Meat yield and quality characteristics of Woori heukdon pigs as affected by dietary amino acids and chromium supplementation

The experimental protocols used in this study were reviewed and approved by the Institutional Animal Care and Use Committee of NIAS (NIAS-2020-437). The experiment was performed between January and July 2023 at the Swine Experimental Farm (Cheonan) and Animal Products Utilization Division (Wanju) of NIAS (Korea). A total of 40 WHD piglets (Duroc sow [62.5%] × KNP sire [37.55%]) at same weaning age (21 days of age and body weight: 31.49 ± 0.24 kg) were randomly divided into two feeding groups. Each group had 20 pigs concluding 10 castrated males and 10 females. All pigs of the control group were fed basal diets that were formulated to meet National Research Council [26] nutrient requirements. The pigs of experimental group were received a basal diet + 4% additional supplementation of lysine, isoleucine, methionine, threonine, valine and tryptophan during the growing phase (30–65 kg body weight), and basal diet + 0.1% (w/w) chromium picolinate during the finishing phase (65–110 kg body weight). The chemical ingredients of the feeding diets at the growing and finishing phases are presented in Table 1. The dose of supplemented amino acids and chromium were based on results of our preliminary study (data not shown) and previous studies [19,27,28]. During the experiment, the animals were housed in different cages (3.5 × 5.0 m, 0.8m²/head) and freely accessed to the feed and water. The pigs were harvested when they reached a common market weight (approximately 180 days-old and 110 kg body weight).

At the end of the feeding trial, the pigs were transported from the experimental farm to a slaughter house with a transporting duration of approximately 2 h. At the abattoir, the pigs were laired in cages for 3 h with full access to water. Slaughter was performed following the commercial process. After removal of internal organs, head, feet and tail, the carcasses were split down the midline, washed using high-pressure washing pumps, and chilled at 2°C. During the slaughter, the live and carcass weights were recorded using a scale installed on the production line.

After 24 h of slaughter, back-fat thickness was measured using a caliper on the midline between 11th and 12th rib, and last rib and first lumber vertebra. Next, both sides of each carcass were dissected into three portions (picnic shoulder, mid-section containing loin and belly, and ham) which were then dissected into 7 cuts (loin, shoulder ribs, shoulder butt, tenderloin, belly, picnic and ham), following the instruction of the Korean Pork Cutting Specification (KPCS) [29]. Thereafter, each cut was manually separated into skin, fat, bone and muscle.

For meat quality properties analysis, three representative cuts: loin (m. longissimus thoracic et lumborum [LTL]), belly and ham (m. semimembranosus) collected from the left carcass sides were used. The cuts were then prepared into sub-samples, and the sampling manners were fixed for all the cuts in each analysis.

pH was measured in triplicate using a pH meter (pH*K 21, NWK-Technology GmbH, Aichach, Germany). Before use, the device was calibrated with provided standard solutions (pH 4.00 and pH 7.00) following the manufacturer’s instruction.

The meat color was measured after 30 min blooming at 5 different locations on the surface of each sample, using a color meter (CR-400, Minolta Camera, Osaka, Japan). The color meter was calibrated against a standard white tile (Y = 86.5, X = 0.3171 and y = 0.3331). The color parameters measured were CIE L*, a* and b*. Cooking loss and shear force value were measured using the procedures as described in our previous study [16].

The chemical composition (protein, fat, moisture and collagen) was determined with a Food Scan™ Lab 78810 (Foss Tecator , Hilleroed, Denmark) as described by Anderson et al. [30].

The fatty acids content was analyzed following the method of Folch et al. [31]. Briefly, lipid content in the pork sample (10 g each) was extracted with chloroform: methanol (2.1, v/v). Following adding with 20 g of Na₂SO₄ and vortexing for 1 min, the lipid layer was carefully collected and placed in Erlenmeyer flask which was concentrated using rotary evaporator in pre-heated 55°C water bath. Thereafter, 1 mL tricosanoic acid and 1 mL of 0.5N NaOH were added to each the sample, thoroughly mixed and placed into vials. A gas chromatography (GC)/flame ionization detector (FID; Varian Technologies, Palo Alto, CA, USA) was used for analyzing the fatty acids. The GC/FID conditions set was same as those shown in our previous study [16].

