curing of salmonella enterica, serovar typhimurium-contaminated cowpea seeds and sprouts with...

PDFlib PLOP: PDF Linearization, Optimization, Protection

Page inserted by evaluation versionwww.pdflib.com – [email protected]

CURING OF SALMONELLA ENTERICA, SEROVARTYPHIMURIUM-CONTAMINATED COWPEA SEEDS AND

SPROUTS WITH VINEGAR AND CHLORINATION

BHOJ RAJ SINGH1, MUDIT CHANDRA, RAVIKANT AGARWAL andNAGARAJAN BABU—

National Salmonella Center (Vet)Bioengineering Building

Division of Bacteriology and MycologyIndian Veterinary Research Institute

Izatnagar-243122, India

Accepted for Publication July 3, 2005—

ABSTRACT

In our attempt to standardize methods to eliminate Salmonella fromcowpea bean sprouts, treatment of cowpea seeds either with 20,000 ppm activechlorine for 1 h or with vinegar (5% acetic acid) for 30 min failed to eliminateS. enterica subspecies enterica serovar Typhimurium (S. typhimurium).Although vinegar eliminated Salmonella in 45 min, it drastically deterioratedthe sprouting rate of the treated seeds. The inoculation of seeds with Salmo-nella reduced germination if the soak water had �7.2 ¥ 108 cfu/mL of Salmo-nella. Salmonella typhimurium rapidly multiplied on germinating cowpeaseeds. Nongerminated seeds exhibited a greater (P � 0.01) number of thepathogen than sprouted seeds. Chlorination and vinegar treatments (up to 3 h)of sprouts from contaminated seeds did not eliminate Salmonella but reducedits populations by 2–4 log10 cfu/g. The study also reports that a 1-h vinegartreatment can also eliminate Salmonella from contaminated sprouts aftergermination.

INTRODUCTION

Although the consumption of sprouts resulted in many infectious diseaseoutbreaks (NACMCF 1999), the popular perception of health benefits fromconsumption of sprouts continues to lead to consumption of many differentkinds of sprouts in different societies. Among the pathogens associated with

1 Corresponding author. National Research Centre on Equines, Sirsa Road, Hisar-125 001, Haryana,India. TEL: 0091-1662-276741 (O), 0091-1662-320997 (R); FAX: 0091-1662-276217; EMAIL:[email protected]

Journal of Food Processing and Preservation 29 (2005) 268–277. All Rights Reserved.© Copyright 2005, Blackwell Publishing268

sprout-borne infections, Salmonella serovars have retained their prominenceover the years, leading to intestinal as well as extra-intestinal infections(Taormina et al. 1999; Backer et al. 2000). Evidence suggests that the sprout-ing of seeds may be a primary source of Salmonella (NACMCF 1999;Taormina et al. 1999). A wide range of chemicals such as ClO2, acidifiedNaClO2, NH4, NaOCl, Na3PO4, Ca(OCl)2, Ca(OH)2, CH3CH2OH, H2O2, citricacid, ascorbic acid, peracetic acid, lactic acid, acetic acid, gaseous acetic acid,ammonia vapors, ozonated water, electrolyzed water, various essential oils anda range of commercial antimicrobial washes (Taormina and Beuchat 1999;Lang et al. 2000; Weissinger and Beuchat 2000; Beuchat et al. 2001; Hima-thongkham et al. 2001; Fett 2002; Sharma and Demirci 2003) are being usedfor treatment of sprouting seeds to eliminate pathogens.

In addition, physical methods such as heat (Suslow et al. 2002), gammairradiation (Rajkowski and Thayer 2000; Rajkowski et al. 2003) or a combi-nation of heat and various chemicals (Suslow et al. 2002) were also evaluated,but mostly on seeds for the production of green sprouts, particularly alfalfa.Despite the popularity of cowpea sprouts in India and neighboring countries,methods of decontamination of cowpea seeds or sprouts are not well explored.The popularity of cowpea sprouts and the availability of vinegar (acetic acid)as a household food preservative (Arya and Rastogi 1993) and the prevalenceof S. typhimurium in foods and animals (NACMCF 1999; Verma et al. 2001;Defra 2002) prompted us to conduct this study to evaluate the effects ofvinegar and chlorine treatments in eliminating Salmonella from cowpea seedsand sprouts.

