kale (p > 0.05).
CPEs from cauliflower, broccoli, cabbage, and Chinese kitam inhibited P. vulgaris, although water and
methanol-water extracts from these vegetables did not. All CPEs exhibited significantly greater antimicrobial effects against S. sonnei than water extracts (p < 0.05). CPEs had greater inhibitory effects against Gram-negative bacteria than against Gram-positive bacteria, as shown by the signifi-cantly larger inhibition zones observed in all four Gram-negative cultures with CPEs, including E. aerogenes, E. coli, S. enterica serotype Typhimurium and S. sonnei, than in Gram-positive cultures (p < 0.05).
Combined effect of CPE from cabbage and other
chemicals on foods
Both TPC and coliform counts on foods were greatly reduced by steeping longer in CPE (Figures 1 and 2). There were significant differences in lethality between 5 and 10 minutes’ steeping in CPE (p < 0.05), but there were no significant differences in lethality between 10 and 30 minutes’ steeping in CPE (p > 0.05). Among all tested samples, lethality, in terms of coliform counts and TPC, was greatest when carrot was steeped in CPE alone. The lethality for the same kind of foodstuff in the triplicate experiments (data not shown) did not differ significantly (p > 0.05) at the same time point.
D
ISCUSSIONAntimicrobial effect of extracts
Many plants are rich in water-soluble compounds, such as tannins, terpenoids, alkaloids, and flavonoids, which have been shown in vitro to have antimicrobial properties [25]. Of all the water extracts from cruciferous vegetables tested in this study, water extracts from cabbage had the highest antimicrobial activity, especially against Gram-negative bacteria. Water extracts from the vegetables tested in this study showed the greatest inhibitory effect against E. aerogenes among all of the tested microorganisms.
The methanol-water extracts of cruciferous vegetables also demonstrated antimicrobial effects in this study, al-though they were significantly lower than the antimicrobial effects of the water extracts. Results from previous studies have also shown that some analogous methanolic and water-soluble compounds in mushroom and many other plants have antimicrobial effects [26,27].
Antimicrobial effects of WSPS from the six kinds of cruciferous vegetables were lower than those of the water extracts, but higher than those of methanol-water extracts. CPEs from the six different kinds of cruciferous vegetables, especially cabbage, showed greater inhibitory effects than those of other extracts, except for water extracts from Chinese
Table.
Antimicrobial effect of extracts of cruciferous vegetables*
Water extract
Methanol-water
extract
Water-soluble polysaccharide
Crude protein extract
CF BC CB CR CK CT CF BC CB CR C K CT CF BC CB CR CK CT CF BC CB CR CK CT
Gram-positive Bacillus cereus
11.1 11.0 12.6 10.5 — 9.6 — — 10.2 — 9.5 9.9 11.1 12.2 11.1 12.1 11.4 11.1 14.3 14.0 16.2 12.6 11.9 12.9 DE F G D D CDE DE DE C CD D CD C D CD C DE E F F DE Bacillus subtilis —9 .2 11.5 9.3 — 9.5 — — 9.6 — 8.9 9.4 — 11.3 11.0 12.0 — 10.9 11.0 12.4 14.3 12.2 — 12.5 F G D D E E E D D C D D E C D E F D E Streptococcus faecalis 11.5 12.3 13.5 — 9.6 9.6 10.0 10.7 10.3 10.1 10.5 10.6 12.9 14.1 15.0 — 11.8 12.1 16.2 17.4 20.6 17.9 14.0 16.4 D CDE EF E D C B C CDE BC BCD CDE B B C C D B C B B C C E C Staphylococcus aureus 14.4 15.6 16.2 16.0 15.2 15.7 9.7 10.4 11.5 10.0 10.9 11.1 13.3 14.1 15.3 — — — 17.3 18.0 22.4 20.4 19.1 19.8 CB C D B C B C D C D A B C B C A B C B CD BB C B B B B C B Listeria monocytogenes 11.3 12.0 13.1 9.7 10.6 10.9 9.1 9.5 11.3 9.2 9.1 10.7 10.4 10.6 11.8 — — — 13.8 14.2 16.5 12.0 12.4 12.2 DD E EFG D E D D D ABCD C E CDE C D D D C D E F EF DE
