Reduction of Campylobacter jejuni and Campylobacter coli in Poultry

477 Journal of Food Protection, Vol. 73, No. 3, 2010, Pages 477–482 Copyright G, International Association for Food Protection

Reduction of Campylobacter jejuni and Campylobacter coli in Poultry Skin by Fruit Extracts DIANA VALTIERRA-RODRI´GUEZ, NORMA L. HEREDIA,* SANTOS GARCIÁ,

AND

´ NCHEZ EDUARDO SA

Facultad de Ciencias Biolo´gicas, Universidad Autońoma de Nuevo Leoń, Apdo. Postal 124-F, Co´digo postal 66451, San Nicola´s de los Garza, N.L., Me´xico MS 09-410: Received 28 September 2009/Accepted 24 November 2009

ABSTRACT Campylobacter spp. are a major cause of foodborne bacterial gastroenteritis in humans, and current methods to control Campylobacter contamination in foods are not completely successful. Plants are a promising source of antimicrobial agents, particularly given the growing interest in ‘‘all natural’’ foods. In this study, the antimicrobial activity of extracts from 28 edible plants against Campylobacter jejuni and Campylobacter coli was evaluated in vitro and in a poultry skin model. Nine of 28 extracts exhibited antimicrobial activity in a diffusion assay, and MBCs were determined for the three most active extracts, i.e., lime, plum, and sour orange peel (MBCs of 2 to 3 mg/ml). Mixtures of the lime, plum, and sour orange peel extracts were applied to chicken skin inoculated with 105 CFU of Campylobacter to test for synergistic or antagonist effects. After incubation (48 h at 4uC) with any extract mixture, no Campylobacter CFUs were detectable. A panel of tasters determined that the mixture of lime and plum gave the best flavor to chicken wings. These active extracts from edible fruits are simple to prepare and are alternatives to reduce or eliminate Campylobacter contamination of chicken products.

Poultry meat, especially from broilers and turkey, is an important vehicle implicated in foodborne illness (25, 38). A strong association has been demonstrated between chicken consumption and sporadic gastrointestinal disease outbreaks caused by Campylobacter spp., one of the most important agents of foodborne diseases in the world (38). Poultry meat is the main reservoir of Campylobacter jejuni (36). Although pork meat is the main reservoir for Campylobacter coli, this bacterium is often found to be associated with poultry (36). Most of the poultry meat production in the world is contaminated with Campylobacter as reflected in the high incidence of Campylobacter isolation from poultry products for retail sale (38). Campylobacter spp. are gram-negative, strictly microaerophilic, curved rods with an optimum growth temperature of 42uC (12). C. jejuni and C. coli are the most important pathogenic species and are responsible for 95% of human campylobacteriosis (12, 38). Campylobacter adheres to the skin of live broilers on poultry farms and is present in large numbers in the early stages of processing (2, 21). Even after scalding and picking, Campylobacter can be recovered in high numbers from whole carcass rinses or skin swabs of broilers at retail (2, 21, 30). During poultry processing, bacteria are removed, destroyed, or controlled by thermal treatments, water, chemical additives (antimicrobials), and mechanical methods (16, 28). For example, after evisceration, carcasses can pass through a chlorinated spray wash and enter a chlorinated chiller. Addition of chlorine (20 to 40 ppm) to the chiller (4uC) reduces Campylobacter populations (30). * Author for correspondence. [email protected].

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However, cooling of carcasses by immersion in the chiller may lead to cross-contamination (38). Irradiation, steam pasteurization, and crust freezing are alternatives to immersion of carcasses in the chlorinated chiller (38). Trisodium phosphate (TSP) treatment is another alternative for controlling microbial loads and has antimicrobial power superior to that of other phosphates (30). Freezing reduces Campylobacter on carcasses (33); however, the consumer preference for fresh, nonfrozen poultry makes this procedure less useful. Although synthetic additives are widely used in the food industry to inhibit microbial contamination, there is a consumer-driven trend toward fewer synthetic food additives and more natural products (7). A possible natural alternative to chemical-based bactericides is the use of plant extracts. Several medicinal plant extracts have antimicrobial activity against Campylobacter (4, 6, 32). Dickens et al. (9) demonstrated that an herbal extract called Protecta II was capable of reducing Campylobacter in broiler carcasses during a simulated chill. In this study, extracts from 28 edible plants were evaluated for their efficacy in inhibiting growth of C. jejuni and C. coli in vitro and in a food model (chicken skin), and sensory analyses were conducted to determine the acceptability of chicken wings treated with the active extracts. Specifically, the antimicrobial activity of whole-fruit extracts of lime and plum and of extract of sour orange peel, against C. coli and C. jejuni is reported. MATERIALS AND METHODS Bacterial cultures. C. jejuni NADC 5653 (kindly provided by Dr. Irene Wesley from the National Center of Animal Diseases, U.S. Department of Agriculture, Ames, IA) and C. coli 19 (isolated in our laboratory from live chicken) were used in this study. Strains

