Abstract

This study evaluated the effectiveness of spray washing with aqueous based solution of cinnamaldehyde (CA) and CA plus lactic acid (LA) in reducing aerobic bacteria, Enterobacteriaceae, E.coli and Salmonella from the grain surface of bovine hide so that the developed solutions can potentially be used to decontaminate the cattle carcasses to ensure meat safety. This study also examined the application impacts of the developed formulations on leather produced from the treated hides recognizing the value of cattle byproducts. Two concentrations (0.5% and 0.75%) of CA and LA were used to develop the formulations for 2 to 5 minutes treatment. Research data revealed the fresh cattle hides washed with water alone (control) resulted in recovery of aerobic bacteria of 7.39 and 9.30; Enterobacteriaceae of 5.43 and 5.42; E.coli of 4.88 and 5.50 and Salmonella of 3.81 and 4.65 log CFU per leather panel at 2 and 5 minutes of treatment respectively. Comparing to control, hides treated with CA solution alone resulted in the highest reduction of aerobic bacteria, Enterobacteriaceae, E.coli and Salmonella up to 2.22, 0.42, 0.72 and 1.61 Log CFU respectively for 2 to 5 minutes treatment. The treatment with the formulations of CA plus LA resulted in the highest reduction of aerobic bacteria, Enterobacteriaceae, E.coli and Salmonella up to 2.12, 3.12, 2.33 and 2.28 log CFU respectively for 2-5 minutes of treatments. From microscopic analysis, mechanical and subjective examinations, it was revealed that the leather produced from the formulation treated hides were comparable to the control in terms of structural integrity.

1. Introduction

Enteric pathogenic bacteria serve as significant hazards and pose a substantial challenge to the meat industry as well as public health. Such pathogens may incorporate into and onto surfaces of cattle to include skin, hair, attached manure and mixed biofilms, thus limiting cleaning and decontamination efficacy ^{1, 2, 3, 4, 5}. Survival of such harbored bacteria can facilitate cross-contamination of the underlying meat and meat processing equipment in an arbitrator ^{2, 6, 7}. Furthermore, washing, cleaning and decontamination with water only has been evaluated and shown to have minimal effects on bacterial populations ⁴. In order to intensify the efficacy of current antimicrobial treatments to overcome contaminations, a concentrated antimicrobial treatments may be adopted but could adversely affect the quality of the outer grain surface of cattle, a valuable byproduct and commodity of the meat industry.

Microbial contamination of meat carcasses occurs during the conversion of live animals to meat ^{8, 9}. Muscles within the animal have been noted to be essentially sterile ¹⁰, however, workers and equipment can spread microbial contamination from the hide to the carcasses meat surface during processing ^{11, 12, 13}. Thus, it is important to decrease pathogens on cattle hides to reduce the risk of human exposure to these pathogens from meat carcasses. Cattle hide contamination with pathogens such as Salmonella, Escherichia coli and other Enterobacteriaceae (ENT), and related gram negative bacilli (gnEB), may arise from environmental exposures including adherent soil, manure and straw during their lifespan where they are continuously exposed to potentially harmful microorganisms, which may become firmly lodged onto their hides and hair ^{1, 2, 4}. The major food-borne pathogens such as Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes colonize the gastrointestinal tract of cattle and are shed in the manure, thereby leading to pathogen contamination and persistence on hides for long lime ¹⁴. While investigating antimicrobial applications on carcass prior removal of hides for the meat industry to ensure food safety it is also important to take into account that the novel formulations have no detrimental impact on the outer grained hide/skin surfaces. The damage on outer grained surface of hide/skin during meat processing may lead to detrimental financial losses because animal hides/skins are value added byproducts for the meat industry which are used to produce items such as leather.

Currently, limited research has been conducted under either experimental or commercial conditions on the antimicrobial effectiveness of hide decontamination treatment although some researchers recognize that hide interventions are the most effective means to reduce pathogens on meat. The reported techniques for decontamination of bovine hides include, pre-slaughter animal washing, ¹⁵, steam condensation ¹⁶, hot water rinse/chemical dehairing ¹⁷, hair removal with wax ¹⁸, antimicrobial washing, ¹⁹, ozonated and electrolyzed oxidizing water ²⁰, hair shaving ²¹ and application of bacteriophages ²². The ultimate goal of all hide treatment techniques is to stop microorganisms transferring from hide surface to the underlying meat.

