Combined Effect of Low-Dose Irradiation and Acidified Sodium Chlorite Washing on Escherichia coli O157:H7 Inoculated on Mung Bean Seeds

Abstract

The effect of low-dose irradiation (0.75 and 1.5 kGy) in combination with acidified sodium chlorite (ASC) on the reduction of Escherichia coli O157:H7 on mung bean seeds was examined. Washing with ASC (0.2, 0.5, 0.8, and 1.2 g/L sodium chlorite and 1.0 g/L citric acid) for 2 h reduced the E. coli O157:H7 population from 5.2 to 2.3–3.3 log CFU/g, depending on the concentrations of sodium chlorite. Gamma ray irradiation at 0.75 and 1.5 kGy resulted in reductions of about 1.8 and 2.8 log CFU/g, respectively. Therefore, a single treatment with ASC washing or gamma ray irradiation at 0.75 or 1.5 kGy could not achieve the complete elimination of E. coli O157:H7 on mung bean seeds. Conversely, low-dose irradiation (0.75 and 1.5 kGy) followed by washing with ASC (0.5–1.2 g/L) reduced the population of E. coli O157:H7 to below the detection limit (<1 log CFU/g). However, E. coli O157:H7 was detected in most samples in the enrichment and germination studies. When the treatment order was reversed (ASC washing followed by low-dose irradiation), the E. coli O157:H7 population was also observed to be below the detection limit. Under this treatment, fewer samples (16.7%) were shown to be positive in the enrichment and germination studies, and complete elimination was not achieved. The germination rates of mung bean seeds were not affected by ASC washing and gamma irradiation; however, the yield and length of sprouts were decreased by gamma irradiation.

Introduction

T he consumption of sprout vegetables has increased in recent decades due to their nutritional value, and many outbreaks of foodborne illnesses have been reported along with this increase in consumption. The source of pathogens, including Salmonella and Escherichia coli O157:H7, are considered to be originated from seeds rather than from the contamination of the final products (Scouten and Beuchat, 2002; Kumar et al., 2006). The contamination levels of seeds are usually low in number (Jaquette et al., 1996); however, the optimal temperature and humidity conditions for seed germination are also suitable for the rapid growth of pathogens (Taormina and Beuchat, 1999; Feng et al., 2007). Accordingly, the decontamination of seeds before germination is important for the safety of sprout vegetables. Sprout seeds have been recommended to be soaked in 20,000 ppm of calcium hypochlorite for 10–20 min before germination (National Advisory Committee on Microbiological Criteria for Foods, 1999). An important problem is that there is an increasing concern about the health hazards and environmental impact associated with the use of high-concentration chlorine (Beuchat, 1998; Liao, 2009). Chlorine loses its effectiveness after reacting with nitrogen compounds in foods, which results in halogenated organic compounds (Aieta et al., 1984; Beuchat, 1998). In addition, chemical treatment has been reported to reduce pathogen levels to some extent, whereas some studies have suggested that the complete elimination of pathogens is difficult to achieve (Jaquette et al., 1996; Lang et al., 2000). The reduction of pathogen to low contamination levels is not always sufficient, as the population of pathogens is likely to reach hazardous levels during germination. Therefore, an alternative disinfection method that is able to completely eliminate pathogens without producing toxic compounds is required to improve the safety of sprouted vegetables.

Irradiation is one of the attractive technologies for reducing the risk of foodborne illness. Although acceptance of irradiated foods is not always high in some consumer communities (Gunes and Tekin, 2006), the irradiation to several kinds of foods such as meat products, fish and seafood, and fresh vegetables is widely recognized and is approved in >50 countries (IAEA, 2009), and the U.S. Food and Drug Administration approved the irradiation at up to 8.0 kGy for decontamination of the seeds (Kim et al., 2006). The mechanism of microbial inactivation in the ionization of radiation is mainly due to the damage of nucleic acids, direct damage or indirect damage by oxidative radicals originating from the radiolysis of water. It is a safe, efficient, environmentally clean, and energy-efficient process being particularly valuable as a decontamination procedure (Farkas, 1998). However, although high decontamination is effective and convenient, high-dose irradiation often decreases the quality of sprouts. Therefore, a combination of low-dose irradiation and chemical sanitizers with higher decontamination efficacy is desirable to eliminate the pathogens from the seeds.

