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J. Milk Food Techno!. Vol. 39. No. 7, Pages 464-466 !,July, 1976) Copyright @ 1976, International Association of Milk, Food, and Environmental Sanitarians

Sulfite Compounds as Neutralizers of Spice Toxicity for Salmonella

CLYDE R. WILSON and WALLACE H. ANDREWS

Division ofMicrobiology, Food and Dn

ABSTRACT spices, and (c) the ability of Salmonella to survive when

The ability of tive inorganic chemical salts (K 2S03, K2S04 , Na2S03, inoculated into these spices.

Na 2S04 , and CaC03) to neutralize the toxicity of six spices (allspice, cinnamon, clove, garlic powder, onion powder, and oregano) for MATERIALS AND METHODS Salmonella was evaluated. Their effect on four spices non-toxic to Source of spices and herbs Salmonella (black pepper, white pepper, rosemary, and thyme) was also Samples of black pepper, white pepper, cinnamon, clove, thyme. and determined. The inhibitory effects of onion and garlic powders were rosemary were available in the laboratory as reserve portions of samples overcome by addition of 0.5% of K S0 or Na,S0 to pre-enrichments 2 3 3 collected for other examinations. Allspice, oregano, onion powder, and of lactose broth, nutrient broth, or trypticase soy broth. Allspice, garlic powder were purchased from a local retail grocery. cinnamon, clove, and oregano remained toxic to Salmonella in all pre-enrichment broths tested, with or without chemical additives. None Salmonella inoculum of the chemical additives had any effect upon isolation of Salmonella S. bredeney, S. glastmp, and S. thompson, all previously isolated from the non-toxic spices. Until a more practical method for analysis of from spices, were grown for 24 h in brain-heart infusion broth, the cells allspice, cinnamon. clove, and oregano is developed, dilution of these collected by centrifugation at 3090 x g for 10 min, washed twice, spices to non-toxic levels is recommended. suspended in Butterfield's phosphate buffer, and refrigerated at 4 C until used. Even though relatively few outbreaks of salmonellosis Aerobic plate counts have been traced to consumption of contaminated spices, Serial dilutions of the spices and herbs were made in Butterfield's numerous isolations of Salmonella from a variety of phosphate buffer, plated in standard plate count agar, incubated 48 h spices, including thyme, paprika, sesame seed, sage, at 35 C, and counted. marjoram, oregano, dehydrated green pepper, and black Lactase pre-enrichment method pepper have been made in our laboratories. Black pepper The method of the Association of Official Analytical Chemists and white pepper have both been implicated as vehicles (A.O.A.C.), modified only by sample size and volume of pre-enrichment broth, was evaluated by pre-enriching 1-g portions of each spice or herb for the spread of Salmonella weltevreden resulting in in 9.0 ml of lactose broth, the pH of the mixture adjusted to 6.8 ± 0.2, several cases of salmonellosis (9). and the inoculum of 1.4 x 106 cells of S. bredeney added to the Julseth and Deibel demonstrated that many spices, pre-enrichment mixture. Subsequent incubation, selective enrichment, including black and white pepper, exhibited little or no and plating were in accordance with the official A.O.A.C. methodology toxicity for Salmonella when examined by conventional (I) with the exception that plating was limited to bismuth sulfite (BS) agar. Since the objective of this experiment was to determine the methods (4). They listed only four of 35 spices (allspice, adequacy or inadequacy of the pre-enrichment and enrichment phases cassia, oregano, and granulated onion) tested as being of the method when applied to the examination of spices, only the toxic to Salmonella at a 1:10 sample/broth ratio. Wei et selective agar with which we usually obtain our best results, BS agar, was used. al. (7) demonstrated that addition of 0.5 o/o K 2SO 3 to an onion-buffer homogenate overcame the bacteriostatic Measurement of inhibitory levels of spices effect of onion and resulted in increased, and presumably Two-fold decreasing amounts (10.0- 0.078 g) of the inhibitory spices more accurate plate counts. Hall (3) showed that (allspice, cinnamon, clove, garlic powder, onion powder, and oregano) were added to 90.0 ml of lactose broth, the pH of the mixture was Salmonella could be recovered from artifically adjusted to 6.8 ± 0.2, and the inoculum of 110-140 cells of S. bredeney contaminated onion powder pre-enriched in lactose and S. thompson added to the pre-enrichment mixture. Subsequent broth at the 1:10 sample/broth ratio when 0.5o/o K 2S03 steps of the examination were as previously described. was added to the enrichment broth, but not in the Addition of inorganic chemicals absence of K 2S03• One-gram portions of the inhibitory spices and herbs were The present study was undertaken to determine: (a) pre-enriched in 9.0-ml amounts of lactose broth, nutrient broth, or the identity and specificity of the cations or anions trypticase soy broth, with and without addition of 0.5% CaC03, K2S03 , K,S04, Na 2S03, and Na2S04• The pH of mixtures was adjusted to needed for reversal of Salmonella inhibition in selected 6.8 ± 0.2, and mixtures were inoculated with 5-10 cells of S. bredeney, spices, (b) the relative efficiency of various S. glostrup, or S. thompson. Subsequent steps of the examination were pre-enrichment broths in recovering Salmonella from as previously described. NEUTRALIZING ANTIBACTERIAL PROPERTIES OF SPICES 465

