Comparison of aqueous chemical treatments to eliminate Salmonella on alfalfa seeds

Several outbreaks of salmonellosis associated with alfalfa sprouts have been documented in the United States since 1995. This study was undertaken to evaluate various chemical treatments for their effectiveness in killing Salmonella on alfalfa seeds. Immersing inoculated seeds in solutions containing 20,000 ppm free chlorine (Ca[OCl]2), 5% Na3PO4, 8% H2O2, 1% Ca(OH)2, 1% calcinated calcium, 5% lactic acid, or 5% citric acid for 10 min resulted in reductions of 2.0 to 3.2 log10 CFU/ g. Treatment with 1,060 ppm Tsunami or Vortex, 1,200 ppm acidified NaClO2, or 5% acetic acid were less effective in reducing Salmonella populations. With the exceptions of 8% H2O2, 1% Ca(OH)2, and 1% calcinated calcium that reduced populations by 3.2, 2.8, and 2.9 log10 CFU/g, respectively, none of treatments reduced the number of Salmonella by more than 2.2 log10 CFU/g without significantly reducing the seed germination percentage. Treatment with 5% acetic, lactic, or citric acids substantially reduced the ability of seeds to germinate. Treatment with 1% Ca(OH)2 in combination with 1% Tween 80, a surfactant, enhanced inactivation by 1.3 log10 CFU/g compared to treatment with 1% Ca(OH)2 alone. Presoaking seeds in water, 0.1% EDTA, 1% Tween 80, or 1% Tween 80 plus 0.1% EDTA for 30 min before treatment with water, 2,000 ppm NaOCl, or 2% lactic acid had a minimal effect on reducing populations of Salmonella. Results indicate that, although several chemical treatments cause reductions in Salmonella populations of up to 3.2 log10 CFU/g initially on alfalfa seeds when analyzed by direct plating, no treatment eliminated the pathogen, as evidenced by detection in enriched samples.     

Evaluation of volatile chemical treatments for lethality to Salmonella on alfalfa seeds and sprouts

A study was done to evaluate natural volatile compounds for their ability to kill Salmonella on alfalfa seeds and sprouts. Acetic acid, allyl isothiocyanate (AIT), trans-anethole, carvacrol, cinnamic aldehyde, eugenol, linalool, methyl jasmonate, and thymol were examined for inhibitory and lethal activity against Salmonella by exposing inoculated alfalfa seeds to compounds (1,000 mg/liter of air) for 1, 3, and 7 h at 60 degrees C. Only acetic acid, cinnamic aldehyde, and thymol caused significant reductions in Salmonella populations (>3 log10 CFU/g) compared with the control (1.9 log10 CFU/g) after treatment for 7 h. Treatment of seeds at 50 degrees C for 12 h with acetic acid (100 and 300 mg/liter of air) and thymol or cinnamic aldehyde (600 mg/liter of air) significantly reduced Salmonella populations on seeds (>1.7 log10 CFU/g) without affecting germination percentage. Treatment of seeds at 50 degrees C with AIT (100 and 300 mg/liter of air) and cinnamic aldehyde or thymol (200 mg/liter of air) did not significantly reduce populations compared with the control. Seed germination percentage was largely unaffected by treatment with gaseous acetic acid, AIT, cinnamic aldehyde, or thymol for up to 12 h at 50 degrees C. The number of Salmonella on seeds treated at 70 degrees C for 80 min with acetic acid (100 and 300 mg/liter of air), AIT (100 mg/liter of air), and cinnamic aldehyde and thymol (600 mg/liter of air) at water activity (a(w)) 0.66 was not significantly different than the number inactivated on seeds at a(w) 0.49. Acetic acid at 200 and 500 mg/liter of air reduced an initial population of 7.50 log10 CFU/g of alfalfa sprouts by 2.33 and 5.72 log10 CFU/g, respectively, within 4 days at 10 degrees C. whereas AIT at 200 and 500 mg/liter of air reduced populations to undetectable levels; however, both treatments caused deterioration in sensory quality. Treatment of sprouts with 1 or 2 mg of AIT per liter of air adversely affected sensory quality but did not reduce Salmonella populations after 11 days of exposure at 10 degrees C.     