Chlorine dioxide is more soluble than chlorine in water and is a strong oxidizing agent; its oxidizing capacity is about 2.5 times higher than that of hypochlorous acid. Its oxidation ability consequently inactivates bacteria, bacterial spores, and viruses (Lillard, 1979; Foegeding et al., 1986; Lukasik et al., 2003). In addition, chlorine dioxide does not form chlorinated organic compounds as easily as chlorine does (Beuchat, 1998). Chlorine dioxide is produced from acidified sodium chlorite (ASC), which is prepared by mixing sodium chlorite with a generally regarded as safe acid (CFR, 2005). The U.S. Food and Drug Administration approved ASC for use in poultry, red meat, comminuted meat products, and processed fruits and vegetables to reduce bacterial contamination (CFR, 2005). However, no study showed the complete elimination of pathogens on vegetable seeds using ASC solutions.

Therefore, the present study made an attempt to combine ASC washing and low-dose irradiation to eliminate the pathogens from mung bean seeds. The objectives were (1) to evaluate the decontamination efficacy of a combination of ASC washing and low-dose (0.75 and 1.5 kGy) gamma irradiation, (2) to clarify whether the treatment order could affect the decontamination efficacy, and (3) to examine the influence of the combined treatment on the seed germination, yield, and sprout length of mung beans.

Materials and Methods

Bacterial strains

Four strains of E. coli O157:H7 (CR-3, MN-28, MY-29, and DT-66) were used in this study. These strains are isolated from bovine feces, since vegetable seeds are possibly contaminated from cattle manure (Breuer et al., 2001). To minimize the growth of microorganisms naturally present on mung bean seeds, all of the test strains of E. coli O157:H7 were adapted to grow in tryptic soy broth (TSB; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with rifampicin (50 μg/mL). Plating on media containing rifampicin greatly minimized interference by naturally occurring microorganisms and facilitated the detection of test pathogen on recovery media (Inatsu et al., 2005a).

Preparation of inocula

Each strain of E. coli O157:H7 was cultured at 37°C in 5 mL of a trypticase soy broth (Nissui Pharmaceutical Co. Ltd.) medium supplemented with 50 μg/mL rifampicin (TSBR). The cultures were transferred to the TSBR by loop at three successive 24-h intervals immediately before they were used as inocula. Cells of each strain were collected by centrifugation (3000 g, 5 min, 20°C) and resuspended in 5 mL of sterile phosphate-buffered saline (PBS, pH 7.2). Equal volumes of cell suspensions were combined to obtain bacterial cocktails, each containing approximately equal populations of each strain. The final suspension, containing 9.0 log CFU/mL, was maintained at 22°C ± 2°C and applied to mung bean seeds within 30 min after preparation.

Procedure for inoculation

Five kilograms of mung bean seeds were soaked in a four-strain suspension of E. coli O157:H7 and mixed gently with a sterile glass rod for 5 min. After the inoculum was decanted, mung bean seeds were placed on a sterile perforated tray and dried in a clean bench at room temperature (22°C ± 2°C) for 8 h. After drying, the inoculated mung bean seeds were mixed well and stored at 4°C until being used for experiments.

Washing protocol

The final concentrations of ASC solutions were adjusted to 0.2, 0.5, 0.8, and 1.2 g/L of sodium chlorite (Nacalai Tesque, Kyoto, Japan) and 1.0 g/L of citric acid (pH 2.5). All of the washing solutions were prepared immediately before application and used within 30 min. The washing treatments were carried out by soaking 200 g of mung bean seeds in 500 mL of the ASC solutions in stainless container (15 × 15 × 10 cm) for 2 h.

Irradiation treatment

Polyethylene bags (280 × 200 mm) containing 200 g of seeds were irradiated at room temperature and received doses of 0.75 and 1.5 kGy at 6.1 kGy/h with a cobalt-60 gamma source (Gamma Cell-220; Nordion International Inc., Ontario, Canada). Dosimetry was performed using 5-mm-diameter of alanine dosimeters (Bruker Instruments, Rheinstetten, Germany), and the free radical signal was measured using an ESR analyzer (EMX-Plus; Bruker Instruments). The actual dose was typically within 2% of the target doses.

Combined treatment of ASC washing and gamma irradiation

The combined treatment was performed in two ways. First, the mung bean seeds were irradiated according to the method described above and washed with ASC solutions. The mung bean seeds were decontaminated in the reverse order for the other way; the washing of mung bean seeds with ASC solutions and the subsequent surface drying for 1 h and irradiation were carried out.

