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°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°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°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°C. The number of Salmonella on seeds treated at 70°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 (aw) 0.66 was not significantly different than the number inactivated on seeds at aw 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°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°C.

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Comparison of Aqueous Chemical Treatments To Eliminate Salmonella on Alfalfa Seeds

Journal of Food Protection ◽

10.4315/0362-028x-63.11.1475 ◽

2000 ◽

Vol 63 (11) ◽

pp. 1475-1482 ◽

Cited By ~ 92

Author(s):

W. R. WEISSINGER ◽

L. R. BEUCHAT

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Tween 80 ◽

The United States ◽

Germination Percentage ◽

Minimal Effect ◽

Direct Plating ◽

Chemical Treatments ◽

Alfalfa Sprouts ◽

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.

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Use of Green Fluorescent Protein Expressing Salmonella Stanley To Investigate Survival, Spatial Location, and Control on Alfalfa Sprouts

Journal of Food Protection ◽

10.4315/0362-028x-64.12.1891 ◽

2001 ◽

Vol 64 (12) ◽

pp. 1891-1898 ◽

Cited By ~ 71

Author(s):

MEGHA GANDHI ◽

SHERENE GOLDING ◽

SIMA YARON ◽

KARL R. MATTHEWS

Keyword(s):

Green Fluorescent Protein ◽

Laser Scanning ◽

Fluorescent Protein ◽

Plasmid Stability ◽

Constitutive Expression ◽

Epifluorescence Microscopy ◽

Initial Population ◽

Alfalfa Sprouts ◽

Green Fluorescent ◽

Alfalfa Seeds

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. 107 CFU/ml) for 5 min at 22°C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12μm within intact sprout tissue. An initial population of ca. 104 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°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°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. 105 CFU/g Salmonella Stanley to the noncontaminated sprouts.

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Comparison of Chemical Treatments to Eliminate Enterohemorrhagic Escherichia coli O157:H7 on Alfalfa Seeds

Journal of Food Protection ◽

10.4315/0362-028x-62.4.318 ◽

1999 ◽

Vol 62 (4) ◽

pp. 318-324 ◽

Cited By ~ 171

Author(s):

PETER J. TAORMINA ◽

LARRY R. BEUCHAT

Keyword(s):

Escherichia Coli ◽

Germination Percentage ◽

Enterohemorrhagic Escherichia Coli ◽

Active Chlorine ◽

Escherichia Coli O157 ◽

Dramatic Decrease ◽

E Coli ◽

Surfactant Treatment ◽

Chemical Treatments ◽

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 ≤ 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 ≥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 ≥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 ≤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 ≥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°C, but not at 5°C. Between 1 and 38 weeks, populations on seeds stored at 5°C remained relatively constant. The pathogen was recovered from alfalfa seeds initially containing 3.04 log10 CFU/g after storage at 25 or 37°C for 38 weeks but not 54 weeks.

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Behavior of Enterohemorrhagic Escherichia coli O157:H7 on Alfalfa Sprouts during the Sprouting Process as Influenced by Treatments with Various Chemicals

Journal of Food Protection ◽

10.4315/0362-028x-62.8.850 ◽

1999 ◽

Vol 62 (8) ◽

pp. 850-856 ◽

Cited By ~ 89

Author(s):

PETER J. TAORMINA ◽

LARRY R. BEUCHAT

Keyword(s):

Escherichia Coli ◽

Cold Storage ◽

Treatment Method ◽

Enterohemorrhagic Escherichia Coli ◽

Escherichia Coli O157 ◽

E Coli ◽

Chemical Treatments ◽

Alfalfa Sprouts ◽

Germinated Seeds ◽

Alfalfa Seeds

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°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 log10 CFU/g of sprouts produced using techniques not dissimilar to those used in the sprout industry.

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Effects of Community Versus Single Strain Inoculants on the Biocontrol of Salmonella and Microbial Community Dynamics in Alfalfa Sprouts†

Journal of Food Protection ◽

10.4315/0362-028x-68.1.40 ◽

2005 ◽

Vol 68 (1) ◽

pp. 40-48 ◽

Cited By ~ 40

Author(s):

