Lauric acid as feed additive – An approach to reducing Campylobacter spp. in broiler meat

The increasing prevalence of Campylobacter spp. within broiler populations is a major problem for food safety and consumer protection worldwide. In vitro studies could already demonstrate that Campylobacter spp. are susceptible to lauric acid. The purpose of this study was to examine in vivo the influence of lauric acid as a feed additive on slaughter parameters, muscle fatty acid profile, meat quality traits and the reduction of Campylobacter coli in inoculated meat of Ross 308 (R308) and Hubbard JA 757 (HJA) broilers in three independent trials (n = 3). Although slaughter parameters did not show any significant differences, the fatty acid profile of both breeds revealed significantly higher lauric acid concentrations (P < 0.0001) in the Musculus pectoralis superficialis of treated broilers. Comparing both tested breeds, R308 test broilers had significantly higher lauric acid concentrations than HJA test broilers (P < 0.0001), indicating a higher conversion rate in those animals. The meat quality traits showed no differences in the R308 breed (P > 0.05), but HJA test broilers had higher values for drip loss, electrical conductivity, CIE color values L* and b*, and lower pH values. The inoculation trials of R308 showed that initial bacterial loads of 5.9 log10 cfu/g were reduced during six days of storage (4°C) to approximately 4.3 log10 cfu/g in the control groups compared to 3.5 log10 cfu/g in the treatment groups (P = 0.0295), which could be due to antimicrobial effects of lauric acid within the muscle. This study therefore suggests that lauric acid as a feed additive has the potential to improve food safety by reducing the numbers of Campylobacter coli in broiler meat. However, this effect seems to be dependent on the breed determining the feed intake capacity, the fat deposition and therefore the ability to incorporate lauric acid in the muscle.


Introduction
Nowadays the infection of animals and the contamination of their relating food products with zoonotic pathogens are attracting an increasing amount of consumer attention. While the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 number of infections with Salmonella spp. is continuously decreasing as a result of many successful control programs, the cases of Campylobacteriosis surged to a particularly high level of about 70,000 infections in Germany in 2015 [1]. Campylobacter spp., mainly C. jejuni, C. coli and C. lari, were responsible for more than 230,000 cases in the European Union in 2014 [2]. Campylobacteriosis is therefore the most frequently identified foodborne bacterial zoonosis. The main cause of infection originates from poultry being heated inadequately or handled without the necessary caution in the kitchen [3]. As cross-contamination and poor hygiene are believed to be the main reasons for contamination with Campylobacter spp. at abattoirs [4,5], many studies deal with approaches to improving the hygiene management or processing technology in those facilities [6][7][8]. In this context, the European Food Safety Authority [2] reported that 38.4% of about 6,700 samples collected from abattoirs, processing plants, and retail shops in 2014 were Campylobacter-positive. Ways to optimize processing methods are, for example, the use of elevated scalding temperatures [9], or improved chilling methods [10] as these two steps are crucial for reducing the bacterial load. With the aim of reducing the prevalence of Campylobacter spp. in broiler flocks, some feeding studies investigated organic acids or medium chain fatty acids (MCFA) as additives in drinking water because of their antimicrobial potential [11][12][13]. Some studies suggested that there might be an impact of feeding medium chain fatty acids on the reduction of Campylobacter jejuni [14,15]. In vitro trials with lauric acid showed that this MCFA has the potential to decrease Campylobacter jejuni [16]. However, to our knowledge, there are no in vivo studies that have investigated the effects of lauric acid as a feed additive regarding Campylobacter spp. prevalence and meat quality. Therefore, the aim of the present study was to investigate the effect of adding palm kernel fatty acid distillates, which contain high amounts of lauric acid, to the diet of two different broiler breeds. The influence on different slaughtering characteristics, meat quality traits as well as the impact on the fatty acid profile of the breast muscle were determined. Regarding antimicrobial effects, an inoculation experiment with Campylobacter coli on the Musculus pectoralis superficialis was conducted.

Material and methods
Animal experiments were performed in accordance with the German Animal Welfare Act, and approved by the Ethics Committee for Care and Use of Laboratory Animals of the Lower Saxony State Office for Consumer Protection and Food Safety Lower Saxony LAVES (approval number 33.9-42502-05-15A500).