Detennination of spices' capacity for self cleansing The extent of the toxicity of allspice, cinnamon, clove, Ten grams of black pepper, allspice, cinnamon, clove, garlic powder, garlic powder, onion powder, and oregano is shown in 7 7 onion powder and oregano were inoculated with 1.2 x 10 to 1.6 x 10 Table 1. Onion powder was the least toxic, allowing cells/g of S. bredeney, S. glostrup, or S. thompson. Salmonella isolations were attempted immediately after inoculation and at Salmonella recovery at a sample/broth ratio of 5.6%. intervals of 24, 48. 72, 96 h, and two weeks, The spices were placed in Clove and garlic powders were most inhibitory, not 90 ml oftrypticase soy broth, and the pH ofthe mixture was adjusted to allowing recovery of Salmonella until the spice level had 6.8 ± 0.2. Onion and garlic powders were examined with and without been decreased to 0.18 o/o. Allspice, cinnamon, and 0.5o/o K S0 in the pre-enrichment broth to verify the ability of K,S0 2 3 3 oregano suppressed recovery at and above the 2.8 o/o level. to overcome the inhibitorv effects of these spices. Subsequent incubation, selective enrich~ent, and plating were in accordance with These results indicate that dependence upon reduction of official A.O.A.C. methods (1). the sample/broth ratio to counter inhibition would lead Identification of isolates either to examination of very small quantities of the more Isolates frol1hselective agar plates were picked to triple-sugar-iron toxic spices or to the use of large volumes of broth to agar slants, incubated at 35 C for 18-24 h, and examined serologically examine even a single 25-g portion. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/39/7/464/2397529/0022-2747-39_7_464.pdf by guest on 28 September 2021 to confirm them as being the same somatic group that had been Three 1-:.roths, with and without chemical additives, inoculated. were comparatively evaluated as pre-enrichment media RESULTS AND DISCUSSION for recovery of Salmonella from spices. Lactose broth was used because of its use in A.O.A.C. methodology for Low aerobic plate counts seemed to indicate that most Salmonella determinations (1). Nutrient broth was of the spices had been subjected to some form of selected because of its use in the investigations of Julseth treatment. Clove, cinnamon, thyme, rosemary, and white and Deibel (4). The inclusion of trypticase soy broth was pepper all had counts of <100/g. Per gram figures for prompted by its demonstrated superiority to lactose black pepper, oregano, and allspice were 170, 5.0 x 10 3, broth as a pre-enrichment broth in recovering 6 and 2.6 x 10 , respectively. Onion powder counts were Salmonella from dried active yeast (8). 4 6.3 x 10 without the addition of 0.5% K2S0 3 to the Inorganic chemical salts used with each pre-enrich­ buffer diluent, and 1.09 x 10 5 with the added K S0 • 2 3 ment broth were K2S0 3, Na2S0 3, K2S04, Na2S04 and Counts of garlic powder were increased from 2.1 x 10 3 to CaC0 • The K S0 was evaluated because of its 3 3 2 3 6.0 x 10 by the use of0.5o/o K2S03 in the buffer diluent. previously proven usefulness for onion examinations (3, The modified A.O.A.C. lactose pre-enrichment 7). Green and Litsky (2) found Na2S03 effective as a procedure was evaluated with six spices inhibitory to neutralizer of the bactericidal effects of iodine, and Salmonella (allspice, cinnamon, clove, garlic powder, because of its close chemical relationship to K2S0 3, the onion powder, and oregano) plus four non-inhibitory effectiveness of Na 2S03 as a neutralizer of toxic spices spices (black pepper, white pepper, rosemary, and thyme). Recovery of Salmonella was obtained at every TABLE 2. Recovery of Salmonella from spices using inorganic chemical salts as additives to the pre-enrichment media level of inoculum, ranging from 14 to 1.4 x 10 6 cells/g, Spice from the non-inhibitory spices. Salmonella was not Inorganic salt (0.5%) All- Cin- Garlic Onion Ore- recovered from the six inhibitory spices, with the spice namon Clove powder powder gano Pre-enrichment exception of onion powder, at the two highest inoculum K,so, a + + 5 6 Lactose brothc levels, 1.4 x 10 and 1.4 x 10 cells/g. Results of our own K2S04 or Na,S03 + + investigations with the above spices, the experiences of Nutrient Na2S04 brothc various other Food and Drug Administration CaC03 laboratories in the examination of spices, and results of None other investigators (4) indicate that most spices may be K,so, + + K,so, + Trypticase examined by conventional analytical procedures with no Na,so, + + soy difficulty. Na,so, + brothc