ANABELLE MATOS ◽

JAY L. GARLAND

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Community Dynamics ◽

Community Level ◽

Microbial Consortia ◽

Single Strain ◽

Acridine Orange Staining ◽

Physiological Profiles ◽

Alfalfa Sprouts ◽

Alfalfa Seeds

Potential biological control inoculants, Pseudomonas fluorescens 2-79 and microbial communities derived from market sprouts or laboratory-grown alfalfa sprouts, were introduced into alfalfa seeds with and without a Salmonella inoculum. We examined their ability to inhibit the growth of this foodborne pathogen and assess the relative effects of the inoculants on the alfalfa microbial community structure and function. Alfalfa seeds contaminated with a Salmonella cocktail were soaked for 2 h in bacterial suspensions from each inoculant tested. Inoculated alfalfa seeds were grown for 7 days and sampled during days 1, 3, and 7. At each sampling, alfalfa sprouts were sonicated for 7 min to recover microflora from the surface, and the resulting suspensions were diluted and plated on selective and nonselective media. Total bacterial counts were obtained using acridine orange staining, and the percentage culturability was calculated. Phenotypic potential of sprout-associated microbial communities inoculated with biocontrol treatments was assessed using community-level physiological profiles based on patterns of use of 95 separate carbon sources in Biolog plates. Community-level physiological profiles were also determined using oxygen-sensitive fluorophore in BD microtiter plates to examine functional patterns in these communities. No significant differences in total and mesophilic aerobe microbial cell density or microbial richness resulting from the introduction of inoculants on alfalfa seeds with and without Salmonella were observed. P. fluorescens 2-79 exhibited the greatest reduction in the growth of Salmonella early during alfalfa growth (4.22 log at day 1), while the market sprout inoculum had the reverse effect, resulting in a maximum log reduction (5.48) of Salmonella on day 7. Community-level physiological profiles analyses revealed that market sprout communities peaked higher and faster compared with the other inoculants tested. These results suggest that different modes of actions of single versus microbial consortia biocontrol treatments may be involved.

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Effects of Indole-3-Acetic Acid on Germination in Lead Polluted Petri Dish of Citrullus lanatus (Thunberg) Matsumura and Nakai, Cucurbitaceae

Aceh International Journal of Science and Technology ◽

10.13170/aijst.4.3.3021 ◽

2015 ◽

Vol 4 (3) ◽

Author(s):

Matthew Chidozie Ogwu ◽

Aiwansoba Raymond Osas ◽

Osawaru Moses Edwin

Keyword(s):

Acetic Acid ◽

Citrullus Lanatus ◽

Combined Treatment ◽

Petri Dish ◽

Germination Percentage ◽

Treatment Level ◽

Optimum Level ◽

Polluted Environment ◽

Tropical Fruit ◽

Indole 3 Acetic Acid

Watermelon, Citrullus lanatus (Thunberg) Matsumura and Nakai is a tropical fruit vegetable. Indole-3-acetic acid (IAA) is a popular phytohormone while lead (Pb) is a common environmental pollutant in urban and sub-urban centers. C. lanatus were obtained from Benin City with a view to study the effects of IAA on their germination in Pb polluted environment. Germination percentage without IAA and Pb treatment in petri dish was significant after ten days. Hastened germination was observed when IAA and lead were used. About 100 % germination was recorded after seven days. This suggests that water melon seeds can initiate growth even in lead polluted environment. Optimum level of 5 ppm IAA with the different levels of lead treatments may be recommended. Most important was that higher concentrations of Pb in the control (without IAA) did not inhibit seedling shoot nor root growth. Longest seedling shoot length (cm) was 10.33 ± 1.24 and 12.13 ± 2.06 on the seventh and eighth day respectively with the combined treatment levels of 1 ppm IAA and 15 ppm Pb. On the ninth day, 15.27 ± 0.96 was obtained from 1 ppm IAA and 20 ppm Pb. Longest seedling root length (cm) values were recorded from the combined treatment levels of 0 ppm IAA and 10 ppm Pb for the seventh (9.10 ± 0.47) and ninth (10.37 ± 1.81) day respectively and 0 ppm and 15 ppm Pb on the eighth (9.37 ± 0.84) day. Significant means were also obtained with the treatment level of 0 and 20 ppm IAA. This present study suggest the germination of C. lanatus under Pb polluted environment may be rescued with optimum IAA.

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Chemical and Irradiation Treatments for Killing Escherichia coli O157:H7 on Alfalfa, Radish, and Mung Bean Seeds

Journal of Food Protection ◽

10.4315/0362-028x-66.5.767 ◽

2003 ◽

Vol 66 (5) ◽

pp. 767-774 ◽

Cited By ~ 63

Author(s):

M. L. BARI ◽

E. NAZUKA ◽

Y. SABINA ◽

S. TODORIKI ◽

K. ISSHIKI

Keyword(s):

Escherichia Coli ◽

Heat Treatment ◽

Mung Bean ◽

Escherichia Coli O157 ◽

E Coli ◽

Chemical Treatments ◽

Dry Heat ◽

Pathogen Populations ◽

Dry Heat Treatment ◽

Alfalfa 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.

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