Rearing
For this study a fast growing (Ross 308) and a slower growing (Hubbard JA 757) chicken breed with a total number of 180 birds were examined. Each breed consisted of 30 broilers being reared under comparable conditions within three independent trials (n = 3) at the Institute for Animal Nutrition, University of Veterinary Medicine, Foundation, Hannover, Germany. To minimize the influences of potential differences in the rearing conditions the groups were randomly assigned to the different compartments in the barn with a density of 23.3 kg/ m 2 mean metabolic bodyweight at the end of the trial. In the first week, the broilers were fed a common starter feed followed by a commercial grower feed for the following two weeks. After that, the breeds were randomly assigned to two subpopulations (15 broilers, respectively) in order to provide specific diets during the finishing period. The basic finisher feed was a common pellet diet which had been reduced by 2% in its fat content, which was supplemented by 5% of a commercial standard fat (C 12 < 6%) in the case of the control group. In contrast, the test group was finished with a supplement of 5% fat enriched with palm kernel fatty acids including high levels of lauric acid (C 12 = 42-53%) ( Table 1). Water and feed were provided ad libitum and controlled twice a day. The feed intake was calculated by subtracting the backweights from the initial weight of food on a daily basis. The project was performed in accordance with the German Animal Welfare Act.

Slaughtering and sampling
At the age of 41 d (first and third trials) or 42 d (second trial), the broilers were slaughtered. Stunning was performed by a percussive blow to the head and bleeding by ventral neck cutting. Live weight was taken before slaughtering. Immediately after slaughtering, the Musculi pectorales superficiales (MPS) were obtained and stored at + 4˚C. During cutting, carcasses, thighs and breast muscles were weighed. Twelve left MPS of each breed and trial (in total 72) were minced with a Grindomix (GM 200, Retsch1, Germany), vacuum packed and stored frozen at -20˚C until further analysis of the fatty acid profile. Drip loss measurements. The drip loss was determined 24 h p. m. After wiping the left PM muscle, they were stored in individual plastic boxes at + 4˚C for 48 hours. The drip loss in percentage was calculated as the weight difference between the samples before and after storage.

Inoculation experiments
Sampling. At 24 h p. m., two samples of 25 g, respectively, were obtained from the right MPS. Both samples were stored in plastic boxes until further processing. One colony of a Campylobacter coli strain (DSM 4689) was transferred to 10 mL Bolton broth (Oxoid GmbH, Wesel, Germany) and incubated 44±4h under microaerophilic conditions. The final amount of bacteria was approximately 8 log 10 colony forming units (cfu) per milliliter. Inoculation. For spiking, 0.1 mL per sample of the inoculum was transferred to the surface. Immediately after inoculation one of the two meat samples was conveyed to a stomacher bag with buffered peptone water in a 1:10 dilution for quantitative analysis as a control for the inoculation dose at the outset of the experiment (day 1). After homogenization in a laboratory paddle blender (Stomacher 1 400 circulator, Seward Ltd., United Kingdom) at 230 rpm for two minutes, 0.1 mL of the suspension was surface-plated in duplicate on modified Charcoal Cefoperazone Deoxycholate Agar (Oxoid GmbH, Wesel, Germany). The plates were incubated for 48 h under microaerophilic conditions (10% CO2, 5% O2, 85% N2) at 41.5˚C. Enumeration was carried out in accordance with ISO 10272-2:2006. The remaining sample was quantitatively analyzed after a storage period of six days at + 4˚C in the same manner as mentioned previously (day 7). Samples below the detection limit of 100 cfu/g were included in the calculation as half of the detection limit (50 cfu/g).

Fatty acid content
The minced meat of the left MPS was freeze-dried in a lyophilization unit (Gamma 1-20, Christ 1 , Osterode, Germany), pulverized in a mixer mill (MM 400, Retsch 1 , Haan, Germany) and analyzed by gaschromatography (GC TRACE 1300, ThermoScientific 1 , Dreieich, Germany; SP-2560 Column, Supelco, Bellefonte, USA; carrier gas: nitrogen) with a modified method according to Lepage and Roy [17]. In brief, a methanol-hexane-tridecanoic-acid mixture was utilized as standard. Subsequently, acetyl chloride was added and the sample was heated, followed by the addition of potassium chloride solution. The measurement was carried out after centrifugation with the superior hexane phase.

