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.     

Efficacy of electrolyzed oxidizing water in inactivating Salmonella on alfalfa seeds and sprouts

Studies have demonstrated that electrolyzed oxidizing (EO) water is effective in reducing foodborne pathogens on fresh produce. This study was undertaken to determine the efficacy of EO water and two different forms of chlorinated water (chlorine water from Cl2 and Ca(OCl)2 as sources of chlorine) in inactivating Salmonella on alfalfa seeds and sprouts. Tengram sets of alfalfa seeds inoculated with a five-strain cocktail of Salmonella (6.3 x 10(4) CFU/g) were subjected to 90 ml of deionized water (control), EO water (84 mg/liter of active chlorine), chlorine water (84 mg/liter of active chlorine), and Ca(OCl)2 solutions at 90 and 20,000 mg/liter of active chlorine for 10 min at 24 +/- 2 degrees C. The application of EO water, chlorinated water, and 90 mg/liter of Ca(OCl)2 to alfalfa seeds for 10 min reduced initial populations of Salmonella by at least 1.5 log10 CFU/g. For seed sprouting, alfalfa seeds were soaked in the different treatment solutions described above for 3 h. Ca(OCl)2 (20,000 mg/liter of active chlorine) was the most effective treatment in reducing the populations of Salmonella and non-Salmonella microflora (4.6 and 7.0 log10 CFU/g, respectively). However, the use of high concentrations of chlorine generates worker safety concerns. Also, the Ca(OCl)2 treatment significantly reduced seed germination rates (70% versus 90 to 96%). For alfalfa sprouts, higher bacterial populations were recovered from treated sprouts containing seed coats than from sprouts with seed coats removed. The effectiveness of EO water improved when soaking treatments were applied to sprouts in conjunction with sonication and seed coat removal. The combined treatment achieved 2.3- and 1.5-log10 CFU/g greater reductions than EO water alone in populations of Salmonella and non-Salmonella microflora, respectively. This combination treatment resulted in a 3.3-log10 CFU/g greater reduction in Salmonella populations than the control (deionized water) treatment.     

The Effectiveness of Sanitizer Treatments in Inactivation of Salmonella spp. from Bell Pepper, Cucumber, and Strawberry

ABSTRACT: Four different postharvest treatments for removal of Salmonella from bell pepper and cucumber were examined, including washes with chlorinated water (HOCl; 200 ppm), acidified sodium chlorite (ASC; 1200 ppm), and peroxyacetic acid (PAA; 75 ppm), and treatment with gaseous chlorine dioxide (ClO₂; total 100 mg). Only ClO₂ gas was evaluated for decontamination of strawberries. Each produce was inoculated with approximately 1.0 × 10⁷ colony-forming units (CFU) of a 5-serovar cocktail of Salmonella on artificially created wounds, smooth surfaces, and stem scar tissue. For tests involving smooth surface inoculation, ASC and PAA treatments decreased contamination to undetectable levels on bell pepper and cucumber, while the chlorine treatment of bell pepper reduced contamination by approximately 2-logs. For stem scar contamination on bell pepper, ASC and PAA treatments both showed >2-log unit reductions, and chlorine treatment showed a <1-log unit reduction. For puncture wounds on bell pepper, HOCl, ASC, and PAA treatments reduced bacterial levels approximately 2-, 3-, and 1-log units, respectively, indicating that HOCl and ASC were more effective than PAA. These aqueous treatments of cucumber with puncture wounds reduced bacterial levels approximately 1-, 2-, and 2-log units, respectively. ClO₂ treatment decreased counts to undetectable levels on all inoculation sites on cucumber and on strawberry smooth surfaces, but failed to completely eliminate Salmonella from bell pepper and from the stem scar and the puncture wounds of strawberry. ASC treatment of bell pepper and ClO₂ gas treatments of cucumber showed the best efficiency for inactivation of Salmonella. ClO₂ treatments effectively reduced Salmonella cells inoculated on the smooth surface and stem scar of strawberries compared with unsanitized control.     

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.     

Efficacy of Sulfuric Acid Scarification and Disinfectant Treatments in Eliminating Escherichia coli O157: H7 from Alfalfa Seeds Prior to Sprouting

ABSTRACT: E. coli O157:H7 reduction on inoculated alfalfa seeds was investigated using acid scarification treatments with or without subsequent application of sanitizers. Scarification with 0.1 to 2N H₂SO₄ for 2.5 to 45 min did not affect (p ≤ 0.05) seed viability. E. coli O157:H7 was reduced by 2.1 to 5.0 logs after treating with 0.1 to 2N H₂SO₄ for 5 to 20 min. Combined scarification (0.5N H₂SO₄) and H₂O₂ or CH₃COOH treatments enhanced microbial destruction by less than 1 log compared to sanitizer alone. Chlorine, Na₂CO₃, or Na₃PO₄ treatments preceded by scarification did not significantly increase microbial destruction compared to sanitizer alone. Appreciable reductions in seed germination were only observed with chlorine treatments.     

