Microbiota of fresh herbs and whole spices used in home food preservation and effectiveness of microbial intervention methodsElizabeth L. Andress1, Isabel C. Blackman2, Elaine M. D’Sa2 and Mark A. Harrison2

1Department of Foods and Nutrition, Extension Family and Consumer Sciences, 2Department of Food Science and Technology, The University of Georgia, Athens, Georgia 30602.

ABSTRACT

Fresh herbs and whole spices may carry a significant microbial load acquired via cultivation and post-harvest processing practices. Their use in minimally processed foods could cause accelerated spoilage or illness, if pathogens are present. Reduction of the microbiota by simple intervention techniques would enhance shelf life and safety of herbs and spices. The microbial profile of selected fresh herbs and whole spices used in home preparation of sauces, flavored oils and vinegars was studied. Effectiveness of a wash treatment, and of washing followed by chlorine dipping (25 ppm solution) were investigated as techniques for decreasing the microbial load. Fresh herbs (basil, cilantro, dill, oregano, parsley, rosemary, tarragon, thyme) and whole spices (allspice, black pepper, mustard) were obtained from two grocery stores and one international farmer’s market location. Aerobic mesophiles, fungi, presumptive Clostridium perfringens, Bacillus cereus, Salmonella spp. and coliform populations from unwashed, washed and chlorine-dipped herb and whole spice samples were enumerated. The microflora of herbs and spices varied with source, with an aerobic mesophilic count (APC) of 2.9 x 102 to 3.2 x 107 CFU/g, a coliform count of 7.9 x 102 to 1.9 x 107 CFU/g and Salmonella levels of 7.9 x 102 to 2.7 x 105 CFU/g. Fungal, Bacillus cereus and Clostridium perfringens populations ranged from undetectable to 1.7 x 107 CFU/g, 1.4 x 106 CFU/g and 8 x 103 CFU/g respectively. Washing with water and chlorine dipping reduced APC numbers by 0.25 – 1.0 log10 and by an

additional 0.2 – 1.0 log10 respectively, the efficiency of the intervention methods being dependent upon the initial

microbial load. This study determined that fresh herbs and whole spices have significant levels of spoilage and potentially pathogenic microorganisms. Consumer intervention steps like washing and chlorine dipping decrease microbial populations and improve the quality of herbs and spices.

REFERENCES

1. Andress, E. and J. Harrison. 1999. Flavored Vinegars. In ‘So Easy to Preserve’. 4th Ed. Cooperative Extension Service, The University of Georgia. 140-143.

2. Baxter, R. and W. Holzapfel. 1982. A microbial investigation of selected spices, herbs, and additives in South Africa. J. Food Sc. 47:570-574.

3. Beckmann, G., D. Koszegi, B. Sonnenschein and R. Leimbeck. 1996. On the microbial status of herbs and spices. Fleischwirtschaft. 76(3): 240-243.

4. De Boer, E., W. Spiegelenberg and F. Janssen. 1985. Microbiology of spices and herbs. Antonie van Leeuwenhoek. 51:435-438.

5. FDA - CFSAN. 1998. Guide to minimize microbial food safety hazards for fresh fruits and vegetables. At http://www.cfsan.fda.gov/~dms/prodguid.html.

6. Garcia, S., F. Iracheta, F. Galvan and N. Heredia. 2001. Microbiological survey of retail herbs and spices from Mexican markets. J. Food Prot. 64(1): 99-103.

7. Jusleth, R. and R. Deibel. 1974. Microbial profile of selected spices and herbs at import. J. Milk Food Technol. 37 (8): 414-419.

8. Kneifel, W. and E. Berger. 1994. Microbiological criteria of random samples of spices and herbs retailed on the Austrian Market. J. Food Prot. 57(10): 893-901.

9. McKee, L. 1995. Microbiological contamination of spices and herbs: A Review. Lebensm. Wiss.u. Technol. 28:1-11.10. Pafumi, J. 1986. Assessment of the Microbiological Quality of Herbs and Spices. J. Food Prot. 49 (12): 958-963.11. Powers, E., R. Lawyer and Y. Masuoka. 1975. Microbiology of Processed spices. J. Milk Food Technol. 39 (11): 683-687. 12. Schwab, A., A. Harpestad, A. Swartzentruber et al. 1982. Microbiological quality of some spices and herbs in retail

markets. Appl. Env. Micro. 44(3):627-630.13. Wu, F., M. Doyle, L. Beuchat et al. 2000. Fate of Shigella sonnei on parsley and methods of disinfection. J. Food Prot.

63(5): 568-572.

