Physical Changes During Post-Mortem Ageing of High-Value Impala (Aepyceros Melampus) Steaks

applied sciences

Article Physical Changes during Post-Mortem Ageing of High-Value Impala (Aepyceros Melampus) Steaks

Tersia Needham 1,2, Retha A. Engels 2 and Louwrens C. Hoﬀman 2,3,*

1 Department of Animal Science and Food Processing, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamýcká 129, 16500 Prague-Suchdol, Czech Republic; [email protected] 2 Department of Animal Sciences, University of Stellenbosch, Private Bag X1, Matieland, Stellenbosch 7602, South Africa; [email protected] 3 Center for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Health and Food Sciences Precinct, 39 Kessels Rd, Coopers, Plains 4108, Australia * Correspondence: louwrens.hoﬀ[email protected]; Tel.: +61-4-1798-4547

Received: 1 June 2020; Accepted: 26 June 2020; Published: 29 June 2020

Featured Application: When harvested under minimal stress, prime impala steaks should be wet-aged in vacuum packaging under refrigerated conditions for eight days to ensure maximum tenderness, uniformity, and quality.

Abstract: Antelope meat production is rapidly growing, not only due to their adaptation to marginal land usage, but also because of its favorable nutritional properties and free-range production. However, limited information is available on the meat quality and processing potential of game meat for commercial consumption. The objective of this study was to determine the ageing period to achieve maximum tenderness of longissimus thoracis et lumborum (LTL) muscles of impala. The LTL muscles of 11 male and 11 female impala were harvested, and divided into eight portions. Each portion was randomly allocated to 1, 2, 4, 6, 8, 10, 12, or 14 days of wet-ageing (4 ◦C) in vacuum packaging. The meat pH, color, weep loss, cooking loss, and Warner–Bratzler shear force were measured throughout ageing. Initially the ageing profile differed depending on the sex of the animal from which the muscle was harvested; however, after 8 days of ageing, maximum tenderness was reached (13.5 0.91 N) and no further sex differences were seen. Ageing improved the surface color of all ± meat until day 8, after which discoloration occurred. Therefore, it is recommended that impala LTL steaks should be wet-aged at 4 ◦C for eight days to achieve maximum tenderness and minimize sex variability.

Keywords: game meat; longissimus thoracis et lumborum; meat processing; tenderness; wet-ageing

1. Introduction The current expansion of the human population is increasing the global demand for meat [1]; however, increasing meat production faces a number of challenges, including limited land availability, livestock feed supply, and climate change. Thus, domesticated livestock species alone may not be capable of meeting the expanding demand for animal protein, and therefore additional underutilized sources should be investigated [2,3]. While beef cattle are particularly vulnerable to desertiﬁcation and bush encroachment brought on by climate change [4], antelope, in comparison, are well adapted to arid African environments, as they may utilize low-quality vegetation and are less susceptible to overgrazing [5]. Therefore, African antelope species represent a practical solution to increasing lean meat production, with promising results already reported for species such as the common eland

Appl. Sci. 2020, 10, 4485; doi:10.3390/app10134485 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 4485 2 of 11

(Taurotragus oryx)[6–9]. With the expansion of the South African game industry and the intensification of production systems for controlled breeding, more animals are available to be culled for meat production [10]. The meat of impala antelope (Aepyceros melampus) produces high carcass yields and dressing percentages, with meat that is high in protein and low in intramuscular fat [11], which makes it an attractive alternative protein source to traditional livestock for meat production [12,13]. However, game meat is considered as tough and dry by consumers, due to low product uniformity in commercially available game meat, as well as a lack of quality standards and knowledge on the proper cooking methods for game meat [14]. However, perceived meat quality is influenced by an array of ante and post-mortem factors, including species, age, sex, environmental and dietary factors, slaughtering conditions, and post-mortem processing of the meat [15]. Sex had minor influences on the meat production potential and physiochemical meat quality parameters of impala [11], but to develop the consumer market for game meat, supply chains must be capable of supplying products with consistently high-quality standards [16], in addition to improving consumer perception concerning the toughness of game meat. It is therefore necessary to investigate methods that may improve the texture and quality of meat from game species, of which wet-ageing has shown potential in other antelope species [8,9,17]. Post-mortem ageing not only improves the tenderness of meat through changes in the micro- and ultrastructure of muscle fibers [18], but it also influences other meat quality attributes such as juiciness, aroma, and flavor [19,20]. Currently, wet-ageing is favored commercially [21], whereby meat is vacuum packaged and stored under refrigerated conditions. The reason for this being that moisture loss and shrinkage during ageing is decreased, and wet-ageing provides a higher convenience during transport and storage of the meat [22,23]. While the fresh meat quality of impala indicates that their meat may be considered as tender after 24 h post-mortem [11], ageing may not only further benefit tenderness, but also the sensory eating quality and transportation of meat from isolated production areas to commercial sale points. However, the influence of post-mortem ageing on the meat quality of impala has not yet been investigated. Thus, the aim of this study was to determine the optimum post-mortem ageing period, based primarily on tenderness and color, of the longissimus thoracis et lumborum (LTL) muscle of impala meat from both sexes.

