Inhibition of Selected Microorganisms in Marinated Chicken by Pimento Leaf Oil and Clove Oleoresin

81999 Applied Pouluy Scienw, Inc

INHIBITION OF SELECTED IN MARINATED MICROORGANISMS CHICKEN BY PIMENTO LEAF O I L AND I

CLOVE OLEORESIN

Primary Audience: Poultry Processors, Researchers

From 1979 to 1989, consumer expenditure for DESCRIPTION OF PROBLEM poultry products rose by 103%. The United States is the number one poultry producer in the world. In Georgia alone, approximately 18 million Ibs of chicken meat are produced on an average day [l]. While annual per capita consumptionof red meat has declined by 10.2 Ibs from 1960 to 1995,poultry consumption increased by 58.9 lbs [2, 31. 1

To whom correspondence should be addressed

The increased demand for convenience foods has resulted in expansion of the processed meat and poultry industry. This has led to the development of further processed poultry products that include items like cut and deboned portions, battered pieces, breaded and pre-cooked cold cuts, nuggets,

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ANGELLA MELISSA A. CARLOS and MARK A. HARRISON' Food Processing Research and Development Laboratoyl Depamnent of Food Science and Technology, m e Universityof Geowk, Food Science Building, Athens, GA 30602 Phone: (706) 542-1088 F B : (706)542-1050 E-mail: [email protected]

Research Report CARLOS and HARRISON

extracts were chosen based on their inhibitory action in previous screening studies [21]. The experimental design used considered the normal bioburden on chicken as well as normal and abusive storage conditions.

MATERIALS AND METHODS PREPARATION O F MARINADE SOLUTIONS Marinade solutions were prepared the day before conducting the experiments and stored at 4°C prior to use. All formulations included 5.0% sodium tripolyphosphate (Rhone-Poulenc, Cranbury, NJ) and 2.5% sodium chloride in water. CLO and PLO (Kalsec, Inc., Kalamazoo, MI) were added at 0, 0.2, and 0.5% levels. The solutions were homogenized for 3-5 min (30 mm generator, 2ST rpm; Pro Scientific Inc., Monroe, CT). PREPARATION OF INOCULA Listeria monocytogenes CA (National Food Processors, Inc., Washington, DC) and V7 (Center for Food Safety and Quality Enhancement, Griffin, GA), Pseudomonas fluorescens, Rputida (Dr. Scott Russell, Department of Poultry Science, The University of Georgia, Athens, GA) and Yarrowia lipolytica (Dr. Yao-wen Huang, Department of Food Science and Technology, The University of Georgia, Athens, GA) were used. Listeria isolates CA and V7 were from Mexican-style cheese and milk, respectively. Both Pseudomonas spp. were from spoiled broiler chickens, and the yeast YiipoZytica was obtained from marinated, spoiled catfish. Each was grown separately in tryptic soy broth (TSB) for 1618 hr to obtain lo8 cells/mL for the bacterial isolates and lo7 cells/mL for the yeast isolate. Immediately before inoculation, each isolate was mixed well and diluted 10-fold with a phosphate buffer (O.lM, pH 7.0). PREPARATION OF CHICKEN BREASTS Fresh skin-on and bone-in chicken breasts were purchased from a local poultry processor. The samples were packed in ice during transit to the laboratory and were subsequently held at 2-4°C prior to injection marination and soakinghmmersion marination.