Microbial analysis

Ten grams of seeds were placed in a stomacher bag, and 90 mL of PBS (pH 7.2) was added; the mixture was pummeled for 60 s. Serial decimal dilutions were prepared with PBS (pH of undiluted and diluted solutions was around 7.0), and the diluted and undiluted samples were pour plated in quadruplicate on tryptic soy agar (TSA) or sorbitol MacConkey (SMAC) agar plates supplemented with 50 μg/mL rifampicin (TSAR and SMACR, respectively) to enumerate the population of E. coli O157:H7. All of the ingredients except rifampicin were combined and sterilized by heating at 121°C for 15 min. The rifampicin solution was added to the molten agar before pouring the medium into Petri dishes. Inoculated enumeration media were incubated at 37°C for 24 h before the presumptive colonies of pathogen were counted.

Detection of surviving pathogens in treated seeds

The survival of E. coli O157:H7 was confirmed according to Bari et al. (2010). The homogenized mixture of 10 g of seeds with 90 mL of peptone water in a stomacher bag was kept in an incubator at 37°C for 24 h for enrichment, and the homogenates (0.1 mL) were plated onto TSAR and SMACR. All of the plates were incubated at 37°C for 24 to 48 h, and the surviving pathogen colonies were counted. In all experiments, E. coli O157:H7 was not recovered from uninoculated seeds.

Determination of germination percentage and growth of sprouts

The germination percentage was determined according to Bari et al. (2003) with some modifications. Approximately 100 control and treated seeds were placed between two pieces of water-saturated 90-mm-diameter filter paper (Whatman International Ltd. Maidstone, United Kingdom) in a Petri dish. The seeds were stored in the dark at 25°C for 2 days, and sterilized water was periodically applied to maintain high-moisture conditions. The germination percentage was calculated by the number of germinated seeds. In addition, the enumeration of E. coli O157:H7 was also carried out according to the method described above.

The length and yield of the sprouts were determined as growth factors to evaluate the quality of the sprouts by using noninoculated seeds. After the decontamination treatments, 10 seeds were weighted and placed in a 100-mL plastic cup. The cup was stored in the dark at 25°C for 6 days, and sterilized water was applied daily. After the storage, the weight of 10 seeds was determined, and the yield was calculated with a weight ratio of sprouts to seeds. Subsequently, the lengths of five sprouts selected randomly from the treated and untreated seeds were measured, and the average values were compared.

Statistical analysis

All experiments were repeated three times, and duplicated samples were used per treatment in each experiment. Significant differences in average values were established by the Tukey–Kramer multiple-comparison method at the 5% level of significance using SPSS (SPSS Inc., Chicago, IL).

Results

Effect of ASC washing on E. coli O157:H7population

The population of E. coli O157:H7 in mung bean seeds after ASC washing was shown in Table 1. Washing with different concentrations of ASC reduced the E. coli O157:H7 population from 4.7–5.2 to 2.0–3.3 log CFU/g, depending on the recovery medium and sodium chlorite concentration. The recovery of E. coli O157:H7 on TSAR was higher than that found for SMACR. This is in agreement with other reports describing the poor performance of selective media in recovering pathogens from treated fresh produce (Inatsu et al., 2005a). Washing with 0.5–1.2 g/L of ASC showed a higher decontamination efficacy than that of washing with 0.2 g/L ASC, and a 2.7–2.9 log CFU/g reduction in the E. coli O157:H7 population was obtained. However, the E. coli O157:H7 population remarkably increased during germination for 2 days, and 6.0–6.9 log CFU/g of E. coli O157:H7 populations were observed, regardless of the treatment conditions and recovery medium. A 0.8 log CFU/g higher population was observed with 0.2 g/L of ASC treated seeds than that found with 0.5–1.2 g/L of ASC in the germination study.

Table 1.