2. Materials and Methods

2.1. Experimental Design, Animals, and Sample Collection Twenty-two subadult (15–18 months old; 11 males and 11 females) impala were culled (ethical approval number 10NP_HOF02) using light caliber rifles (.22 or .243) fitted with a suppressor. The impala were kept within a 200 ha semiextensive production system at a stocking density of 250–300 animals, within the Savanna biome of Modimolle (Limpopo, South Africa). The natural vegetation [24] formed the primary feed source, and a supplementary feed was provided daily according to the recommended dietary requirements for this species [25]. Feed samples were taken throughout the study and pooled before analysis according to the Association of Official Analytical Chemists International (AOAC 934.01, 992.15, 942.05, 962.0) [26] for moisture (8.3%), crude protein (91.7%), ash (27.9%), and fiber components (crude fiber: 27.9%, neutral detergent fiber: 47.7% and acid detergent fiber 30.5%). Immediately after being shot, each impala was exsanguinated and transported to the slaughter facilities on-farm, where they were eviscerated and processed according to standard guidelines [27]. Dressed carcasses were then hung in a chiller set to 4 1 C to undergo rigor mortis. After 24 h, ± ◦ all impala carcasses were deboned, and the left longissimus thoracis et lumborum (LTL) muscles were excised. Each LTL muscle was divided into eight equal steaks with a thickness of approximately two centimeters each, cut at a right angle to the longitudinal axis of the muscle. Each steak was randomly allocated to one of the following ageing days: 1, 2, 4, 6, 8, 10, 12, and 14. The samples Appl. Sci. 2020, 10, 4485 3 of 11 allocated to Day 1 (D1) were analyzed on the day of deboning (24 h post-mortem), whereas each steak allocated to the remaining days was weighed, vacuum sealed in composite plastic bags (70 µm nylon and polyethylene; oxygen permeability of 30 cm3/m2/day/atm, carbon dioxide permeability of 105 cm3/m2/day/atm and moisture vapor transfer rate of 2.2 g/m2/day/atm) with a 5 mb residual pressure (Multivac, Model C200, Sepp Haggenmuller, Wolfertschwenden, Germany) and stored in a chiller at 4 1 C until the allocated day of physical analysis. ± ◦ 2.2. Physical Meat Quality Evaluation throughout Ageing At each of the aforementioned time points, the relevant steaks were assessed for physical meat quality parameters. Firstly, moisture loss was determined by removing the steak, blotting it dry, and then weighing it to determine the weep loss, as a percentage of the steak’s original weight. Thereafter, the pH was measured with a two-point calibrated (using standard buffers of pH 4 and pH 7) Crison pH25 portable pH meter (Crison instruments, Barcelona, Spain) with a glass electrode. Measurements were taken by inserting the electrode at an angle as close to the center of each steak as possible. Between each measurement, the electrode was cleaned by rinsing with distilled water and blotted dry with absorbent paper. The steaks were then allowed to bloom for 30 min, after which color measurements were taken with a calibrated Color-guide 45◦/0◦ colorimeter (4 mm aperture, 2◦ observer angle; BYK-Gardner GmbH, Gerestried, Germany) at five random positions, and averaged per sample. The International Commission on Illumination (CIE) system was used, where hue angle (color definition) and chroma values (saturation/color intensity) were calculated from L* (lightness), a* (red-green spectrum) and b* (blue-yellow spectrum) values according to the following equations: ! 1 b∗ Hue angle (◦) = tan− (1) a∗ q 2 2 Chroma (C ) = (a + b∗ ) (2) ∗ ∗ Subsequently, the cooking loss of each sample was determined by placing a weighed piece of steak inside a thin plastic bag and cooking in a preheated water bath (80 ◦C) for a period of 60 min [6]. All steaks from the relevant ageing day were cooked in one batch and each steak was fully submerged for the duration of the cooking period. After 60 min, the cooked steaks were removed from the water bath and excess water was drained before chilling at 4 1 C for approximately six hours until cooled. ± ◦ Thereafter, the steaks were blotted dry with absorbent paper and weighed, and moisture loss was expressed as a percentage of the initial weight of the uncooked steak [28]. Following the cooking loss determination, the Warner–Bratzler shear force (WBSF) was measured using six cylindrical core samples (diameter: 1.27 cm) taken from the center of each steak, while excluding visible connective tissue. The cores were placed within the Warner–Bratzler blade such that they were cut at a right angle to the longitudinal axis of the fibers (blade speed: 3.33 mm/s) and the peak force during cutting was recorded in Newtons (N). The mean force was determined from the six samples per steak, with lower shear force values indicating higher tenderness.