Gram-negative Proteus vulgaris
——— — — — — — — — — — — 11.3 11.3 — — 11.5 10.6 11.2 11.2 — — 10.8 DD D E D F E Pseudomonas aeruginosa 11.2 13.1 15.8 12.5 14.2 15.1 — — 10.0 — 1 0. 6 10.6 — — 12.3 11.2 11.1 11.6 16.1 17.4 20.1 15.1 16.5 17.4 DC D C D C C D B C D E B C D CDE D D C D BC B B C D D C Pseudomonas fluorescens 9.7 13.7 14.9 12.2 13.1 14.2 — — 10.5 — 9.8 10.0 — — 12.5 11.7 11.1 12.1 13.1 14.2 18.2 12.1 12.4 13.1 EC DE C D C BCDE CDE DE D C D C D D C C D F EF D Enterobacter aerogenes 20.5 20.7 21.9 19.3 18.9 18.9 10.2 11.6 12.2 10.7 11.6 12.5 13.5 13.1 15.8 13.1 11.8 12.3 23.0 25.1 25.2 24.0 22.7 23.2 AA A B A A A B C A B A AB AB AB B B C B C C C D A A A A A A Escherichia coli 19.1 19.5 20.2 18.2 17.3 18.6 11.2 11.7 11.7 10.0 10.5 12.6 17.0 17.7 20.0 19.1 18.1 19.2 23.2 25.3 26.6 21.2 20.4 21.7 BA B A BA A A B A B B C B C D A A AA AAA AA A B B C A B Salmonella serovar 18.3 19.8 20.4 18.0 17.1 18.6 11.2 11.8 12.5 11.2 12.0 12.2 17.3 17.5 20.3 18.0 17.4 17.7 24.7 25.5 27.4 24.2 21.7 23.1 Typhimurium B A B A B A A A A A A A B A A A AB A B A A A A AB A Shigella sonnei 18.2 20.2 22.6 — — 15.7 — — 9.6 — 9.1 10.2 15.9 16.8 17.5 17.2 14.9 14.2 25.1 26.5 27.5 21.3 21.2 22.9 BA A B E E D E A A BBB C A A A B A B A
Yeast Candida albicans
11.2 11.6 16.8 12.5 10.3 1 0. 9 11.0 12.2 12.4 11.2 10.6 11.8 11.2 10.6 11.7 11.1 11.3 11.5 14.5 14.9 18.1 14.4 12.4 13.6 DD E C C E D A B AAA B C D A B C C D D D C D C D C C D D E E F D
*Inhibition zone in mm (including 8 mm disk) as the mean of triplicate of experiments. Capital letters A–G: within the same column, values denoted by different capital letters are significantly different at
p
< 0.05.
CF = cauliflower; BC = broccoli; CB = cabbage; CR = Chinese radish; CK = Chinese kale; CT = Chinese kitam; — = no inhibitory ac
tivity.
Figure 1. Combined effect of crude protein extract (CPE) and citric acid on handling foods. Beef, carrot, and celery were steeped in a final concentration of 500 ppm CPE in the presence of 1% citric acid for 5, 10, or 30 minutes. The data are represented in terms of lethality, defined as the logarithmic ratio of microbial counts before and after treatment.
Lethality 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4
Steeping time (min)
5 10 30 Lethality 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 5 10 30 ▲ ▲ ■ ■ ■ ■ ▲ ▲ ◆ ◆ ◆ ◆ ▲ ■ ■ ■ ▲ ▲ ◆ ◆ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆ ■ ■ ■ ◆ ◆ ◆ ▲ ▲ ▲ ■ ■ ■ ◆ ◆ ◆ ▲ ▲ ▲ ■ ■ ■ ◆ ◆ ◆ ▲ ▲ ▲ ▲ ■ ◆ Lethality 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 Lethality 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 Lethality 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4
Total aerobic plate count
Beef Coliform Beef Lethality 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 CPE and citric acid
citric acid CPE
CPE and citric acid citric acid CPE
Carrot Carrot
Celery Celery
Total aerobic plate count ▲ ■ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆ ▲ ■ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆ ■ ■ ■ ▲ ▲ ▲ ◆ ◆ ◆
Figure 2. Combined effect of crude protein extract (CPE) and chlorine on handling foods. Beef, carrot, and celery were steeped in a final concentration of 500 ppm CPE in the presence of 10 ppm chlorine for 5, 10, or 30 minutes. The data are represented in terms of lethality, defined as the logarithmic ratio of microbial counts before and after treatment.
