Composite versus single sampling of spent irrigation water to assess the microbiological status of sprouting mung bean beds

BACKGROUND: Spent irrigation water testing has been recommended in sprouted seed production to detect the presence of pathogens. However, because of the heterogeneous distribution of contamination within batches of sprouted seed, taking single samples of spent irrigation water may return false‐negative results. The following evaluated whether spent irrigation water collected from multiple points provided a more representative assessment of the microbiological status of the sprouting mung bean bed compared to when single samples were taken. RESULTS: Generic Escherichia coli or Aeromonas was recovered in one and 10 of the 160 sprout samples taken from 32 sprouting mung bean batches, respectively. Composite spent irrigation water samples tested positive for generic E. coli on 19 occasions compared to 12 when single samples were taken. Mesophilic Aeromonas was detected in 13 composite spent irrigation water samples which compared to eight single samples. The prevalence of either target bacterium in composite spent irrigation water samples was not significantly (P > 0.05) different compared to when a single sample was collected. CONCLUSIONS: Sampling spent irrigation water from multiple points under sprouting mung bean beds does not significantly increase the probability of detecting contamination, if present. The findings of the study should be considered when devising sampling plans for spent irrigation water testing in bean sprout production. Copyright © 2008 Society of Chemical Industry     

A rapid and automated fiber optic-based biosensor assay for the detection of Salmonella in spent irrigation water used in the sprouting of sprout seeds

Recent outbreaks of foodborne illness have been linked to the consumption of contaminated sprouts. The spent irrigation water used to irrigate sprouts can carry many microorganisms, including pathogenic strains of Escherichia coli and Salmonella enterica. These pathogens are believed to originate from the seeds. The U.S. Food and Drug Administration recommends that sprout producers conduct microbiological testing of spent irrigation water from each production lot at least 48 h after seeds have germinated. Microbial analysis for the detection of Salmonella is labor-intensive and takes days to complete. A rapid and automated fiber-optic biosensor assay for the detection of Salmonella in sprout rinse water was developed in this study. Alfalfa seeds contaminated with various concentrations of Salmonella Typhimurium were sprouted. The spent irrigation water was assayed 67 h after alfalfa seed germination with the RAPTOR (Research International, Monroe, Wash.), an automated fiber optic-based detector. Salmonella Typhimurium could be positively identified in spent irrigation water when seeds were contaminated with 50 CFU/g. Viable Salmonella Typhimurium cells were also recovered from the waveguides after the assay. This biosensor assay system has the potential to be directly connected to water lines within the sprout-processing facility and to operate automatically, requiring manual labor only for preventative maintenance. Therefore, the presence of Salmonella Typhimurium in spent irrigation water could be continuously and rapidly detected 3 to 5 days before the completion of the sprouting process.     

Microbiological analysis of seed sprouts in Norway

As part of larger survey of microbial contamination of fruits and vegetables in Norway, four different sprouted seed products were analysed for bacterial and parasitic contaminants (n = 300 for bacterial analyses and n = from 17 to 171 for parasite analyses, depending on parasite). Escherichia coli O157, Salmonella, Listeria monocytogenes, Cyclospora oocysts, Ascaris eggs and other helminth parasites were not detected in any of the sprout samples. Thermotolerant coliform bacteria (TCB) were isolated from approximately 25% of the sprout samples, with the highest percentage of TCB positive samples in alfalfa sprouts. Most TCB were Enterobacter spp. and Klebsiella. E. coli was isolated from 8 of 62 TCB positive mung bean sprout samples. Cryptosporidium oocysts were detected in 8% of the sprout samples and Giardia cysts were detected in 2% of the samples. All the Cryptosporidium positive samples, and most of the Giardia positive samples, were mung bean sprouts. Parasite concentrations in positive samples were low (between 1 and 3 oocysts/cysts per 50 g sprouts). Sprout irrigation water was also analysed for microbial contaminants. E. coli O157 and L. monocytogenes were not detected. TCB were isolated from approximately 40% of the water samples. Salmonella reading was isolated from three samples of spent irrigation water on 3 consecutive days. Cryptosporidium and Giardia were also isolated from spent irrigation water. Additionally, eight samples of unsprouted mung bean seed were analysed for Cryptosporidium oocysts and Giardia cysts. One or both of these parasites were detected in six of the unsprouted seed samples at concentrations of between 1 and 5 oocysts/cysts per 100 g unsprouted seed. Whilst our results support spent irrigation water as the most suitable matrix for testing for bacteria, unsprouted seed is considered the more useful matrix for analysing for parasite contaminants.     

