2.1. Product Procurement
Cattle were harvested on one of four days within a two-week period at a commercial abattoir in Rockhampton, Australia. Tropical breed content and implant usage was monitored and reported for cattle according to their accompanying MSA vendor declaration when they were transferred to an MSA-licensed abattoir [16
]. All cattle had substantial and equal Bos indicus
influence, none had received hormone growth promotants, approximately 36% were grass-fed and 63% were grain-fed, and 60% were male and 40% of cattle were female. Carcasses were subjected to MSA grading, and data were recorded and utilized for sorting purposes. MSA marbling was scored from 100 to 1190 in increments of 10 based on the amount and distribution of marbling in the longissimus dorsi [17
]. Ossification was scored from 100 to 590 in increments of 10 using the AUS-MEAT Carcase Maturity Chart [17
]. In addition, hot carcass weight (HCW; kg), 12th rib fat thickness (mm), eye muscle area (cm2
), and hump height (mm) were collected and recorded for each carcass. In the MSA grading system, hump height serves as an indicator of tropical breed content. AUS-MEAT fat and meat color scores were also recorded [17
]. Finally, longissimus
muscle ultimate pH and temperature values were recorded using a hand-held, probe-type pH meter (model WP-80, TPS Pty Ltd, Springwood, Brisbane, Australia). Carcass data were used to select carcasses representative of four different predicted eating quality categories: 2 = unsatisfactory; 3* = “Selected”; 4* = “Classic”; and 5* = “Premium”.
Five subprimals were collected from the right side of each carcass: outside skirt [Institutional Meat Purchase Specification (IMPS) 121C; diaphragm], inside skirt (IMPS 121D; transversus abdominis), inside round cap (IMPS 169B; gracilis), bottom sirloin flap (IMPS 185A; obliquus abdominis internus), and flank steak (IMPS 193; rectus abdominis). Subprimals were vacuum packaged individually, held in chilled storage at 0 to 1 °C until 14 d postmortem, and frozen at −20 °C. All subprimals were combined into a single consignment from Brisbane, Australia, to Texas Tech University, Lubbock, TX, USA, via cargo freight and road transport. Subprimals were held at frozen temperatures (−20 °C) during shipment and storage upon arrival at Texas Tech University, Lubbock, TX, USA.
2.2. Sample Processing
The muscles were thawed for 48 h at 2 to 4 °C before sample processing. Excess fat and sinew were removed from all the muscles prior to processing. All the muscles were cut in half parallel to the muscle fibers, with each half being alternatively assigned to enhancement treatment (control, clean, and phosphate). All the muscle halves were weighed to obtain the green weight. The control muscle halves were untreated. The clean muscle halves were enhanced with sodium chloride (NaCl; Morton Salt Inc., Chicago, IL, USA), food grade sodium bicarbonate (NaHCO3; Church & Dwight Co. Inc., Ewing Township, NJ, USA), and water. The brine was prepared in 5 °C tap water with 4.16% NaCl and 4.00% NaHCO3−. Brine was poured over the muscles in the vacuum tumbler for a target pickup of 112% (111.68% ± 3.82) of fresh muscle weight. All the muscles were placed in a vacuum tumbler (Koch Industries, Wichita, KS, USA) and a vacuum (508 mm Hg) was pulled. The muscles were batch tumbled at 10 RPM for 20 min. The samples were allowed to rest for 10 min, and the tumbled weight was obtained and recorded for each muscle half. The pickup percentage was calculated by taking the tumbled weight divided by the green weight, multiplied by 100.
The phosphate muscle halves were enhanced with NaCl (Morton Salt Inc., Chicago, IL, USA), sodium tripolyphosphate (STPP; Carfosel 408, Prayon Inc., Augusta, GA, USA), and water. The brine was prepared in 5 °C tap water with 4.16% NaCl and 4.00% STPP. Brine was poured over the muscles in the vacuum tumbler for a target pickup of 112% (111.59% ± 4.00) of fresh muscle weight. The muscles were tumbled as previously described for clean enhancement. The samples were allowed to rest for 10 min, and the tumbled weight was obtained and recorded for each muscle half. The pickup percentage was calculated by taking the tumbled weight divided by the green weight, multiplied by 100.
Regardless of enhancement treatment, a sample was obtained (weighing approximately 10 g) after treatment application and before vacuum packaging. This sample was vacuum packaged individually and frozen at −20 °C until analysis of final pH. All the remaining muscle samples were vacuum packaged individually and held at 2 to 4 °C. The samples were sorted into one of six testing days, and the muscles were used in consumer sensory testing within 9 days of processing.
2.4. Consumer Sensory Evaluation
The Texas Tech University Institutional Review Board approved procedures for use of human subjects for consumer panel evaluation of meat sensory attributes (IRB#: 2017-598).
