1. Introduction
The global population is expected to grow in coming years, which will result in an increase of global food consumption. Specifically, meat consumption is estimated to rise by around 14% by 2030 [
1]. The necessity to produce more meat in the present and the near future requires the search for strategies to make the use of ingredients in diets of animals producing the meat more efficient. In this regard, high quantities of metabolizable protein (MP) in the diets are needed to maximize lean tissue deposition in animals, which often surpasses the protein supply in the diet [
2].
Including bypass amino acids in the diets of cattle improves productive performance [
3]. This dietary strategy helps to limit the amino acid degradation in the rumen by ruminal microorganisms so they can instead be used for muscle deposition [
4]. Efficient utilization of all resources in meat production is important. Livestock feed additives contribute to high environmental and economic costs, either by gas production or/and excretion of ammonia in feces to the environment. Certain ingredients as additives in the ruminant diets like amino acids can improve their performance, productivity, and product quality. Nevertheless, these types of additives must be protected from ruminal microorganisms to be beneficial to the animal. One way to protect these ingredients is through microencapsulation. For instance, arginine supplementation increases intramuscular fat (IMF) and fatty acid (FA) content deposition without affecting the proportions and ratios of the most important FAs, such as n-6:n-3 in pork [
5,
6]. In addition to increasing the marbling, dietary arginine has shown to decrease cooking and drip losses in pork [
7]. L- arginine in the diet also enhances the expression of genes related to the growth of muscle fibers, and the accumulation of fat among muscle bundles [
8]. Further, supplementation of dietary arginine at 1% in ruminants (sheep) has been reported to improve the productive performance and trigger the muscle protein deposition by expression of myogenin, myostatin, and muscle atrophy F-box, which directly impact on the increase in the loin eye area in lamb [
9]. This could be counterproductive for IMF deposition because the change in metabolism reduces the lipogenesis through an effect of myostatin expression depletion. The impact of arginine on gene expression rather than an improved use of nutrients by the ruminal microflora has been confirmed in studies reporting no effect of 360 or 180 mg rumen protected L-arginine on acetate, propionate, or butyrate concentrations in the bovine rumen, despite an increased intestinal arginine flow and digestibility of the amino acid [
10]. The improvement of gene expression by arginine in different species leads to higher meat quality. Nevertheless, the effects of rumen bypass arginine on meat quality remain unclear.
Microencapsulation involves embedding a substance in another material using physical or mechanical processes [
11]. Environmental factors such as oxygen, water, pH, and interactions with other ingredients can impact the stability of active compounds [
12]. Encapsulation processes are used in chemical, pharmaceutical, and food industries. They protect active compounds and achieve controlled release. They also reduce adhesion during storage and transportation and prevent changes in properties [
13]. The encapsulation process in animal nutrition was introduced during the 1990s, with a wide exploration during the last 20 years [
14]. Nonetheless, its applications seem very distant from the real potential compared to other industries such as the pharmaceutical industry. The actual uses of microencapsulation in animal nutrition aim to improve the delivery of helpful substances in the intestine, such as fatty acids, amino acids, antioxidants, and enzymes. Additionally, this can also include live microorganisms such as probiotics [
15]. Yet, the use of microencapsulation in the animal industry remains vastly unexplored and it is necessary to expand the possibilities of the application that benefit the efficient production of food from animal origin in a more ecological approach.
Due to its hydrophobic characteristics, Carnauba wax (
Copernicia prunifera) has shown to efficiently encapsulate amino acids and other nitrogen components such as urea cores against ruminal microbial degradation, as bypass nutrients. Carnauba wax has as advantages its high availability in the market, low cost, and it is nontoxic to animals. It is an encapsulant highly utilized in the medicine, dentistry, automobile, and food industries [
16,
17].
The objective of this exploratory study was to evaluate the effects of a low cost microencapsulation of arginine within carnauba wax at two different proportions (wax–arginine, 3:1 and 2:1) supplemented in bovine diets (Angus, Hereford, and Angus × Hereford) on the physicochemical quality of the meat, before and after retail simulation of 7 d.
