Potential Use of Caprylic Acid in Broiler Chickens: Effect on Salmonella Enteritidis

Abstract

The effect of dietary caprylic acid (CA) on Salmonella Enteritidis, as well as the surface treatment of chicken skin contaminated with Salmonella Enteritidis was evaluated. To evaluate the dietary effect of CA on Salmonella Enteritidis, the individually housed broiler chickens (n=48) were divided into 4 groups (positive control, negative control, 2.5 g/kg of CA in the feed, and 5 g/kg of CA in the feed). The feed of all groups, except the negative control, was artificially contaminated with Salmonella Enteritidis ATCC 13076 (10⁷ colony-forming units/100 g of feed). Both concentrations of dietary CA significantly decreased counts of Salmonella Enteritidis in the crop and cecum of experimental chickens (p<0.05). The effect of CA in the crop contents was more pronounced than in the cecum. Surface treatment of chilled chicken halves with CA at 1.25 and 2.5 mg/mL significantly decreased Salmonella Enteritidis contamination of chicken skin (p<0.05). The sensory evaluation of the skin and meat showed that treatment of the skin with 1.25 mg/mL of CA worsened odor and appearance of the chicken skin, while sensory traits of chicken meat were not significantly affected. Taste and overall acceptability was not influenced by CA in both meat and skin. Treatment of the skin with 2.5 mg/mL of CA resulted in more pronounced changes of the skin odor and appearance. In conclusion, dietary CA reduced carriage of Salmonella Enteritidis in chickens, whereas surface-treatment reduced or eliminated Salmonella Enteritidis contamination in the processed bird.

Introduction

Despite the implementation of Salmonella control programs in European poultry production, Salmonella enterica remains one of the most frequent zoonotic pathogens (EFSA, 2014). Salmonella enterica serovar Enteritidis has been identified as the most common serotype isolated from poultry products (Johny et al., 2009). The ingestion of the pathogen via contaminated feed is the major source of Salmonella infections in poultry (Suzuki, 1994; Al-Natour and Alshawabkeh, 2005). Aside from affecting the welfare and performance of animals, the presence of salmonellas can result in their transfer to retail products (Tamblyn and Conner, 1997). Chicken meat contaminated with salmonellas represents a considerable hazard for public health. Within the European Union, 91,034 confirmed human cases of salmonellosis were reported in 2012, with broiler and turkey meat representing the main sources (EFSA, 2014). A primary colonization site of Salmonella Enteritidis in chickens is the cecum (Allen-Vercoe and Woodward, 1999). Furthermore, Salmonella Enteritidis appears to have a strong ability to colonize and reach extraintestinal sites, such as the liver or spleen (Han et al., 2013). The cross-contamination of carcasses can easily occur in the processing plant. As cited by Lillard (1989), out of 4% Salmonella-positive broilers entering the processing plant, 35% Salmonella-positive carcasses were leaving.

Fatty acids and their derivatives are reported to have bacteriostatic and bactericidal properties against a wide range of microorganisms, together with affecting the immune response of the host (reviewed by Harrison et al., 2013). Antibacterial effects of fatty acids were observed in Listeria monocytogenes (Monk et al., 1996; Nobmann et al., 2009), Staphylococcus aureus (Monk et al., 1996; Nobmann et al., 2010), Bacillus cereus (Karlova et al., 2010), Escherichia coli (Smith et al., 2008; Karlova et al., 2010), or food spoilage microorganisms (Yang et al., 2003; Nobmann et al., 2009; Polakova et al., 2010). Medium-chain fatty acids are natural compounds of palm kernel oil, coconut oil, milk, or colostrum of mammals (Hulánková et al., 2013). Among livestock, goat milk is a richer source of caproic, caprylic, and capric acids than cow milk (Marounek et al., 2012). As reported by the Joint FAO/WHO Expert Committee on Food Additives, caprylic acid (CA) is considered safe when used as a flavor (JECFA, 1999). CA is one of the least cytotoxic fatty acids, as reported for example by Lima et al. (2002). In the United States, CA has been recognized as safe (Generally Recognized as Safe [GRAS] by the U.S. Food and Drug Administration, ID Code 124-07-2) and approved as a chemical decontaminant used in poultry processing (USDA-FSIS, 2012).