2.3. Statistical Analysis The experimental design was a randomized block split plot, with the animal as the block, sex as the main plot factor and ageing period (days post-mortem) as the split plot factor. Both main effects (sex and ageing period) were tested, as well as their interaction. The parameters of the physical analysis (pH, weep loss, cooking loss, Warner–Bratzler shear force, and color) were analyzed using general linear models to establish univariate ANOVAs (analysis of variance) in SAS (Version 9.4; SAS Institute Inc., Cary, USA). Normality of the data was ensured using Shapiro–Wilk tests. No outlier values were seen within the data. Fisher’s least significant difference post hoc testing was used to compare sex, muscle, and ageing period means. A significance level of 5% was considered throughout. Appl. Sci. 2020, 10, 4485 4 of 11

3. Results Main effects are summarized in Table1; however, significant interactions were observed between sex and ageing period for pH (p = 0.028), cooking loss percentage (p = 0.020), Warner–Bratzler shear force (p < 0.001), CIE L* value (p = 0.004), CIE a* value (p = 0.001), and chroma value (p = 0.001) of impala meat. Thus, these interactions and not their main effects will be discussed. No interactions were recorded between sex and ageing period for weep loss percentage, CIE b* value, or the hue angle of impala meat (Table1).

Sex didAppl. not Sci. have 2020, 10 an, x FOR influence PEER REVIEW on the weep loss percentage of impala meat (Table1 of 111 ). However, weep loss was influenced (p < 0.001) by the ageing period, with the lowest weep loss percentage Appl. Sci. 2020, 10, x FOR PEER REVIEW 1 of 11 recorded on daySex 2 (3.7 did not0.27%). have an influence Thereafter, on the weepweep loss loss percentage percentage of impala increased meat (Table until 1). However, day 6 (5.9 0.28%), weep loss was± influenced (p < 0.001) by the ageing period, with the lowest weep loss percentage ± after which arecorded plateauSex did on was notday have 2reached (3.7 an ± 0.27%).influence until Thereafter, on the the endweep weep ofloss lo the sspercentage percentage ageing of periodincreased impala meat on until day(Table day 14 6 1). (5.9 (6.4 However, ± 0.28%),0.27%). ± Male impalaweepafter whichloss meat was a plateau hadinfluenced a was significantly reached(p < 0.001) until by the higher the end agei ofng pH the period, ageing (p = with0.028) period the on thanlowest day 14 female weep (6.4 ±loss 0.27%). impala percentage meat on days 2, recorded Male on impala day 2 meat(3.7 ± had0.27%). a significantly Thereafter, higherweep lo pHss percentage(p = 0.028) than increased female until impala day meat6 (5.9 on ± 0.28%), days 2, 4, 6, 10, and 14, with the highest pH recorded for male impala meat on day 14 (5.9 0.03) (Figure1). after4, 6, which10, and a 14,plateau with wasthe highest reached pH until recorded the end for of malethe ageing impala period meat on day 14 (6.4(5.9 ±± 0.27%).0.03) (Figure ± 1). However, no However, significant Male impala no significant di meatfferences had differences a significantly between between higher sexes sexes pH werewere (p = recorded 0.028) recorded than for female days for 1, days impala 8, or 12 1, meat for 8, orpH. on 12 days for 2, pH. 4, 6, 10, and 14, with the highest pH recorded for male impala meat on day 14 (5.9 ± 0.03) (Figure 1). However, no significant differences between sexes were recorded for days 1, 8, or 12 for pH. Female Male