2. Materials and Methods
2.1. Characteristics and Origin of the Animals and Samples
This study was carried out in the Biochemistry laboratory and feedlots of the School of Animal Science and Ecology of the Universidad Autonoma de Chihuahua (UACH). Fifteen heifers in total (Angus, Hereford, and Angus × Hereford crossbreed, 357 ± 24.6 kg initial weight) produced in the Teseachi experimental ranch from UACH were used in this experiment. The handling and housing, as well the experimental procedures with the animals complied with the institutional Bioethics code and Animal Welfare Guidelines, approved by the Institutional Bioethics and Animal Welfare Committee with authorized number P/302/2017. All animals were weighed at the start and end of the trial and daily weight gain was calculated to determine if the addition of microcapsules affected the productive performance.
2.2. Evaluated Treatments
The heifers were assigned randomly to one of the three treatments: T1, control without arginine; T2, microcapsules 3:1 wax–arginine ratio; and T3, microcapsules 2:1 wax–arginine ratio. Fifteen animals in total were evaluated considering five animals in each treatment. Every treatment had heifers from the three breeds. The animals were supplemented daily for 28 d prior to sacrifice with 50 g of microencapsulated arginine. The diet was formulated according to nutritional requirements by the Committee on Nutrient Requirements of Beef Cattle [
18] and it was offered daily ad libitum to the animals. The composition is presented in
Table 1.
The animals were slaughtered at a Federal Inspection slaughterhouse in Chihuahua city. The slaughter procedure followed Official Mexican regulations (NOM-033-SAG/ZOO-2014) [
19]. The
Longissimus lumborum muscle caudal to the 13th rib was dissected directly from the carcass to be used in the analysis of variables. The muscles (20 cm, aprox.) were vacuum packed and transported with a refrigerated vehicle (20 min at 4 °C) to the meat laboratory at UACH, twenty-four hours postmortem. Once received, the samples were sliced into 2.54 cm cross sections. The
Longissimus lumborum sections were re-vacuum packed in a vacuum bag (70 µm thickness, polypropylene) and aged for 28 d (4 °C, 55% humidity) and physical–chemical measurements were carried out. After 28 d aged some samples were analyzed while others were packed in commercial polystyrene trays (21 cm × 18 cm) with commercial meat soaker pads and wrapped with commercial polyvinylchloride film permeable to oxygen (Food grade- 6 cm
3 m mm m
−2 d atm
−1 at 23 °C) to simulate a test of shelf-life in retail display conditions, and left refrigerated at 4 °C, in darkness for 7 d. After the simulation of retail exposure, the physical–chemical measurements were carried out again. The variables evaluated were pH, water retention capacity (WHC), shear force, CIEL*a*b* space, and intramuscular fat content.
2.3. Microcapsules Production
The microcapsules production was performed at the Biochemistry laboratory of the School of Animal Science and Ecology of the UACH. The carnauba wax was purchased from “Ceras universales S.A.” a Mexican distribution center (
https://cerasuniversales.com/ accessed on 12 April 2024). The L-arginine (CAS No. 1119-34-2) was obtained from ”Encapsuladoras de México, S.A. de C.V.” (
https://encapsuladoras.com accessed on 12 April 2024), which conforms to the standards FCC11 and USP41.
The melt emulsion technique was performed to encapsulate arginine in carnauba wax. The wax was melted at 90 °C and the arginine was added with constant stirring at 500 rpm until complete homogenization. Span 80 ® (Sorbitan Monooleate, Supelco ®, Bellefonte, PA, USA, 2.5% of the volume of water) was added into 200 mL of distilled water heated at the same temperature than wax. Water was added to the arginine dispersion with the wax and sped at 500 rpm for 3 min. The mix was allowed to cool to room temperature and crushed with a mortar.
2.4. Morphological Characterization
To evaluate and characterize the microencapsulated systems, micrographs were obtained by scanning electron microscopy at 100×, 500×, 1500×, and 5000× (SEM). SEM micrographs of the samples were obtained using a JSM-6390 SEM scanning electron microscope (Jeol, Tokyo, Japan). The size of the microcapsules of the different microencapsulated systems was determined with the Imagej program.