In in vitro experiments, CA was the most effective fatty acid against Salmonella enterica serotypes Enteritidis, Infantis, and Typhimurium (Skrivanová et al., 2004). Vasudevan et al. (2005) estimated the effect of CA (50 and 100 mM) on autoclaved chicken cecal contents, artificially infected with Salmonella Enteritidis in vitro. Both concentrations of CA reduced numbers of Salmonella Enteritidis by 10⁵ colony-forming units (CFU)/g within 1 min of treatment. Medium-chain fatty acids have also been proved to possess the antibacterial activity in the course of experimental infection. In broiler chickens, experimentally infected with Salmonella Enteritidis, caproic acid (C_6:0, 3 g/kg of feed) led to a significant decrease in the level of colonization of ceca and internal organs by the pathogen (Van Immerseel et al., 2004). The use of dietary CA at 7 g/kg and 10 g/kg was found to be inhibitory on salmonellas in broiler chickens (Johny et al., 2009).

Fatty acids may not only be administered to the feed or water, but also an ability to decontaminate the chicken skin should be considered. Bacterial flora was reduced by a single washing, or successive washings of chicken skin by potassium hydroxide and lauric acid (Hinton et al., 2007; Hinton and Cason, 2008). Bacterial population, including Enterobacteriaceae, was also successfully reduced by the treatment of chicken skin with oleic acid (Hinton and Ingram, 2000).

The aim of this study was to evaluate the effect of CA on counts of Salmonella Enteritidis in chickens that were reared on feed that was experimentally contaminated with Salmonella Enteritidis. Furthermore, the effect of surface treatment of chicken skin by CA on Salmonella Enteritidis attached to broiler skin during the refrigerated storage was tested.

Materials and Methods

Effect of CA in chickens fed Salmonella Enteritidis-contaminated feed

One-day-old male Ross 308 chickens (n=48) purchased from a commercial hatchery (Rabbit, Czech Republic) were used. Animals were randomly divided into 4 groups of 12 animals (positive control, negative control, treated group I [2.5 g/kg CA in the feed], treated group II [5 g/kg CA in the feed]), and housed in 4 floor pens. Room temperature was 32°C in the first week, 30°C in the second week, and 27°C for the rest of the experimental time. The chickens were kept in the floor pens for the first 14 days of their lives. At 14 days of age (D0), all chickens were moved to individual metabolic cages. Animals of both control groups were fed with a wheat–corn based, granulated diet, free of antimicrobials (Biopharm, Jilove u Prahy, Czech Republic), containing dry matter, crude protein, and crude fat at 883, 218, and 61 g/kg, respectively; nitrogen-corrected apparent metabolizable energy was 12.59 MJ/kg. Animals of treated groups received the same diet supplemented with 2.5 or 5 g/kg of CA (Sigma Aldrich, Czech Republic). CA was added instead of rapeseed oil of the basal diet.

On the 16^th, 18^th, and 20^th day of the chickens' life (D2, D4, D6), the feed of positive control and both treatment groups was contaminated with 5 mL of overnight-grown culture of Salmonella Enteritidis ATCC 13076 (Oxoid, Czech Republic; 10⁷ CFU/100 g of feed; method similar to Andino et al., 2014). Prior to the feed contamination, the bacterial strain was made resistant to 20 μg/mL of rifampicin by serial subculturing colonies of pure bacterial culture in 24-h intervals on XLD agar plates (Oxoid) containing increasing concentrations of rifampicin (Sigma-Aldrich, Czech Republic). The starting concentration of rifampicin was 0.32 μg/mL, followed by sequential twofold increases up to 20 μg/mL). Fresh bacterial culture used for the feed contamination (18-h growth at 37°C) was mixed slowly and thoroughly in feed until complete homogenization. To avoid the cross-infections, caged chickens of the negative control group were separated with a plastic barrier from other cages.

Chickens had ad libitum access to their own feed and water supply. Health status of animals was checked daily. Body weights and feed intake were monitored weekly. On the 22^nd day of age (D8), animals were euthanized with inhalation of isofluranum (isoflurane) (Torrex Chiesi CZ Ltd., Czech Republic), followed by cervical dislocation. Immediately after euthanasia, the gastrointestinal tract was completely removed, and crop and cecum contents were taken for microbiological analyses. One gram of the content was aseptically added to 9 mL of sterile peptone water, and serially diluted (10²–10⁸ CFU/mL). The number of viable, rifampicin-resistant bacteria was determined by streaking 0.1 mL of an appropriate dilution on XLD agar plates containing 20 μg/mL of rifampicin. Inoculated plates were incubated aerobically at 37°C for 24 h. Typical colonies (H₂S-positive salmonellas, resistant to rifampicin) were counted and means and SD were calculated. Results were expressed as log₁₀ CFU/g of digesta. Differences in bacterial counts between groups were compared using analysis of variance, followed by Scheffe's test. All data were analyzed using the SAS program (SAS Institute, 2001). The experiment was performed under the supervision of the Ethical Committee of the Institute of Animal Science (Prague, Czech Republic) and Central Commission for Animal Welfare of the Ministry of Agriculture of the Czech Republic.

