EFFICACY OF AN HERBAL EXTRACT, AT VARIOUS CONCENTRATIONS, ON THE MICROBIOLOGICAL QUALITY OF BROILER CARCASSES AFTER SIMULATED CHILLING 1

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1 2001 Poultry Science Association, Inc. EFFICACY OF AN HERBAL EXTRACT, AT VARIOUS CONCENTRATIONS, ON THE MICROBIOLOGICAL QUALITY OF BROILER CARCASSES AFTER SIMULATED CHILLING 1 J. A. DICKENS 2 and K. D. INGRAM USDA-ARS Poultry Processing and Meat Quality Research Unit, Russell Research Center, Athens, GA Phone (706) FAX: (706) adickens@saa.ars.usda.gov Primary Audience: Poultry Plant Managers, Food Safety Personnel, Researchers SUMMARY Bacterial contamination of raw processed poultry continues to be of concern to consumers, as well as regulatory and health officials. For the past 40 yr, scientists have been working on suitable and acceptable decontamination methods to reduce or eliminate spoilage organisms and human enteropathogens from raw processed meat and poultry products. Protecta Two [1], an herbal extract on a sodium chloride carrier, was evaluated in a 30-min, 1 C, simulated chill for its effectiveness in lowering microbial counts on broiler carcasses. Sampling was performed on plantrun control (PRC) carcasses and carcasses subjected to a 30-min chill (1 C) in tap water or a 1 or 0.5% solution of Protecta Two. After treatment, carcasses were subjected to the whole-carcass rinse procedure and were examined for total aerobes, coliforms, Campylobacter, and generic E. coli. Results are reported as mean Log 10 colony-forming units per milliliter of rinse fluid recovered. The water treatment reduced counts significantly from those of the PRC, and the Protecta II treatments further reduced the counts to the lower limits of detection for the coliforms, Campylobacter, and generic Escherichia coli. Key words: Broilers, Campylobacter, chilling, herbal extract, microbiology 2001 J. Appl. Poult. Res. 10: DESCRIPTION OF PROBLEM Chilling is a required and regulated step in processing of all forms of poultry. It is mandated that the carcass internal temperature be below 4 C within 4 h of the start of the processing operation to stop the growth of spoilage organisms, as well as pathogenic organisms associated with poultry. Because chilling is the last step in the processing chain in which the whole carcass can be treated, increasing emphasis has been directed to bacterial reductions during this processing step. Water chilling alone reduces counts 1 Mention of specific brand names does not imply endorsement by the authors or the institutions at which they are employed to the exclusion of others not mentioned. 2 To whom correspondence should be addressed.