INTRODUCTION Commercially available fresh herbs and whole spices have been known to carry various microorganisms including aerobic spoilage bacteria, yeasts and molds, and potential pathogens (2, 8, 9, 12). Use of herbs without an appropriate intervention step to eliminate microflora could result in contamination and spoilage of the end product, or survival of the pathogens, with the potential for the product to cause foodborne illness. The endproducts of concern in home-food preservation, like flavored vinegars and oils, may be refrigerated (4°C) or stored at room temperature (20-27°C). The latter temperature range may permit growth of surviving mesophilic facultative flora, though the likelihood of microbial proliferation is lower in low pH vinegar-type products. Home-food preservation guides (1) recommend the sanitation of herbs to be used in herb-flavored products, using a combination ‘wash plus chlorine-bleach dip’ process. Chlorine-based disinfectants have been recommended by the FDA (5) to treat produce prior to large-scale processing by the food industry and are also included on produce treatment advisories for consumers, for home-use. The objective of this study was: to study the bacterial and fungal profiles of selected fresh herbs and whole spices; to determine the efficacy of currently recommended home herb-disinfection procedures; and to determine the effects of various combinations of variables in the ‘wash plus chlorine-bleach dip’ process for reduction of microflora on herbs and spices.

MATERIALS AND METHODS Part A: Fresh herbs (basil, cilantro, dill, oregano, parsley, rosemary, tarragon, thyme) and whole spices (allspice, mustard seed, black pepper) were obtained from two local grocery stores and one farmer’s market, and stored at 4°C or room temperature (whole spices) until used. Composites of six bunches of herbs were prepared, and 25 g representative samples were obtained. Untreated (U) samples were directly analyzed by the addition of 225 ml 0.1% peptone water, stomached for 1 min, appropriate dilutions were prepared and portions were plated/inoculated onto various media.In order to monitor the reduction in microbial numbers after a preliminary “wash” step, 500 ml tap water was added to each washed (W) sample in a stomacher bag, the sample was washed “gently but thoroughly” by manipulating the stomacher bag for 1 min, and the wash water was drained from the bag.Chlorine-treated (C) samples were initially treated in a manner similar to the washed (W) samples. Then, 500 ml of diluted household bleach (Clorox® at a concentration of 1 teaspoon in 6 cups of water, equivalent to a 25 ppm solution), was added to the stomacher bag, swirled, poured out and the bag was drained. The herbs were then rinsed twice with 250 ml amounts of tap water for 15 s each. Both (W) and (C) samples were patted dry on paper towels, as recommended (1), and analyzed in a manner similar to the (U) samples. Microbiological analyses (10): Sample dilutions were plated onto each of the following media, incubated, and examined for presence of “typical” colonies or growth patterns.Aerobic plate count (APC): Standard plate count agar (SPC) incubated at 28°C for up to 72 h.Yeast and mold: Dichloran Rose Bengal Chloramphenicol (DRBC) agar incubated at 20°C for up to 4 days.Coliforms: An MPN (Most Probable Number- 4 tube series) analysis was performed using Brilliant Green Lactose Bile broth incubated at 37°C/48 h. MPN results were noted in terms of coliforms/g sample. Sample dilutions were also plated onto Levine EMB agar and incubated at 37°C/24 h.Presumptive Clostridium perfringens: Sulfite Polymixin Sulfadiazine (SPS) agar incubated under anaerobic conditions in GasPak jars at 37°C/24 h. Bacillus cereus: Bacillus cereus agar incubated at 30°C/48 h.Salmonella: Bismuth Sulfite (BSA) agar incubated at 37°C/48 h. In addition, 1 ml of each sample was inoculated into lactose broth pre-enrichment tubes and on subsequent growth, into appropriate selective enrichment tubes and each incubated at 37°C/ 24h. Xylose Lysine Desoxycholate (XLD) and BSA agar plates were streaked with inoculum from each selective enrichment tube and incubated at 37°C/24h to determine the presence of typical colonies. Part B: Parsley was the herb of choice for this study. Ten g samples from 12 bunch composites were treated as unwashed (U), washed (W) or chlorine-dipped (C), as described in Part A above. The reductive effects of various combinations of treatment variables on aerobic mesophilic microflora of parsley, with respect to wash-times, chlorine-dip times, and chlorine concentrations were examined. Wash times and chlorine (Clorox®) contact times studied were 30s, 1 or 2 min; at chlorine concentrations of 50, 100 or 200 ppm; appropriate controls were also included. Sample dilutions were plated onto Plate Count agar and incubated at 28°C for up to 72 h.

CONCLUSIONS

The results of our studies have determined that, as noted in similar studies by other researchers (9), the microbial profile of fresh herbs and whole spices is highly variable. Some of this variability may be accounted for by differences in growing and harvesting conditions, type of herb (which may make microenvironments available for microbial retention), age and shelf life of the samples, and post-processing environmental exposure. Overall, the microbial quality of whole spices studied was higher than that of the fresh herbs, the APCs being 1-2 log10 lower and the levels of other microbial types being significantly lower than the fresh herbs studied. This may be due to irradiation or treatment with an antimicrobial gas which is generally commercially used to bring about a microbial reduction in whole spices. High aerobic mesophilic counts (which may accelerate product spoilage) and relatively significant fungal counts may be a problem, especially if the molds present are Aspergillus spp. and the herbs are used in long-term storage-type products. Intervention steps like washing and chlorine-dipping reduce microbial numbers on herbs and spices. At lower concentrations of free chlorine (25 ppm, as recommended in food preservation guides), this reduction ranges from 0.25 – 1.0 log10, and may be due mainly to the effects of mechanically manipulating the sample. At higher

concentrations of free chlorine (up to 200 ppm, for up to 2 min), disinfection effects due to chlorine are observed, but the process, at the currently recommended levels does not bring about a large reduction in microflora, that would assure product safety.