Surface-treatment of chilled chicken carcasses by CA

Ten chilled broiler chicken carcasses (1725±201 g) were obtained at the chilling stage of the slaughter. After delivery to the laboratory, carcasses were washed under cold potable tap water for 10 s to remove any potential surface dirt and debris (Kim and Marshall, 2000). Inoculation of carcasses with bacterial cultures was performed according to Zhao et al. (2009). Briefly, chicken carcasses were submerged in a sterile beaker containing a freshly diluted culture of rifampicin-resistant Salmonella Enteritidis ATCC 13076 in sterile saline (10⁶ CFU/mL) for 60 s. Inoculated carcasses were kept in a laminar flow hood for 20 min at room temperature, to allow the attachment of bacteria. The treatment of chicken carcasses with CA was performed as described by Doležalová et al. (2010) as follows: each carcass was divided into halves. One half of the first five chicken carcasses were dipped into 1000 mL of a sterile solution of CA at 1.25 mg/mL for 60 s, while the other half was dipped in sterile distilled water for the same time. The remaining five carcasses underwent the same procedure, except the concentration of CA used for the surface treatment was 2.5 mg/mL. All samples were air-dried for 20 min in a laminar flow hood. The method of evaluation was previously used in the experiment estimating the effect of organic acids on Arcobacter butzleri (Skřivanová et al., 2011). The experiment was repeated twice.

pH values of skin were measured at ambient temperature (pH meter 3520 Jenway; P-lab, Prague, Czech Republic); three readings per skin were performed and averaged. Chicken skin was sampled after 0, 1, 2, and 3 days of storage at 4±2°C. For the “day 0” sampling, the samples were taken before the treatment and drying. For microbiological analyses, 10 g of a skin sample each day was aseptically dissected and shaken for 15 min in 90 mL of sterile peptone water (Özdemir et al., 2006). After extraction, decimal dilutions were made using sterile peptone water, and 0.1 mL of appropriate 10-fold dilutions (10²–10⁷ CFU/mL) were surface-plated on XLD agar plates containing 20 μg/mL of rifampicin and incubated aerobically at 37°C for 24 h. All samples were incubated in triplicates. Typical colonies were counted and expressed as log₁₀ CFU/g skin. Differences in pH values and bacterial counts (among groups and days of storage) were compared by analysis of variance followed by the Sheffe's test. All data were analyzed using the SAS program (SAS Institute, 2001).

For sensory evaluation, 30 chilled chicken carcasses (1828 g±152) were obtained from the aforementioned supplier. Prior to the surface treatment with CA, chilled carcasses were washed under running cold potable water for 10 s to remove any potential surface dirt and debris (Kim and Marshall, 2000), then carcasses were randomly divided into three groups and dipped into the respective solution (sterile CA 1.25 mg/mL, 2.5 mg/mL, or distilled water) for one min. Treated chicken carcasses were dried for 20 min in a laminar flow hood and transported to the laboratory. For sensory evaluation, the procedure described by Doležalová et al. (2010) was applied. Chicken breasts with skin were evaluated by a panel of 10 selected assessors (employees of the Institute of Animal Science and the Czech Agriculture and Food Inspection Authority) trained according to ISO 8586-1: 1993. The evaluation was performed in a sensory laboratory equipped with booths. The samples were baked (180°C, 2 h), coded, and served at 50°C. Appearance, odor, flavor, and overall acceptability were scored. No spices or other ingredients were added, except water. Both meat and skin were evaluated. A seven-point scale was used for the assessment (1–very undesirable, 7–very desirable). Data were analyzed using the SAS statistical program for applying general linear model (GLM) procedures (SAS, 2001). Differences between treatment means were tested by Tukey's method (Bures et al., 2006).

Results

Experimental infection

The effect of dietary CA on counts of Salmonella Enteritidis in both crop and cecum is shown in Table 1. The average number of Salmonella Enteritidis in the crop and cecum reached 4.9 and 3.7 log₁₀ CFU/g, respectively. Addition of CA to the diet reduced Salmonella Enteritidis in the crop and cecum below the detection threshold (2 log₁₀ CFU/g).