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or marinated portions [4]. Application of a marinade to meat or poultry enriches flavor [4, increases moisture retention and tenderness [6], inhibits warmed-over flavor [7], and preserves color and flavor [SI. This is especially important in the production of marinated poultry where the processor wants to produce a more favorable product. Different ingredients, usually with spices or spice extracts included, can be incorporated into the solution depending on the application. About 500 million lbs of spice are consumed annuallyin the United States alone, with about 20% of the spice used by the meat and poultry industry [9]. Spices and their extracts also possess preservative and natural antioxidant properties. With the current trend in the industry of employing robotics for marination (i.e., using injector machines), spice extracts are becoming more popular than the spice itself since they are more compatible with such systems and they do not cause fouling or clogging. Extracts offer several advantages: they have better solubility with the right carrier, more top and back flavor notes, more concentrated aromatics, and are free from microorganisms [lo]. Furthermore, several documented reports show that certain extracts have antibacterial, antifungal, and antiviral activities [ l l , 12, 13, 14, 153. Such activities have been observed for both spoilage and pathogenic microorganisms. Poultry meat is known to harbor different types of microorganisms, including spoilage bacteria like Shewanella putrefaciens A, B, and D, Pseudomonasfluorescens A, B, and D, and E fragi [161 as well as pathogenic ones like Campylobacter jejuni [17], Salmonella [18], Listeria monocytogenes [191, Clostridiumperfingens, Staphylococcus aureus and Yersinia enterocolitica [20]. While numerous in vitro studies on the antimicrobialactivities of spices and their extracts have been carried out, only a limited number have been done on food systems. Food application studies are needed since concentrations that are effective in culture media are usually not sufficient to cause the same degree of activity in foods. Therefore, our goal was to determine the inhibitory action of clove oleoresin (CLO) and pimento leaf oil (PLO) against certain spoilage and pathogenic microorganisms on chicken marinated by injection and soaking. These spice

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Y lipolytica. Since the medium was not differential, these plate counts were described as total yeast counts. Total psychrotrophiccounts were enumerated on tryptic soy agar (Difco Laboratories, Division of Becton Dickinson and Co., Sparks, MD) and incubated at 7°C for 10 days. MARINATION BY SOAKING/ IMMERSION Chicken breast samples were inoculated and dried as stated above. These were then placed in sterile plastic bins containing the different marinade formulations. Contact time was set for 1 hr, after which the samples were drained for 15 min and finally placed on plastic foam trays layered with poultry pads (one/tray). The same storage, sampling times, and procedures were employed as in injection marination, except that time zero sampling was done immediately after draining. STATISTICALANALYSES The statistical design used for this study was a 2 (temperature) x 5 (microorganism) x 2 (marination method) x 4 (sampling time) x 6 (spice extract concentration) complete factorial design. Three replicates were provided for each treatment. Results were analyzed using General Linear Model (GLM) procedures and Duncan's Multiple Range Test (DMRT) from the SAS Institute (CW, NC) ~ 3 1 .

RESULTS AND DISCUSSION Initially or at time zero, the population levels of the different test organisms were similar, except for the uninoculated, unmarinated control samples that either had similar, undetectable, or lower (by approximately 1-2 loglo) population levels. For the samples stored at 4"C, inhibition of growth was still observed for some spice extract treatments through 7 days when compared to the inoculated control samples (Figures 1-4).Reduction in final populations by approximately 0.5 to > 2 loglo was observed for treatments containing the spice extracts at 4°C. This was not so for the ones incubated at 12"C, which started manifesting spoilage after 3 or 4 days of storage. CLO and PLO at 0.5% level significantly (P < .OS) inhibited the pseudomonads. The total yeast count was significantly (P< .05)