6 Female Male ab a 5.9 6 bc abc abc 5.8 ab a 5.9 cde cdef abc 5.7 bc abccd 5.8 fghi pH cde cdefefg defg cdef 5.6 cd 5.7 fghiih ghi i i pH 5.5 efg defg 5.6 cdef 5.4 ih 5.5 ghi i i 5.3 5.4 12468101214 5.3 Days post-mortem 12468101214 Figure 1. Effect of sex and ageing (days post-mortemDays post-mortem) on the pH of female and male impala longissimus Figure 1. Effect of sex and ageing (days post-mortem) on the pH of female and male impala longissimus a–i thoracis et lumborum muscles. Meansa–i with different superscripts differ from one another (p 0.05). thoracis et lumborum muscles. Means with different superscripts differ from one another (p ≤ 0.05). ≤ Error bars representFigureError bars 1. Effect therepresent of standard sex theand standard ageing error (days error of thepost-mortem of the means. means.) on the pH of female and male impala longissimus thoracis et lumborum muscles. a–iMeans with different superscripts differ from one another (p ≤ 0.05). Error bars represent the standard error of the means. Female impalaFemale meat impala was meat found was found to have to have significantly significantly higher higher cooking cooking loss percentages loss percentages than that than that of of males on 1, 2, 4, 8, and 14 days post-mortem (p = 0.020; Figure 2). The highest cooking loss percentage males on 1, 2,(33.3 4,Female 8,± 0.46%) and impala 14was days recorded meatpost-mortem was on found day 4 to for have (femalep =significantly0.020; impala Figuremeat higher and2 cooking ).the The lowest loss highest cooking percentages cookingloss percentage than that loss percentage (33.3 0.46%)of(29.5 males was ± 0.46%) on recorded 1, 2, was 4, 8, recorded and on 14 day days on 4 post-mortemday for 1 femalefor male (p = impala 0.020; Figure meat. meat 2).No The differences and highest the lowestcookingwere recorded loss cooking percentage between loss percentage ± (33.3 ± 0.46%) was recorded on day 4 for female impala meat and the lowest cooking loss percentage (29.5 0.46%)the was sexes recorded for days 6, on 10, dayand 12. 1for A general male trend impala can meat.be observed No diwithfferences female impala were meat recorded having between the ± (29.5slightly ± 0.46%) higher was overall recorded LTL muscle on day cooking 1 for male loss impala percentages meat. thanNo differences males for the were duration recorded of the between ageing sexes for daystheperiod. 6, sexes 10, for and days 12. 6, A10,general and 12. A trend general can trend be can observed be observed with with female female impala impala meat meat having having slightly higher overallslightly LTL higher muscle overall cooking LTL muscle loss cooking percentages loss percentages than than males males for for the the duration of the of ageing the ageing period. period. Female Male

35 Female Male a 34 ab 35 abc ab abcd 33 abcde bcde a 3432 ab cdefg abc ab abcd 3331 abcde bcde cdefg bcdef 3230 defg efg gh fgh efg 3129 bcdef gh h defg Cooking loss (%) 30 efg 28 gh fgh efg gh 2927 h Cooking loss (%) 2826 27 12468101214 26 Days post-mortem 12468101214

Days post-mortem Figure 2. Effect of sex and ageing (days post-mortem) on the cooking loss percentage of female and Figure 2. Effectmale of impala sex and longissimus ageing thoracis (days et lumborumpost-mortem muscles.) on a–hMeans the cooking with different loss superscripts percentage differ of from female and male Figure 2. Effect of sex and ageing (days post-mortem) on the cooking loss percentage of female and impala longissimusone another thoracis (p ≤ 0.05). et lumborum Error bars representmuscles. the standarda–hMeans errorwith of the dimeans.fferent superscripts differ from one male impala longissimus thoracis et lumborum muscles. a–hMeans with different superscripts differ from another (p one0.05). another Error (p ≤ bars0.05). Error represent bars represent the standard the standard error errorof of the means. means. ≤ Appl. Sci. 2020, 10, 4485 5 of 11

Table 1. Effect of sex and ageing period (days post-mortem) on the physical parameters of impala longissimus thoracis et lumborum meat. Significant interactions were found for pH, cooking loss, shear force, L*, a*, and chroma values, and thus main effects are not interpreted but depicted within the table only.