Population of Escherichia coli O157:H7 After Washing with Acidified Sodium Chlorite

	Population of Escherichia coli O157:H7 (log CFU/g) ^a
	After treatment		After germination
	Recovery medium		Recovery medium
Concentration (ppm)	TSAR	SMACR	TSAR	SMACR
Control	5.2 ± 0.2^A	4.7 ± 0.3^A
200	3.3 ± 0.3^B	2.9 ± 0.2^B	6.9 ± 0.1^A	6.8 ± 0.2^A
500	2.7 ± 0.2^C	2.4 ± 0.3^C	6.2 ± 0.3^B	6.0 ± 0.3^B
800	2.4 ± 0.1^C	2.0 ± 0.3^C	6.2 ± 0.3^B	6.1 ± 0.3^B
1200	2.3 ± 0.3^C	2.0 ± 0.3^C	6.3 ± 0.6^B	6.1 ± 0.6^B

The population data are represented by means of three independent trials with two samples and standard deviations (n = 6). Within individual columns, the values followed by different letters are significantly different (p < 0.05).

TSAR, tryptic soy agar supplemented with rifampicin; SMACR, sorbitol MacConkey supplemented with rifampicin.

Effect of low-dose irradiation and in combination with ASC washing

The population of E. coli O157:H7 treated with irradiation alone or in combination with ASC wash was shown in Table 2. Irradiation at 1.5 kGy significantly reduced the E. coli O157:H7 population from 4.7 and 5.2 to 1.9 and 2.5 log CFU/g on SMACR and TSAR, respectively (p < 0.05). A lower irradiation dose at 0.75 kGy also reduced the E. coli O157:H7 population, and a reduction of about 1.4 log CFU/g was obtained. However, irradiation at 0.75 kGy followed by washing with ASC (0.5–1.2 g/L) was able to decrease the E. coli O157:H7 population to an undetectable level, but E. coli O157:H7 was detected in all of the samples in the enrichment and germination studies. E. coli O157:H7 was also detected in 4 out of 6 samples (66.7%) after irradiation at 1.5 kGy and subsequent ASC washing. Therefore, the complete elimination of E. coli O157:H7 was not achieved with these combinations of treatments.

Table 2.

Effect of Low-Dose Gamma Irradiation Followed by Acidified Sodium Chlorite Washing on Population of Escherichia coli O157:H7

		Population of E. coli O157:H7 (log CFU/g) ^a
		After treatment		After germination ^b
		Recovery medium		Recovery medium
Dose (kGy)	Concentration (ppm)	TSAR	SMACR	TSAR	SMACR
Control		5.2 ± 0.2	4.7 ± 0.3	nd^c	nd
0.75		3.8 ± 0.4	3.3 ± 0.3	nd	nd
	200	2.2 ± 0.7 (6/6)	1.8 ± 0.5 (6/6)	6/6 (6.3 ± 0.6)	6/6 (5.6 ± 0.2)
	500	ND (6/6)^d	ND (6/6)	6/6 (6.2 ± 0.5)	6/6 (5.3 ± 0.5)
	800	ND (6/6)	ND (6/6)	6/6 (5.9 ± 0.8)	6/6 (5.2 ± 0.6)
	1200	ND (6/6)	ND (6/6)	6/6 (6.1 ± 1.0)	6/6 (5.3 ± 0.6)
1.5		2.5 ± 0.2	1.9 ± 0.2	nd	nd
	200	ND (6/6)	ND (6/6)	6/6 (6.1 ± 0.3)	6/6 (5.6 ± 0.2)
	500	ND (5/6)	ND (5/6)	5/6 (6.3 ± 0.6)	5/6 (5.3 ± 0.7)
	800	ND (5/6)	ND (5/6)	5/6 (5.9 ± 0.6)	5/6 (5.4 ± 0.6)
	1200	ND (4/6)	ND (4/6)	4/6 (5.9 ± 0.3)	4/6 (5.8 ± 0.3)

The number of positive samples for E. coli O157:H7 after germination at 25°C for 2 days and the population in this case are provided.

nd, not done, as the presence of E. coli O157:H7 was apparent in these conditions.

ND indicates a level below the detection limit (<1 log CFU/g). The number of positive samples for E. coli O157:H7 following enrichment is provided.

Effect of treatment order on population of E. coli O157:H7

The mung been seeds were irradiated at 0.75 and 1.5 kGy after ASC washing to determine the effect of the treatment order on the reduction of E. coli O157:H7 population (Table 3). The E coli O157:H7 population was reduced to a below-detectable level (<1 log CFU/g) just after treatments regardless of the irradiation dose and the ASC concentration used. After the treatment with ASC (1.2 g/L) washing followed by irradiation at 0.75 kGy, 3 out of 6 (50.0%) and 2 out of 6 (33.3%) samples were found to be positive in the enrichment and germination studies, respectively. Therefore, these results showed a better reduction efficiency than the 0.75 kGy treatment followed by ASC (1.2 g/L) wash (Table 2). On the other hand, after washing the mung bean seeds with 1.2 g/L of ASC followed by irradiation at 1.5 kGy, 1 out of 6 samples (16.7%) were found positive for E. coli O157:H7 in the enrichment and germination studies. Therefore, these study results showed the highest reduction efficacy of the E coli O157:H7 population compared to all other single and/or combination treatments. (Tables 1 –3).