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J. Food Prot., Vol. 73, No. 3

TABLE 1. Antimicrobial effect of plant extracts with the agar well diffusion method against C. jejuni and C. coli Antimicrobial action againsta: Plant

Poblano pepper Green pepper morroń Pineapple Chayote Cilantro Asparagus Nopales Green tomato Tomato Plum Dried plum Lemon Lime Seedless lime Sour orange peel Blueberry Field pumpkin Coconut Peach Spearmint Cantaloupe Turnip Papaya Garlic Broccoli Green bell pepper Red onion Jıćama a

Scientific name

Capsicum annuum L. Capsicum annuum L. Ananas comosus (L.) Merr. Sechium edule (Jacq.) Sw. Coriandrum L. Asparagus L. Opuntia spp. Physalis L. Solanum lycopersicum L. Prunus L. Prunus L. Citrus | limon (L.) Citrus aurantifolia Christm. Citrus aurantifolia Christm. Citrus aurantium L. Vaccinium spp. Cucurbita pepo L. Cocos nucifera L. Prunus persica L. Mentha spicata L. Cucumis melo L. Brassica napus L. Carica papaya L. Allium sativum L. Brassica oleracea L. var. italica Plenck Capsicum annuum L. Allium cepa L. Pachyrhizus erosus

Extraction solvent

Resuspension solvent

C. jejuni 5653

C. coli 19

Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol

Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

X X z X z X X X X zzz zz zz zzz z zzz X X X X X X X X X X

X X z X z z X X X zzz zz zz zzz z zzz X X X X X X X X X X

Ethanol Ethanol Ethanol

PBS PBS PBS

X X X

X X X

X, no inhibition; z, little inhibition (0- to 5-mm diameter); zz, medium inhibition (5- to 10-mm diameter); zzz, high inhibition (.10-mm diameter).

were maintained at 280uC in brain heart infusion broth (Bioxon, Mexico City) containing 20% (vol/vol) glycerol. Campylobacter strains were grown in brain heart infusion broth supplemented with 0.6% yeast extract and incubated for 48 h at 42uC under microaerobic conditions (CO2 chamber, 10% CO2). An aliquot from this culture was spread in Mueller-Hinton agar supplemented with 5% horse blood and incubated for 48 h at 42uC under microaerobic conditions. Resulting colonies were isolated and suspended in saline solution and adjusted to 1.5 | 108 CFU/ml. Plant extracts. Edible plants (Table 1) were purchased from retail markets in the metropolitan area of Monterrey, Nuevo Leoń, Mexico. Plant material was washed first with tap water and then with distilled water. After that, 20-g samples were immersed in 100 ml of 96% ethanol and then ground with a mortar and pestle to extract soluble material. Extracts were held (48 h at room temperature), and suspensions were filtered through Whatman no. 1 paper and placed in glass plates (20 cm in diameter) to evaporate the ethanol (for about 48 h at room temperature). Dried extracts were resuspended in 10 to 15 ml of phosphate-buffered saline (PBS) or ethanol, filter sterilized with nitrocellulose membranes (0.45-mm pore size; Millipore), and collected in sterile amber flasks. Reconstituted sterile extracts were maintained at 4uC until they were used (maximum of 6 months) (10). An aliquot was used to determine dry weight.