This study was designed to evaluate the effectiveness of low concentrations of cinnamaldehyde (CA)/ Lactic acid (LA) spray washing formulations for food safety by reducing pathogens on the haired surface of cattle hides while evaluating the effects on value added byproducts of the meat industry. Lactic acid is generally recognized as safe (GRAS) by the Food and Drug Administration and has been approved by the Food Safety and Inspection Services (FSIS) for usage in a hazard analysis and critical control points (HACCP) design plan up to 5% as an antimicrobial to decrease pathogenic contamination on surfaces of meat including whole or cut meat, carcasses, parts, trim, and organs, as a wash, spray, rinse, dip, chiller water or scald water, pre and post chill ²³. CA is a natural oil phenolic aldehyde antimicrobial obtained from cinnamon bark that can interfere with biological processes. The carbonyl bond in CA is highly polar and due to the polarity of this bond carbon atom becomes electrophilic and reactive toward nucleophiles such as nitrogen containing structures (DNA, proteins) through their amine groups and thus disrupting the metabolic function of microorganism ²⁴. Such natural oils reduce activity and replication rates, changes in the cell morphology, motility and extrusion of intracellular material of bacteria. Furthermore, CA has shown to inhibit the formation of biofilms at below minimum inhibitory concentrations ^{24, 25, 26}.

2. Materials and Methods

Fresh de-fleshed cattle hides were acquired from a local meat packing and processing facility, JBS Packerland (Souderton, PA). For the experiments, hide pieces were cut into 12 in x12 in panels from backbone area of a whole bovine hide for subsequent spray wash treatment with the individual formulations. For bacterial recovery from treated hides, random 10 in x 5 in surfaces of the hide panels were designated and swab samples were collected after 2 min and 5 min of treatment. To illustrate structural characteristics which facilitate bacterial survival and attachment a metal stamp was used to punch out a 0.5in stamps from cattle hides. One set of stamps was non-inoculated while one set was inoculated with 1 microliter of a 10⁸ Log/ml cocktail of 3 Salmonella spp. (S. anatum, S. saint-paul and S. Typhimurium) and allowed to sit for 20 mins.

Commercial grade chemicals used for all testing formulations. cinnamaldehyde ≥95% and LA ≥ 85%, were purchased from Aldrich Chemical (Milwaukee, WI). All additional reagents used for preparations of formulations were of the highest purity available from commercial suppliers. Tap water was used as a control in addition for formulations where prepared of 0.50% CA in 0.20% Tween-20 aq. solution brought to a pH of 6.89; 0.75% CA in 0.20% Tween-20 aq. solution brought to a pH of 6.75; 0.50% CA + 0.50% LA in 0.20% Tween-20 aq. solution brought to a pH of 2.77; and 0.75% CA + 0.75% LA in 0.20% Tween-20 aq. solution brought to a pH of 2.77. All formulations where dissolved in tap water at room temperature (~21°C) and prepared ~24 hours prior to experimental spray applications on hides.

For antimicrobial testing the following formulations, and (control) tap water, (Solution A) of 0.50% CA in 0.20% Tween-20 aq.; (Solution B) 0.75% CA in 0.20% Twee-20 aq.; (Solution C) 0.50% CA + 0.50% LA in 0.20% Twee-20 aq.; (Solution D) 0.75% CA + 0.75% LA in 0.20% Twee-20 aq. were applied to the haired surface of individual hide panel using a hand held 1000 ml polyethylene spray bottle. A certain amount of 20 mL (20 puffs) of individual formulation was sprayed on each panel, adequate enough to cover the 12 in x 12 in surface area (panel size). The individual formulation was allowed to sit for ~5min before two separate 10 cm x 5 cm surface areas were swabbed from the same panel at 2 and 5 min respectively for microbial testing. After treatment, all hide panels were washed separately in a 6-in-1 drum set-up (Dose Maschinenbau GmbH, Lichtenau, Germany) for 2h using the USDA hide washing protocol (100% water, 0.15% Boron TS, and 0.10% Proxel) as a first step of leather processing.