Table 3.

Effect of Acidified Sodium Chlorite Washing Followed by Low-Dose Gamma Irradiation on the Population of Escherichia coli O157:H7

		Population of E. coli O157:H7 (log CFU/g) ^a
		After treatment		After germination ^b
		Recovery medium		Recovery medium
Dose (kGy)	Concentration (ppm)	TSAR	SMACR	TSAR	SMACR
Control		5.2 ± 0.2	4.7 ± 0.3	nd^c	nd
0.75		3.8 ± 0.4	3.3 ± 0.3	nd	nd
	200	ND(5/6)^d	ND (5/6)	4/6 (6.5 ± 0.6)	4/6 (6.0 ± 0.7)
	500	ND (3/6)	ND (3/6)	3/6 (6.7 ± 0.5)	3/6 (6.2 ± 1.2)
	800	ND (3/6)	ND (3/6)	3/6 (6.1 ± 1.5)	3/6 (6.0 ± 0.7)
	1200	ND (3/6)	ND (3/6)	2/6 (6.1 ± 0.7)	2/6 (5.8 ± 0.6)
1.5		2.5 ± 0.2	1.9 ± 0.2	nd	nd
	200	ND (5/6)	ND (5/6)	4/6 (6.1 ± 1.1)	4/6 (5.9 ± 1.3)
	500	ND (2/6)	ND (2/6)	2/6 (6.5 ± 0.0)	2/6 (6.5 ± 0.1)
	800	ND (2/6)	ND (2/6)	2/6 (6.6 ± 1.0)	2/6 (6.0 ± 0.4)
	1200	ND (1/6)	ND (1/6)	1/6 (6.4 ± 0.0)	1/6 (6.3 ± 0.0)

The number of positive samples for E. coli O157:H7 after germination at 25°C for 2 days and the population in this case are provided.

nd, not done, as the presence of E. coli O157:H7 was apparent in these conditions.

ND indicates a level below the detection limit (<1 log CFU/g). The number of positive samples for E. coli O157:H7 following enrichment is provided.

Effect of ASC washing and gamma irradiation on germination of mung bean seeds

The germination rates of mung bean seeds treated with ASC solutions and/or gamma irradiation were shown in Table 4. The germination rates ranged from 96.7% to 98.2% with ASC washing, gamma irradiation, and combinations of ASC and gamma irradiation. No significant change in the germination rates of mung bean seeds (p > 0.05) was found for any set of treatment conditions.

Table 4.

Effect of Acidified Sodium Chlorite Washing and Gamma Irradiation on Germination Rate and Growth of Mung Bean Sprout