Use of spent irrigation water for microbiological analysis of alfalfa sprouts

Numerous outbreaks of foodborne illness have been linked to the consumption of raw sprouts. Sprout producers have been advised by the Food and Drug Administration to include microbiological testing of spent irrigation water during production as part of an overall strategy to enhance the safety of sprouts. Alfalfa sprouts and irrigation water were analyzed to show the feasibility of using irrigation water for monitoring the microbiological safety of sprouts. Sprouts and water were produced and harvested from both commercial-scale (rotary drum) and consumer-scale (glass jars) equipment. Rapid increases of aerobic mesophiles occurred during the first 24 h of sprouting, with maximum levels achieved after 48 to 72 h. The counts in irrigation water were on average within approximately 1 log of their respective counts in the sprouts. Similar results were obtained for analysis of Escherichia coli O157:H7 in irrigation water and sprouts grown from artificially inoculated seeds. Testing of spent irrigation water indicated the contamination status of alfalfa sprouts grown from seeds associated with outbreaks of Salmonella infection.     

Hot water treatments to inactivate Escherichia coli O157:H7 and Salmonella in mung bean seeds

The majority of the seed sprout-related outbreaks have been associated with Escherichia coli O157:H7 and Salmonella. Therefore, an effective method is needed to inactivate these organisms on the seeds before they are sprouted. This study was conducted to assess the effectiveness of various hot water treatments to inactivate E. coli O157:H7 and Salmonella populations on mung beans seeds intended for sprout production and to determine the effect of these treatments on seed germination after the seeds were dipped in chilled water for 30 s. Mung bean seed inoculated with four-strain cocktails of E. coli O157:H7 and Salmonella were soaked into hot water at 80 and 90 degrees C with shaking for various periods and then dipped in chilled water for 30 s. The treated seeds were then assessed for the efficacy of the treatment for reducing populations of the pathogens and the effects of the treatment on germination. After inoculation and air drying, 6.08 +/- 0.34 log CFU/g E. coli O157:H7 and 5.34 +/- 0.29 log CFU/g Salmonella were detected on the seeds. After hot water treatment at 90 degrees C for 90 s followed by dipping in chilled water for 30 s, no viable pathogens were found and no survivors were found in the enrichment medium and during the sprouting process. The germination yield of the seed was not affected significantly. Therefore, hot water treatment followed by dipping in chilled water for 30 s could be an effective seed decontamination method for mung bean seeds intended for sprout production.     

Industry practices and compliance with U.S. Food and Drug Administration guidelines among California sprout firms

Since 1995, raw vegetable sprouts have been implicated as the vehicle of infection in 15 foodborne outbreaks involving Salmonella and 2 foodborne outbreaks involving Escherichia coli O157:H7. To reduce the numbers of sprout-related outbreaks, the U.S. Food and Drug Administration (FDA) published Guidance for Industry: Reducing Microbial Food Safety Hazards for Sprouting Seeds in 1999. Between October 2000 and April 2001, 61.5% (16 of 26) of the known commercial sprout firms in California were enrolled in a survey to evaluate the industry practices of California sprouting operations and to determine compliance with FDA guidelines. A standardized questionnaire was used to collect data on firm demographics and seed disinfection practices. Additionally, free chlorine levels in seed disinfection solutions were measured, and 48-h spent irrigation water samples were collected from each firm. The irrigation water was screened for Salmonella and E. coli O157:H7 with FDA-recommended test kits. Free chlorine levels in the treatment solutions ranged from 50 to 35,000 mg/liter (ppm), with a median of 14,000 mg/liter (ppm). Free chlorine levels were higher for firms producing alfalfa sprouts than for those producing only mung bean or soybean sprouts (P=0.03). Levels of free chlorine tended to be higher for firms using a calcium hypochlorite treatment solution than for firms using a sodium hypochlorite treatment solution (P=0.067). All 32 irrigation water samples screened for Salmonella tested negative. Of the irrigation water samples tested for E. coil O157:H7, 75% (24 of 32) tested negative, and 25% (8 of 32) tested presumptive positive. The eight presumptive positive samples were found to be negative after further testing. These results indicate that producers of alfalfa sprouts are generally achieving the FDA-recommended calcium hypochlorite level of 20,000 mg/liter (ppm), whereas mung bean sprout producers are not.     