2 DICKENS and INGRAM: HERBAL EXTRACT DURING CHILL 195 on carcasses by simply removing superficial bacteria from the carcasses with the washing action of commercial chillers. This reduction in counts can be negated through the cross contamination of carcasses by bacteria that are washed off of other carcasses during agitation. Pietzsch and Levetzow [2] demonstrated a 50% reduction in the incidence of Salmonella-positive carcasses in heavily contaminated flocks during immersion chilling, but subsequent Salmonella-free flocks were contaminated with the Salmonella during the chill as well as some earlier processing steps. Thomson et al. [3] and Dickens and Cox [4] evaluated the effects of air agitation on moisture pick-up, chilling time and temperature, and microbiological quality of processed poultry. The research of Dickens and Cox [4] demonstrated that air agitation improved the microbiological quality but increased moisture pick-up above regulatory standards. The reasons for the reductions in bacterial counts due to air agitation are not known, but Dickens and Cox [4] suggested that it could be due to the scrubbing action of the bubbles produced by air being forced through the water under pressure. Dickens and Whittemore [5] incorporated acetic acid with air injection into the chiller. This procedure lowered the moisture uptake and significantly reduced incidence of inoculated Salmonella but had only limited effectiveness on other bacterial populations. Recently, researchers have been trying natural means of reducing microbial contamination. The use of natural antimicrobials produced from herbs and spices lends itself to more favorable acceptance by the general public as well as by countries that restrict the importation of products. Lis-Balchin et al. [6] demonstrated the effectiveness of essential oils derived from the steam distillation of leaves of Pelargonium species and cultivars on numerous organisms including Salmonella enteriditis and Listeria innocua. These oils showed greater efficacy on the L. innocua than did commercial thyme oil. Hao et al. [7] applied alcohol extracts of angelica root, banana puree, bay, caraway seed, carrot root, marjoram, pimento leaf, and thyme to cooked chicken to determine antimicrobial activity against Aeromonas hydrophilia and Listeria monocytogenes. An active ingredient found in clove (eugenol) and pimento extract reduced these bacterial populations by 4 log after 14 d of storage when compared to control samples. Smith-Palmer et al. [8] found that the antimicrobial properties of 21 plant essential oils and two essences were effective in reducing counts of Salmonella enteritidis, Campylobacter jejuni, Escherchia coli, Staphylococcus aureus, and L. monocytogenes. The oils of bay, cinnamon, cloves, and thyme were the most effective oils tested. Concentrations ranging from 0.05 to 1% were determined to be bactericidal, depending on the temperature of the treatment. These oils were most effective at 35 C and decreased in effectiveness as the temperature was lowered to 4 C. With trends moving toward natural product development, the objective of this research was to determine the effects of various concentrations of an herbal extract, Protecta Two, after a simulated 30-min chill (1 C) on the microbiological quality of processed poultry carcasses. MATERIALS AND METHODS GENERAL All carcasses for the experiments were purchased from a local processor and were removed from the evisceration line immediately after final wash but before chill. Carcasses were placed into insulated containers to maintain carcass temperature and were transported to the research facility for testing. All treatments were performed within 30 min of removal from the processing line in identical prototype paddle chillers containing 114 L water. Treatment solutions were tap water or 1 or 0.5% Protecta Two for 30 min at 1 C; plant-run control (PRC) carcasses collected after the final wash before chilling were included to delineate a baseline for carcasses prior to chilling. The chilling solutions were prepared by overfilling the chillers and adding ice to the water to equilibrate the temperature to 1 C and then lowering the water level to 114 L. The treatment solutions were prepared by adding 1.14 or 0.57 kg Protecta Two to the chiller or an equal amount of water on a weight basis to the other chiller. Temperature and ph measurements were made with a Schott Gerate model CG 837 ph meter [9] prior to placing the carcasses into the treatment baths. One hundred eight carcasses, three treatments, and 54 PRC were examined. After treat-

3 196 ment, carcasses were rinsed with a tap water spray to remove any residual treatment solution, allowed to drip, placed into individual bags containing 100 ml sterile water (ph 7.6), and subjected to the low-volume whole-carcass rinse [10] using an automated shaker sampler [11]. The carcasses were removed from the shaker and allowed to drip for 30 s before the diluent was decanted into sterile specimen cups; the collected diluent was transported to the laboratory for preparation of dilutions and plating procedures. MICROBIOLOGICAL PROCEDURES Serial dilutions of the rinse diluent were prepared in sterile peptone water. Total aerobic bacterial populations were enumerated using plate count agar [12]. One-tenth of a milliliter from a serial dilution of the rinse diluent was plated in duplicate on the surface of the agar, spread with a sterile bent glass rod, and incubated at 35 C for 48 h prior to counting the colonyforming units. Campylobacter were enumerated by plating in duplicate onto the surface of Campy-cefex agar [13]. One-tenth of a milliliter of a serial dilution of the rinse diluent was spread on the surface of each plate with a sterile bent glass rod; plates were then incubated at 42 C for 48 h in a microaerophilic environment (5% O 2, 10% CO 2, and balance N 2 ). After incubation, colony-forming units characteristic of Campylobacter were counted. All colonies counted as Campylobacter from each sample were confirmed as a member of the genus by examination of cellular structure and motility on wet mount under phase contrast microscopy. Each colony type was further characterized as a member of the species jejuni, coli, orlari by a positive reaction with a latex agglutination test [14]. Coliform and generic E. coli counts were made by plating 1 ml serial dilution from the rinse diluent onto duplicate E. coli petrifilm plates [15]. Petrifilm plates were incubated at 35 C for 24 to 48 h, and colony types characteristic of coliform and E. coli were counted. STATISTICAL ANALYSIS The general linear model (GLM), least squares means, and Tukey s studentized range test of SAS software [16] were used to analyze all microbiological data with treatment as the JAPR: Research Report main effect and treatment by replication as the error term. RESULTS AND DISCUSSION Herbs, by definition, are flowering plants whose stem above ground does not become woody and persistent. Most herbs contain various chemicals within their cells that have demonstrated the ability to heal wounds and decrease inflammation as well as being effective treatments for enteric diseases such as cholera [17]. It is speculated that these chemicals have evolved over time to give the plants resistance to specific insects, fungi, bacteria, and viruses. Unpublished data of Gruenau Corp., Germany [18] demonstrates that the herbal extracts of Protecta Two are bacterial inhibitors that kill bacteria and reduce or inhibit surface bacterial growth. The basis for the bactericidal action of the compound is the synergistic effect of the polyphenols produced from the extracts and the salt carrier. The bactericidal effect is contingent upon surface contact with the sample. Protecta Two herbal treatment, generally recognized as a safe (GRAS) bactericide, has been cleared by the USDA Property Mix Committee to require no labeling when used as a processing aid. Preliminary work with the product demonstrated a decrease in effectiveness as the temperature of the product increased above 5 C, with best results noted between 1 and 4 C. Because this product is proprietary, no further information can be made available on the chemical composition or bactericidal action. Results from the experiments demonstrated that chilling with water alone significantly reduced the microbial loads on carcasses (Table 1). Tap water with less than 3 ppm chlorine was used as the chilling medium. Log 10 counts per milliliter of rinse diluent were lowered by 1.1, 1.1, 1.4, and 1.1 Log for total aerobes, coliforms, Campylobacter, and generic E. coli, respectively. Commercially processed broilers are chilled in water with 20 to 50 ppm total chlorine. Therefore, in standard commercial practices, the reduction in the microbial counts would be expected to be even greater. Due to the logistics of ensuring all carcasses in each replication were from the same flock, chilled PRC were not evaluated.