2.5. Physicochemical Evaluation
Evaluation of the physicochemical meat characteristics was performed after the ageing period (28 d post-mortem) and after the simulation of retail display (7 d).
pH. The pH was evaluated with a digital potentiometer for meat (Sentron, Model 1001, The Netherlands). The measurements were taken directly on the meat according to the method of Honikel [
20]. The electrode was inserted perpendicular to the muscle to a depth of 2 cm, avoiding contact with the remaining fat and connective tissue. Three readings were taken in different areas of the sample and the average was calculated.
Water Holding Capacity (WHC). Exudate release was determined by the compression method proposed by Tsai and Ockerman [
21], using 0.3 g of sample compressed 20 min. An analytical balance with a resolution of ±0.05 g, filter paper (#1 Whatman
®, Maidstone, UK), and 2.25 kg methacrylate plates were used. The results were expressed as a percentage of exudate released, according to the following expression: % exudate = (weight of the sample after compression) − (weight of the sample before compression)/weight of the sample before compression × 100. The determinations were performed by triplicate on each sample.
Shear Force. The samples were prepared for the shear stress according to the AMSA methodology [
22]. The samples were cooked on electric griddles (George Foreman
®, Beachwood, OH, USA) until reaching an internal temperature of 71 ± 0.1 °C, they were stored for 12 h at 4 °C and eight 10 mm diameter cylinders were obtained using a manual punch. The cylinders were obtained parallel to the longitudinal orientation of the muscle fibers. These were cut using a Warner–Bratzler blade (60 ° triangular opening) at a speed of 100 mm/min into 30 mm lengths. The peak force (expressed in kg-force) for the transverse cut in each cylinder was determined on a TA-XT plus texture analyzer (Stable Micro Systems Ltd., Godalming, UK).
Color (CIEL*a*b*). The color space was determined by the CIEL*a*b* parameters, where L* is luminosity, a* (+) is the red tendency and b* (+) expresses the yellow tendency. The measurements were obtained with a colorimeter (Konica Minolta CR400, Ramsey, NJ, USA. Illuminant C. D
65. 2° observer angle of measurement. Aperture of 8 mm) according to the CIE (Commission Internationale Pour I`Eclarige) reference system [
23] and the AMSA methodology [
24]. For this, connective tissue and visible fat were removed from the muscle surface and the samples were exposed to air oxygen for 30 min to allow oxygenation of myoglobin (blooming). Three readings were taken for each sample in different areas and averages were obtained for the values of L*, a*, b*.
Total lipid extraction. The extraction was carried out with the Soxhlet method described by the Association of Analytical Chemists, 1995 [
25].
2.6. Statistical Analysis
A completely randomized experimental design was used. The evaluated variables were analyzed with Analysis of Variance (ANOVA) using PROC GLM of the SAS System v.9.0 statistical package. Differences among treatments were evaluated using Tukey’s mean comparison test (p < 0.05). Values were reported as means ± standard deviation.
5. Conclusions
The results of this work showed that the simple and low-cost microencapsulation of arginine in carnauba wax may work to introduce the amino acid into the animal system. Hence, the inclusion of microencapsulated arginine treatments in bovine diets may improve the quality of aged beef. The best results were obtained in arginine supplemented animals (both 2:1 and 3:1 ratio), by reducing the shear force and increasing the intramuscular fat content of the beef. Nevertheless, the exposure of aged beef to oxygen for 7 days can be detrimental to those positive effects. The results of this study show a potential use of a low-cost encapsulation of the amino acid for a practical and immediate use as an additive in cattle diets. Nevertheless, further research is recommended to elucidate if some factors such as different doses, longer times of supplementation, and higher experimental units can help to obtain stronger and more conclusive results, as well as improve the productive performance of the cattle. Additionally, it is recommended to further investigate the intestinal absorption of the amino acid. Supplementation with microencapsulated arginine could be a viable strategy for the livestock industry to improve meat quality.