SAMPLES OF FRESH HERBS AND WHOLE SPICES USED IN THE STUDY

RESULTS AND DISCUSSION Part A: Represented below are the aerobic plate counts (APCs, Fig. 1) and fungal counts (Fig. 2) of selected fresh herbs and whole spices studied.

Fig.1: Aerobic plate counts of herb and spice samples from grocery stores (A,C) and farmer's market (B)

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The APC values range between 2.64 log10 – 7.65 log10, and the counts from samples obtained from Market A

are higher than the corresponding sample counts obtained from markets B and C, with the exception of black pepper and mustard. The APC values, in general, are in agreement with published literature, wherein counts up to 106-7 CFU/g have been reported for black pepper, thyme, dill, basil and other herbs (3, 4, 9). APCs for allspice were somewhat lower as compared to the other samples studied, while a spike was observed in the counts for black pepper, particularly the sample obtained from market B. This too, is in agreement with published studies on both white and black peppercorns (whole and ground), with counts ranging from 102 – 107 CFU/g (6,7). In one published study, storage temperature (20, 37, 55° C) did not appear to affect the APC of pepper samples (9), while temperature of storage had a very pronounced effect on the counts of e.g. Shigella sonnei on chopped parsley (13), with temperatures of 21°C promoting higher bacterial numbers as compared to 4°C. This temperature effect observed with fresh herbs may have contributed to the differences in APCs observed in our study, together with other environmental factors like herb source and frequency of automated wetting/watering of shelf-displayed fresh herbs.

Fig. 2: Fungal counts of herb and spice samples obtained from grocery stores (A,C) and farmer's market (B)

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The fungal (mold and yeast) counts demonstrated that yeasts were the predominant fungi in the samples studied. No fungi were detectable in whole spices (allspice, black pepper) while extremely low numbers (2.6 – 2.7 log10) were detected in two of the three mustard seed samples. For the fresh herbs, counts ranged

between 4.54-7.24 log10. Overall, samples from market C had higher fungal counts than those from A or B.

Published studies have reported fungal counts ranging from 3.9 x 104 to 1.1 x 108 CFU/g (9) the major fungi identified being Aspergillus spp., especially A. glaucus and A. flavus. Beckman (3) reported fungal counts in parsley, sage and thyme that exceeded local standards of 1 x 105 CFU/g.

Coliform counts closely paralleled APC numbers with a range of 7.9 x 102 (allspice) to 1.9 x 107 (dill) CFU/g while Salmonella levels were lower, ranging from 7.9 x 102 (black pepper) to 2.7 x 105 ( cilantro, basil) CFU/g. Bacillus cereus populations ranged from undetectable (mustard, allspice) to low (black pepper, 103), to 1.4 x 106 CFU/g (oregano); and Clostridium perfringens populations ranged from undetectable in most samples from all three markets, to 8 x 103 CFU/g in oregano from market A.

In this preliminary study, it was also determined that a “wash” step, carried out in accordance with recommended guidelines reduced APC numbers by 0.25 – 1.0 log10, while a “chlorine-dip” step reduced

numbers by an additional 0.2 – 1.0 log10. Additionally, the chlorine-dip brought about a greater reduction (1-2

log10) in numbers of yeasts and molds as compared to other microflora being studied.

Part B: The results of this part of the study have determined that the use of a preliminary “wash” step reduces the numbers of aerobic mesophilic flora on parsley samples, depending on the length of the wash time used. While there was no significant difference between initial numbers (6.0-7.0 log10) and treated samples with a

wash time of 30s, a reduction of 1.2-1.7 log10 and 1.5 – 2.0 log10 was observed with wash times of 1 min and 2

min, respectively. Similarly, while a chlorine concentration of 50 ppm free chlorine did not bring about a significant reduction in bacterial counts, higher concentrations of 100 and 200 ppm free chlorine did bring about a reduction in numbers ranging from 1.0-1.5 log10 and 1.5-2.5 log10, respectively. Increasing the chlorine contact

time from 30s to 1 or 2 min also brought about a significant reduction in microbial numbers. Thus, it can be concluded, that water-wash and chlorine-dip treatments, at free chlorine concentrations up to 200 ppm for up to 2 min each, used in tandem, do bring about a reduction in aerobic microflora on parsley. Additionally, no residual disinfectant odor was detectable on the samples, under the treatment conditions studied.

This material is partially funded through the Cooperative State Research, Education, and Extension Service of the U.S. Department of Agriculture, Project Number 00-51110-9762.