Sex Days Post-Mortem Parameter Sex Days Post-Mortem p Female Male SEM p 1 2 4 6 8 10 12 14 SEM p × pH 5.6 5.8 0.04 0.050 5.6 c 5.7 b 5.7 b 5.7 bc 5.7 b 5.8 a 5.8 a 5.8 a 0.02 <0.001 0.028 Weep loss (%) 6.0 5.5 0.46 0.427 * 3.7 c 4.7 b 5.9 a 6.4 a 6.5 a 6.5 a 6.4 a 0.27 <0.001 0.468 Cooking loss (%) 32.4 30.7 0.54 0.034 31.1 b 31.9 ab 31.8 ab 31.2 ab 31.6 ab 32.1 a 31.5 ab 31.1 b 0.33 0.222 0.020 Shear force (N) 18.3 14.9 1.00 0.026 25.5 a 21.9 b 20.5 b 16.9 c 13.5 d 12.2 ed 11.3 ed 10.8 e 0.91 <0.001 <0.001 L* 33.6 33.0 1.11 0.692 31.8 c 33.8 ab 33.2 ab 33.0 b 33.2 ab 34.0 a 33.9 ab 33.4 ab 0.32 <0.001 0.004 a* 12.7 12.7 0.46 0.945 12.5 bc 11.3 d 12.4 c 13.4 a 13.1 a 13.1 a 12.9 ab 13.0 ab 0.18 <0.001 0.001 b* 8.9 8.3 0.77 0.557 7.6 d 7.9 cd 8.4 bc 8.7 ab 8.8 ab 9.2 a 9.1 a 9.3 a 0.23 <0.001 0.201 Chroma 15.7 15.3 0.80 0.010 14.8 b 13.9 c 15.1 b 16.1 a 15.9 a 16.1 a 15.9 a 16.1 a 0.22 <0.001 0.001 Hue angle 36.5 32.8 1.81 0.384 30.5 c 34.5 a 33.4 ab 32.2 bc 33.2 ab 34.5 a 34.6 a 34.7 a 0.73 <0.001 0.481 Abbreviations: SEM: standard error of the mean, L*: lightness from 0 (black) to 100 (white), a*: lightness from red (–) to green (+) spectrum, b*: lightness from blue (–) to yellow (+) spectrum. *No weep loss was measured on day one as physical analysis occurred prior to packaging. a–eMeans with different letters within each main effect differ from each other (p 0.05). ≤ Appl. Sci. 2020, 10, 4485 6 of 11

FemaleAppl. impala Sci. 2020 meat, 10, x FOR had PEER higherREVIEW (p = 0.004) L* values than male impala on day2 of 118 and day 10 of ageing. ThereAppl. Sci. were2020, 10, nox FOR significant PEER REVIEW differences between sexes for the remaining2 of six 11 time points Female impala meat had higher (p = 0.004) L* values than male impala on day 8 and day 10 of (Figure3). FemaleFemale impala impala also meat had had higher higher (pp = =0.004)0.001) L* values a* values than male on impala day 4 on (13.0 day 8 and0.26) day than 10 of male impala ageing. There were no significant differences between sexes for the remaining six time± points (Figure (11.8 0.26),3).ageing. whereasFemale There impala maleswere also no had significant had higher higher (differencesp = 0.001) (p a* betwee0.05) valuesn a*on sexes day values for 4 (13.0 the onremaining ± 0.26) day than 12six male time (13.4 impala points0.26) (11.8(Figure ± than females ± 3). Female impala also had higher (p = 0.001)≤ a* values on day 4 (13.0 ± 0.26) than male impala± (11.8 ± (12.5 0.26)0.26), (Figure whereas4). Nomales significant had higher (p di ≤ 0.05)fferences a* values between on day 12 sexes(13.4 ± 0.26) were than recorded females (12.5 for ± days0.26) 1, 2, 6, 8, 10, ± (Figure0.26), whereas 4). No significant males had differences higher (p ≤ between 0.05) a* valuessexes were on day recorded 12 (13.4 for ± days0.26) 1,than 2, 6, females 8, 10, or (12.5 14. ± 0.26) or 14. (Figure 4). No significant differences between sexes were recorded for days 1, 2, 6, 8, 10, or 14. Female Male Female Male 36 36 ab a 35 abc ab a abc abc 35 abcd abcd abc 34 abc abc abcd abcd 34 33 efg abc 33 efg bcde bcde 32 cdef abc cdef bcde defg bcde CIE L* CIE 32 cdef cdef 31 defg fg CIE L* CIE 31 g fg 30 g 30 29 29 28 28 1 2 4 6 8 10 12 14 1 2 4 6 8 10 12 14 Days post-mortem Days post-mortem

Figure 3. EffFigureect of 3. sex Effect and of sex ageing and ageing (days (dayspost-mortem post-mortem) )on on the the L* (lightness) L* (lightness) values of values the surface of color the surface color Figure 3. Effect of sex and ageing (days post-mortem) on the L* (lightness) valuesa–g of the surface color of impala longissimus thoracis et lumborum meat from females and males. Means a–gwith different of impala longissimus thoracis et lumborum meat from females and males.a–g Means with different superscriptsof impala longissimus differ from thoracis one another et lumborum (p ≤ 0.05). meat Error from bars females represent and the males. standard Means error of with the means.different superscripts disuperscriptsffer from differ one from another one another (p (0.05).p ≤ 0.05). Error Error bars bars represent represent the standard the standard error of the error means. of the means. ≤ Female Male Female Male 16 ab 1416 abcd abc a ab ab abcd abcd abc a ab ab ab abcd 1214 fg ab fg abcd bcd abc 12 def ef ab cde 10 g abcd bcd abc def ef cde 108 g