Table 1.

Influence of Caprylic Acid (CA) on Numbers * of Rifampicin-Resistant Salmonella (log ₁₀ Colony-Forming Units/g) in Crop and Cecum Contents of Chickens Submitted to Either a Salmonella-Free Diet (Group 1) or a Contaminated Diet (Groups 2–4)

Group	Treatment	Crop contents	Cecum contents
1	Negative control	<2a	<2a
2	Positive control	4.9±0.4b	3.7±0.3b
3	CA 2.5 g/kg	<2a	<2a
4	CA 5.0 g/kg	<2a	<2a

Means±SD.

a,b

Values in the same column with the different superscript are significantly different (p<0.05). Cell concentration <100/mL was considered as 100/mL in statistical calculations.

Antimicrobial activity of CA against Salmonella Enteritidis attached to chicken skin

In our experiment, CA (1.25 and 2.5 mg/mL) was tested against Salmonella Enteritidis attached to broiler skin to assess its potential as a poultry surface decontaminant. Treatment with CA did not significantly decrease the pH of the skin (data not shown; p>0.05). The differences regarding pH between samples treated with CA and the corresponding controls decreased during storage, with a 0.42-units difference just after treatment and 0.10 difference within the 3 days (average values).

The effect of CA on the number of Salmonella Enteritidis attached to the chicken skin inoculated with Salmonella Enteritidis is shown in Table 2. Treatment of Salmonella Enteritidis–contaminated samples with CA significantly decreased numbers of Salmonella Enteritidis after 1, 2, and 3 days of storage in both tested concentrations (p<0.05). The inhibitory effect (and numbers of bacteria within each group) was consistent during the 3 days of the storage.

Table 2.

Effect of Caprylic Acid on Number of Salmonella a Recovered from Chicken Skin During 3-Day Storage

	Storage (day)
Concentration of caprylic acid (mg/mL)	1	2	3
Salmonellas
0^b	6.58±0.45c	6.31±0.39c	6.38±0.40c
1.25	5.63±0.42d	5.32±0.33d	5.08±0.57d
2.5	4.32±0.38e	4.12±0.41e	4.86±0.66e

Mean of three measurements in triplicates±SD, log₁₀ colony-forming units/g skin.

Samples were treated with sterile distilled water instead of the acid.

c,d,e

Values in the same column with the same superscript are not significantly different (p≥0.05). Cell concentration <100/mL was considered as 100/mL in statistical calculations.

Sensory evaluation of baked chicken meat revealed no significant differences in appearance, odor, taste, and overall acceptability between control chicken carcasses and those treated with CA (Table 3). Scores of odor and appearance of chicken skin revealed some statistically significant changes (p>0.05), in both tested concentrations of CA. Taste and overall acceptability, however, decreased only nonsignificantly (p>0.05).

Table 3.

Sensory Evaluation of Baked Chicken Meat and Skin Treated with Caprylic Acid (CA) ^*

	Odor	Appearance	Taste	Overall acceptability
Meat
Control	5.9^a	5.5^a	5.5^a	5.6^a
CA 1.25 mg/mL	5.5^a	5.4^a	5.1^a	5.2^a
CA 2.5 mg/mL	5.6^a	5.5^a	5.0^a	5.0^a
SEM	0.5	0.4	0.4	0.5
Skin
Control	5.3^a	5.1^a	4.5^a	4.6^a
CA 1.25 mg/mL	4.3^b	4.0^b	4.3^a	4.3^a
CA 2.5 mg/mL	4.0^b	3.8^b	4.0^a	4.2^a
SEM	0.3	0.3	0.5	0.4

All traits were assessed by a 10-member panel on a scale of 1=very undesirable and 7=very desirable. Values are expressed as least-square means.

a,b

Values in the same column with different superscripts are significantly different (p≤0.05).

SEM, standard error of the mean.