MATERIALS AND METHODS

Preparation of Culture for Inoculation

An S. typhimurium (S. enterica subspecies enterica serovar Typhimu-rium) E-2391 strain isolated from the spleen of a poultry bird that died in amaize grain-associated outbreak of salmonellosis was grown in Tryptic SoyBroth (Becton, Dickinson and Co., Sparks, MD) at 37C for 18 h. The cellpellet from 100 mL of the culture (centrifuged at 4000 ¥ g for 15 min at 4C)was suspended in 100 mL of sterile water to achieve an OD540 nm of 0.6 (about7.2 ¥ 109 cfu/mL). The colony-forming units of Salmonella in the inoculawere determined by the spread-agar method (Edwards and Ewing 1986). Theinocula were prepared fresh from the same stock, as required.

Cowpea Seeds

Certified cowpea seeds (cv. UPC 607), obtained from GB Pant Universityof Agriculture and Technology, Pantnagar, India, were tested for the presence

269CURING OF COWPEA SPROUTS WITH VINEGAR AND CHLORINATION

of Salmonella by homogenizing three 25-g lots of seeds in 225 mL of sterileaqueous 0.85% NaCl (NSS) using an Ultra Turrax homogenizer (IKA-WerkeGmbH & Co., Staufen, Germany) for 3 min. Dilutions of the homogenate wereprepared in sterile NSS to determine the presence of Salmonella (colony-forming units per milliliter) by the spread-agar method (Edwards and Ewing1986). Ten milliliters of undiluted homogenate was processed to detect Sal-monella through pre-enrichment, selective enrichment and plating (Edwardsand Ewing 1986).

Contamination of Cowpea Seeds with Salmonella

Cowpea seeds (400 g) were soaked in 1 L of bacterial suspension of S.typhimurium (7.2 ¥ 107 cfu/mL) for 4 h with gentle agitation (30 rpm) at roomtemperature ([RT] 30 � 2.8C). Thereafter, an excess inoculum was decanted;the seeds were washed twice with sterile distilled water, placed on a sterileperforated tray lined with six layers of cheesecloth and dried in a laminar flowhood at RT (30 � 2.8C) for 24 h to reduce the moisture content to a maximumof 15%. The control seeds were similarly soaked in sterile distilled water for4 h then dried. The cowpea seeds were then used for selected treatments.

Sprouting of Cowpea Seeds

Twenty-five grams of cowpea was soaked in 100 mL of sterile tap waterwith gentle agitation (30 rpm) on a shaking platform kept at an ambienttemperature (RT) of approximately 30 � 2.8C for 6 h to initiate sprouting.Thereafter, the excess fluid was decanted, the cowpea seeds were placed on asterile perforated tray lined with six layers of wet cheesecloth for germinationat RT (30 � 2.8C) in a dark and humid (85%) enclosure for 48 h with regularspraying at 5-min intervals with 5.0–7.0 mL of sterile tap water.

Inoculation of the Sprouts

Sprouts from Salmonella-free seeds were soaked with gentle agitation(30 rpm) for 30 min in 10 volumes of water containing 7.2 ¥ 107 cfu/mL of S.typhimurium at RT. The excess inoculum was decanted; the sprouts werewashed twice with sterile distilled water, placed on a sterile perforated traylined with sterile cheesecloth and subjected to selected treatments.

Salmonella and Germination of Cowpea Seeds

To determine the effect of Salmonella on the germination of cowpeaseeds, 25-g lots of seeds were soaked for 4 h at RT in 250 mL of watercontaminated with S. typhimurium E2391 at levels of 7.2 ¥ 107, 7.2 ¥ 108 and

270 B.R. SINGH ET AL.

7.2 ¥ 109 cfu/mL. After draining the excess water, the sprouting of the seedswas initiated at RT in a dark location as previously described. The germinatedcowpea seeds were counted after 24, 30, 48, 96 and 168 h, and the populationof Salmonella was determined at 48, 96 and 168 h as previously described.

Disinfectant Solutions

Vinegar (Mohan Meakin, Ghaziabad, India) containing 5% acetic acidwas used without dilution. A solution of 20 mg/mL active chlorine was pre-pared by dissolving 6.14 g of Ca(ClO)2 (S. D. Fine Chemicals, Mumbai, India)in 100 mL of sterile tap water. The active chlorine concentration was verifiedby iodometric analysis (Arya and Rastogi 1993).