4 DICKENS and INGRAM: HERBAL EXTRACT DURING CHILL 197 TABLE 1. Mean Log 10 colony-forming units per milliliter (± SD) of rinse from whole-carcass rinse samples for plantrun control (PRC), water control, and 0.5 or 1% Protecta-Two-treated carcasses in a 30-min simulated chill (n = 162 total carcasses; 54 PRC, and 108 treated) A ORGANISM PRC TAP WATER ONLY 0.5% PROTECTA TWO 1.0% PROTECTA TWO Total aerobes 3.7 ± 1.1 a 2.3 ± 0.6 b 1.7 ± 0.6 c 1.4 ± 0.6 c Coliforms 2.4 ± 0.7 a 1.1 ± 0.4 b 0.5 ± 0.2 c 0.4 ± 0.1 c E. coli 2.0 ± 0.4 a 0.5 ± 0.1 b 0.1 ± 0.0 c 0.1 ± 0.0 c Campylobacter 1.5 ± 0.6 a 0.4 ± 0.1 b 0 ± 0.0 c 0 ± 0.0 c a,b,c Numbers in rows with different superscripts are significantly different (P 0.05). A Six replications of six carcasses each with 54 PRC; n = 162. The addition of a 0.5% solution of the herbal extract, Protecta Two, demonstrated further reductions in the Log 10 counts on all carcasses when compared to tap water alone. These reductions were dramatic, with all mean counts being less than the actual detection limit. There was no visual color or physical change to the skin of treated carcasses, as has been observed with the use of organic acids [19, 20]. The 0.5% solution of the herbal extract lowered the ph (from 7.8 to 6.7) of the standard tap water chilling medium. The after-treatment rinse procedure used in this experiment was found to be adequate in removing any residual bactericide from the carcass after treatment. This finding was validated by spiking the rinse diluent from Protecta- Two-treated carcasses with a marker organism and plating as the standard rinse diluents were plated. All of the spiked plates, with natural flora and marker organisms, showed extensive growth supporting the assumption that none of the extract was found after the carcasses were rinsed with potable water. Previous work with Protecta Two [21] has demonstrated the ability of this herbal extract to reduce the microbial flora of broiler carcasses when used at a 2% concentration but was found to be economically unfeasible. Work is continuing on developing the optimum parameters for bactericidal and economic aspects to make the product a feasible alternative for the poultry industry. CONCLUSIONS AND APPLICATIONS 1. The chilling step of processing significantly reduces the number of microorganisms associated with processed broiler carcasses. 2. Protecta Two at concentrations of 0.5 or 1% is an effective bactericidal agent that significantly reduces the number of microorganisms on chilled broiler carcasses. 3. The use of Protecta Two during the chilling of broiler carcasses reduces the levels of the pathogen Campylobacter to undetectable levels. 4. Economic constraints will make the use of this product unrealistic in chillers, due to the massive amount of water used in the chilling procedure, until costs of the product can be lowered. 5. New parameters and protocols are being investigated to facilitate the use of Protecta Two as a viable processing aid to ensure a safer food product. REFERENCES AND NOTES 1. Protecta Two, Bavaria Corporation, Apopka, FL Pietzsch, O., and R. Levetzow, The problem of salmonellosis of poultry as related to poultry chilling. Fleischwirtschaft 54: Thomson, J.E., A.J. Mercuri, J.A. Kinner, and D.H. Sanders, Effect of time and temperature of continuous chilling of fryer chickens on bacterial counts. Poult. Sci. 44: Dickens, J.A., and N.A. Cox, The effect of air scrubbing on moisture pickup, aerobic plate counts, Enterobacteriaceae, and the incidence of salmonellae on artificially inoculated broiler carcasses. Poult. Sci. 71: Dickens, J.A., and A.D. Whittemore, The effect of acetic acid with and without air injection on moisture pickup and microbiological quality of pre-chilled broiler carcasses. Poult. Sci. 71(Suppl. 1):91.