Treatment	Germination rate (%) ^a	Length (mm) ^b	Yield (−) ^a,c
Control	97.8 ± 1.0^A	12.8 ± 1.8^A	7.7 ± 0.5^A
ASC washing
200 ppm	97.9 ± 0.9^A	12.8 ± 1.9^A	7.5 ± 0.3^A
500 ppm	98.2 ± 1.0^A	13.5 ± 1.7^A	7.4 ± 0.5^A
800 ppm	97.8 ± 0.9^A	13.2 ± 1.5^A	7.5 ± 0.7^A
1200 ppm	98.0 ± 1.0^A	13.0 ± 2.1^A	7.6 ± 0.5^A
ASC after gamma irradiation
0.75 kGy	98.2 ± 1.3^A	6.7 ± 1.1^B	4.9 ± 0.3^BC
+ 200 ppm	96.9 ± 1.5^A	6.2 ± 1.1^BC	4.9 ± 0.2^BC
+ 500 ppm	97.4 ± 0.5^A	6.3 ± 1.3^BC	4.8 ± 0.4^BC
+ 800 ppm	96.8 ± 1.2^A	6.6 ± 1.3^B	5.1 ± 0.4^B
+ 1200 ppm	97.7 ± 1.7^A	6.4 ± 0.9^BC	5.0 ± 0.3^BC
1.5 kGy	97.2 ± 1.5^A	5.4 ± 0.9^C	4.3 ± 0.4^BC
+ 200 ppm	98.1 ± 1.6^A	5.4 ± 0.8^C	4.2 ± 0.4^C
+ 500 ppm	97.6 ± 1.1^A	5.6 ± 0.8^BC	4.5 ± 0.5^BC
+ 800 ppm	97.7 ± 0.8^A	5.7 ± 0.7^BC	4.3 ± 0.3^BC
+ 1200 ppm	96.7 ± 1.1^A	5.6 ± 0.7^BC	4.2 ± 0.5^C
Gamma irradiation after ASC
0.75 kGy	98.2 ± 1.3^A	6.7 ± 1.1^B	4.9 ± 0.3^BC
+ 200 ppm	97.9 ± 0.7^A	6.4 ± 0.9^BC	4.9 ± 0.4^BC
+ 500 ppm	97.7 ± 1.0^A	6.4 ± 0.8^BC	4.8 ± 0.2^BC
+ 800 ppm	98.2 ± 0.8^A	6.5 ± 0.8^B	4.8 ± 0.4^BC
+ 1200 ppm	97.1 ± 1.0^A	6.2 ± 0.6^B	4.4 ± 0.5^BC
1.5 kGy	5.4 ± 0.9^C	4.3 ± 0.4^BC
+ 200 ppm	98.1 ± 0.7^A	5.7 ± 0.7^BC	4.5 ± 0.1^BC
+ 500 ppm	97.6 ± 0.4^A	5.8 ± 0.6^BC	4.4 ± 0.5^BC
+ 800 ppm	97.7 ± 0.3^A	5.6 ± 0.6^BC	4.4 ± 0.5^BC
+ 1200 ppm	96.7 ± 0.4^A	5.8 ± 0.7^BC	4.6 ± 0.2^BC

The data are provided as means and standard deviations of three replications with two samples (n = 6). Within individual columns, the values followed by different letters are significantly different (p < 0.05).

The data are provided as means and standard deviations of three replications with ten sprouts (n = 30). Within individual columns, the values followed by different letters are significantly different (p < 0.05).

The yield was calculated by the weight ratio of sprouts to seeds (sprouts weight/seeds weight).

ASC, acidified sodium chlorite.

Effect of ASC washing and gamma irradiation on growth of mung bean seeds

The length and yield of mung bean sprouts grown for 6 days were shown in Table 4. There were no significant differences in the length between controls and samples washed with 0.2–1.2 g/L of ASC solutions (p > 0.05), and the average length was recorded as 12.8–13.5 cm. On the other hand, gamma-irradiated samples showed significantly shorter lengths than those of the controls and the ASC-washed samples (p < 0.05). The lengths of sprouts irradiated at 0.75 and 1.5 kGy were 6.2–6.7 mm and 5.4–5.8 mm, respectively. As with the sprout length, no significant differences in yields between controls and ASC-washed samples were observed (p > 0.05). However, the yields of sprouts were significantly decreased by gamma irradiation at 0.75 and 1.5 kGy (p < 0.05).

Discussion

The effectiveness of ASC solutions in eliminating pathogens has been reported for spinach (Lee and Baek, 2008; Stopforth et al., 2008; Nei et al., 2009), Chinese cabbage (Inatsu et al., 2005b), shredded carrots (Ruiz-Cruz et al., 2006), and lettuce (Allende et al., 2008, Keskinen et al., 2009). However, there have been limited studies reporting the reduction of pathogens on mung bean seeds by ASC washing. In this study, an ∼1.8–2.9 log CFU/g reduction of E. coli O157:H7 was obtained from 4.7 to 5.2 log CFU/g of control seeds. The difficulty of complete elimination through treatment by washing was reported by other studies (Taormina and Beuchat, 1999; Gandhi and Matthews, 2003; Liao, 2009). Considering that the microbial population rapidly increases during seed germination (Saroj et al., 2007), the washing treatment itself does not always assure the safety of the sprout vegetables.