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.     

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.     

APPLICATION OF CHLORINE TO REDUCE POPULATIONS OF ESCHERICHIA COLI ON BEEF

The effects of chlorine against 2 strains of E. coli attached to the surface of beef carcass tissue (BCT) were examined using a model carcass washer. Lean and adipose BCT with approximately 5 log ₁₀ CFU/cm ² E. coli bacteria were spray-treated with water and sodium hypochlorite (NaOCl) to give chlorine concentrations of 50, 100, 250, 500, or 800ppm, incubated for 24 h, 4C, and E. coli populations enumerated. Spray treatments with water did significantly (P < 0.05) reduce the bacterial populations of either organism attached to lean or adipose BCT, as compared to populations of controls; however, reductions were less than 0.60 log ₁₀ CFU/cm ². Treatments with 500 and 800 ppm chlorine against E. coli ATCC 25922 attached to BCT resulted in the greatest reductions of 1.22 and 1.28 log ₁₀ CFU/cm ², respectively. At 800 ppm chlorine , E. coli O157:H7 ATCC 43895 attached to BCT was reduced by 1.04 log ₁₀ CFU/cm ², whereas spray treatments with 50, 100, 250, and 500 ppm chlorine resulted in reductions of < 1 log ₁₀ CFU/cm ². Spray treatments with chlorine from sodium hypochlorite solutions reduced populations of E. coli, however, these reductions were not sufficient to completely inactivate the bacteria attached to red meat .     

Decontamination Efficacy of Combined Chlorine Dioxide with Ultrasonication on Apples and Lettuce

ABSTRACT:  The bacterial reduction of Salmonella and Escherichia coli O157:H7-inoculated apples and lettuce by ClO₂ at 0, 5, 10, 20, and 40 ppm with and without 170-kHz ultrasonic treatments for 3, 6, and 10 min, respectively, have been studied. The treatments of ClO₂ at 20 and 40 ppm for 3, 6, and 10 min or at 5 and 10 ppm for 6 and 10 min with 170-kHz ultrasonication caused 3.115 to 4.253 log reductions in Salmonella and 2.235 to 3.865 log reduction in E. coli O157:H7 on inoculated apples. Using combined ClO₂ and ultrasonication to treat 4.48 × 10⁴ CFU/g Salmonella and 1.07 × 10⁵ CFU/g E. coli O157:H7-inoculated lettuce, the bacterial reductions were 2.257 to 2.972 and 1.357 to 2.264 log, respectively. The residual ClO₂ decreased with increasing treatment times, over 80% of ClO₂ was detected after the 3-min treatment, and more than 70% remained after the 10-min treatment time. No bacteria were recovered from the posttreatment solutions of ClO₂ or ClO₂ combined with ultrasonication. The temperature of the ClO₂ treatment was 20.1 °C, and it increased to 40.1, 44.9, and 50.3 °C, with 170-kHz ultrasonic treatments for 3, 6, and 10 min, respectively, on apples.     

Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting

This study investigated novel two-step organic acid/hypochlorite treatments as alternatives to 20000 ppm active chlorine (from calcium hypochlorite) for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting. Commercially available alfalfa seeds were inoculated with a five-strain E. coli O157:H7 mixture and dried to attain ca. 10(6) CFU/g of seeds. Seeds then underwent one of several soak treatments including: (1) 5% (v/v) lactic acid for 10 min at 42 degrees C; (2) 5% acetic acid (v/v) for 10 min at 42 degrees C; (3) 2.5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine (from calcium hypochlorite) for 15 min at 25 degrees C; (4) 5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine for 15 min at 25 degrees C; or (5) 20000 ppm active chlorine for 15 min at 25 degrees C. Each treatment reduced numbers of inoculum cells by about 6.0 log10 CFU/g as determined by plating on Sorbitol MacConkey agar (SMac). Plating on non-selective brain heart infusion agar (BHI) showed that treatments 1-4 reduced counts by 2.3-4.1 log10 CFU/g, thus indicating a large proportion of injured cells. Successive lactic acid and hypochlorite treatments (3 and 4) were more lethal than either organic acid alone (1 and 2). No surviving cells were detected on SMac or BHI following treatment with 20000 ppm active chlorine (treatment 5). Regardless of the previous treatment, E. coli O157:H7 counts increased to 10(7)-10(8) CFU/g during sprouting. Germination of seeds was not adversely affected by any of the treatments (germination > 90%). Results of this study show that: (a) non-lethal cell injury must be considered when evaluating intervention treatments against E. coli O157:H7 on alfalfa seeds; (b) reductions of 2-4 log10 CFU/g can be attained without using 20000 ppm active chlorine; (c) successive lactic acid and hypochlorite treatments have greater lethality than organic acid treatments alone; and (d) none of the treatments tested can prevent regrowth of surviving E. coli O157:H7 during sprouting.     