Discussion

Intestinal presence of Salmonella Enteritidis in chickens is a common phenomenon, and although it is rarely associated with clinical signs of illness, it can be the source of salmonellosis in humans, via the contaminated meat (Hinton, 1986). Feed is the major source of Salmonella Enteritidis in poultry (Al-Natour and Alshawabkeh, 2005). The artificial contamination of chicken feed was chosen as a source of the infection, to mimic the natural conditions. A comparable method was used, for example, by Fan et al. (2014). The authors used polymerase chain reaction (PCR) to detect Salmonella Enteritidis in tissues and in formation of eggs in hens, with the highest percentage of Salmonella Enteritidis–positive samples found in cecum. However, bacterial numbers were not estimated by the authors. In the study by Andino et al. (2014), real-time quantitative PCR was used, followed by relative quantification. The authors found strain-specific variation in Salmonella survival in feed. Again, bacterial numbers were not determined by plating.

It has been proposed that fatty acids supplementation in feed will provide an antibacterial effect in the crop, but will have no effect further down in the gastrointestinal tract (Thompson and Hinton, 1997). In our experiments, the numbers of Salmonella Enteritidis decreased below the detection limit both in the crop and cecum (Table 1). It is not possible to specify whether the antibacterial activity of CA against Salmonella Enteritidis was stronger in crop or in cecum; however, its effect was apparent along the whole gastrointestinal tract. The ability of CA added to broiler-chicken feed to reduce bacterial counts along the gastrointestinal tract was also observed by Johny et al. (2009), where the addition of higher concentrations of CA (7 g/kg and 10 g/kg) to feed by crop gavage decreased the numbers of Salmonella Enteritidis in the gastrointestinal tract of chickens infected with Salmonella Enteritidis.

An antibacterial effect of CA was observed in previous in vitro observations (Skrivanova et al., 2004), where the susceptibility of Salmonella spp. to 15 fatty acids was determined. CA was the only acid that inhibited bacterial growth. Inhibitory activity of CA on Salmonella Enteritidis in autoclaved chicken cecal contents was studied by Vasudevan et al. (2005). Concentrations of 50 and 100 mM (7.2 and 14.4 mg/mL, respectively) reduced the bacterial population by 5 orders of magnitude (5 log₁₀ CFU/mL) within 1 min of incubation, with a complete inactivation of the pathogen within 24 h. Emulsions of 1.25 mM monoglyceride of capric acid at pH 4–5 caused a 6–7 log₁₀ reduction of Salmonella spp. in 10 min; however, no reduction was observed at neutral pH (Thormar et al., 2005). The mechanism of action of medium-chain fatty acids has not yet been fully elucidated. However, the experiments that aimed to estimate the effect of medium-chain fatty acids on the gastrointestinal microbial community revealed that even though the mechanism of antibacterial action remains unclear, the changes of a bacterial population are not confined to one specific group of bacteria, but rather affects a number of species (Skrivanová et al., 2010; Solís de los Santos et al., 2010).

Broiler chickens entering the processing facilities possess a wide variety of microorganisms on the skin (Kotula and Pandya, 1995; Hinton and Cason, 2008). In some countries, chlorine water is used as a sanitizer in poultry processing (Directive 6355.1 of the Food Safety and Inspection Service of the U.S. Department of Agriculture dated September 23, 1996) (USDA-FSIS, 1996). However, the widespread use of easily available chlorine as a disinfectant in food processing has raised safety concerns regarding its carcinogenic byproducts and their potential incorporation into the food (Russell and Keener, 2007). In the United States, organic-acid rinses have been approved by the Food Safety and Inspection Service of the U.S. Department of Agriculture (Directive 6340.1 dated November 24, 1992) (USDA-FSIS, 1992). CA is a nontoxic, naturally occurring constituent of many foods, belonging to the GRAS substances.

It has been shown previously that CA can be used for reduction of salmonellas in meat products. Moschonas et al. (2012) observed that treatment with 0.5 and 1.0 % (wt/vol) of CA significantly reduced counts of salmonellas in surface-browned but uncooked frozen breaded chicken products. However, there was no mention of a sensory evaluation being performed in the aforementioned study. The alternative for a surface treatment with CA, without affecting the sensory traits, can be the use of CA monoacylglycerol, its sucrose ester, or other corresponding derivative.

Conclusions

With the aim of preventing a foodborne salmonellosis in humans, CA can be used as a dietary supplement for broiler chickens. Another option, in some countries, can be the surface-treatment of chilled broiler carcasses with CA. A possible use of CA derivatives has been proposed to reduce the risk of affecting the sensory traits of the chicken skin.

Footnotes

Acknowledgments

This study was supported by the projects MZeRO07014 (Ministry of Agriculture of the Czech Republic) and CIGA20142014 (Czech University of Life Sciences in Prague).

Disclosure Statement

No competing financial interests exist.

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