The independently 10cm x 5cm spray washed areas treated with the selected formulations were aseptically swabbed with a sterile sponge and placed into a corresponding sampling bag with 25ml of buffered peptone water for analysis (Nasco Meat and Turkey Carcass Sampling Kit, Salida, California). The sample bags were then hand massaged for ~2 min. Samples were serially diluted and spread-plated on Tryptic Soy Agar (TSA), Xylose-Lysine-Tergitol 4 (XLT-4) Agar, MaConkey Agar (MAC), Sorbitol MacConkey Agar, with Cefixime and Tellurite (CT-SMAC) for aerobic bacteria, Enterobacteriaceae (ENT) and related gram-negative bacilli (gnEB), Salmonella and E. coli counts, respectively (all agar was obtained from Fisher Scientific, Pittsburg, PA). After spread plating, samples were incubated between 24-48 h at 37°C and bacterial colonies were enumerated for bacterial recovery with the lowest detection level at 1 CFU per 10cm x 5cm area.

After washing separately, the 12in x 12in formulation treated panels were placed in a dehairing drum and the control panel was placed into a separate individual drum. All hide panels were de-haired per the USDA tanning protocol ²⁷. Subsequent to dehairing, all panels were combined into one drum for pickling, tanning, re-tanning, coloring, and fat liquoring steps. The samples were tanned into crust upper shoe leather following the standard USDA tanning procedures ²⁷. The resulting panels were remained at temperature (21°C) and humidity (50% relative humidity) in a controlled environmental chamber (Caron Environmental Chamber, Marietta, OH) until further subjective and mechanical testing and stereo microscopy analyses were conducted.

To assess the effects of the developed formulations on byproduct quality, the mechanical properties of the processed leather were measured. The mechanical properties including tensile strength, Young’s Modulus (“stiffness”), elongation (“stretchability”), and fracture energy (“energy required to open unit area of crack surface”) were conditioned and tested as per the ASTM methods D1610 and D2209. Five dumbbell shaped leather samples were cut from each leather panel following the protocol in ASTM D2209 parallel to the backbone. The average thickness of the leather sample ranged from 2.0 mm to 2.7 mm. An Insight-5 test frame and Testworks-4 data acquisition software (MTS Systems Corp., Minneapolis, MN) were used to evaluate the mechanical properties of the leather samples. The strain rate and the grip distance for this study were set to 24.5 cm/min and 10.16 cm respectively. Samples were tested in a room set at 23±3 oC and 50±5 % relative humidity. Leather subjective tests (break, handle, fullness, color and general appearance) were conducted by an experienced tanner in our labs. Each value was graded from 1 to 5 for each testing parameter, with 1 being the lowest quality and 5 being the highest quality. From these ratings, an overall evaluation (general appearance) was determined from 1 to 5.

Representative leather panels subjected to spray treatment with individual formulation were inspected under a stereo microscope (Nikon Digital Microscope SMZ-2T, Melville, NY) to determine any detectible changes in the hide grain structure from the treatment. Scanning electron microscope (SEM) images were taken to identify potential finer structural changes in the surface and fibers of the leather. For SEM images, samples were mounted on stubs and sputter gold coated for 1 minute (EMS 150R ES, EM Sciences, Hatfield, PA). Samples were viewed with a FEI Quanta 200 F Scanning Electron Microscope (SEM), (Hillsboro, OR, USA) with an accelerating voltage of 10KV in high vacuum mode.

Based on a minimum of three replications per treatment, log-values of microbial populations were analyzed by One-way analysis of variance, using SPSS software (version 14.0, SPSS Inc., Chicago, IL). To compare treatment group differences against the control group (water treatment alone), Dunnett’s post-hoc analyses were conducted.

3. Results and Discussion

Figure 1A illustrates an SEM image of an un-inoculated stamp of the outer grained hide of cattle. The image displays rough jagged edges and biofilm formulations on the surface and hair of the cattle. Further, Figure 1B shows the outer grained hide of cattle inoculated with a cocktail of Salmonella spp. This image illustrates the capability of pathogens such as Salmonella ability to attach to hide surfaces and incorporating into preexisting biofilms. Hygiene of the haired surface of cattle will continue to challenge the effort for meat safety. As meat carcasses are essentially sterile it is understood that meat-borne illness cases are highly associated with hide contamination where the pathogens can transfer from the hide surface to the underlying meat during processing. Bacteria are known to attach to various topographies, which may include hides, may be riddled with filth which maybe harboring pathogens ^{2, 28, 29}. To increase the effectiveness of antimicrobials and overcome the protective nature of the environmental materials which harbor the bacteria, concentrations of antimicrobials may be increased but need to be properly evaluated. Such increases may cause damage to the hide and result in high levels of antimicrobial residuals.