CIE a* CIE 68 CIE a* CIE 46 24 02 0 12468101214 12468101214 Days post-mortem Days post-mortem

Figure 4. Effect of sex and ageing (days post-mortem) on the a* values (red-green spectrum) of the surface colorFigure of impala 4. Effectlongissimus of sex and ageing thoracis (days etpost-mortem lumborum) onmeat the a* fromvalues females(red-green andspectrum) males. of thea–g Means with surfaceFigure color4. Effect of impala of sex longissimusand ageing thoracis (days post-mortemet lumborum) meaton the from a* values females (red-green and males. spectrum) a–gMeans ofwith the different superscriptssurface colordi offf impalaer from longissimus one another thoracis et lumborum (p 0.05). meat Errorfrom females bars and represent males. a–gMeans the standard with error of different superscripts differ from one another (p ≤ 0.05).≤ Error bars represent the standard error of the the means. means.different superscripts differ from one another (p ≤ 0.05). Error bars represent the standard error of the means. The interaction (p < 0.001) between sex and ageing period for the chroma values of impala meat The interactionis presentedThe interaction (p in< Figure0.001) ( p5. < Female 0.001) between between impala sex had sex and higherand ageingageing chroma period period values for thethan for chroma males the chroma onvalues day of1, valuesimpala2, 4, and meat of14 impala meat is presentedpost-mortem inis presented Figure, while5 in. Figure Female males 5. hadFemale impala higher impala chroma had had higher values higher than chromachroma female values values impala than on thanmales day 12.on males day 1, on 2, 4, day and 14 1, 2, 4, and 14 post-mortem, while males had higher chroma values than female impala on day 12. post-mortem, while males had higher chroma values than female impala on day 12. No interactions were recorded between sex and ageing period for the b* values or hue angles of impala LTL muscles, and there were no differences between sexes (Table1). However, the post-mortem ageing period had a significant influence on the b* values of impala LTL meat. The lowest b* values were recorded on day 1 (7.6 0.23), with a steady increase as the ageing period progressed until a ± plateau was reached at the highest b* values from day 10 onwards (Table1). The post-mortem ageing period also had an influence (p < 0.001) on the hue angle, which was lowest on day 1, after which it fluctuated between day 2 to 8, where after it reached a plateau from day 10 until day 14. Female impala meat had higher (p < 0.001) shear force values than males at day 1, 2, and 4, while no differences were recorded between the sexes from day 6 to day 14 (Figure6). All muscles, regardless of sex, showed a gradual decline in shear force values until day 8, after which a plateau was reached until the end of the ageing period at day 14. Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 11

Female Male

20 a 18 abc abcd abc ab de abcd 16 ef abcd 14 abcd abcd cd bcd fg f 12 g 10

Chroma 8 6 Appl. Sci. 2020, Appl.10, 4485 Sci. 2020, 10, x FOR PEER REVIEW 3 of 11 7 of 11 4 2 0 Female Male 12468101214 20 Days post mortem a 18 abc abcd abc ab de abcd 16 ef Figure 5. Effect of sex and ageing (days post-mortemabcd ) on the chroma values of the surface color of 14 abcd abcd cd bcd fg f a–g impala12 longissimus thoracisg et lumborum meat from females and males. Means with different superscripts10 differ from one another (p ≤ 0.05). Error bars represent the standard error of the means.

Chroma 8 No 6interactions were recorded between sex and ageing period for the b* values or hue angles of impala LTL4 muscles, and there were no differences between sexes (Table 1). However, the post- mortem ageing2 period had a significant influence on the b* values of impala LTL meat. The lowest b* values were0 recorded on day 1 (7.6 ± 0.23), with a steady increase as the ageing period progressed until a plateau was12468101214 reached at the highest b* values from day 10 onwards (Table 1). The post-mortem ageing period also had an influence (p < 0.001)Days on post the mortemhue angle, which was lowest on day 1, after which it fluctuated between day 2 to 8, where after it reached a plateau from day 10 until day 14.