To assess whether the dietary supplementation affected the flavor properties, two representative cut types (loin and belly) were used. The analysis of flavor volatile compounds was done following the procedure of Van BA et al. [32] with suitable modifications. The pork samples were manually chopped and cooked at around 180°C on a frying-pan with continuously turning for about 2 min. Afterward, the samples (2 g each) were taken, placed into 20-mL vial and tightly capped with magnetic cap. Extraction of volatiles was carried out at 60°C for 50 min using a SPME auto-sampler (PAL RSI 85, Agilent, Santa Clara, CA, USA), and were then analyzed using a GC and mass spectrophotometry (5977B MS, Agilent) under the conditions as described by Hoa et al. [16]. All flavor volatiles were identified by using Wiley library (Agilent) and further confirmed by external standards. The concentration of identified compounds was calculated using a concentration-known internal standard (2-methyl-3-heptanone).

Data was analyzed using the Statistical Analysis System (SAS) Enterprise software (version 7.1, SAS Institute, Cary, NC, USA). The General Linear Model procedure of the SAS was used in which the feeding diet was set as the main effect while, the carcass traits, meat yield, color traits, chemical composition, shear force, fatty acids and flavor volatiles were set as random variables. Means comparison was carried out using the Duncan’s test, and a p-value of < 0.05 was considered as statistically significant difference.

Table 1 shows that no differences in the carcass traits were observed between the animal groups (p > 0.05). However, it was observed that the back-fat thickness tended to decrease in the pigs which were received the dietary supplementations. Compared to market weight of commercial pig breeds such as ([Landrace × Yorkshire] ♀ × Duroc ♂) finished at the same age (180 days old) reported by Van Ba et al. [33], the WHD pigs in the present study exhibited a similar body weight. This signifies that the growth potential of WHD pigs in this study was comparable to that of the commercial pig breeds. Furthermore, the results indicating no differences in the live weight between the pig groups could be related to the same dietary energy levels (3,300 kcal/kg). Regarding this, a numerous studies have also reported that feeding diets have no effects on pig’s growth rate as they meet the required energy for growth [34–36].

Regarding the carcass composition, no effects of the dietary supplementations were observed on the total meat, fat, bone and skin weights (p > 0.05). This indicated a similar rate of protein and fat (subcutaneous and intermuscular fat depots) deposition in both the control and experimental groups. Similar to the present results, Hu et al. [19] found no effects of dietary amino acids supplementation on the total meat, fat, bone and skin yields of commercial pigs finished at 110–120 kg body weight. A study reported by Park et al. [28] showed a reduction in back-fat thickness and increased meat yield of pigs supplemented with 200 ppb chromium.

The chemical composition of WHD meat fed the control and experimental diets are presented in Table 2. IMF content is recognized to be the most important constituent determining eating quality of meat because it contributes to tenderness, juiciness and flavor [3,4]. Therefore, producing pork with increased IMF content to meet the consumer’s demand is critical task for the pig industry [19]. Results showed that the pigs received the dietary supplementation had a significantly higher IMF content in both loin (increased by 1.55%) and ham (increased by 0.94%) than the control group (p < 0.05). The representative images showing the transverse cuts of loins are shown in Fig. 1. Our results align with those of Ma et al. [18] and Hu et al. [19], who reported an increase in IMF content of pork LTL muscle fed dietary amino acids (arginine and glutamic acid) supplementation. Tan et al. [37] also found an approximately 70% greater IMF content of longissimus dorsi (LD) muscles of growing-finishing pigs received dietary amino acids supplementation compared to non-supplemented pigs. In contrast to the increase of IMF in the loin and ham cuts, a significant decrease (by approximately 7%) of fat content was observed in the belly cut of pigs fed the experimental diet (41.05%) compared to the control diet (47.48%). In the present study, the belly cuts were fabricated according to the KPCS [29], where only the skin and ribs are removed. Therefore, the fat content, is comprised of all subcutaneous, intermuscular and IMF depots. This signifies that supplementation with the amino acids and chromium picolinate effectively reduced the subcutaneous and/or intermuscular fat deposition on the belly cut. We have recently observed that the belly cut of WHD pigs has a much higher fat content (over 40%) compared to that of commercial LYD pig (around 30%) [16]. Such the high fat content may result in a higher trimming loss and reduced consumer preference for the belly. In the present study, the dietary supplementations with amino acids and chromium effectively increased the desirable fat (IMF) in the lean cuts (loin and ham) and decreased the undesirable fat (e.g., subcutaneous and/or intermuscular fat) in the high-fat content cut like belly.

Fig. 1. Representative images show. (A) Finishing-Woori heukdon (WHD) pigs fed the dietary supplementation before slaughter, (B) cross-sectioned loin of non-supplemented WHD pigs, and (C) cross-sectioned loin of supplemented WHD pigs.