Statistical methods
Statistical analysis was performed by using SAS (Statistic Analyzing Software, version 7.1, Cary, NC, USA). The normal distribution was tested by the Shapiro-Wilk-Test. The significant differences concerning the breeds and fat additions or interactions in between were determined by a two-way analysis of variance (ANOVA). Unless otherwise stated, P-values below 0.05 were considered significant.

Slaughtering characteristics
Both R308 and HJA showed no differences in the slaughtering characteristics between the control and the test group (P > 0.05) ( Table 2). These results are in accordance with Zeitz, Fennhoff [18], who also found that feeding lauric acid had no negative impact on performance characteristics. In contrast, van der Hoeven-Hangoor, van der Vossen [19] found that it is possible to enhance broiler performance by adding medium chain fatty acids (capric and lauric acid) to the diet. However, when comparing both breeds, R308 had significantly higher values for the live, carcass, thigh and breast muscle weights (P < 0.001) ( Table 2). This was due to the higher feed intake capacity of R308 (56017 g ± 4216 for the control and 54267 g ± 3773 for the test group) compared to the feed intake capacity of HJA (40017 g ± 2705 and 40197 g ± 5000 for control and test group, respectively) ( Table 3). These findings are in line with Acar, Moran [20] and Mehaffey, Pradhan [21], who also found that different breeds have altering performance characteristics.

Meat quality traits
There were no differences between the test and the control group of the R308 breed for the meat quality traits drip loss, EC, pH, L Ã , a Ã , and b Ã (P > 0.05) ( Table 4). Concerning the HJA breed, drip loss and EC (1.30 ± 0,90% and 9.40 ± 3,01 mS, respectively) of the test group were higher compared to the control group (1.03 ± 0,28%, 7.38 ± 2,8 mS) (P < 0.05), while the pH values of the test broilers was lower than the pH values of the control group (P < 0.05) ( Table 4). Several studies [22][23][24] postulated that drip loss is positively correlated to EC and negatively correlated to pH, which is in line with the present observations. The water-holding capacity is influenced by pH because of the protein denaturation, which occurs p. m. as a consequence of the release of H + -ions [25] and because of the physiologic, and enzymatic degradation of cell membranes [26]. Correspondingly, EC is also related to the relocating of ions and cell fluids by the degradation of cell membranes [22]. The observation of differences between the diet groups in the HJA breed was unexpected because R308 had higher feed intake and consequently higher intake of lauric acid than HJA. Thus, the assumption was that, if there were significant differences they would have been found in the R308 breed. The addition of lauric acid appears to have only a significant influence on the pH value and therefore on the other meat quality parameters in the HJA breed. Reasons for this difference in the lauric acid effect between the two breeds are not known and further investigations are necessary. The MPS of the test group had higher L Ã and b Ã values than the controls (P < 0.05), but there was no difference observed for a Ã values (P > 0.05) ( Table 4). Otto and Roehe [24] found that paler meat is related to higher drip loss, which is in keeping with our results. This lighter color is due to the fact that higher drip loss and the protein denaturation alters the light scattering on the surface of meat.