Reduction of Escherichia coli O157:H7 and Salmonella spp. on laboratory-inoculated mung bean seed by chlorine treatmentt

Three U.S. outbreaks of foodborne illness due to consumption of contaminated raw mung bean sprouts occurred in the past 2 years and were caused by Salmonella Enteritidis. The original source of the pathogens is thought to have been the seed. The aim of this study was to determine whether treatment with aqueous chlorine would eliminate the pathogens from mung bean seed inoculated in the laboratory with four-strain cocktails of Escherichia coli O157:H7 and Salmonella spp. Treatments (for 5, 10, or 15 min) with buffered (500 mM potassium phosphate, pH 6.8) or unbuffered solutions containing 0.3 or 3.0% (wt/vol) Ca(OCl)2 were tested. In order to mimic common commercial practice, seed was rinsed before and after treatment with sterile tap water. Treatment for 15 min with buffer (500 mM potassium phosphate, pH 6.8) or sterile water in combination with the seed rinses resulted in maximum reductions of approximately 3 log10 CFU/g. The largest reductions (4 to 5 log10 CFU/g) for the chlorine treatments in combination with the rinses were obtained after treatment with buffered 3.0% (wt/vol) Ca(OCl)2 for 15 min. Treatment of mung bean seed for 15 min with unbuffered or buffered 3.0% (wt/vol) Ca(OCl)2 did not adversely affect germination. Even though treatments with 3% (wt/vol) Ca(OCl)2 in combination with the water rinses were effective in greatly reducing the populations of both bacterial pathogens, these treatments did not result in the elimination of the pathogens from laboratory-inoculated seed.     

Comparison of chlorine and a prototype produce wash product for effectiveness in killing Salmonella and Escherichia coli O157:H7 on alfalfa seeds

Outbreaks of Salmonella and Escherichia coli O157:H7 infections associated with alfalfa and other seed sprouts have occurred with increased frequency in recent years. This study was undertaken to determine the efficacy of a liquid prototype produce wash product (Fit), compared with water and chlorinated water, in killing Salmonella and E. coli O157:H7 inoculated onto alfalfa seeds. We investigated the efficacy of treatments as influenced by seeds from two different lots obtained from two seeds suppliers and by two methods of inoculation. The efficacy of treatments was influenced by differences in seed lots and amount of organic material in the inoculum. Significant (alpha = 0.05) reductions in Salmonella populations on seeds treated with 20,000 ppm of chlorine or Fit for 30 min ranged from 2.3 to 2.5 log10 CFU/g and 1.7 to 2.3 log10 CFU/g, respectively. Reductions (alpha = 0.05) in E. coli O157:H7 ranged from 2.0 to 2.1 log10 CFU/g and 1.7 to more than 5.4 log10 CFU/g of seeds treated, respectively, with 20,000 ppm of chlorine or Fit. Compared with treatment with 200 ppm of chlorine, treatment with either 20,000 ppm of chlorine or Fit resulted in significantly higher reductions in populations of Salmonella and E. coli O157:H7. None of the treatments eliminated these pathogens as evidenced by their detection on enrichment of treated seeds. Considering the human health and environmental hazards associated with the use of 20,000 ppm of chlorine, Fit provides an effective alternative to chlorine as a treatment to significantly reduce bacterial pathogens that have been associated with alfalfa seeds.     

Reduction of Salmonella enterica on alfalfa seeds with acidic electrolyzed oxidizing water and enhanced uptake of acidic electrolyzed oxidizing water into seeds by gas exchange