Figure 5. EffectFemale of sex impala and meat ageing had (dayshigher post-mortem(p < 0.001) shear) on force the values chroma than values males at of day the 1, surface 2, and 4, color while of impala longissimusno differences thoracisFigure 5. etEffect were lumborum of recordedsex andmeat ageing between from (days the femalespost-mortem sexes from and) on the males.day chroma 6 toa–g dayvaluesMeans 14 of(Figure the with surface 6). di ffAllcolorerent muscles, of superscripts regardlessimpala oflongissimus sex, showed thoracis a gradual et lumborum decline meat in shearfrom femalesforce values and males.until daya–gMeans 8, after with which different a plateau differ from one another (p 0.05). Error bars represent the standard error of the means. was superscriptsreached until differ the ≤from end oneof the another ageing (p ≤period 0.05). Errorat day bars 14. represent the standard error of the means.

No interactions were recorded between sex and ageing period for the b* values or hue angles of impala LTL muscles, and there were no differencesFemale betweenMale sexes (Table 1). However, the post- mortem35 ageing period had a significant influence on the b* values of impala LTL meat. The lowest b* values were recordeda on day 1 (7.6 ± 0.23), with a steady increase as the ageing period progressed 30 a until a plateau was reached at the highest b* values from day 10 onwards (Table 1). The post-mortem 25 b ageing period also had an influence (p < 0.001)cde on the hue angle, which was lowest on day 1, after 20 b which it fluctuated between day 2 to 8, where after it reacheddef a plateaudefg from day 10 until day 14. 15 fg fg Female impala meat hadcde higher (pc < 0.001) shearcd force values than males at day 1, 2, and 4, while 10 efg no differences were recorded between the sexes from day 6 to dayfg 14 (Figurefg 6). All muscles, 5 g regardless of sex, showed a gradual decline in shear force values until day 8, after which a plateau 0 was reached until the end of the ageing period at day 14. 1 2 4 6 8 10 12 14