Mathematical modeling and assessment of microbial migration during the sprouting of alfalfa in trays in a nonuniformly contaminated seed batch using Enterobacter aerogenes as a surrogate for Salmonella Stanley

Raw seed sprouts have been implicated in several food poisoning outbreaks in the past 10 years. The U.S. Food and Drug Administration recommends that sprout growers use interventions (such as testing of spent irrigation water) to control the presence of pathogens in the finished product. During the sprouting process, initially low concentrations of pathogen may increase, and contamination may spread within a batch of sprouting seeds. A model of pathogen growth as a function of time and distance from the contamination spot during the sprouting of alfalfa in trays has been developed with Enterobacter aerogenes. The probability of detecting contamination was assessed by logistic regression at various time points and distances by sampling from sprouts or irrigation water. Our results demonstrate that microbial populations and possibility of detection were greatly reduced at distances of > or = 20 cm from the point of contamination in a seed batch during tray sprouting; however, the probability of detecting microbial contamination at distances less than 10 cm from the point of inoculation was almost 100% at the end of the sprouting process. Our results also show that sampling irrigation water, especially large volumes of water, is highly effective at detecting contamination: by collecting 100 ml of irrigation water for membrane filtration, the probability of detection was increased by three to four times during the first 6 h of seed germination. Our findings have quantified the degree to which a small level of contamination will spread throughout a tray of sprouting alfalfa seeds and subsequently be detected by either sprout or irrigation water sampling.     

Heat treatments to enhance the safety of mung bean seeds

Salmonella enterica serovars and Escherichia coli O157:H7 have been associated with contaminated seed sprout outbreaks. The majority of these outbreaks have been traced to sprout seeds contaminated with low levels of pathogens. E. coli O157:H7 strains can grow an average of 2.3 log CFU/g over 2 days during seed germination, and Salmonella can achieve an average growth of 3.7 log CFU/g. Therefore, it is important to find an effective method to reduce possible pathogenic bacterial populations on the seeds prior to sprouting. Our objective was to assess the effectiveness of various dry heat treatments on reducing E. coli O157:H7 and Salmonella populations on mung beans intended for sprout production and to determine the effect of these treatments on seed germination. Mung beans were inoculated with five-strain cocktails of E. coli O157:H7 and of Salmonella serovars harboring the green fluorescent protein gene and then air dried overnight. Heat treatments were performed by incubating the seeds at 55 degrees C for various periods of time. Heat-treated seeds were then assessed for the efficacy of the heat treatment and the effects of heat treatment on germination rates. After inoculation and drying, 6 log CFU/g E. coli O157:H7 and 4 log CFU/g Salmonella were detected on the seeds. Following heat treatment, pathogenic bacterial populations on the seeds were below detectable levels (<1 log CFU/g), but the germination rate of the seed was not affected. Thus, the risk of contamination and the presence of pathogens in the finished sprouts were greatly reduced via the seed heat treatment process.     

Quantitative analysis of the growth of Salmonella stanley during alfalfa sprouting and evaluation of Enterobacter aerogenes as its surrogate

Raw seed sprouts have been implicated in several food poisoning outbreaks in the last 10 years. Few studies have included investigations of factors influencing the effectiveness of testing spent irrigation water, and in no studies to date has a nonpathogenic surrogate been identified as suitable for large-scale irrigation water testing trials. Alfalfa seeds were inoculated with Salmonella Stanley or its presumptive surrogate (nalidixic acid-resistant Enterobacter aerogenes) at three concentrations (-3, -30, and -300 CFU/g) and were then transferred into either flasks or a bench top-scale sprouting chamber. Microbial concentrations were determined in seeds, sprouts, and irrigation water at various times during a 4-day sprouting process. Data were fit to logistic regression models, and growth rates and maximum concentrations were compared using the generalized linear model procedure of SAS. No significant differences in growth rates were observed among samples taken from flasks or the chamber. Microbial concentrations in irrigation water were not significantly different from concentrations in sprout samples obtaihed at the same time. E. aerogenes concentrations were similar to those of Salmonella Stanley at corresponding time points for all three inoculum concentrations. Growth rates were also constant regardless of inoculum concentration or strain, except that lower inoculum concentrations resulted in lower final concentrations proportional to their initial concentrations. This research demonstrated that a nonpathogenic easy-to-isolate surrogate (nalidixic acid-resistant E. aerogenes) provides results similar to those obtained with Salmonella Stanley, supporting the use of this surrogate in future large-scale experiments.     