Alfalfa sprouts have been implicated in several salmonellosis outbreaks in recent years. The disinfectant effects of acidic electrolyzed oxidizing (EO) water against Salmonella enterica both in an aqueous system and on artificially contaminated alfalfa seeds were determined. The optimum ratio of seeds to EO water was determined in order to maximize the antimicrobial effect of EO water. Seeds were combined with EO water at ratios (wt/vol) of 1:4, 1:10, 1:20, 1:40, and 1:100, and the characteristics of EO water (pH, oxidation reduction potential [ORP], and free chlorine concentration) were determined. When the ratio of seeds to EO water was increased from 1:4 to 1:100, the pH decreased from 3.82 to 2.63, while the ORP increased from +455 to +1,073 mV. EO water (with a pH of 2.54 to 2.38 and an ORP of +1,083 to +1,092 mV) exhibited strong potential for the inactivation of S. enterica in an aqueous system (producing a reduction of at least 6.6 log CFU/ml). Treatment of artificially contaminated alfalfa seeds with EO water at a seed-to-EO water ratio of 1:100 for 15 and 60 min significantly reduced Salmonella populations by 2.04 and 1.96 log CFU/g, respectively (P < 0.05), while a Butterfield's buffer wash decreased Salmonella populations by 0.18 and 0.23 log CFU/g, respectively. After treatment, EO water was Salmonella negative by enrichment with or without neutralization. Germination of seeds was not significantly affected (P > 0.05) by treatment for up to 60 min in electrolyzed water. The uptake of liquid into the seeds was influenced by the internal gas composition (air, N2, or O2) of seeds before the liquid was added.     

Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts.

Laser scanning confocal microscopy (LSCM) was used to observe the interaction of Salmonella Stanley with alfalfa sprouts. The green fluorescent protein (gfp) gene was integrated into the chromosome of Salmonella Stanley for constitutive expression, thereby eliminating problems of plasmid stability and loss of signal. Alfalfa seeds were inoculated by immersion in a suspension of Salmonella Stanley (ca. 10(7) CFU/ml) for 5 min at 22 degrees C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12 microm within intact sprout tissue. An initial population of ca. 10(4) CFU/g seed increased to 7.0 log CFU/g during a 24-h germination period and then decreased to 4.9 log CFU/g during a 144-h sprouting period. Populations of Salmonella Stanley on alfalfa seeds decreased from 5.2 to 4.1 log CFU/g and from 5.2 to 2.8 log CFU/g for seeds stored 60 days at 5 and 22 degrees C, respectively. The efficacy of 100, 200, 500, or 2,000 ppm chlorine in killing Salmonella Stanley associated with sprouts was determined. Treatment of sprouts in 2,000 ppm chlorine for 2 or 5 min caused a significant reduction in populations of Salmonella Stanley. Influence of storage on Salmonella Stanley populations was investigated by storing sprouts 4 days at 4 degrees C. The initial population (7.76 log CFU/g) of Salmonella Stanley on mature sprouts decreased (7.67 log CFU/g) only slightly. Cross-contamination during harvest was investigated by harvesting contaminated sprouts, then directly harvesting noncontaminated sprouts. This process resulted in the transfer of ca. 10(5) CFU/g Salmonella Stanley to the noncontaminated sprouts.     

Acidified Sodium Chlorite as an Alternative to Chlorine for Elimination of Salmonella on Alfalfa Seeds

ABSTRACT:  The health and environmental hazard associated with the use of chlorine for food processing has been documented previously. This study was conducted to determine if acidified sodium chlorite (ASC) could be used to replace calcium hypochlorite (Ca[OCl]₂) for disinfection of alfalfa seeds. Contaminated seeds containing approximately 1.5 × 10⁷ CFU/g of Salmonella were treated with ASC or Ca(OCl)₂ at different concentrations and for different periods of time. Results showed that the efficacy of ASC and Ca(OCl)₂ for elimination of Salmonella on contaminated seeds could be improved greatly by extending the treatment time from the traditional 15 to 45 min. Treatment of seeds with 800 ppm of ASC for 45 min reduced the number of Salmonella by 3.9 log units, approximately 1.2 log units higher than that treated with 20000 ppm of Ca(OCl)₂. Treatment of seeds with a lower concentration (100 to 400 ppm) of ASC for 45 min reduced the number of Salmonella by 1.3 to 2.2 log units. Soaking alfalfa seeds in 800 ppm of ASC for 45 min did not affect seed germination. However, soaking seeds in 20000 ppm of Ca(OCl)₂ for 45 min reduced seed germination by 20%. Unlike Ca(OCl)₂, antimicrobial efficiency of ASC was not affected by pre-exposure to alfalfa seeds. Data presented also showed that Salmonella on newly inoculated seeds that had been stored at 4 °C for less than 7 d were more sensitive to sanitizer treatment than those on seeds that had been stored for 4 wk or longer.     