Disinfection of Seeds and Sprouts

Twenty-five grams of Salmonella-inoculated cowpea bean seeds orsprouts was treated with 250 mL of disinfectant solution with continuousagitation on a rotary shaker (50 rpm) for 15, 30, 45, 60, 90, 120 or 180 min at30C. Just after treatment, the seeds/sprouts were transferred to 250 mL ofsterile phosphate buffer (pH 7.0) to neutralize the effect of residual treatmentsolution. The population of Salmonella was determined before and after eachtreatment as previously described.

Viability of Cowpea Seeds

Approximately 10 g of cowpea seeds was set aside for germination aftertreatment with selected disinfectant solutions. Cowpea seeds exhibiting aruptured seed coat, protruding root radicles or opened hypocotyls after 48 hwere considered sprouted.

Statistical Analysis

The disinfection treatments were replicated three times under equivalentconditions of growth, and the data were analyzed with Microsoft Excel 2000to evaluate the effect of selected treatments with the Student’s t test (Rickmersand Todd 1967).

RESULTS AND DISCUSSION

The cowpea seeds were free from Salmonella and exhibited a mean92 � 2.0% germination rate. The maximum germination was achieved in 48 hof incubation. The inoculation of the seeds with 7.2 ¥ 107 cfu/mL of Salmo-


nella in the soak water exhibited no significant effect on the sprouting rate(Table 1). However, when S. typhimurium concentrations were greater than7.2 ¥ 107 cfu/mL in the soak water, the reduction in germination was signifi-cant (P � 0.01). The reduction in germination was attributed to the number ofSalmonella that adhered to the seeds and not merely on the presence ofSalmonella. The inhibition of cowpea seed germination rarely attributed to anyzoonotic pathogen may be because of contamination with the S. typhimuriumstrain, the cowpea cultivar or the method of inoculation used in this study.

The methods used for soaking cowpea seeds and for inoculation are veryclose to the methods practiced in India and in neighboring countries forsprouting cowpea seeds and are substantially different from earlier studieswhere the seeds were exposed to a pathogen (Taormina et al. 1999; Weissingerand Beuchat 2000) for only a few minutes. The equivalent S. typhimuriumstrain E-2391 was shown to reduce germination and growth of maize (Singhet al. 2004) and mung bean seeds (Singh et al. 2005).

The seeds inoculated with S. typhimurium at 7.2 ¥ 107 cfu/mL harbored5.8 ¥ 107 cfu/g seeds (Table 1). The populations of Salmonella significantlyincreased on germinating as well as on nongerminating seeds. Similar Salmo-nella growth was observed on seeds treated with chlorinated water or vinegarprior to sprouting (Table 2). Initially, the populations of Salmonella werereduced by 3.5 and 7.8 log10 cfu/g after 1 h of treatment with chlorinated wateror 30 min of treatment with vinegar, respectively (Table 2). Thereafter, anincrease in Salmonella by 3.2 and 4.9 log10 cfu/g on germinated and nonger-

TABLE 1.GERMINATION OF COWPEA SEEDS SOAKED IN THE PRESENCE OF SALMONELLA

ENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUM

Observation S. typhimurium in soak water

7.2 ¥ 107 cfu/mL 7.2 ¥ 108 cfu/mL 7.2 ¥ 109 cfu/mL Control (No Salmonella)

% Germination at24 h 27.1 (�1.57) 10.3 (�1.24)* 3.3 (�1.67)* 28.3 (�1.57)30 h 57.9 (�0.36)† 28.7 (�2.61)* 9.3 (�2.01)* 62.5 (�1.65)48 h 89.4 (�2.50)‡ 29.3 (�1.63)* 11.7 (�1.67)* 92.3 (�0.95)

Salmonella (log10 cfu/g) at0 h 5.8 (�0.31) 7.4 (�0.12) 7.8 (�0.22) 0.0 (�0.0)48 h 6.2 (�0.02) 7.6 (�0.15) 8.3 (�0.02) 0.0 (�0.0)96 h 8.3 (�0.03) 8.3 (�0.04) 9.6 (�0.03) 0.0 (�0.0)168 h 9.1 (�0.04) 9.4 (�0.13) 9.8 (�0.07) 0.0 (�0.0)

* Significantly different at P � 0.01 level of significance.† Significantly different at P � 0.05 level of significance.‡ Significantly different at P � 0.10 level of significance.


minated seeds, respectively, was evident on chlorinated seeds, and an increasein Salmonella by 3.2 and 6.5 log10 cfu/g on germinated and nongerminatedseeds, respectively, in vinegar-treated seeds was evident (Table 2). The growthof Salmonella inside the cowpea seeds during germination was observed asreported earlier for Escherichia coli and Salmonella during the germination ofother seeds (Weissinger and Beuchat 2000; Singh et al. 2004, 2005).