Inactivation of escherichia coli 0157:H7 and Salmonella on mung beans, alfalfa, and other seed types destined for sprout production by using an oxychloro-based sanitizer

The efficacy of a stabilized oxychloro-based food grade sanitizer to decontaminate seeds destined for sprout production has been evaluated. By using mung bean seeds as a model system, it was demonstrated that the sanitizer could be used to inactivate a five-strain cocktail of Escherichia coli O157:H7 or Salmonella introduced onto beans at 10(3) to 10(4) CFU/g. Salmonella was more tolerant to stabilized oxychloro than was E. coli O157:H7, with sanitizer levels of >150 and >50 ppm, respectively, being required to ensure pathogen-free sprouts. The decontamination efficacy was also found to be dependent on treatment time (>8 h optimal) and the seed-to-sanitizer ratio (>1:4 optimal). Stabilized oxychloro treatment did not exhibit phytotoxic effects, as germination and sprout yields were not significantly (P > 0.05) different as compared with untreated controls. Although human pathogens could be effectively eliminated from mung beans, the aerobic plate count of native microflora on sprouts grown from treated seed was not significantly (P > 0.05) different from the controls. The diversity of microbial populations (determined through 16S rRNA denaturing gradient gel electrophoresis analysis) associated with bean sprouts was not significantly affected by the sanitizer treatment. However, it was noted that Klebsiella and Herbasprillum (both common plant endophytes) were absent in sprouts derived from decontaminated seed but were present in control sprouts. When a further range of seed types was evaluated, it was found that alfalfa, cress, flax, and soybean could be decontaminated with the stabilized oxychloro sanitizer. However, the decontamination efficacy with other seed types was less consistent. It appears that the rate of seed germination and putative activity of sanitizer sequestering system(s), in addition to other factors, may limit the efficacy of the decontamination method.     

Dynamic changes in phytochemical composition and antioxidant capacity in green and black mung bean (Vigna radiata) sprouts

The mung bean (Vigna radiata) sprout is a popular fresh vegetable in many parts of the world. In this study, the dynamic change of ascorbic acid, phenolic composition and antioxidant capacity were evaluated in green and black mung bean sprouts. Germination increased ascorbic acid, total phenolic content and antioxidant capacity in both mung beans. The green mung bean sprout generally had higher ascorbic acid, total phenolic content and antioxidant capacity than the black mung bean sprout. Most phenolic compounds, such as caffeic acid, catechin, ferulic acid, gallic acid and rutin, were found to gradually increase after germination. Therefore, mung bean sprouts, especially green mung bean sprout possessing high level of antioxidant phytochemicals, can be valuable functional vegetables and good sources of natural antioxidants.     

Radiation processing for elimination of Salmonella typhimurium from inoculated seeds used for sprout making in India and effect of irradiation on germination of seeds

The effect of radiation processing on the germination of the sprout seeds mung (Phaseolus aureus), matki (Phaseolus aconitifolius), chana (Cicer arietinum), and vatana (Pisum sativum) in terms of percent germination, germination yield, sprout length, vitamin C content, and texture was investigated. Gradual decreases in the percent germination, germination yield, and sprout length with increases in radiation dose (0.5 to 2.0 kGy) were observed. Vitamin C content and texture remained unaffected for the seeds treated with doses of up to 2 kGy. To determine the efficacy of radiation treatment in elimination of foodborne pathogens, seeds inoculated with 4 log CFU/g of Salmonella Typhimurium were treated with radiation doses of 1 and 2 kGy. A reduction in counts of Salmonella Typhimurium in inoculated seeds after radiation treatment was observed. A radiation dose of 2 kGy resulted in the complete elimination of 4 log CFU/g of Salmonella Typhimurium from the inoculated seeds. However, on sprouting for 48 h, the count of Salmonella Typhimurium reached 8 log CFU/g for the control seeds and the seeds treated with a 1-kGy radiation dose. The aerobic plate counts for seeds were 2.0 to 2.6 log CFU/g, which were reduced to 0.9 to 1.2 log CFU/g on treatment with a 2-kGy radiation dose. On sprouting for 48 h, the aerobic plate count reached 8 log CFU/g for both the control and radiation-treated seeds. The study demonstrates that irradiation can control bacterial levels on seeds but not contamination introduced during posttreatment handling. Therefore, radiation processing of the final product (sprouts) is recommended, rather than of the seeds.     