5 198 JAPR: Research Report 6. Lis-Balchin, M., G. Buchbauer, T. Hirtenlehner, and M. Resch, Antimicrobial activity of Pelargonium essential oils added to a quiche filling as a model food system. Lett. Appl. Microbiol Hao, Y.Y., R.E. Brackett, and M.P. Doyle, Efficacy of plant extracts in inhibiting Aeromonas hydrophila and Listeria monocytogenes in refrigerated, cooked poultry. Food Microbiol. 15: Smith-Palmer, A., J. Stewart, and L. Fyfe, Antimicrobial properties of plant essential oils and essences against five food-borne pathogens. Lett. Appl. Microbiol. 26: Schotte Gerate, D6238 Hofheim, Germany. 10. Cox, N.A., J.E. Thomson, and J.S. Bailey, Sampling of broiler carcasses for Salmonella with the low volume rinse. Poult. Sci. 60: Dickens, J.A., N.A. Cox, J.S. Bailey, and J.E. Thomson, Automated microbiological sampling of broiler carcasses. Poult. Sci. 64: Difco, (BBL), Sparky, MD Stern, N.J., K. Wojton, and B. Kwiatek, A differential selective medium and dry ice generated atmosphere for recovery of Campylobacter jejuni. J. Food Prot. 55: Intergrated Diagnostics Inc., Baltimore, MD M Health Care, St Paul, MN SAS Institute, SAS Users Guide: Statistics. Version 6 Edition. SAS Institute Inc., Cary, NC. 17. Direkbusarakom, S., Y. Ezura, M. Yoshimizu, and A. Herunsalee, Efficacy of Thai traditional herb extracts against fish and shrimp pathogenic bacteria. Fish Pathol. 33: Nau, F., Gruenau Corp. Post fach 1063, Illertissen, Germany, D Personal communication. 19. Dickens, J.A., B.G. Lyon, A.D. Whittemore, and C.E. Lyon, The effect of an acetic acid dip on carcass appearance, microbiological quality, and cooked breast meat texture and flavor. Poult. Sci. 73: Juven, B.J., N.A. Cox, A.J. Mercuri, and J.E. Thomson, A hot acid treatment for eliminating Salmonella from chicken meat. J. Milk Food Technol. 37: Dickens, J.A., N.A. Cox, and M.E. Berrang, Efficacy of an herbal extract on the microbiological quality of broiler carcasses during a simulated chill. Poult. Sci. 79: ACKNOWLEDGMENTS The authors thank V. A. Savage for technical assistance with the engineering and planning and L. K. Orr for technical assistance in the microbiology laboratory.

APPENDIX. 1. On use of chlorine, from FAO/WHO Consultation. References cited

APPENDIX. 1. On use of chlorine, from FAO/WHO Consultation. References cited APPENDIX The draft report of the Joint FAO/WHO Expert meeting on the benefits and risks of the use of chlorine-containing disinfectants in food production and food processing. (Ann Arbor, USA, 27 30 May