CaC03 + None + TABLE 1. Recovery of S. bredeney and S. thompson .from six in­ hibitory spices at various sample/broth ratios a Salmonella not recovered b Salmonella recovered Grams of spice per 90 ml All· Cin- Ore- Cinoculated with 5-10 Salmonella organisms per gram of spice of lactose broth spice namon Clove Garlic Onion gano

10.00 a was evaluated. Na2S04 and K2S04 were included to 5.00 +b 2.50 + demonstrate that the basis of the neutralization resided 1.25 + + + + in the sulfite anion rather than the sodium or potassium 0.63 + + + + cation. CaC0 3 was added in an attempt to counter the 0.31 + + + + 0.16 + + + + + + low pH of some spices and possibly neutralize some of 0.08 + + + + + + the toxic factors. 0.00 + + + + + + Results obtained using the three pre-enrichment aNo recovery of S. bredeney or S. thompson broths and five additives are shown in Table 2. When bRecovery of S. bredeney and S. thompson onion powder was inoculated with 5-10 Salmonella 466 WILSON AND ANDREWS cells/g, recovery was accomplished in the trypticase soy K 2S0 3, which had leached into the pre-enrichment broth regardless of additive or lack of additive. In medium from the garlic. With oregano, only S. thompson nutrient broth and lactose broth, recovery of Salmonella was recovered, and only from the examination made was possible only from pre-enrichment broths containing immediately after inoculation of the material. No