Chemical and irradiation treatments for killing Escherichia coli O157:H7 on alfalfa,radish, and mung bean seeds

In this study, the effectiveness of dry-heat treatment in combination with chemical treatments (electrolyzed oxidizing [EO] water, califresh-S, 200 ppm of active chlorinated water) with and without sonication in eliminating Escherichia coli O157:H7 on laboratory-inoculated alfalfa, radish, and mung bean seeds was compared with that of dry-heat treatment in combination with irradiation treatment. The treatment of mung bean seeds with EO water in combination with sonication followed by a rinse with sterile distilled water resulted in reductions of approximately 4.0 log10 CFU of E. coli O157:H7 per g. whereas reductions of ca. 1.52 and 2.64 log10 CFU/g were obtained for radish and alfalfa seeds. The maximum reduction (3.70 log10 CFU/g) for mung bean seeds was achieved by treatment with califresh-S and chlorinated water (200 ppm) in combination with sonication and a rinse. The combination of dry heat, hot EO water treatment, and sonication was able to eliminate pathogen populations on mung bean seeds but was unable to eliminate the pathogen on radish and alfalfa seeds. Other chemical treatments used were effective in greatly reducing pathogen populations on radish and alfalfa seeds without compromising the quality of the sprouts, but these treatments did not result in the elimination of pathogens from radish and alfalfa seeds. Moreover, a combination of dry-heat and irradiation treatments was effective in eliminating E. coli O157:H7 on laboratory-inoculated alfalfa, radish, and mung bean seeds. An irradiation dose of 2.0 kGy in combination with dry heat eliminated E. coli O157:H7 completely from alfalfa and mung bean seeds, whereas a 2.5-kGy dose of irradiation was required to eliminate the pathogen completely from radish seeds. Dry heat in combination with irradiation doses of up to 2.0 kGy did not unacceptably decrease the germination percentage for alfalfa seeds or the length of alfalfa sprouts but did decrease the lengths of radish and mung bean sprouts.     

Growth of Salmonella during sprouting of alfalfa seeds associated with salmonellosis outbreaks

Growth of Salmonella was assessed during sprouting of naturally contaminated alfalfa seeds associated with two outbreaks of salmonellosis. Salmonella was determined daily in sprouts and sprout rinse water samples by a three-tube most probable number (MPN) procedure and a commercial enzyme immunoassay (EIA). Growth of Salmonella in the sprouts was reflected in the rinse water, and the MPNs of the two samples were generally in agreement within approximately 1 log. The results from EIA testing of sprouts and water samples were also in agreement. The pathogen was present in the seed at less than 1 MPN/g, and it increased in number to maximum population levels of 102 to 10(3) MPN/g in one seed lot and 10(2) to 10(4) MPN/ g in the other seed lot. Maximum populations of the pathogen were apparent by day 2 of sprouting. These results show the ability of the pathogen to grow to detectable levels during the sprouting process, and they provide support for the recommendation to test the sprout water for the presence of pathogens 48 h after starting seed sprouting. The effectiveness of a 10-min, 20,0000-microg/ml (ppm) calcium hypochlorite treatment of the outbreak-associated seeds was studied. For both seed lots, the hypochlorite treatment caused a reduction, but not elimination, of Salmonella contamination in the finished sprouts. These results confirm the need to test each production batch for the presence of pathogens, even after 20,000 microg/ml (ppm) hypochlorite treatment of seeds, so that contaminated product is not distributed.     

Reduction of Escherichia coli O157:H7 and Salmonella typhimurium in artificially contaminated alfalfa seeds and mung beans by fumigation with ammonia.