The U.S. Food and Drug Administration has approved the use of irradiation at doses of up to 8.0 kGy to control bacterial pathogens on seeds to be used for sprouting (Kim et al., 2006). Thayer et al. (2003) reported the elimination of Salmonella Mbandaka from alfalfa seeds by irradiation at 4 kGy. Saroj et al. (2009) indicated the reduction of Salmonella Typhimurium inoculated on mung bean seeds from 4.6 to 1.8 log CFU/g by gamma irradiation at 1 kGy and the achievement of elimination at a dose of 2 kGy. In this study, gamma irradiation at 0.75 and 1.5 kGy was tested to eliminate the pathogens at lower doses than other studies. As a result, although a significant reduction in E. coli O157:H7 population was achieved by gamma irradiation at 0.75 and 1.5 kGy (p < 0.05), the complete elimination of the pathogen could not be achieved with low-dose (≤1.5 kGy) irradiation.

Many combination treatments have been investigated over the years. These include chemical treatments plus irradiation (Bari et al., 2003), high-pressure processing followed by heat treatment (Koseki et al., 2008), and soaking in water and subsequent high pressure processing (Neetoo et al., 2009). It was suggested that the decontamination effect of combination treatment is additive or synergistic depending on combinations, and in some circumstances an antagonistic effect may result (Raso and Barbosa-Cánovas, 2003). In addition, additive or synergistic effect depends on treatment order (Kim and Thayer, 1996). The present study revealed the effectiveness of gamma irradiation combined with ASC. When the effectiveness of the combined treatment is tested, the treatment order may affect the results of the experiments. The obtained results indicated that gamma irradiation after ASC washing was more effective than that of the reverse order in eliminating E. coli O157:H7 from mung bean seeds. This conclusion was mainly confirmed from an enrichment study; the positive ratio of E. coli O157:H7 was apparently lower for gamma-irradiated seeds after ASC washing. Although a reasonable cause for this result is difficult to articulate, soaking in ASC solutions induced a higher moisture content of mung bean seeds, and a higher amount of free radicals were expected to be produced by gamma irradiation. This higher amount of free radicals would effectively eliminate the pathogen from the mung bean seeds. In addition, when the mung been seeds were irradiated and subsequently washed by ASC solutions, survivors of the irradiation located in the crevices of the seeds would not be attacked by ASC solutions. On the other hand, radiation energy can penetrate the crevices of the seeds. Therefore, a low-dose of irradiation was considered to be suitable to be used for the final treatment of decontamination processes to control the pathogen levels when several decontamination methods were combined.

Liao (2009) reports that a sanitization treatment with 800 ppm of ASC for 45 min did not affect the germination rate of alfalfa seeds. Taormina and Beuchat (1999) indicated slight decreases in the germination rate of alfalfa seeds treated with 500–1200 ppm of ASC. Consequently, several articles have reported on the relationship between ASC treatment and the germination rates of alfalfa seeds. However, there has been limited data on the germination rate of mung bean seeds after ASC treatment. The present study has indicated no significant changes in germination rates as a result of 0.2–1.2 g/L of ASC treatments (p > 0.05). Additionally, no significant changes in germination rates could be obtained for mung bean seeds treated with gamma irradiation at 0.75 and 1.5 kGy (p > 0.05). These results were considered to be reasonable, as there have been several reports indicating similar results. Saroj et al. (2007) have observed no significant changes in the germination rates of mung bean seeds irradiated at 0.5–2.0 kGy (p > 0.05). The germination rate is among the important factors used to optimize the decontamination method, and this information is expected to be utilized by sprout growers.

Although germination rate is important factor to evaluate the quality changes of mung bean seeds, it is not sufficient for the total evaluation of the seeds' quality. Rajkowski et al. (2003) reported decreases in the yield ratio and size of broccoli sprouts along with the increase in irradiation doses. Similarly, other researchers have noted decreases in the length of mung bean sprouts produced from irradiated seeds (Bari et al., 2003; Bari et al., 2009). The present study also observed a decrease in the yield and length of mung bean sprouts as a result of the gamma irradiation of seeds. However, ASC treatments resulted in no significant changes in the yields and lengths of the sprouts. Striking an ideal balance between the quality and safety of final products is an important task faced by sprout growers. Considering that decreases in the growth of sprouts is dose dependent (Saroj et al., 2007), a low dose of irradiation combined with other decontamination treatments, such as ASC washing, represents one possible option of decontamination. However, more efforts to achieve both high decontamination efficacy and high quality will be required, since the pathogens on mung bean seeds could not be completely eliminated and growth of the sprouts was inhibited. In addition, storage test will be needed to assess the seeds' quality properly.

Footnotes

Disclosure Statement

No competing financial interests exist.

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