Lethality 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 5 10 30 Lethality Lethality Lethality Lethality Beef Coliform Beef Lethality
CPE and chlorine chlorine CPE
CPE and chlorine chlorine CPE Carrot Carrot Celery 1.4 Celery 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 5 10 30 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4
radish, Chinese kale, and Chinese kitam against P. fluorescens. Proteins or peptides isolated from various bioresources have previously been reported to have antimicrobial effects [14,25,28]. The antimicrobial activities of proteins may be associated with their three-dimensional structures [29]. Our results showed that CPEs from cruciferous vegetables have greater antimicrobial effects than those of other extracts. The mechanisms involved in these greater antimicrobial effects of CPEs are worthy of further investigation.
Combined effect of CPE from cabbage and other
chemicals on foods
Generally, salad vegetables and meats are cleaned by steeping these foodstuffs in water for a short period of time (e.g. 5 minutes). Preliminary tests conducted in this study indicated that steeping carrot, celery, and beef in water for 5 minutes only resulted in a decrease in viable coliform counts of 21%, 22%, and 27%, and in TPC of 42%, 38%, and 24% on carrot, celery, and beef, respectively. In order to improve the efficiency of disinfection, the steeping water was supplemented with CPE from cabbage alone and selective combinations of CPE with citric acid or chlorine. The antimicrobial effect of combined chemicals is much greater than that with one chemical alone [24]. Francis and O’Beime found that treating ready-to-use vegetables with various reagents such as chlorine, citric acid, and ascorbic acid helped to reduce the counts of both Listeria innocua and E. coli on vegetables [30]. Some plant extracts have also been used to treat livestock disease in South Africa [31]. The shelf-life of guava juice can be extended by adding combined extracts of corni fructus, cinnamon, and Chinese chive [21]. In this study, the lethality of citric acid and CPE with beef was slightly greater than that of CPE alone with beef after the same duration of treatment (Figure 1). This means that citric acid does not increase the lethality of CPE. Similar results were obtained for carrot and celery. The combination of CPE and chlorine had the greatest lethality, in terms of coliform counts and TPC, followed by CPE alone and chlorine alone (Figure 2). This means that chlorine enhances the lethality of CPE, especially in terms of TPC. The antimicrobial effect of citric acid might be related to its acidity, while that of chlorine might be related to its ability to denature protein [24]. These results indicate the feasibility of using CPE for food treatment.
Direct application of CPE or water extracts to food is recommended for sanitizing purposes. Pure active anti-microbial compounds isolated from food products have been recommended as food preservatives and for use in antiseptic processes [14]. The combination of vanillin,
ascorbic acid, and adjustment of water activity may be promising for natural strawberry preservation [32]. Bagamboula et al also reported that various combinations of low temperature, pH, and sodium chloride inhibited Shigella spp. [4]. These naturally occurring, active anti-microbial compounds may help to alleviate the pressures associated with long-term use of antimicrobial chemicals, such as the emergence of drug-resistant strains [33,34], and they can be used in combination with physical treatments such as pH, temperature control, and regulation of water activity in the food manufacturing process to extend the shelf-life of food products [35].
C
ONCLUSIONSThis study demonstrated that different extracts from cruciferous vegetables have different degrees of antimi-crobial activity. Four different extracts from each of the six cruciferous vegetables exhibited greater inhibitory ef-fects against negative bacteria than against Gram-positive bacteria. CPEs exhibited the greatest antimicrobial effects compared with other extracts. Both TPC and coliform counts on the surfaces of beef, carrot, and celery decreased significantly after steeping in CPE. This hints at the possibility that naturally occurring compounds can be used as food preservatives and might be applicable in antiseptic processes.
A
CKNOWLEDGMENTWe thank the Tajen Institute of Technology for financial assistance of this work.
R
EFERENCES1. Park CJ, Park CB, Hong SS, et al. Characterization and cDNA cloning of two glycine- and histidine-rich antimicrobial peptides from the roots of shepherd’s purse, Capsella
bursa-pastoris. Plant Mol Biol 2000;44:187–97.
2. Hashem FA, Saleh MM. Antimicrobial components of some cruciferae plants (Diplotaxis harra Forsk. and Erucaria microcarpa Boiss.). Phytother Res 1999;13:329–32.
3. Morimitsu Y, Hayashi K, Nakagawa Y, et al. Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, wasabi. Biofactor 2000;13:271–6.
4. Bagamboula CF, Uyttendaele M, Debevere J. Antimicrobial effect of spices and herb on Shigella sonnei and Shigella flexneri.
J Food Prot 2003;66:668–73.
protein. J Sci Food Agr 1997;28:786–90.
6. van Popple G, Verhoeven DT, Verhagen H, et al. Brassica vegetables and cancer prevention. Adv Exp Med Biol 1999;472: 159–68.