Sprouts eaten raw are increasingly perceived as hazardous foods because they have been vehicles in outbreaks of foodborne disease, often involving Escherichia coli O157:H7 and Salmonella Typhimurium. Although the source of these pathogens has not been established, it is known that the seeds usually are already contaminated at the time sprouting begins. Earlier studies had shown that ammonia was lethal to these same pathogens in manure, so it seemed reasonable to determine whether ammonia was effective against them when associated with seeds to be used for sprouting. Experimentally contaminated (10(8) to 10(9) CFU/g) and dried seeds, intended for sprouting, were sealed in glass jars in which 180 or 300 mg of ammonia/liter of air space was generated by action of ammonium sulfate and sodium hydroxide. Samples were taken after intervals up to 22 h at 20 degrees C. Destruction of approximately 2 to 3 logs was observed with both bacteria associated with alfalfa seeds, versus 5 to 6 logs with mung beans. Greater kills are apparently associated with lower initial bacterial loads. Germination of these seeds was unaffected by the treatment. It appears that this simple treatment could contribute significantly to the safety of sprout production from alfalfa seeds and mung beans.     

Evaluation of chemical and physical (high-pressure and temperature) treatments to improve the safety of minimally processed mung bean sprouts during refrigerated storage

The objective of this study was to compare the effects of combined high hydrostatic pressure and temperature treatments with different chemical sanitation treatments (water, sodium hypochlorite, and hydrogen peroxide) on the microbiological properties of mung bean sprouts. In a first study, the raw product was subjected to several combined high-pressure and temperature treatments for calculating a mathematical model by a response surface methodology. The number of pressure-temperature (150 to 400 MPa; 20 to 40 degrees C) combinations was limited to 10. In addition, a model system consisting of mung bean sprout juice was inoculated with Listeria monocytogenes (CECT 4032). Microbial inactivation with this model system was also investigated by a response surface methodology. The highest aerobic mesophilic bacteria and L. monocytogenes inactivation was achieved at maximum pressure and temperature (5.5 and 1.8 log cycles, respectively). In a second study, the effect of five different processing lines on the microbial load reduction of minimally processed mung bean sprouts during refrigerated storage was studied. All treatments reduced the initial population of aerobic mesophilic bacteria and fecal coliforms, with the physical treatment of 400 MPa and 40 degrees C being the most effective, showing initial reductions of 5.8 and 7.8 log CFU/ g, respectively. Recovery of bacteria from sprouts treated under these conditions was not observed during storage. However, the sprouts that received washing treatments with water, sodium hypochlorite, and hydrogen peroxide exhibited increases in aerobic mesophilic and fecal coliform counts after 3 days of storage at 4 degrees C.     

Occurrence of parasites on fruits and vegetables in Norway.

Between August 1999 and January 2001, samples of various fruits and vegetables obtained within Norway were analyzed by published methods for parasite contamination. Neither Cyclospora oocysts nor Ascaris (or other helminth) eggs were detected on any of the samples examined for these parasites. However, of the 475 samples examined for Cryptosporidium oocysts and Giardia cysts, 29 (6%) were found to be positive. No samples were positive for both parasites. Of the 19 Cryptosporidium-positive samples. 5 (26%) were in lettuce, and 14 (74%) in mung bean sprouts. Of the 10 Giardia-positive samples, 2 (20%) were in dill, 2 (20%) in lettuce, 3 (30%) in mung bean sprouts, 1 (10%) in radish sprouts, and 2 (20%) in strawberries. Mung bean sprouts were significantly more likely to be contaminated with Cryptosporidium oocysts or Giardia cysts than the other fruits and vegetables. Concentrations of Cryptosporidium and Giardia detected were generally low (mean of approximately 3 [oo]cysts per 100 g produce). Although some of the contaminated produce was imported (the majority, if sprouted seeds are excluded), there was no association between imported produce and detection of parasites. Crvptosporidium oocysts and Giardia cysts were also detected in water samples concerned with field irrigation and production of bean sprouts within Norway. This is the first time that parasites have been detected on vegetables and fruit obtained in a highly developed. wealthy country, without there being an outbreak situation. These findings may have important implications for global food safety.     