The observed 1.7–3.3 log10 larger counts of Salmonella in nongerminatedseeds compared to the germinated seeds (sprouts) of equivalent groups indi-cated that either excessive multiplication of S. typhimurium inhibited thegermination of cowpea seeds, or S. typhimurium proliferated rapidly in poorer-quality seeds. Additional studies are necessary to elucidate the inhibitingmechanism of S. typhimurium for the germination of cowpea seeds.

The treatment of Salmonella-inoculated seed with 20,000 ppm chlorinesignificantly (P � 0.01) increased germination, but treatment with vinegardrastically reduced (P � 0.01) germination. The reduction in Salmonellapopulations was less marked following the chlorine treatment than followingthe vinegar treatment. Therefore, the vinegar treatment, although more effi-cient in eliminating Salmonella from seeds, cannot be recommended for thedecontamination of cowpea seeds. This recommendation is in concurrence toearlier findings with other seeds (NACMCF 1999; Taormina et al. 1999;Weissinger and Beuchat 2000). Previous studies with the use of various chemi-cal, physical and combination methods evaluated the effective elimination of

TABLE 2.GERMINATION AND POPULATION OF SALMONELLA AFTER TREATMENT OF COWPEASEEDS INOCULATED WITH SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR

TYPHIMURIUM (7.2 ¥ 108 cfu/mL)

Treatment of cowpea seed Germination(%)

Salmonella (log10 cfu/g) on cowpea seeds

Beforesprouting

Sprouted(48 h)

Not sprouted(48 h)

Salmonella-inoculated seeds20,000 ppm chlorine (1 h) 4.3 (�0.04) 67.0 (�4.0)* 7.5 (�0.04)† 9.2 (�0.08)*Vinegar (30 min) AE+ 6.0 (�1.0)* 3.2 (�0.2)* 6.5 (�0.04)*Sterile tap water 7.8 (�0.02) 35.0 (�2.0) 7.8 (�0.05) 9.8 (�0.07)

Control seeds20,000 ppm chlorine (1 h) 0.0 (�0.0) 90.7 (�2.1) 0.0 (�0.0) 0.0 (�0.0)Vinegar (30 min) 0.0 (�0.0) 5.0 (�2.0)* 0.0 (�0.0) 0.0 (�0.0)Sterile tap water 0.0 (�0.0) 92.0 (�2.0) 0.0 (�0.0) 0.0 (�0.0)

* Significantly different at P � 0.01 level of significance.† Significantly different at P � 0.10 level of significance.AE+, Salmonella detected only after enrichment.


Salmonella from selected sprouts (NACMCF 1999; Weissinger and Beuchat2000).

The inactivation of Salmonella on sprouts was comparatively rapid in thefirst 15 min of treatment with 20,000 ppm chlorine or vinegar than during thetreatment times greater than 15 min (Table 3), indicating that Salmonellapopulations from isolated sprouts were not exclusively on the surface of thesprouts as previously reported. Fett (2002), Fett and Cooke (2003) and Singhet al. (2004, 2005) reported the penetration of Salmonella into sprouts of otherseeds. The maximum reduction in Salmonella counts in sprouts with chlorineor vinegar treatments was 3.3 or 5.0 log10 cfu/g, respectively. Salmonella thatcontaminates sprouts after harvest was rapidly eliminated by vinegar treatmentand was reduced by 5.3 log10 cfu/g with chlorinated water during treatment for60 min.