Preliminary antimicrobial activity test. The agar diffusion well test (11) was used for preliminary screening. Petri dishes (150 mm) were filled with 25 ml of Mueller-Hinton agar (BDBioxon) supplemented with 5% lysed horse blood (obtained from a local slaughterhouse). Plates were incubated at 37uC for 24 h for sterility testing and immediately used. Aliquots (100 ml) of the bacterial culture (adjusted to 1.5 | 108 CFU/ml) were spread homogeneously onto the agar. Five holes (6 mm in diameter) were then made in the seeded agar plate by using an inverted culture tube (6 by 50 mm) and filled with 100 ml of extract or with PBS or 96% ethanol as controls. The plates were incubated at 42uC for 48 h under microaerobic conditions (23). Inhibitory activity was visualized as a loss of bacterial growth in the area surrounding the holes filled with the plant extracts and quantified by measuring the diameters of the inhibition zones. MBC. Sterile 96-well polystyrene U-microtiter plates (Costar, Corning Inc., Corning, NY) were filled with 50 ml of 2| MuellerHinton broth (Difco) plus 50 ml of varying concentrations of the extracts (22). The plates were inoculated with 5 ml of a fresh culture (adjusted to 1 | 108 CFU/ml) and incubated at 42uC for 48 h in a CO2 chamber (23). After incubation, the content of each well was spread plated for enumeration in Mueller-Hinton agar supplemented with 5% lysed blood and incubated at 42uC for 48 h in a CO2


REDUCTION OF CAMPYLOBACTER BY FRUIT EXTRACTS

chamber. The MBC was defined as the lowest concentration of the extract that resulted in no visible bacterial colony growth on the Mueller-Hinton agar plate after the incubation period. To determine whether the extracts had synergistic effects, the most effective extracts were mixed and their effect against Campylobacter was determined by the checkerboard method described by Orhan et al. (29) with minor modifications. The mixtures were lime (L) and plum (P) (LzP), L and sour orange peel (SOP) (LzSOP), and PzSOP. Sterile 96-well microtiter plates were filled with 50 ml of 2| Mueller-Hinton broth plus 50 ml of the extract mixtures. The mixtures contained different combinations of extracts at different amounts (100, 75, 50, and 25% of the MBC of each extract). The plates were inoculated, incubated, and plated as described above. The effects of mixtures and synergism were evaluated by the fractional inhibitory concentration index (FIC) (20). The FIC is defined as the sum of the MBC of each extract when used in combination, divided by the MBC of each extract used alone (29). An FIC value of ,0.5 indicates a synergistic effect; an FIC value between 0.5 and 2 is considered indifferent; and an FIC value of $2 indicates an antagonist effect (29). Food model. Raw chicken skin was bought in a local supermarket in the metropolitan area of Monterrey, Nuevo Leoń, Mexico. The skin was separated from any visible fat layer with a sterile knife and sterile scissors and cut into pieces (2 by 2 cm). The microflora was removed from the skin as follows. The cut pieces were first rinsed 10 times with tap water and then twice with sterile distilled water in 1-liter Nasco sterile bags. The pieces were then separated and exposed to UV irradiation at a wavelength of 254 nm in open sterile petri dishes for 30 min each side in a sterile chamber. The pieces were then frozen at 220uC for 24 h, a process known to reduce Campylobacter numbers (3). The skin was then confirmed to be free of Campylobacter by assessing the number of CFU on Campy-cefex agar supplemented with 5% of lysed blood (34). Individual skin samples were then inoculated with 100 ml of 1 | 106 CFU/ml of a cocktail of both Campylobacter strains and left to rest for 10 min at room temperature to allow bacterial adherence. After that, 2-ml aliquots of the different extracts or their mixtures (L, LzP, LzSOP, and LzPzSOP) were added, each at their MBC. All the samples were incubated at 4uC. Samples were withdrawn at 0, 48, and 120 h and placed in a Nasco sterile bag with 9 ml of sterile saline and vigorously homogenized (by hand) for 2 min. The suspensions were serially diluted (1021 to 1025) in sterile saline solution, viable counts were determined on Campycefex agar plates supplemented with 5% lysed blood (37), and the plates were incubated as described above. Inoculated skin samples treated with 2 ml of (10%) TSP or saline were used as positive and negative controls, respectively (31). Skin samples in which the inoculum was replaced with 2 ml of sterile saline were also used as controls. Sensory analysis. Chicken wings were purchased in a local supermarket of the metropolitan area of Monterrey. The wings were washed with potable water, the water was drained, and the wings were left to sit for 2 min at room temperature and then placed in plastic containers (25 by 20 by 10 cm; 35 wings per container) with 300 ml of the following extracts or mixtures: L, LzP, LzSOP, and LzPzSOP (each at their own MBC). Wings (similar in number) were also placed in containers with potable water and used as negative control. The containers were vigorously mixed and refrigerated (4uC) for 48 h. The treated wings were then placed in aluminum baking pans, covered with aluminum foil, and baked for 80 min at 180uC. The sensory analysis was done with a