Fatty acid profile
The analysis of the fatty acid profile clearly showed higher contents of lauric acid (C 12) within the breast muscles of the test groups than both control groups (P < 0.0001) ( Table 5). This is most probably the result of the test diet containing more than forty times higher concentrations (20.9 g/kg feed dry matter) of lauric acid than the control diet (0.5 g/kg feed dry matter) which was incorporated in the muscle matrix. Our results are consistent with those reported by Roth and Ristic [27] who also showed that it is possible to alter the fatty acid profile of broilers by providing a particular diet of rapeseed oil.
Regarding the potential of utilizing lauric acid, R308 broilers seem to have a higher conversion rate as they had two-fold higher levels of absolute lauric acid content in their MPS compared to the HJA breed (P < 0.0001), whereas no difference between the control groups occurred (P = 0.8751) ( Table 5). This could be a result of the higher feed intake, as described above, and thereby related higher intake of lauric acid of R308, being a fast growing breed with exalted feed intake performance compared to HJA. In addition, the R308 test group had a higher total fat content (82.27 g/kg) compared to the HJA test group (51.14 g/kg) and therefore higher absolute lauric acid levels (7.7% and 6.7% for R308 and HJA, respectively) ( Table 5). This leads to the assumption that lauric acid could be more effective in breeds that are inclined to have higher fat depositing abilities because the higher the total fat content in the muscle, the higher the absolute lauric acid levels could be. Table 5. Mean values (± standard deviation) of the fatty acid profile (left MPS) of Ross 308 (R308) and Hubbard JA 757 (HJA) broilers fed with 5% commercial fat addition (control group) and broilers fed with 5% supplement of fat with high lauric acid content (test group) (n = 3).

Inoculation
As expected the starting values of day 1 did not vary (P > 0.05) between the test and control group, with an average value of approximately 6 log 10 cfu/g in both breeds (Fig 1). This bacterial count of day 1 served as a control for the adequate procedure of this inoculation experiment. The value was approximately 1 log 10 higher than average counts that are usually found in commercial broiler slaughterhouses on naturally contaminated carcasses of Campylobacter positive flocks [28]. After six days of storage (day 7), the microbial loads of C. coli of the R308 breed control group dropped from approximately 5.7 log 10 to 4.3 log 10 cfu/g, while the test group showed a higher reduction of initially 5.9 log 10 to 3.5 log 10 cfu/g (Fig 1). As a result, the bacterial loads of the test groups were significantly lower after storage time (P = 0.0295). As for the HJA breed, there was also a clear decrease from initially 6 log 10 cfu/g (both control and test groups) to levels of 3.5 log 10 cfu/g (control) and 2.8 log 10 cfu/g (test) (Fig 1). However, in this case, only a tendency of lower values in the test group after storage time was distinguishable (P = 0.0685). The sensitivity of Campylobacter spp. towards their growth conditions explains the drop in germ counts during refrigerated storage. This observation is in accordance with investigations by Chan, Tran [29] and Oyarzabal, Oscar [30] who showed a reduction of Campylobacter spp. at low temperatures (4˚C) as well. Our study shows that it is possible to reduce these bacteria on the meat by approximately 1 log 10 cfu/g by adding lauric acid to the diet. This could be attributed to the higher content of lauric acid in the breast muscle meat. These results are in line with the in vitro described ability of lauric acid to reduce Campylobacter jejuni [16]. The exact antimicrobial mechanism of lauric acid cannot be clarified in this study. This medium chain fatty acid is assumed to have the ability to destabilize the cell-membrane, followed by cell-degeneration. For Salmonella spp. and Escherichia coli this was visible in disorganized cytoplasm, while Clostridium perfringens showed a detachment between the inner side and outer surface of the cell-membrane. A change in the permeability of K + -ions was not at the beginning (day 1) and end (day 7) in log 10 cfu/g of Ross 308 (R308) and Hubbard JA 757 (HJA) broilers fed with 5% commercial fat addition (control group) and broilers fed with 5% supplement of fat with high lauric acid content (test group). The asterisk (*) marks significant differences (P < 0.05). There were no significant differences between day 1 and 7 and within the feeding groups (P > 0.05) (n = 3). https://doi.org/10.1371/journal.pone.0175693.g001 detectable [31,32]. Similar findings were made concerning Campylobacter jejuni by Molatova, Skrivanova [16], who also revealed disorganization of cytoplasm of C. jejuni cells after treatment with lauric acid.

Conclusion
We therefore conclude that lauric acid as a feed additive could possibly improve the food safety of broiler meat because of its ability to reduce the bacterial load with Campylobacter coli. However, broiler breed specific characteristics have to be taken into account as there were considerable differences between the breeds tested in the present study. Further research has to investigate these influences and clarify whether these observations are transferable to other food-borne pathogens like Salmonella spp. or Listeria spp. which are able to grow during refrigerated storage [33].