The postharvest inoculation of cowpea sprouts resulted in most of theSalmonella remaining on the surface of the sprouts, and thus more susceptibleto chlorine or vinegar treatments. After 1, 1.5, 2 or 3 h of treatment of cowpeasprouts with vinegar, Salmonella was not isolated even with pre-enrichmentculture followed by a selective enrichment culture. Vinegar treatments weremore effective in eliminating Salmonella from cowpea sprouts than chlorinetreatments. These conclusions corroborate earlier reports with other sprouts(Lang et al. 2000). Treatment of sprouts with 20,000 ppm chlorine will notresult in Salmonella-free sprouts even after 3 h of soaking, probably becauseof lack of chlorine penetration into sprout tissues where many Salmonella maybe located. Active chlorine may be inactivated by sprout proteins or other

TABLE 3.SURVIVAL OF SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR

TYPHIMURIUM ON COWPEA SPROUTS

Time ofexposure(min)

Salmonella (log10 cfu/g) onsprouts grown from contaminatedseed after treatment with

Salmonella (log10 cfu/g) oninoculated sprouts treateddirectly with

20,000 ppm chlorine Vinegar 20,000 ppm chlorine Vinegar

Control 8.3 (�0.05) 8.3 (�0.05) 7.5 (�0.08) 7.5 (�0.08)15 7.8 (�0.05) 5.9 (�0.03) 5.9 (�0.05) 4.9 (�0.03)30 7.6 (�0.03) 5.7 (�0.01) 3.6 (�0.06) 2.9 (�0.03)45 6.9 (�0.04) 4.6 (�0.10) 2.8 (�0.05)* 2.1 (�0.20)*60 6.5 (�0.05) 4.2 (�0.08) 2.3 (�0.04) 0.0 (�0.00)90 6.2 (�0.12) 4.1 (�0.13) 2.2 (�0.09) 0.0 (�0.00)

120 5.9 (�0.03) 3.8 (�0.07) 2.2 (�0.04) 0.0 (�0.00)180 5.2 (�0.07) 3.3 (�0.03) 2.1 (�0.07) 0.0 (�0.00)

* Significantly different at P � 0.01 level of significance.


organic contaminants, leading to the loss of disinfection capacity and inad-vertent protection of microorganisms. Similar observations of the failure ofchlorinated water to eliminate S. typhimurium from clover seeds that resultedin a sprout-borne Salmonella outbreak was reported by Brooks et al. (2001).

Although chemical disinfectants can eliminate Salmonella from seeds,the full germination of seeds is not guaranteed (NACMCF 1999), and fewattempts are published that evaluate selected disinfection treatments of maturesprouts. The present study on mature cowpea sprouts suggests that Salmonellacan be eradicated from cowpea sprouts with 1-h vinegar treatment. However,when the sources of Salmonella contamination were the seeds, neither thevinegar nor chlorine was effective, and the boiling of cowpea sprouts asrecommended for other sprouts (NACMCF 1999) is the safest and the bestoption.

CONCLUSIONS

The vinegar treatment of cowpea seeds, although more effective thanchlorination for the reduction of S. typhimurium, exhibited an unacceptableadverse effect on cowpea seed germination and cannot be used for the disin-fection of cowpea seeds. Salmonella that contaminates cowpea sprouts aftermaturation can be inactivated by exposure to vinegar for 1 h. Chorine wasineffective for the inactivation of Salmonella that contaminates mature cowpeasprouts.

ACKNOWLEDGMENTS

We thank Mr. J.D. Joshi and Mr. K.K. Arya of the National SalmonellaCenter (Vet) for technical assistance and the Director, Indian VeterinaryResearch Institute, Izatnagar, for providing funds.

REFERENCES

ARYA, P.R. and RASTOGI, P.P. 1993. Practical Fruit and Vegetable Pres-ervation, pp. 124–127, Directorate of Publication GB Pant University ofAgriculture and Technology, Pantnagar, India.

BACKER, H.D., MOHLE-BOETANI, J.C., WERNER, S.B., ABBOTT, S.L.,FARRAR, J. and VUGIA, D.J. 2000. High incidence of extra-intestinalinfections in a Salmonella Havana outbreak associated with alfalfasprouts. Public Health Rep. 115, 339–345.


BEUCHAT, L.R., WARD, T.E. and PETTIGREW, C.A. 2001. Comparison ofchlorine and a prototype produce wash product for effectiveness in killingSalmonella and Escherichia coli O157:H7 on alfalfa seeds. J. Food Prot.64, 152–158.

BROOKS, J.T., ROWE, S.Y., SHILLAM, P., HELTZEL, D.M., HUNTER,S.B., SLUTSKER, L., HOEKSTRA, R.M. and LUBY, S.P. 2001. Sal-monella typhimurium infections transmitted by chlorine-pretreated cloversprout seeds. Am. J. Epidemiol. 154, 1020–1028.