Aerobic bacterial recovery of 7.38 ± 0.43 and 9.30 ± Log/CFU was enumerated from 50 cm² surface area when un-inoculated hide panels were spray washed with tap water alone (control) at 2 and 5 minutes, respectively (Figure 2). On hide panels treated with 0.50 % CA alone aerobic bacterial recovery was enumerated at 6.87 ± 0.17 and 7.17 ± 0.18 log/CFU at 2 and 5 mins of treatments respectively. When CA treatment was increased to 0.75%, aerobic bacterial recovery reduced to 6.57 ± 0.15 and 5.32 ± 0.15 respectively at 2 and 5 mins of treatment (Figure 2). In a spray wash combined formulation of 0.50% CA and 0.50% LA aerobic bacteria recovery was 5.48 ± 0.08, 7.08 ± 0.01 log/ CFU. Furthermore, when hide panels were spray washed with a combined formulation of 0.75% CA and 0.75% LA bacterial recovery further reduced to 5.26 ± 0.16 and 5.59 ± 0.04 log/ CFU at 2 and 5 minutes (Figure 2). Within each treatment, time was a factor on bacterial recovery except at the 0.50% CA (P ≤ 0.001). At a concentrations of 0.50 % CA, time was not a significant factor (P = 0.60). However, at 2 and 5 mins of treatment, each formulation were significantly different than its corresponding control (P ≤ 0.003). Results indicate that CA and CA + LA formulations have the ability to effectively reduce the overall number of aerobic bacteria on hide surfaces.

This test revealed that spray washing with tap water alone resulted in ENT/gnEB recovery of 5.43 ± 0.38 and 5.43 ± 0.29 Log/CFU from un-inoculated hide at 2 and 5 minutes, respectively (Figure 3). Panels treated with 0.50% CA alone ENT/gnEB recovery was enumerated at 5.69 ± 0.07 and 5.00 ± 0.00 log/ CFU at 2 and 5 minutes of treatments respectively. CA treatment of 0.75% alone resulted in recovery ENT/gnEB of 5.10 ± 0.17 and 5.53 ± 0.34 respectively at 2 and 5 mins of treatment (Figure 3). For a combined formulation of 0.50% CA and 0.50% LA, ENT/gnEB recovery was enumerated at 2.71. ± 0.12 and 3.79 ± 0.69 Log/CFU at 2 and 5 mins respectively. When 0.75% CA and 0.75% LA combination formulation was used bacterial recovery was enumerated at 2.31 ± 0.25 and 3.78 ± 0.69 Log/ CFU at 2 and 5 minutes respectively (Figure 3). When water alone was used time was not a significant factor in bacterial recovery (P = 0.68). At 2 minute of treatment no significant difference was observed between the control and treatments of 0.50% CA and 0.75% CA (P ≥ 0.54). Similarly, at 5 minutes of treatment no significant difference in ENT/gnEB recovery was observed between the control and 0.75% CA formulation (P ≥ 0.58). However, both combined formulations of 0.50% and 0.75% CA + LA resulted significant reduction in bacterial recovery in compare to control. It was interesting to see that in some cases, at 5 minutes treatment higher recovery was observed than at 2 minutes of treatment. This is probably either due to the individuality of naturally collected hide panels or reducing action of antimicrobials with time.

In this study spray washing with tap water alone resulted in E.coli recovery of 4.88 ± 0.10 and 5.50 ± 0.08 Log/CFU from un-inoculated hide at 2 and 5 minutes, respectively (Figure 4). Panels treated with 0.50% CA alone E. coli recovery was enumerated at 5.46 ± 0.11 and 5.39 ± 0.34 Log/CFU at 2 and 5 mins of treatments respectively. CA treatment of 0.75% alone resulted in E.coli recovery of 4.16 ± 0.34 and 5.36 ± 0.10 respectively at 2 and 5 mins of treatment (Figure 4). For a combined formulation of 0.50% CA and LA, E.coli recovery was enumerated at 5.34. ± 0.24 and 5.48 ± 0.48 log/ CFU at 2 and 5 mins respectively. When 0.75% CA and 0.75% LA combination formulation was used bacterial recovery was enumerated at 2.55 ± 0.16 and 3.48 ± 0.60 log/ CFU at 2 and 5 minutes respectively (Figure 4). Additionally, results show that treatment with the concentrations of 0.50% CA and 0.50% CA + 0.50% LA, exposure time did not result in significant change in reduction (P ≥ 5.90) of E.coli. A significant difference in reduction of Ecoli was observed between the control and formulation treatments of 0.75% CA + 0.75% LA (P ≤ 0.001) at both time frames. The overall results indicate CA alone may have limited ability to reduce E.coli. However, the formulation of CA combining LA has an increased ability to suppress E.coli growth this can be further improved by increasing the concentration of the formulation.