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.     

Survival and Growth of Vibrio cholerae O1, Salmonella typhi, and Escherichia coli O157:H7 in Alfalfa Sprouts

The survival and growth of Vibrio cholerae O1, Salmonella typhi , and Escherichia coli O157:H7 during germination and sprouting of disinfected alfalfa seeds and alfalfa sprouts was determined. All pathogens showed ability to grow during germination and sprouting, reaching counts of approximately 6.0 log₁₀ CFU/g after 24 h. No growth was observed for any pathogen when the sprouts were inoculated after 24 h of seed germination. At this time, the background microflora was abundant. Numbers of pathogens inoculated on alfalfa sprouts decreased less than 1 log10 CFU/g over 15 d of refrigeration. Alfalfa sprouts can be an important factor contributing to the endemicity for typhoid fever and cholera in México.     

SUPERCRITICAL CARBON DIOXIDE TREATMENT TO INACTIVATE AEROBIC MICROORGANISMS ON ALFALFA SEEDS

The supercritical carbon dioxide (SC-CO₂) process involves pressurizing CO₂ in a chamber which generates liquid phase of carbon dioxide. Pressurized liquid CO₂ has a strong extraction capability of organic and inorganic compounds. The recent studies have also demonstrated that antimicrobial effect of SC-CO₂ due extraction some cellular components of microorganisms. The efficacy of a supercritical carbon dioxide treatment on alfalfa seeds contaminated with Escherichia coli K12 was tested at 2000, 3000, and 4000 psi at 50C. Samples were treated for 15, 30, and 60 min at each pressure. After pummeling the seed samples in 0.1% peptone water, the initial and final Escherichia coli and total aerobic bacteria on the seeds were determined by plating on 3M Petri Films. After 48 h of incubation at 37 C, the colonies were enumerated. Treated seeds were evaluated in terms of germination characteristics. For aerobic plate count, the effect of pressure in the range of 2000–4000 psi was not statistically significant (p > 0.05) even though 85.6% inactivation was achieved at 4000 psi for 60 min. For E. coli, the reductions for 2000, 3000, and 4000 psi treatments for 15 min were 26.6, 68.1, and 81.3%, respectively. As the time was increased from 15 to 60 min at 4000 psi, the percent E. coli reduction increased from 81.3% to 92.8%. The percent germination for all treatments was over 90%. There was no significant difference (p > 0.05) in the germination rate of treated and untreated seeds. Supercritical carbon dioxide treatments demonstrated a reduction of E. coli K12 and total aerobic counts without affecting the germination characteristics of alfalfa seeds (p < 0.05). This study was a step in the direction of improving safety of alfalfa seeds used to produce fresh sprouts, which have been the cause of several outbreaks.     

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.     

Inactivation of Escherichia coli O157:H7 and Salmonella typhimurium DT 104 on alfalfa seeds by levulinic acid and sodium dodecyl sulfate

Studies were conducted to determine the best concentration and exposure time for treatment of alfalfa seeds with levulinic acid plus sodium dodecyl sulfate (SDS) to inactivate Escherichia coli O157:H7 and Salmonella without adversely affecting seed germination. Alfalfa seeds inoculated with a five-strain mixture of E. coli O157:H7 or Salmonella Typhimurium were dried in a laminar flow hood at 21°C for up to 72 h. Inoculated alfalfa seeds dried for 4 h then treated for 5 min at 21°C with 0.5% levulinic acid and 0.05% SDS reduced the population of E. coli O157:H7 and Salmonella Typhimurium by 5.6 and 6.4 log CFU/g, respectively. On seeds dried for 72 h, treatment with 0.5% levulinic acid and 0.05% SDS for 20 min at 21°C reduced E. coli O157:H7 and Salmonella Typhimurium populations by 4 log CFU/g. Germination rates of alfalfa seeds treated with 0.5% levulinic acid plus 0.05% SDS for up to 1 h at 21°C were compared with a treatment of 20,000 ppm of calcium hypochlorite or tap water only. Treatment of alfalfa seeds with 0.5% levulinic acid plus 0.05% SDS for 5 min at 21°C resulted in a >3.0-log inactivation of E. coli O157:H7 and Salmonella.     