DEFRA, R.T. 2002. Salmonella Live Stock Production in GB-2002, pp. 127–142, Department of Environment, Food and Rural Affairs, VeterinaryLaboratory Agency, Surrey, U.K.

EDWARDS, P.R. and EWING, W.H. 1986. Identification of Enterobacteri-aceae, 4th Ed., pp. 146–207, Elsevier Science, New York, NY.

FETT, W.F. 2002. Reduction of Escherichia coli O157:H7 and Salmonellaspp. on laboratory-inoculated mung bean seeds by chlorine treatment. J.Food Prot. 65, 84–852.

FETT, W.F. and COOKE, P.H. 2003. Reduction of Escherichia coli O157:H7and Salmonella on laboratory-inoculated alfalfa seed with commercialcitrus-related products. J. Food Prot. 66, 1158–1165.

HIMATHONGKHAM, S., NUANUALSUWAN, S., RIEMANN, H. andCLIVER, D.O. 2001. Reduction of Escherichia coli O157:H7 and Sal-monella typhimurium in artificially contaminated alfalfa seeds and mungbeans by fumigation with ammonia. J. Food Prot. 64, 1817–1819.

LANG, M.M., INGHAM, B.H. and INGHAM, S.C. 2000. Efficacy of novelorganic acid and hypochlorite treatment for eliminating Escherichia coliO:157:H7 from alfalfa seeds prior to sprouting. Int. J. Food Microbiol.58, 73–82.

NATIONAL ADVISORY COMMITTEE ON MICROBIOLOGICALCRITERIA FOR FOODS/FOOD AND DRUG ADMINISTRATION(NACMCF/FDA). 1999. Microbiological safety evaluations and recom-mendations on sprouted seeds. Int. J. Food Microbiol. 52, 123–153.

RAJKOWSKI, K.T. and THAYER, D.W. 2000. Reduction of Salmonella spp.and strains of Escherichia coli O157:H7 by gamma radiation of inocu-lated sprouts. J. Food Prot. 63, 871–875.

RAJKOWSKI, K.T., BOYD, G. and THAYER, D.W. 2003. IrradiationD-values for Escherichia coli O157:H7 and Salmonella sp. on inoculatedbroccoli seeds and effects of irradiation on broccoli sprout keepingquality and seed viability. J. Food Prot. 66, 760–766.

RICKMERS, A.D. and TODD, H.N. 1967. Statistics: An Introduction, pp.62–91, McGraw-Hill, New York, NY.


SHARMA, R.R. and DEMIRCI, A. 2003. Treatment of Escherichia coliO:157:H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidiz-ing water. Int. J. Food Microbiol. 86, 231–237.

SINGH, B.R., AGARWAL, R.K. and CHANDRA, M. 2004. Pathogeniceffects of Salmonella enterica subspecies enterica serovar Typhimuriumon sprouting and growth of maize. Indian J. Exp. Biol. 42, 1100–1106.

SINGH, B.R., CHANDRA, M. and AGARWAL, R.K. 2005. A study oninteraction of Salmonella enterica subspecies enterica serovar Typhimu-rium and mung bean (Phaseolus aureus) plants. J. Food Prot. 68, 476–481.

SUSLOW, T.V., WU, J., FETT, W.F. and HARRIS, L.J. 2002. Detection andelimination of Salmonella mbandaka from naturally contaminated alfalfaseeds by treatment with heat or calcium hypochlorite. J. Food Prot. 65,452–458.

TAORMINA, P.J. and BEUCHAT, L.R. 1999. Behavior of enterohemorrhagicEscherichia coli O157:H7 on alfalfa sprouts during the sprouting processas influenced by treatments with various chemicals. J. Food Prot. 62,318–324.

TAORMINA, P.J., BEUCHAT, L.R. and SLUTSKER, L. 1999. Infectionsassociated with eating seed sprouts: An international concern. Emerg.Infect. Dis. 5, 626–634.

VERMA, J.C., SINGH, V.P., SINGH, B.R. and GUPTA, B.R. 2001. Occur-rence of Salmonella serotypes in animals in India. Indian J. Comp.Microbiol. Immunol. Infect. Dis. 22, 51–55.

WEISSINGER, W.R. and BEUCHAT, L.R. 2000. Comparison of aqueouschemical treatments to eliminate Salmonella on alfalfa seeds. J. FoodProt. 63, 1475–1482.


curing of salmonella enterica, serovar typhimurium-contaminated cowpea seeds and sprouts with...

Documents