7. Chang CT, Hseueh YL, Shung HY. Purification and properties of chitinase from cabbage stems with roots. Biochem Mol Biol
Int 1996;40:417–25.
8. Fahey JW, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci USA 1997;94: 10367–72.
9. Nestle M. Broccoli sprouts in cancer prevention. Nutr Rev 1998;56:127–30.
10. Datta A, Gopal M. Safety evaluation of the fungicide iprodione on cauliflower. Bull Environ Contamination Toxicol 1999;62: 496–501.
11. Rhoades J, Roller S. Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods. Appl Environ Microbiol 2000;66:80–6.
12. Roller S, Covil N. The antifungal properties of chitosan in laboratory media and apple juice. Int J Food Microbiol 1999;47: 67–77.
13. Wang GH. Inhibition and inactivation of five species of foodborne pathogens by chitosan. J Food Prot 1992;55:916–9. 14. Lucca AJD, Walsh TJ. Antifungal peptides: novel therapeutic
compounds against emerging pathogens. Antimicrob Agents
Chemother 1999;43:1–11.
15. Roe S. Protein Purification Techniques, A Practical Approach, 2nd edition. New York: Oxford University Press, 2001.
16. Copeland RA. Methods for Protein Analysis. A Practical Guide to
Laboratory Protocols. London: Chapman & Hall, 1994.
17. Wang JC, Lee TM, Lai MN. Studies on water-soluble polysaccharides produced by Hericium spp. with submerged culture. Food Sci (Taiwan) 1998;25:714–26.
18. Chaplin MF, Kennedy JF. Carbohydrate Analysis: A Practical
Approach, 2nd edition. New York: IRL Press, 1994.
19. Andrews JM. The development of the BSAC standardized method of disc diffusion testing. J Antimicrob Chemother 2001; 48:29–42.
20. Menon T, Ponnuvel KM. Disc diffusion test in the identification of Candida species. Mycoses 2000;43:165–8.
21. Hsieh PC, Mau JL, Huang SH. Antimicrobial effect of various combinations of plant extracts. Food Microbiol 2001;18:35–43. 22. Ginn RE, Packard VS, Fox TL. Evaluation of the 3M dry medium culture plate (PetrifilmTM SM) method for determining numbers of bacteria in raw milk. J Food Prot 1984;47:753–8. 23. Nelson CL, Fox TL, Busta FF. Evaluation of dry medium film
(Petrifilm VRB) for coliform enumeration. J Food Prot 1984;47: 520–6.
24. Mossel DAA, Corry JEL, Corry BS, et al. Essentials of the
Microbiology of Foods, 1st edition. New York: John Wiley & Sons, 1995.
25. Cowan MM. Plant products as antimicrobial agents. Clin
Microbiol Rev 1999;12:564–82.
26. Alzoreky NS, Nakahara K. Antimicrobial activity of extracts from some edible plants commonly consumed in Asia. Int J
Food Microbiol 2003;80:223–30.
27. Lin CM, Preston JF 3rd, Wei CI. Antibacterial mechanism of allyl isothiocyanate. J Food Prot 2000;63:727–34.
28. Hiroyuki W, Susumu T, Yoshitaka T. Increased killing activity of an antimicrobial peptide, lactoferrin B, with minocyclin and monoacylglycerol. Biosci Biotechnol Biochem 2002;66:211–6.
29. Tydell CC, Yount N, Tran D, et al. Isolation, characterization, and antimicrobial properties of bovine oligosaccharide-binding protein. A microbicidal granule protein of eosinophils and neutrophils. J Biol Chem 2002;277:19658–64.
30. Francis GA, O’Beime D. Effects of vegetable type and anti-microbial dipping on survival and growth of Listeria innocua and E. coli. Int J Food Sci Technol 2002;37:711–8.
31. Masika PJ, Afolayan AJ. Antimicrobial activity of some plants used for the treatment of livestock disease in the Eastern Cape, South Africa. J Ethnopharmacol 2002;83:129–34.
32. Cerruti P, Alzamora SM, Vidales SL. Vanillin as an antimicro-bial for producing shelf-stable strawberry puree. J Food Sci 1997;62:608–10.
33. Hogan D, Kolter R. Why are bacteria refractory to antimicro-bials? Curr Opin Microbiol 2002;5:472–7.
34. Perreten V, Schwarz F, Cresta M, et al. Antibiotic resistance spread in food. Nature 1997;398:801–2.
35. Pszczola DE. Antimicrobials: setting up additional hurdles to ensure food safety. Food Technol 2002;56:99–107.