Efficacy of electrolyzed water in inactivating Salmonella enteritidis and Listeria monocytogenes on shell eggs

The efficacy of acidic electrolyzed (EO) water produced at three levels of total available chlorine (16, 41, and 77 mg/ liter) and chlorinated water with 45 and 200 mg/liter of residual chlorine was investigated for inactivating Salmonella Enteritidis and Listeria monocytogenes on shell eggs. An increasing reduction in Listeria population was observed with increasing chlorine concentration from 16 to 77 mg/liter and treatment time from 1 to 5 min, resulting in a maximal reduction of 3.70 log CFU per shell egg compared with a deionized water wash for 5 min. There was no significant difference in antibacterial activities against Salmonella and Listeria at the same treatment time between 45 mg/liter of chlorinated water and 14-A acidic EO water treatment (P > or = 0.05). Chlorinated water (200 mg/liter) wash for 3 and 5 min was the most effective treatment; it reduced mean populations of Listeria and Salmonella on inoculated eggs by 4.89 and 3.83 log CFU/shell egg, respectively. However, reductions (log CFU/shell egg) of Listeria (4.39) and Salmonella (3.66) by 1-min alkaline EO water treatment followed by another 1 min of 14-A acidic EO water (41 mg/liter chlorine) treatment had a similar reduction to the 1-min 200 mg/liter chlorinated water treatment for Listeria (4.01) and Salmonella (3.81). This study demonstrated that a combination of alkaline and acidic EO water wash is equivalent to 200 mg/liter of chlorinated water wash for reducing populations of Salmonella Enteritidis and L. monocytogenes on shell eggs.     

INCIDENCE AND GERMICIDE SENSITIVITY OF SALMONELLA TYPHI AND VIBRIO CHOLERAE O1 IN ALFALFA SPROUTS

The prevalence of some enteric bacteria in alfalfa sprouts obtained from public markets and supermarkets in Queretaro City was determined. In addition, the antimicrobial effect of several commercial germicides was tested on alfalfa sprouts for reduction of native coliforms and inoculated Vibrio cholerae Ol or Salmonella typhi. Escherichia coli and Salmonella sp. were detected in 74% and 1.1% of 90 of samples, respectively, and no sample tested positive to V. cholerae Ol. Coliforms ranged from 7.3 to 8.5 log₁₀ CFU/g. Treatment of alfalfa sprouts with 200 mg/L of hypochlorite, of a commercial iodophor, or of chlorine dioxide, or with 100 mg/L of Citricidal® for 5 min, reduced native coliforms only by 1–2 log₁₀ CFU/g. Reductions of S. typhi and V. cholerae O1 with 200 mg/L of sodium hypochlorite and 100 mg/L Citricidal® were also no more than J.5 log₁₀ CFU/g. Sprouts from seeds contaminated with V. cholerae O1 that were irrigated daily with water containing 100 mg/L chlorine dioxide, showed significantly lower V. cholerae Ol counts than seeds irrigated with tap water (p<0.05). However , V. cholerae O1 persisted after 8 days of sprouting, when the sprouts are ready for marketing. Treatment of seeds and sprouts with antimicrobials does not appear to be effective for reducing pathogens to safe levels.     

Behavior of enterohemorrhagic Escherichia coli O157:H7 on alfalfa sprouts during the sprouting process as influenced by treatments with various chemicals.