The effect of calcinated calcium and chlorine treatments on Escherichia coli O157:H7 87-23 population reduction in radish sprouts

Abstract: The effect of calcinated calcium spray on Escherichia coli O157:H7 87–23 population reduction during radish sprout production was studied. Artificially inoculated radish seeds were soaked in sodium hypochlorite (NaOCl) solutions (200 and 20000 ppm), rinsed in distilled water, and sprayed with water or a calcinated calcium solution during sprouting. Microbial plate count was obtained at each step of the process and germination rate was determined after 72 h of sprouting. Scanning electron microscopy (SEM) was done on treated seeds and sprouts to locate which parts were populated by the E. coli cells. The results showed that the active compound in the calcinated calcium was calcium oxide. The treatment of 200 ppm NaOCl soaking followed by 0.04% calcinated calcium spray resulted in no microbial growth after a 72‐h sprouting, while maintaining a high germination rate. The 0.4% calcinated calcium spray significantly reduced the germination rate and is therefore not recommended. Soaking the seeds in a 20000 ppm chlorine solution achieved the highest E. coli count reduction (1.65 log CFU/g). However, the E. coli cells that survived the 20000 ppm chlorine soak grew to 6 log CFU/g sprouts after a 72‐h sprouting, significantly higher than the initial count on the seeds. The SEM microimages showed that the bacteria were mostly located in the roots of the radish sprouts and all across the seed surface. The E. coli O157:H7 87–23 cells appeared to be located in biofilms or embedded into the radish sprout tissues during sprouting. Practical Application: The seed sanitation treatment with 20000 ppm chlorine solution that is currently used by the sprout industry was once again found to be ineffective in eliminating inoculated pathogenic cells. More importantly, the remaining cells that have survived the chlorine wash would grow during sprouting to reach an alarmingly high cell concentration. The new observation of E. coli cells and sprout tissue interaction manifested as embedding of the cells in sprout tissues, if confirmed, will have a significant impact on the microbial safety intervention strategies used in the sprout industry. This research demonstrated the importance of eliminating all pathogens on the seeds before germination and sprouting.     

RAPID DETECTION OF OUTBREAK ESCHERICHIA COLI O157 AND SALMONELLA ON ALFALFA SPROUTS BY IMMUNOMAGNETIC CAPTURE AND TIME‐RESOLVED FLUORESCENCE1

Commercially available alfalfa seeds were inoculated with low levels (～ 4 CFU/g) of pathogenic bacteria and sprouted at 25C. At 48 h, the spent irrigation water and sprouts were separately transferred to brain heart infusion (BHI) broth and enriched for 4 h at 37C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E. coli O157 or Salmonella in the enriched media. Separation and concentration of captured pathogens were achieved using magnetic particle concentrators (MPC). IMB captured E. coli O157:H7 and Salmonella spp. then formed sandwiched complexes with europium (Eu) labeled anti‐E. coli O157 antibodies and samarium (Sm) labeled anti‐Salmonella antibodies, respectively. After washing the complexes, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent Eu‐ and Sm‐chelates. The specific time‐resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and many Salmonella spp. from spent irrigation water or sprouts grown from contaminated seeds. Nontargeted bacteria, e.g., native microflora present on the untreated seeds and inoculated Aeromonas and Citrobacter, exhibited no crossreactivity and counts were not significantly different from background fluorescence of the IMB alone. Since pathogen detection was achieved within 6 h, the assay could detect contamination levels as low as 4 CFU/g of seeds and it showed no cross‐reactivity with nonpathogenic microflora present on the sprouts, the developed methodology could be used as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella spp. in sprouts and their irrigation water.     

外源锌浸种处理对绿豆芽生长和营养品质的影响

为开发一种富锌食品,采用不同浓度硫酸锌(0²00 mg Zn²⁺/L)溶液浸泡绿豆种子8 h并水培96 h后,研究外源锌浸种对绿豆芽生长指标、主要营养成分、植酸及锌积累的影响。结果显示:25⁷5 mg Zn²⁺/L锌溶液浸种可以提高绿豆芽下胚轴长、根长、生物产量和主要营养成分的含量,其中以50 mg Zn²⁺/L最显著,该浓度锌溶液浸种后培育的绿豆芽中游离氨基酸总量、维生素C、可溶性蛋白、可溶性糖的含量分别达到8.96 mg/g、23.76 mg/100 g、56.82 mg/g、6.62 mg/g;在实验考察的浓度范围内,绿豆芽中锌含量增加了90.9%⁶17.0%;萌发和外源锌浸种分别使植酸含量降低了61.5%和62.0%,但两组间无显著差异（p>0.05）。本研究表明,适当浓度的外源锌(<75 mg Zn²⁺/L)浸种处理对绿豆的萌发和生长是有利的,不仅能增加微量元素锌的营养价值,同时又能提高绿豆芽的营养品质。