479

FIGURE 1. Preliminary antimicrobial activity test in agar diffusion wells showing inhibitory activity of L, P, and SOP against C. coli. PBS was used as control.

semitrained panel (n ~ 32) composed of faculty and student volunteers. The sensory test was a preference ranking test (24). A value of 1 was recorded for the least palatable and a value of 5 for the tastiest. Each person drank from a glass of water between every sample (35). A single sensory evaluation was conducted. Statistical analysis. The results were evaluated with an analysis of variance test using the SigmaStat program, and the sensory analysis was evaluated with a Friedman test (24).

RESULTS AND DISCUSSION Several products from plants have been proposed as preservatives because they contain compounds that exhibit antimicrobial activity. However, the susceptibility of a microorganism to a plant extract depends on both the properties of the extract and the microorganism itself (15). In the present work, the antimicrobial effect of ethanolic extracts against C. jejuni and C. coli was determined for a total of 28 extracts. These extracts were obtained in ethanol, finally resuspended in PBS, and then analyzed. Some of the extracts were not completely dissolved in PBS; these were resuspended in ethanol and analyzed. Nine of the extracts showed different levels of antimicrobial activity (Table 1). The extracts that showed the greatest antimicrobial activity against C. jejuni and C. coli were L, P, and SOP (Fig. 1). These active extracts were obtained in ethanol, finally resuspended in PBS, and then analyzed for both their MBC and their activity in a food model. The MBCs (means ¡ standard deviations [SD]) were as follows: 2 ¡ 0.1 mg/ml for L in both strains of Campylobacter; 2 ¡ 0.08 mg/ml for P in C. jejuni and of 3 ¡ 0.6 mg/ml for P in C. coli; 2 ¡ 0.4 mg/ml for SOP in both strains of Campylobacter (Table 2). The antimicrobial activity of lemon extract was previously reported by Conte et al. (7) against microorganisms other than Campylobacter, including Bacillus licheniformis, and the yeasts Saccharomyces cerevisiae and Pichia subpelliculosa. Preliminary studies conducted by Adeleye and Opiah (1) showed that extracts of lime were active


480


TABLE 2. MBCs of plant extracts against C. jejuni and C. coli Mean MBC ¡ SD (mg/ml) Strain

L

P

SOP

C. jejuni 5653 C. coli 19

2 ¡ 0.1 2 ¡ 0.1

2 ¡ 0.08 3 ¡ 0.6

2 ¡ 0.4 2 ¡ 0.4

against Staphylococcus aureus, Streptococcus faecalis, Candida albicans, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Shigella dysenteriae. Antimicrobial activity of citrus peel was previously reported against Escherichia coli and S. aureus (14); commercial orange oil fractions also have demonstrated activity against Campylobacter and Arcobacter (27), and products from plum have exhibited antimicrobial activity against Salmonella, E. coli O157:H7, and Bacillus cereus (5, 19). To establish their efficacy, natural antimicrobials were evaluated alone or in combination. Synergistic effects would be useful, since lower amounts of compounds could be used to inhibit growth (17) and the sensory properties of foods would be less altered. Furthermore, combining extracts could give new flavors or mask unwanted ones. However, the FICs for the different mixtures were between 1.0 and 1.3 for both strains, indicating an indifferent effect, rather than synergy or antagonism (Table 3). Although no synergism was demonstrated, the absence of antagonistic effects means that extracts can be mixed to achieve desirable sensory properties without diminishing their antimicrobial activities. Here tests were conducted using L alone, LzP, LzSOP, and LzPzSOP, each at their respective MBC. Lime was chosen as the main extract because it showed a slightly higher activity, is easily available in the market, and is often used in cooking. Since most of the microbial contamination of chicken carcasses is in the skin (13), studies to determine the effectiveness of antimicrobial treatments were conducted with skin. Skin was initially treated to reduce or eliminate Campylobacter by washing with sterile water, exposure to UV, and freezing. Only skin samples that gave no viable counts of Campylobacter after these treatments were used. To simulate the worst case, high numbers of Campylobacter cells were added to the skin (105 CFU per sample). The inoculated chicken skin was exposed to 4uC to simulate normal refrigeration conditions for commercial chicken products (8). Exposure of Campylobacter in the skin samples to the extracts produced a significant (P , 0.05) reduction in TABLE 3. Fractional inhibitory concentration index resulting from mixtures of extracts FICa Strain