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INJECTION MARINATION Chicken breast samples were injected with the different marinade solutions using a pilot scale injector (Schroeder Maschinenbau GMBH, D-33824 Werther, Germany) with pressure and speed set at 2.0 atm and 6.5, respectively. Marinated samples were placed on plastic foam trays layered with poultry pads (onekray). The bacterial and yeast isolates were inoculated onto areas of the chicken breast tissue (25 cm2/isolate) by spotting 0.1 mL of diluted cell suspensions onto the surfacewith a micropipet. The same pattern of inoculation was followed for each piece. This resulted in an initial inoculum level of approximately 102 and l@/cm2 for the bacterial and yeast cultures, respectively. The pieces were then allowed to dry for 30 min under a laminar flow hood to allow absorption of the moisture into the meat and attachment of the microbial cells to the surface. The first set of pieces was sampled and designated as time zero using the rinse method [22] and spiral plating procedures (Autoplate 4000 Spiral Biotech, Bethesda, MD). Uninoculated, m a r i n a t e d pieces were also included as controls to determine the effects of the extracts on the natural microflora present on the chicken samples. Prior to storage at 4 and 12"C, the rest of the poultry samples were completely overwrapped on trays with Saran plastic film (oxygen permeability: 1(cc) (mL)/(645.2 cm2) (atm) (24 hr) @ 256°C; DowBrands L.P., Indianapolis, IN). Succeeding sampling times were done after 2,4, and 7 days. Populations were enumerated on Listeria selective agar, Oxford Formulation (Unipath Ltd., Hampshire, England) incubated at 32°C for 48 hr, Psatdomonus selective agar (Unipath Ltd., Hampshire, England) incubated at 25°C for 48 hr and plate count agar (PCA; Difco Laboratories, Division of Becton Dickinson and Co.,Sparks, MD) + lOOp(g/mL chloramphenicol (Sigma Chemical C o . , St. Louis, MO) incubated at 25°C for 48 hr for L. monocytogenes, pseudomonads and total yeast counts, respectively. The PCA + chloramphenicol did not allow for the differentiation of Y lipolytica from other yeasts that may have been naturally present on the chicken. Based on colonial appearance and the calculated numbers inoculated onto the meat surface, the initial population of yeasts was determined to be primarily the inoculated

MICROBIAL INHIBITION BY SPICES

Research Report CARLOS and HARRISON

confirms those made by Evert Ting and Deibel [28] in that 1.0% cloves in sterile meat slurries were not effective in inhibiting L. monocytogenes Scott A. Storing the chicken breast samples at the abuse temperature resulted in significantly higher counts (P< .OS) than in those kept at 4"C, which was expected. Except in the case of yeasts, soaking or immersion was a more effective means of marination from the microbiological standpoint. We speculate that this is due to more intimate contact between the antimicrobials present in the marinade solution and the microorganismson the tissue surface. Moreover, in the case of injection, the chicken pieces did not receive the same amount of marinade because they were not uniform in size, shape, muscle thickness, and ratio of bone to muscle. Such variations were inevitable with the food product used in this study. Although the data we gathered from our in vitro study [21] suggested that a higher concentration may be necessary for antimicrobial effects in chicken, concentrations of 0.2% and 0.5% spice extracts were chosen for flavor and aroma considerations. Also, these levels may find application for the food system employed [29]. At these concentrations, only limited inhibitory effects were observed, and at some point, microbial populations overcame the antimicrobial system(s). Chicken skin contains fat, and it has been suggested that a high lipid fraction in meat absorbs the spice extract, thereby reducing the concentration in the aqueous phase [30]. Thus, bactericidal effects are also negatively affected. The behavior of the different test organisms in the food system employed is difficult to predict and assess because of the interaction of so many variables or factors that may be inherent in the product. Statistical analysis revealed that the test organisms responded differently to the different treatments, so it is not possible to make a single general conclusion. These studies indicate, however, that PLO and CLO can exert significant antimicrobial activity against pseudomonads and yeasts. Combining these findings with other approaches, such as low-temperature storage, would be a promising approach in developing antimicrobial systems for marinated poultry in an industrial setting.