The behavior of Escherichia coli O157:H7 on alfalfa seeds subjected to conditions similar to those used commercially to grow and market sprouts as it is affected by applications of NaOCl, Ca(OCl)2, acidified NaClO2, acidified ClO2, Na3PO4, Vegi-Clean, Tsunami, Vortexx, or H2O2 at various stages of the sprouting process was determined. Application of 2,000 ppm of NaOCl, 200 and 2,000 ppm of Ca(OCl)2, 500 ppm of acidified ClO2, 10,000 ppm of Vegi-Clean, 80 ppm of Tsunami, or 40 and 80 ppm of Vortexx to germinated seeds significantly reduced the population of E. coli O157:H7. With the exception of acidified NaOCl2 at 1,200 ppm, spray applications of these chemicals did not significantly reduce populations or control the growth of E. coli O157:H7 on alfalfa sprouts during the sprouting process. Populations of E. coli on alfalfa sprouts peaked at 6 to 7 log10 CFU/g 48 h after initiation of the sprouting process and remained stable despite further spraying with chemicals. The population of E. coli O157:H7 on sprouts as they entered cold storage at 9 +/- 2 degrees C remained essentially unchanged for up to 6 days. None of the chemical treatments evaluated was able to eliminate or satisfactorily reduce E. coli O157:H7 on alfalfa seeds and sprouts. Observations on the ability of E. coli O157:H7 to grow during production of alfalfa sprouts not subjected to chemical treatments are similar to those from a previous study in our laboratory on the behavior of Salmonella Stanley. Our results do not reveal a chemical treatment method to eliminate the pathogen from alfalfa sprouts. We have demonstrated that currently recommended procedures for sanitizing alfalfa seeds fail to eliminate E. coli O157:H7 and that the pathogen can grow to populations exceeding 7 1og10 CFU/g of sprouts produced using techniques not dissimilar to those used in the sprout industry.     

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.     

E. coli o157:H7 population reduction from alfalfa seeds with malic acid and thiamine dilauryl sulfate and quality evaluation of the resulting sprouts

It has been reported that washing seeds with a 20000 ppm Ca(OCl)(2) solution as recommended by the U.S. Food and Drug Administration is unable to eliminate E. coli cells attached to seed surfaces, and the bacterial cells that have survived a sanitation wash can proliferate during sprouting to a high population. The objectives of this research were to examine the efficacy of malic acid (MA) and thiamine dilauryl sulfate (TDS) combined treatments on the inactivation of E. coli O157:H7 on alfalfa seeds, to study the growth of the remaining E. coli cells during sprouting, and to evaluate the sprout quality. When 10 g of inoculated alfalfa seeds were washed in a 10% MA-1% TDS solution, a complete elimination of E. coli was achieved. The same result was observed by washing the seeds in a 20000 ppm Ca(OCl)(2) solution. However, when the seed size was increased to 50 g while maintaining the same seed-to-sanitizer ratio, both the MA + TDS and the 20000 ppm chlorine washes failed to completely inactivate the E. coli cells on the seeds. Nevertheless, the 10% MA-1% TDS solution was significantly more effective in E. coli count reduction compared to the 20000 ppm chlorine wash. The E. coli O157:H7 cells remaining on the seeds after treatments with both sanitizers grew up to 7 to 8 log CFU/g sprout after 96 h of sprouting. Under the treatment conditions used in this study, none of the treatments resulted in significant differences in germination rate, yield, or quality of the sprouts. PRACTICAL APPLICATION: The malic acid (MA) and thiamine dilauryl sulfate (TDS) combined treatment may provide a new solution to secure the microbial safety of seeds and sprouts. An important finding of this study is that seed sample size has a significant impact on the inactivation of E. coli O157:H7 on alfalfa seeds. The microbial inactivation results obtained in a laboratory set-up cannot be directly applied to a large scale operation. A validation test on the large scale has to be performed to evaluate the efficacy of the sanitizer.     

Comparison of chemical treatments to eliminate enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds.