K 2S0 3 or Na2S03• Inoculated garlic powder examined by recoveries at all were made from allspice, cinnamon, or the same procedure yielded Salmonella only from clove. It is not known if this lack of recovery was due to pre-enrichment broths containing K 2S03 or Na2S03• the bactericidal properties of these spices or to Examination of allspice, cinnamon, clove, and oregano inadequate methodology. Black pepper was included as a by the same procedures recovered no Salmonella from non-toxic spice control, and recovery was obtained with any pre-enrichment broth, with or without an additive. all three Salmonella serotypes at each time interval These results indicate toxic agents or mechanisms checked. different from those found in onion and garlic. On the This study shows that the sulfite anion is responsible basis of its ability to neutralize the inhibitory effect of for reversal of Salmonella inhibition by onion and garlic Downloaded from http://meridian.allenpress.com/jfp/article-pdf/39/7/464/2397529/0022-2747-39_7_464.pdf by guest on 28 September 2021 onion powder without the presence of chemical additives, powders and that trypticase soy broth is the preferred trypticase soy broth appears to be the superior of the pre-enrichment broth of those tested. The practical three broths evaluated. implications of the study are that onion and garlic Table 3 illustrates that vapors from onion and garlic powders may be examined for Salmonella using TABLE 3. Survival of' Salmonella after various intervals in selected conventional methodology modified only by the addition spices having bactericidal properties of O.So/o K 2S03 or Na2S03 to the pre-enrichment Period of exposure to spice medium. Allspice, cinnamon, clove, and oregano, Spice o• 24 h 48 h 72 h 96h 2wks however, must be examined by diluting them beyond Black pepper +c + + + + + their toxic levels until a means to neutralize their toxicity _d Cinnamon can be found. Clove Allspice Oregano +e Garlic powder ACKNOWLEDGMENT Garlic powderg + + + + + + Onion powder + + + f + + +f The authors thank Aida Romero and Paul L. Poelma, Division of Onion powderg + + + + + + Microbiology. for supplying the Salmonella cultures isolated from spices. a Examined immediately after inoculation with 1.2 x 107-1.6 x 107 Salmonella cells REFERENCES bNon-inhibitory control 1. Association of Official Analytical Chemists. 1975. Official methods cRecovery of S. bredeney, S. glostrup and S. thompson except as noted of analysis 12th ed .. Assoc. Offic. Anal. Chern., Washington, D.C., dsalmone/la not recovered sees. 46.013-46.026. es. thompson only 2. Green, B. L., and W. Litsky. 1974. The use of sodium sulfite as a fs. glostrup and S. thompson only neutralizer for evaluating providone-iodine preparations. Health gTrypticase soy broth pre-enrichment contained 0.5% K S0 2 3 Lab. Sci. 11:188-194. powders were not able to cleanse these products of 3. Hall, J. R. 1969. Enhanced recovery of Salmonella montevideo from relatively large numbers of Salmonella (1.2 x 107 to onion powder by the addition of potassium sulfite to lactose broth. 1.6 x 107 /g) over a 2-week period despite reported J. Assoc. Offic. Anal. Chern. 52:940-942. 4. Julseth, R. M., and R. H. Deibel. 1974. Microbial profile of selected bactericidal qualities of onion and garlic vapors (5, 6). spices and herbs at import. J. Milk Food Techno!. 37:414-419. These previous investigations, however, used fresh onion 5. Lovell. T. H. 1937. Bactericidal effects of onion vapors. Food Res. and garlic and only qualitatively showed toxicity by ob­ 2:435-438. serving the prevention or reduction in amount of 6. Walton, L., M. Herbold, and C. C. Lindegren. 1936. Bactericidal etTects of vapors from crushed garlic. Food Res. 1:163-169. bacterial growth. The observations were made with 7. Wei. L. S., J. A. Siregar, M.P. Steinberg, and A. I. Nelson. 1967. glycerin agar plates which had been exposed to vapors of Overcoming the bacteriostatic activity of onion in making standard minced onion or garlic for various intervals of time plate counts. J. Food Sci. 32:346-349. before inoculation. Recovery of all three Salmonella 8. Wilson, C. R .. P. L. Poelma, and W. H. Andrews. 1974. Compari­ serotypes from garlic powder with trypticase soy broth son of culture methods for detection of Salmonella in dried active yeast. J. Assoc. Off. Anal. Chern. 57:696-700. containing 0.5%K S0 , but not without it, indicates that 2 3 9. World Health Organization. 1974. Salmonella surveillance. Weekly the organisms survived the garlic vapors, but were epidemiological record: No. 42, 351-352. World Health Organiza­ inhibited by a substance, capable of being neutralized by tion, Geneva, Switzerland.