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reduced by 0.2% PLO. The lower concentration was found more effective toward the yeast than the higher concentration for PLO. This may be attributed to the dispersion problem encountered with this essential oil. Even after homogenizing, some globules were observed floating on top of the marinade solution containing this oil. However, it was still effective as was observed in our in vitro studies [21]. Application of this antimicrobial agent in the industry thus necessitates use of oil-soluble carriers like vegetable oil, which is in fact used in some commercial marinade formulations. Both the aforementioned spices contain eugenol in the volatile fractions, a substance that has been shown to possess antimicrobial activity both in vitro and in food systems [24, 251. Miyao [26] reported that this compound controlled the growth of six microbial strains isolated from Vienna sausage. They observed that the shelf life of this food product was prolonged by 12 days at 10°C by dipping in a 50% ethanol solution + 2% eugenol for 10 sec. Furthermore, the high tannin content in cloves (1&13%) may provide additional antagonistic effects [27l. As was mentioned, the chicken breast samples stored at 12°C were spoiled after about 4 days of storage. At this time, the pseudomonads and total psychrotro hic populations were approximately 1 0 7 or greater (Figures 2 and 3) and the products had off-odors characteristic of spoiled poultry products. Furthermore, the pH of the rinse gathered from samples stored at 12°C was much higher than from those obtained at 4°C. From an initial pH of approximately 6.0, it increased to about 7.0-7.5 and 6.7-7.10 for pieces stored at 12°C and 4"C, respectively. Since the predominant psychrotrophic organisms found on chicken are typically pseudomonads [ 161, the population trends obtained for pseudomonads were similar to those of total psychrotrophic counts. However, for some treatments, slightly lower counts were obtained for the total psychrotrophic counts because of the more permissive incubation temperature (25°C) for the plating medium used to obtain total Pseudomonas counts. No significant inhibition was observed for any of the treatments against L. monocytogenes CA or V7. This observation

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CONCLUSIONS AND APPLICATIONS 1. Growth of pseudomonads on product stored at 4°C was inhibited by 0.5% PLO and 0.5% CLO, while 0.2% PLO was effective against the yeasts. Pseudomonads are considered the

REFERENCES AND NOTES 1. Ceolgia Poultry Federation, 1996. Georgia Agricultural Statistics Service. Georgia Department of Agnculture, Atlanta, GA. 2. American Meat Institute, 1991. Annual Report. Washington, DC. 3. USDA, 1994. The Livestock and Poultry Meat Situation. U.S. Department of Agriculture, Washington, DC. 4. Baker, RC and C.k Bruce, 1989. Further process-

ing of oultry. Pa

251-282 in: Processing of Poultry. G.C. &ad, ed. EE.4er Science Publishers Ltd., Essex, England.

5. Chen, T.C., 1982. Studies on the marination of chicken parts for deep-fat frying. J. Food Sci. 4710161019.

6.Aog,C.Y.W.andLLYoung, 1987.Effectofmarination with sodium py-rophosphate solution on oxidative stability of frozen cooked broiler leg meat. Poultry Sci. a676-678. 7. Mahon, J.H., 1963. U.S. Patent No. 3,104,170. Food Tech. 18101-102. 8. Cassidy, J.P., 1977. Phosphates in meat processing. Food Prod. Dev. 11:74-77. 9. Weiser, H.H., CJ. Mountney, and W.A. Gould, 1971. Practical Food Microbiology and Technology. 2nd Edition. AVI Publishing Co.,Westport, CT.

10. Farrell, K.T., 1990. Spices, Condiments, and Seasonings. 2nd Edition. AVI, New York, NY. 11. Farag, RS., Z.Y. Daw, and S.H. Abo-Rays, .1989. . Influence of some essential oils on e ' SltlCUS growth and production of a f l a e a y p t h e t i c medium. J. Food Sci. 54:74-76. 12. Deans, S.G. and G.A. Ritchie, 1987. Antibacterial activity of French tarragon (m 'dracunculus Linn.) essential oil and its constituents during ontogeny. J. Hortic. Sci. 6350S508.

13. Sharma, A, G.M. Tewari, A.J. Shrikhande, S.R Padwal-Desai, and C. Bandyopadhyay, 1979. Inhibition of aflatoxin-producing fungi by onion extracts. J. Food Sci. 44:1042-1044.