Spray washing with tap water alone resulted in Salmonella recovery of 3.81 ± 0.02 and 4.65 ± 0.10 Log/CFU from un-inoculated hide at 2 and 5 minutes, respectively (Figure 5). Hide panels treated with 0.50% CA alone Salmonella recovery was enumerated at 5.10 ± 0.09 and 3.97 ± 0.08 log/ CFU at 2 and 5 mins of treatments respectively. CA treatment of 0.75% alone resulted in Salmonella recovery of 3.04 ± 0.02 and 3.37 ± 0.04 respectively at 2 and 5 mins of treatment (Figure 5). At combined formulation of 0.50% CA and 0.50% LA, Salmonella, recovery was enumerated at 3.24. ± 0.24 and 2.98 ± 0.28 Log/CFU at 2 and 5 mins respectively. When 0.75% CA and 0.75% LA combined formulation was used bacterial recovery was enumerated at 2.60 ± 0.30 and 2.37 ± 0.30 log/ CFU at 2 and 5 minutes respectively (Figure 5). In compare to control a significant difference in reduction of Salmonella from hide surface was observed for 0.75% of CA and combined formulation of CA + LA. This trend indicates further reduction of the pathogen can be potentially achieved by washing with the developed formulation at a concentration over 0.75% and also combined formulation has stronger ability than CA alone to reduce Salmonella on hide surfaces.

Chemical treatments of hides may lead to microscopic defects in produced leather quality such as sueding which is resulted from losing of fibers on the grain surface of the prepared leather or abrasions of the grain surface. Such defects reduce the tightness and quality of the prepared leather. To assess the quality of leather produced from the formulation treated hides, leather panels were examined using a stereo microscope (Figure 6). Produced leather panels were folded, and a stereo microscopic image was taken at the crease to enhance the surface features. Leather samples 6A and 6B (Figure 6) which were produced from 0.5% CA and 0.75% CA treated hides respectively revealed less sueding than the control. Figure 6C and Figure 6D had similar visual amount of sueding with no discernable difference from the control. Surface stereo microscope images (Figure 7) revealed that there was no discernable difference between the grain structure of leather made from control and formulation treated hides. SEM images of the surface of crust leather produced from hides treated with water (Figure 8, Control) reveled uneven or rough surface, whereas the leather from formulations treated hides (Figure 8A - Figure 8D) appeared to have smoother and homogeneous surfaces. Transactional SEM images releveled no noticeable unraveling of bundle fibers with no apparent difference between the control and the treated panels (Figure 9, Control – 9D).

Leathers produced from formulation treated hides were assessed for softness, fullness, grain tightness (break), color and general appearance by hand and visual examination and found at similar quality to the control (Table 1). In addition, the overall mechanical properties (tensile strength, elongation, Young’s Modulus, and fracture energy) of the resulting leather products from the formulation treated hides were comparable to that produced from the control (Table 2). This indicates that the application of the solutions did not have any detrimental effect on the properties of the final produced leather.

4. Conclusion

Bacterial contamination is a major issue in the meat industry which may lead to food related illnesses. In this study, two natural products have been tested for their antimicrobial efficacy in reducing microorganisms including pathogens form the haired surface of cattle hides. Formulation of CA alone is found effective in reducing swab recoveries of aerobic bacteria and Salmonella from treated hides with better results for increased concentration. The limited effectiveness of CA on Ent/gnEB and E.coil reduction can be overcome by using a combined formulation of CA and LA. In most cases for this study, the higher concentration of combined formulation works more effectively in limiting microorganism than the lower concentration as expected. Additionally, at the concentrations used no detrimental impacts on final leather was observed due to the spray formulation treatments. In future research, increased concentrations and time frames may be implemented to further increase reduction of pathogens on hide surfaces.

Acknowledgements

The authors would like to thank Vincent Hui, an undergraduate student at Drexel University for his help in microbial analysis. We would also like to thank Nick J. Latona and Joe Uknalis for preparing and taking Stereo microscopic and SEM micrographs of leather samples. This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-AC05-06OR23100. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE.

References