The focus of this study was to determine the efficacy of various chemicals in eliminating 2.04 to 3.23 log10 CFU/g of Escherichia coli O157:H7 from alfalfa seeds and to determine the survivability of the pathogen on seeds stored for prolonged periods at three temperatures. Significant (P < or = 0.05) reductions in populations of E. coli O157:H7 on inoculated seeds were observed after treatments with 500 and 1,000 ppm of active chlorine (as Ca[OCl]2) for 3 but not 10 min and with > or =2,000 ppm of Ca(OCl)2 regardless of pretreatment with a surfactant. Treatment with 20,000 ppm of active chlorine failed to kill 2.68 log10 CFU/g of seeds. Acidified NaClO2 (500 ppm) was effective in reducing populations of the pathogen by >2 logs per g. Acidified ClO2 significantly reduced populations of E. coli O157:H7 on seeds at concentrations > or =100 ppm, and 500 ppm of ClO2 reduced the pathogen from 2.7 log10 CFU/g to <0.5 CFU/g. Chlorine (as NaOCl) was not effective at concentrations < or =1,000 ppm; significant reduction was achieved only after treatment with 2,000 ppm for 3 or 10 min. Notable reduction in populations was observed after treatment with 30 or 70% C2H3OH, but there was a dramatic decrease in germination percentage. Treatment with 0.2% H2O2 significantly reduced populations, and the organism was not detected by direct plating after treatment with > or =1% H2O2. Significant reduction in population of E. coli O157:H7 occurred after treatment with 1% trisodium phosphate, 40 ppm of Tsunami and Vortexx, and 1% Vegi-Clean. A significant decrease in the number of E. coli O157:H7 on dry seeds was observed within 1 week of storage at 25 and 37 degrees C, but not at 5 degrees C. Between 1 and 38 weeks, populations on seeds stored at 5 degrees C remained relatively constant. The pathogen was recovered from alfalfa seeds initially containing 3.04 log 10 CFU/g after storage at 25 or 37 degrees C for 38 weeks but not 54 weeks.     

Treatment of Escherichia coli O157:H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidizing water

Electrolyzed oxidizing water is a relatively new concept that has been utilized in agriculture, livestock management, medical sterilization, and food sanitation. Electrolyzed oxidizing (EO) water generated by passing sodium chloride solution through an EO water generator was used to treat alfalfa seeds and sprouts inoculated with a five-strain cocktail of nalidixic acid resistant Escherichia coli O157:H7. EO water had a pH of 2.6, an oxidation-reduction potential of 1150 mV and about 50 ppm free chlorine. The percentage reduction in bacterial load was determined for reaction times of 2, 4, 8, 16, 32, and 64 min. Mechanical agitation was done while treating the seeds at different time intervals to increase the effectiveness of the treatment. Since E. coli O157:H7 was released due to soaking during treatment, the initial counts on seeds and sprouts were determined by soaking the contaminated seeds/sprouts in 0.1% peptone water for a period equivalent to treatment time. The samples were then pummeled in 0.1% peptone water and spread plated on tryptic soy agar with 5 microg/ml of nalidixic acid (TSAN). Results showed that there were reductions between 38.2% and 97.1% (0.22-1.56 log(10) CFU/g) in the bacterial load of treated seeds. The reductions for sprouts were between 91.1% and 99.8% (1.05-2.72 log(10) CFU/g). An increase in treatment time increased the percentage reduction of E. coli O157:H7. However, germination of the treated seeds reduced from 92% to 49% as amperage to make EO water and soaking time increased. EO water did not cause any visible damage to the sprouts.     

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.     

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 on laboratory-inoculated alfalfa seed with commercial citrus-related products

Alfalfa sprouts contaminated with the bacterial pathogens Escherichia coli O157:H7 and Salmonella have been the source of numerous outbreaks of foodborne illness in the United States and in other countries. The seed used for sprouting appears to be the primary source of these pathogens. The aim of this study was to determine whether the efficacy of commercial citrus-related products for sanitizing sprouting seed is similar to that of high levels of chlorine. Five products (Citrex, Pangermex, Citricidal, Citrobio, and Environné) were tested at concentrations of up to 20,000 ppm in sterile tap water and compared with buffered chlorine (at 16,000 ppm). Alfalfa seeds were inoculated with four-strain cocktails of Salmonella and E. coli O157:H7 to give final initial concentrations of ca. 9.0 and 7.0 CFU/g, respectively. Treatments (10 min) with Citrex, Pangermex, and Citricidal at 20,000 ppm and chlorine at 16,000 ppm produced similar log reductions for alfalfa seed inoculated with four-strain cocktails of E. coli O157:H7 and Salmonella (3.42 to 3.46 log CFU/g and 3.56 to 3.74 log CFU/g, respectively), and all four treatments were significantly (P<0.05) more effective than the control treatment (a buffer wash). Citrobio at 20,000 ppm was as effective as the other three products and chlorine against Salmonella but not against E. coli O157:H7. Environné was not more effective (producing reductions of 2.2 to 2.9 log CFU/g) than the control treatment (which produced reductions of 2.1 to 2.3 log CFU/g) against either pathogen. None of the treatments reduced seed germination. In vitro assays, as well as transmission electron microscopy, confirmed the antibacterial nature of the products that were effective against the two pathogens and indicated that they were bactericidal. When used at 20,000 ppm, the effective citrus-related products may be viable alternatives to chlorine for the sanitization of sprouting seed pending regulatory approval.     

Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts

A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were also determined. The treatment (10 or 20 min) of seeds in water (4 degrees C) containing an initial concentration of 21.8 +/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05) reduction in populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for 20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/- 0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05) reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water. Treatments (2 min) with up to 23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and natural microbiota compared to soaking in water (control) but did not enhance the lethality compared to the sprouts not treated with continuous sparging. The treatment of sprouts with ozonated water (20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the sensory quality during subsequent storage at 4 degrees C for 7 to 11 days. Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth, and biofilm formation that provide evidence of factors contributing to the difficulty of killing microorganisms by treatment with ozone and other sanitizers.     

Antimicrobial effect of electrolyzed water for inactivating Campylobacter jejuni during poultry washing

The effectiveness of electrolyzed (EO) water for killing Campylobacter jejuni on poultry was evaluated. Complete inactivation of C. jejuni in pure culture occurred within 10 s after exposure to EO or chlorinated water, both of which contained 50 mg/l of residual chlorine. A strong bactericidal activity was also observed on the diluted EO water (containing 25 mg/l of residual chlorine) and the mean population of C. jejuni was reduced to less than 10 CFU/ml (detected only by enrichment for 48 h) after 10-s treatment. The diluted chlorine water (25 mg/l residual chlorine) was less effective than the diluted EO water for inactivation of C. jejuni. EO water was further evaluated for its effectiveness in reducing C. jejuni on chicken during washing. EO water treatment was equally effective as chlorinated water and both achieved reduction of C. jejuni by about 3 log10 CFU/g on chicken, whereas deionized water (control) treatment resulted in only 1 log10 CFU/g reduction. No viable cells of C. jejuni were recovered in EO and chlorinated water after washing treatment, whereas high populations of C. jejuni (4 log10 CFU/ml) were recovered in the wash solution after the control treatment. Our study demonstrated that EO water was very effective not only in reducing the populations of C. jejuni on chicken, but also could prevent cross-contamination of processing environments.     

Inactivation of Escherichia coli O157:H7 and Salmonella by gamma irradiation of alfalfa seed intended for production of food sprouts

Inonizing irradiation was determined to be a suitable method for the inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seed to be used in the production of food sprouts. The radiation D (dose resulting in a 90% reduction of viable CFU) values for the inactivation of Salmonella and E. coli O157:H7 on alfalfa seeds were higher than the D-values for their inactivation on meat or poultry. The average D-value for the inactivation of Salmonella on alfalfa seeds was 0.97 +/- 0.03 kGy; the D-values for cocktails of meat isolates and for vegetable-associated isolates were not significantly different. The D-values for nonoutbreak and outbreak isolates of E. coli O157:H7 on alfalfa seeds were 0.55 +/- 0.01 and 0.60 +/- 0.01 kGy, respectively. It was determined that the relatively high D-values were not due to the low moisture content or the low water activity of the seed. The D-values for Salmonella on alfalfa seeds from two different sources did not differ significantly, even though there were significant differences in seed size and water activity. The increased moisture content of the seed after artificial inoculation did not significantly alter the D-value for the inactivation of Salmonella. The results of this study demonstrate that 3.3- and 2-log inactivations can be achieved with a 2-kGy dose of ionizing radiation, which will permit satisfactory commercial yields of sprouts from alfalfa seed contaminated with E. coli O157:H7 and Salmonella, respectively.