Effectiveness of Individual or Combined Sanitizer Treatments for Inactivating Salmonella spp. on Smooth Surface, Stem Scar, and Wounds of Tomatoes

ABSTRACT: Unwaxed, green tomatoes ('Florida 47' cultivar) were contaminated with Salmonella and then treated with aqueous solutions of sodium hypochlorite (HOCl; 200 ppm), acidified sodium chlorite (ASC; 1200 ppm), peroxyacetic acid (PAA; 87 ppm), or chlorine dioxide gas (ClO₂; total 100 mg). Additionally, a combined treatment of immersion in HOCl, followed by immersion in ASC and then exposure to ClO₂ gas was investigated. Tomatoes were spot inoculated with a 5-strain Salmonella cocktail on smooth surfaces, stem scar tissue, or puncture wounds. A 3 replicate set of each of the sample groups was stored at 20 °C and 95% relative humidity (RH) and retested after 5 d. Greater than 4.0-log unit reductions of Salmonella spp. inoculated on the smooth surface of the tomatoes were seen for all aqueous sanitizer treatments, with Salmonella populations below the detection limit after 5 d of storage. All aqueous treatment groups showed > 1.0-log unit reductions in Salmonella at the stem scar and >2.0-log unit reduction at puncture wounds. The ClO₂ gas treatment reduced Salmonella to undetectable levels at the stem scar, but had no apparent effect on populations inoculated in puncture wounds. The combined treatment resulted in a 3.0-log unit reduction of inoculated Salmonella at puncture wounds. In all cases except for treatment with chlorine, surviving Salmonella populations did not increase after the 5 d of storage. Results of this study suggest the combined treatment was most effective for minimizing the risk of Salmonella contaminated on tomatoes.     

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.     

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium DT104, and Listeria monocytogenes on inoculated alfalfa seeds with a fatty acid-based sanitizer

Alfalfa seeds were inoculated with a three-strain cocktail of Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Typhimurium DT104, or Listeria monocytogenes by immersion to contain approximately 6 to 8 log CFU/g and then treated with a fatty acid-based sanitizer containing 250 ppm of peroxyacid, 1,000 ppm of caprylic and capric acids (Emery 658), 1,000 ppm of lactic acid, and 500 ppm of glycerol monolaurate at a reference concentration of 1X. Inoculated seeds were immersed at sanitizer concentrations of 5X, 10X, and 15X for 1, 3, 5, and 10 min and then assessed for pathogen survivors by direct plating. The lowest concentration that decreased all three pathogens by >5 log was 15. After a 3-min exposure to the 15X concentration, populations of E. coli O157:H7, Salmonella Typhimurium DT104, and L. monocytogenes decreased by >5.45, >5.62, and >6.92 log, respectively, with no sublethal injury and no significant loss in seed germination rate or final sprout yield. The components of this 15x concentration (treatment A) were assessed independently and in various combinations to optimize antimicrobial activity. With inoculated seeds, treatment C (15,000 ppm of Emery 658, 15,000 ppm of lactic acid, and 7,500 ppm of glycerol monolaurate) decreased Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes by 6.23 and 5.57 log, 4.77 and 6.29 log, and 3.86 and 4.21 log after 3 and 5 min of exposure, respectively. Treatment D (15,000 ppm of Emery 658 and 15,000 ppm of lactic acid) reduced Salmonella Typhimurium by >6.90 log regardless of exposure time and E. coli )157:H7 and L. monocytogenes by 4.60 and >5.18 log and 3.55 and 3.14 log after 3 and 5 min, respectively. No significant differences (P > 0.05) were found between treatments A, C, and D. Overall, treatment D, which contained Emery 658 and lactic acid as active ingredients, reduced E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes populations by 3.55 to >6.90 log and may provide a viable alternative to the recommended 20,000 ppm of chlorine for sanitizing alfalfa seeds.     

Effect of pan-frying in extra-virgin olive oil on odour profile, volatile compounds and vitamins

Changes in odour of Arauco (ARA) and Arbequina (ARB) extra-virgin olive oil (OO) were monitored during frying by electronic nose (EN) and solid-phase microextraction–gas chromatography methodologies. Degradation of α- and γ-tocopherols was monitored by HPLC. Electronic nose data and volatile compounds were analysed at intervals of 15 min ( t ₁₅) during 60 min of frying ( t ₆₀). α- and γ-tocopherols were determined at intervals of 5 min ( t ₅) during 30 min of frying ( t ₃₀). Principal components analysis applied to EN data showed one component, PC₁ which accounted 96.6% of the total odour variation. SnO₂ sensors had a positive correlation with PC₁. ARA variety corresponding to frying t ₆₀ had the highest positive correlation with PC_1. Analysis of variance results for volatile compounds showed an increase on production for: 3-methyl butanal, n -pentanal, n -hexanal, n -heptanal and n -nonanal at 15 min of frying for ARB OO and at 30 min for ARA OO. α-tocopherol and γ-tocopherol showed an important decrease after the first 5 min of frying for ARB OO and at 15 min for ARA OO.