Each muscle sample was removed from packaging and weighed individually to obtain a raw weight. The muscles were cooked individually to 74 °C on a George Foreman clamshell grill (Model GRP99, Spectrum Brands. Inc., Middleton, WI, USA) with the lid closed and a plate temperature set to 218 °C. The muscle temperature was monitored using a digital, instant read Thermapen thermometer (Model Mk4, ThermoWorks, American Fork, UT, USA). When the muscles reached the required temperature, they were removed from the heat source. The peak temperature and cooked weight were recorded. The cooking loss percentage was calculated by subtracting the cooked weight from the raw weight, dividing by the raw weight, and multiplying by 100. In addition, total cooking time was recorded. The muscles were rested for at least 3 min prior to slicing. The muscles were sliced into 13 mm strips perpendicular to the muscle fibers, and the strips were cut in half lengthwise, resulting in strips that were approximately 5 cm long. The strips were transferred to pre-heated rectangular stainless-steel pans, which were maintained in insulated water bath warming units (Model W-3Vi; American Permanent Ware Company; Dallas, TX, USA) at ~60 °C throughout the test session. Each warming unit held nine pans.
Consumer panels were conducted in the Texas Tech University Animal and Food Sciences Building. Consumer panelists (n = 360) were recruited from Lubbock, Texas, and the surrounding local communities by scheduling community groups with populations of regular red meat eaters within the age range of 18–75. Each consumer was monetarily compensated and was only allowed to participate one time. Each session consisted of 60 people and lasted approximately 60 min.
Consumer testing was conducted according to MSA protocols [15
], with previously described modifications for cooking method. Each consumer evaluated seven samples, including one warm-up sample, which was excluded from analysis, to orient consumers to the sample format. The warm-up samples were always served in the first position, followed by six test samples served in a predetermined, balanced order. The serving order of the six test products was controlled by a 6 × 6 Latin square design, ensuring that all the products were presented an equal number of times in each serving order position and before and after each other product. The test products were selected from the five muscles that were or were not enhanced. The predicted eating quality score (MSA grade) was also used for selection and allocation into the consumer testing design. Those products were equally represented and evenly distributed among the 60 consumers each evening. The testing occurred over the course of six evenings, with a similar and even product distribution in each testing session. Software-controlled routines ensured that the samples from each individual muscle were served in five different order positions and within different subsets of 12 consumers within each group of 60.
Each panelist was seated at a numbered booth and was provided with a ballot, plastic utensils, a toothpick, unsalted crackers, a napkin, an empty cup, a water cup, and a cup with diluted apple juice (10% apple juice and 90% water). Each ballot consisted of a demographic questionnaire, seven sample ballots, and a post-panel survey regarding beef purchasing habits. Before beginning each panel, consumers were given verbal instructions by Texas Tech personnel about the ballot and the process of testing samples. The panels were conducted in a large classroom that has standard fluorescent lighting (i.e., no red filters were used) with tables that were divided into individual sensory booths.
Each sample had 10 consumer observations (i.e., each muscle half yielding at least 20 strips served in duplicate to 10 predetermined consumers). Consumers scored palatability traits, including tenderness, juiciness, flavor liking, and overall liking, on 100-mm line scales verbally anchored at 0 (not tender, not juicy, dislike extremely) and 100 (very tender, very juicy, like extremely). Consumers were asked to rate the quality of each sample as unsatisfactory, good everyday quality, better than everyday quality, or premium quality. The 10 individual scores for each trait were averaged to generate mean sensory scores for each palatability trait and satisfaction prior to analysis. A composite score (MQ4) was calculated using the following equation: (tenderness × 0.3) + (juiciness × 0.1) + (flavor liking × 0.3) + (overall liking × 0.3) [15
]. Weightings for tenderness have decreased and flavor liking increased from original weightings by [15
] for a balanced contribution to the MQ4 value. The weightings give an indication of the relative importance of the four sensory attributes (tenderness, juiciness, flavor, and overall satisfaction) to the final meat quality score. According to the fixed weightings above, when a consumer eats beef, their satisfaction is influenced equally by tenderness, flavor liking, and overall liking, with a lesser contribution by juiciness.
All remaining pieces that were not consumed were vacuum packaged and chilled overnight at 2–4 °C. The samples were then snap frozen in liquid nitrogen. Frozen and cubed samples were homogenized in a food processor (Model Blixer 3 Series D, Robot Coupe, Ridgeland, MS, USA), blended into an ultrafine powder, and transferred into a labeled Whirl-Pak bag. The bags were stored in a freezer at −80 °C until subsequent analysis for percent moisture.
2.6. Statistical Analysis
The data were analyzed in SAS using PROC GLIMMIX (version 9.4, SAS Inst. Inc., Cary, NC, USA). For compositional analyses, the enhancement treatment, the muscle, and their interaction were included as fixed effects. For consumer sensory analyses, the enhancement treatment, the muscle, the MSA grade, and their interactions were included as fixed effects. The percent pickup was considered and tested as a potential covariate but was decided against due to its relationship both to the muscle (one of the treatment factors) and the enhancement treatment, as that would violate the statistical guidelines for covariate usage. Treatment least squares means were separated with the PDIFF option of SAS using a significance level of P ≤ 0.05. Mean separation tests for all pairwise comparisons were performed using the PDIFF function, which requests that P-values for differences of all least squares means be produced. The PROC CORR of SAS was used to assess the relationship between compositional traits and consumer eating quality traits by generating Pearson correlation coefficients. The PROC FREQ of SAS was used to summarize consumer demographic information.