Days post-mortem Warner-Bratzler shear force (N) force shear Warner-Bratzler Female Male Figure 6. Effect of sex and ageing (days post-mortem) on the Warner–Bratzler shear force (N) of female 35 a–h and male impalaFigurelongissimus 6. Effecta of sex thoracisand ageing et (days lumborum post-mortemmuscles.) on the Warner–BratzlerMeans with shear diforcefferent (N) of superscriptsfemale differ 30 a and male impala longissimus thoracis et lumborum muscles. a–hMeans with different superscripts differ from one another25 (p 0.05). Error barsb represent the standard error of the means. from one another≤ (p ≤ 0.05). Error bars represent the standard error of the means. cde 20 b def defg 4. Discussion4. Discussion15 fg fg cde c cd 10 efg fg fg Despite the slight5 fluctuations in meat pH throughout the ageing period, a generalg increase in pH can be observed over0 ageing period. A similar overall increase in pH occurred in eland LTL steaks from day 2 to day 21 [8]1 and for 2 springbok 4 from 6 day 1 until 8 day 10 5 [17]. 12 Boakye 14 and Mittal [29] also Days post-mortem recorded an overall (N) force shear Warner-Bratzler pH increase in vacuum-aged beef until day 16 post-mortem, and attributed this increase to fluctuations in the charge of meat proteins as a consequence of proteolytic enzyme activity. Whilst post-mortemFigure 6. Effectageing of sex and was ageing shown (days post-mortem to improve) on the meat Warner–Bratzler tenderness, shear force ageing (N) of female is often accompanied a–h by weep loss, anand unfavorablemale impala longissimus effect thoracis caused et lumborum by moisturemuscles. Means lost with from different raw superscripts meat whichdiffer is considered from one another (p ≤ 0.05). Error bars represent the standard error of the means. unattractive to consumers [30]. Weep loss is affected by changes in the muscle pH post-mortem, as well as the rate4. Discussion and extent of proteolysis and protein oxidation, as the degradation of cytoskeletal proteins results in shrinkage of the muscle cells and the reduction of their ability to retain water [30]. In the present study, the changes in the weep loss percentage of impala LTL steaks were as expected, with a plateau being reached on day 6 post-mortem. This plateau in weep loss percentage as the ageing period progresses is similar to observations in previous studies in game [8,17], and is the consequence of a limited amount of unbound water in the meat for exudation. For vacuum-aged meat, a weep loss of one to two percent is considered acceptable by consumers, while more than four percent may be considered excessive [31]. Whilst the weep loss for impala meat over the ageing period exceeded this at every time point, other factors such as compression during vacuum packaging can influence this significantly [32], with up to 13% of the moisture lost being accountable to losses from vacuum sealing [8]. Nonetheless, the weep loss from impala steaks in the current study were comparable to that of springbok during ageing [17], which is of similar body size and muscle yield to impala, but the Appl. Sci. 2020, 10, 4485 8 of 11 use of absorbent pads to minimize the visual appearance of the weep could be considered for wet-aged cuts from these species sold in vacuum packaging. The surface color of meat is an important quality attribute that influences the consumer acceptability at the point of purchase, as color is often equated to meat “freshness” [33,34]. Discoloration in meat is thus a primary cause of lost retail sales which force producers to discard or devalue substantial amounts of retailed meat [35]. Discoloration can occur on the surface of meat due to oxidative processes resulting in increased metmyoglobin formation and thus a brown surface color that is considered unacceptable by consumers [36]. Therefore, it is important to monitor the color stability of meat throughout the post-mortem ageing process, to ensure that discoloration does not occur and that a consistent, high-quality and valuable game meat product remains [37]. The color parameters for the impala LTL steaks in the present study indicates that the surface color becomes lighter, redder and more saturated as the ageing period progresses. However, the a* values of the aged impala LTL steaks remained well above the established minimum of 12 for consumer acceptability [38] from 6–14 days post-mortem. A significant increase was observed for both the b* and hue angle values of the aged impala LTL steaks from day 1 until day 8, after which a plateau was reached from day 10 onwards. The increase in hue angle shows that the impala LTL steaks in the present study became duller and browner after 10 days of ageing, with this discoloration potentially indicating microbial spoilage [39]. Thus, it is recommended that impala steaks should not be aged more than 8 days post-mortem to prevent discoloration. The cooking loss of LTL steaks from female impala was at a maximum of 3.3% higher than that of males on some ageing days in the present study, which is likely due to the changes in pH, but did not have a significant influence on the shear force of the meat. Tenderness and its variability are the most important sensory meat quality characteristics for consumers influencing the repurchase of such products [15]. The variability in tenderness is largely influenced by differences in its structural components. The concentration of the collagen, and the extent of collagen cross-linking, determines the baseline toughness of the meat, the latter of which can only be altered to a limited extent [40,41]. Meat tenderness is therefore primarily improved by means of post-mortem proteolysis of the structural proteins within muscle fibers during ageing [41]. The shear force (tenderness) of the impala meat in the present study was improved by ageing, where the largest influence of sex was seen between days 1 to 4 post-mortem. This result may be due to the female impala in the present study being older (estimated at 24 to 36 months old) than the male impala ( 15–18 months old) as a result of difficulty estimating ± the age of female impala in the field, as they have no horns. Nonetheless, after 6 days of ageing, the variation in shear force values between the sexes was negated. Overall, post-mortem ageing decreased the shear force values of impala LTL steaks from 25.5 0.91 N on day 1 to 13.5 0.91 N on day 8, where ± ± after no further benefit was seen for tenderness or color. A similar decrease in shear force (13.8 N) was observed from springbok under similar ageing conditions, after five days of ageing [17]. However, in beef, up to 14 days of ageing is required to realize this decrease in shear force (10–16 N) [42,43]. The ultimate or maximum tenderness of meat is determined by the rate and extent that myofibrillar proteins are degraded during post-mortem proteolysis [18], and is recorded to be higher in certain game species than beef due to increased activity of proteolytic enzyme [44].

5. Conclusions Wet-ageing decreased the variability of shear force values in impala meat from different sexes after eight days post-mortem, thereby improving product uniformity and its marketability. Ageing further than this provided no further benefit in tenderness, and resulted in discoloration. It is recommended that sensory evaluation be performed to determine whether aroma, flavor, and texture attributes are altered by this ageing period, to ensure optimal consumer acceptability.

Author Contributions: Conceptualization, methodology, funding acquisition, and supervision, L.C.H.; investigation, data collection, and project execution, R.A.E.; data interpretation, original draft preparation, Appl. Sci. 2020, 10, 4485 9 of 11 and editing, T.N.; review and editing, L.C.H. and R.A.E. All authors have read and agree to the published version of the manuscript. Funding: This research is supported by the South African Research Chairs Initiative (SARChI) and partly funded by the South African Department of Science and Technology (UID number: 84633), as administered by the National Research Foundation (NRF) of South Africa, and partly by the Department of Trade and Industry’s THRIP program (THRIP/64/19/04/2017) with Wildlife Ranching South Africa as partner and by Stellenbosch University. Any opinions, findings and conclusions or recommendations expressed in this material are that of the author(s) and the National Research Foundation does not accept any liability in this regard. Acknowledgments: The authors would like to acknowledge the Internal Grant Agency (IGA) FTZ CZU, Prague (no. IGA20205005) for their support. The authors would also like to thank Castle de Wildt (South Africa, http://www.castledewildt.co.za/wildlife-breeding/) for the donation of the animals. Conflicts of Interest: The authors declare no conflict of interest.

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