14. Larrondo, J.V.,M.&ut, and M.A. Calvo-Toms, 1995. Antimicrobial activity of essences from Labiates. Microbios. 82171-172. 15. Siddiqui, Y.M., M. Ellayebi, A.M.Haddad, and M.N. AI-Ahdal, 1996. Effect of essential oils on the enveloped viruses: Antiviral activity of oregano and clove oils on herpes simplexvirus type 1and Newcastle disease virus. Med. Sci. Res. 24:185-186. 16. Russell, S.M.,D.L. Fletcher, and N.A. Cox, 1996. Spoilage bacteria of fresh broiler chicken carcasses. Poultry Sci. 75:2041-2047. 17. Christopher, F.M.,G.C. Smilh, and C.Vandem ant, 1982. hamination of poultry giblets, raw milk, and meat for -fetus subsp.&. J. Food Prot. 45:260-262. 18. Lahellec, C. and P. Colin, 1985. Relationship between serotypes of S & n s u l k from hatcheries and rearing farms and those from processed poultycarcasses. Br. Poultry Sci. 26:179-186. 19. Bailey, J.S., D.L. Fletcher, and N.k Cox, 1989. Recovery and serotype distribution of monocvtoeenes from broiler chickens in the southeastern United States. J. Food Prot. S2:148-150.

20. Cunningham, F.E, 1987. Types of micmrganisms associated with poultry carcasses. Pages 2 9 4 2 in: The Microbiology of Poultry Meat Products. Academic Press, Inc., New York, NY. 21. Carlos, AM.& 1997. Antimicrobial potential of selected natural ingredients for use with mannated poultry products. Master's Thesis, The Universityof Georgia, Athens, GA. 22. American Public Health Association, 1992. Compendium of Methods for the Microbiological Examination of Foods. 3rd Edition. C. Vandenant and D.F.

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primary cause of spoilage of poultry meat stored at refrigerated temperature. Yeasts, though not a primary concern for fresh refrigerated meats, may also cause spoilage of these products if they receive treatments that would suppress bacterial growth, such as addition of acidulants. 2. Soaking proved to be a more effective method of marination from the microbiological standpoint. 3. We determined the concentration of the spice extracts required to inhibit the test organisms of concern from in vitro studies.We then applied these antimicrobialsto marinated chicken at slightly higher concentrations that would still be applicable for such products. Application of these spice extracts yielded reduced growth rates of the test organisms on samples stored at 4°C. Even after 1wk of storage inoculated samples did not manifest attributes of spoiled meats such as formation of off-odors, off-colors,and slime. Thus, these compounds have potential for use in marinated poultry products. Further studies, however, should be done on sensory aspects as well as the antimicrobial activity of spice blends and other marinade formulations.

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CARLOS and HARRISON Splittstoesser, eds. h e r . Public Health Assn., Washington, DC. 23. Data were subjected to statistical analysis using the General Linear Model GLM) of the Statistical Analysis System (SAS, Caw, Nd) with spice levels, temperature, marination method, and sampling time as the main variables. Interactions of these variables were also analyzed. Separation of means was also done using Duncan's Multiple Range Test.

24. Aktug, S.E and M.Karapinar, 1987. Inhibition of foodborne patho ens by thymol, eugenol, menthol, and anethole. Intl. J. k o d Microbiol. 4:161-166.

25. Bderman, LB., F.Y.Lieu, and SA.Seier, 1977.

26. Miyao, S., 1975. Inhibitory effects of eugenol and of ethanol extract of mace on the wth of microorganisms isolate from Vienna sausage.%ood Hyg. Soc. Japan. 16412416.

28. Evert Ting, W.T. and K E Deibel, 1992. Sensitivity of M v to spices at two temperatures. J. Food Safety 12.129-137. 29. DeibeL ICE and GJ. Banwart, 1984. Effect of is;iUni at three temperatures. spices on J. Food Safety. 6241-251. 30. Farbood, M.I., J.H. MacNeil, and IC Ostovar, 1976. Effect of rosemary spice extractive on microorganisms in meats. J. Milk Food Tech. 3

ACKNOWLEDGEMENTS This study was su ported in part by the Georgia Experiment Station, h e University of Georgia, and the Georpa Food Processing Advisory Council.

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Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldehyde, and eugenol. J. Food Sci. 421107-1109.

27. Nkanga, EJ. and N. Uraih, 1981. Prevalence of in meat samples from traditional lococcu~ s t s in Benin City, Nigeria and ossible control by use of condiments. J. Food Prot. 4 4:d.