C. jejuni 5653 C. coli 19 a

A, indifferent.

LzP

LzSOP

PzSOP

1.1 ¡ 0.1 (A) 1.3 ¡ 0.3 (A) 1.0 ¡ 0.3 (A) 1.3 ¡ 0.3 (A) 1.2 ¡ 0.3 (A) 1.3 ¡ 0.4 (A)

FIGURE 2. Effect of the extract formulations against a cocktail of C. jejuni and C. coli in chicken skin. Inoculated skin was treated with L, LzP, LzSOP, and LzPzSOP. Ctr, inoculated skin treated with phosphate buffer. All the extracts were added at their MBC as follows: L, 2 mg/ml; P, 3 mg/ml; SOP, 2 mg/ml. TSP was added at 10%. Experiments were performed in duplicate a total of three times.

bacterial number when compared to the negative control (Fig. 2). At 48 h of incubation, the population of Campylobacter in the treated samples diminished to undetectable levels (,10 CFU/ml). These results indicate that the MBC of the extracts, which achieves a minimum of a 4-log reduction, when applied in the food model was similar in effectiveness to those determined in studies in vitro. TSP, at concentrations used in the broiler industry, was very effective in eliminating Campylobacter from chicken skin; skin treated with TSP yielded no detectable CFUs of Campylobacter at any of the incubation times (Fig. 2). For the sensory analysis in chicken wings, a test of preference was carried out using the same mixtures used in the food model. Adding the mixture of LzP to the chicken wings gave the best acceptable flavor (rated at 3.8), similar in preference to the control with no extract added (rated 3.6; P , 0.05); whereas the other treated chicken samples were slightly less attractive in taste (significantly different) than the control (Fig. 3). Although there are several effective synthetic compounds available to control Campylobacter in chicken during processing, consumers are asking for fresher and more natural products, free of both microorganisms and synthetic preservatives (18). It is therefore important to offer new natural antimicrobial alternatives for use in foods. Since Campylobacter causes the highest number of cases of foodborne illnesses in many countries and raw poultry is one of the most common vehicles of contamination, it is important to find alternatives to control Campylobacter before it reaches the consumer’s kitchen. Furthermore, raw chicken is consumed as sashimi in several countries and carries an additional risk of causing disease (26). Thus, a procedure to control the microorganism in raw product is also necessary. In this study, the selected extracts from


REDUCTION OF CAMPYLOBACTER BY FRUIT EXTRACTS

9.

10.

11.

FIGURE 3. Gustatory preference for chicken wings with and without fruit extracts. The sensory analysis was done with a semitrained panel (n ~ 32), using five wings per treatment. All the extracts were added at their MBC as follows: L, 2 mg/ml; P, 3 mg/ ml; SOP, 2 mg/ml. A value from 1 to 5 was recorded, 1 for lower preference and 5 for greater preference. CTRL, chicken wings with no extract added.

edible fruits and their mixtures were able to reduce the viability of C. jejuni and C. coli in vitro and in chicken skin by .4 log, and one of the proposed formulas was organoleptically acceptable for application to chicken (LzP). These active extracts, which come from edible fruits and are simple to prepare, could be an alternative to reduce or eliminate contamination of chicken products before home processing, thus lowering the risk of illnesses associated with Campylobacter. Work is in progress in our laboratory on the isolation and identification of the antimicrobial compounds and their mechanism of action against this pathogen.

12.

13.

14.

15. 16.

17.

18.

19.

ACKNOWLEDGMENTS This research was supported by the Consejo Nacional de Ciencia y Tecnologıá de Me´xico (CONACYT) and PAICYT-UANL. We are thankful to CONACYT for the scholarship granted to Diana Valtierra.

20.

21.

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