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The adipose tissues of pork carcass may be deposited from (i), diet-derived fatty acids and (ii), the DFS pathway [38]. In the DFS pathway, the fatty acids are synthesized by converting glucose into triglycerides through the glycolysis cycle [1]. The possible mechanisms underlying the phenomena observed in the present study could be due to: (i) the supplemented amino acids promoted the lipogenesis gene’s expression in the muscle tissues [19], resulting in the increased IMF content, and (ii) the chromium could amplify the insulin action, resulting in increased glucose converting into energy required for pig’s metabolic activities rather than for the DFS process [23]. Mooney and Cromwell [25] found a significant reduction in total carcass fat in growing-finishing pigs supplemented chromium picolinate or chromium chloride. In the present study, the dietary supplementations with combined amino acids and chromium did not affect the total carcass fat (Table 3), but it effectively decreased in the fat level of belly cut. This could be related to the synergetic effect of both the supplemented amino acids and chromium picolinate.

For the other composition, the dietary supplementations did not affect the moisture content but it reduced the protein content of loin and ham cuts, this was probably due to the increased IMF content in these cuts. The supplementations also caused an increase in the moisture content of belly, this could be associated with the decreased fat level in this cut, because the fat and moisture content in meat content are inversely related to each other [39].

It is well recognized that cooking loss (reflecting the water holding capacity), pH and shear force, are important quality traits of meat. The dietary supplementations did not affect on all these traits in the loin and belly cuts (Table 4) (p > 0.05). However, compared with the control group, the dietary supplementations reduced the cooking loss of belly (p < 0.05). This indicates that the dietary supplementations improved the water holding capacity of belly cuts. Color is an important quality trait of meat [40]. The dietary supplementations did not influence the lightness, redness and yellowness of all the three cuts examined (Table 5). Similar to the present fining, Tan et al. [37] and Hu et al. [19] reported no effects of dietary supplementations with amino acids (e.g., arginine and glutamic acid) on pH, cooking loss and color traits of pork LD muscles. Until now, there is limited research on the dietary supplementation with chromium on pork quality. Studies by Boleman et al. [41] and Wang et al. [42] also showed that dietary supplementation with chromium chloride or picolinate did not influence the meat quality of LD muscles of finishing pigs.

To examine whether the dietary supplementations influence the fatty acids composition of meat, two representative cuts (loin and belly) were used, and the results are shown in Table 6. For the loin cut, the dietary supplementations only affected the C18:0, C18:2n6 and C18:3n3 contents. Compared with the experimental diet, the pigs fed the control diet had a lower C18:0 content, and higher C18:2n6 and C18:3n3 contents, which contributed to the higher total polyunsaturated fatty acids (PUFA), n3 and n6 PUFA contents in this cut (p < 0.05). For the belly cut, only C18:0 content was affected by the dietary supplementations. Oleic acid (C18:1n9) is well recognized as the most predominant and important for cooked meat flavor development [43]. We observed that the dietary supplementations did not alter the C18:1n9 content in both the cuts. However, under the current experimental conditions, the alternation of C18:0, C18:2n6 and C18:3n3 contents might be related to the supplemented amino acids or chromium alone and their combined effects on the absorbing rate of these fatty acids from the feeding diets and/or activity amplification of fatty acid synthase that converts malonyl-CoA to palmitate and subsequently elongated to C18:0 by elongase enzyme in the de novo fatty acid synthesis pathway [1]. Also, the results indicating the decreased PUFA content of loin in the dietary supplementation group could be related to the decreased desaturation of saturated fatty acids (SFAs) into unsaturated fatty acids (UFAs) by the desaturase enzymes in the DFS pathway. Another possible mechanism responsible for the change of fatty acids composition by the dietary amino acids supplementation is the increased production of nitric oxide from these amino acids, which reduces the uptake of glucose by stimulating glucose-oxidation in muscle tissues, and subsequently reduces the DFS [37]. In general, it was observed that dietary supplementations apparently showed a negligible effect on the fatty acids compositions of the pork. In agreement with our results, numerous studies have also found that dietary amino acids or 200 ppm chromium picolinate supplementation do not alter total SFA and UFA contents in LD muscles of growing-finishing pigs [19,44].

The concentration of aroma volatiles of cooked loin and belly samples are presented in Table 7. It is well recognized that aroma flavor sensed by smell buds is a very important eating quality trait [45,46]. The aroma flavor of meat is composed of a variety of volatiles which are generated as a result of thermal oxidation of lipids, Maillard reaction, and the interaction between lipid-thermally oxidized products with the products of Maillard reaction [45,47]. Under the current analytic conditions, a total of forty-four aroma volatiles included: 18 aldehydes, 8 alcohols, 3 ketones, 4 sulfur-and nitrogen-containing compounds, 5 pyrazines and 5 hydrocarbons, were detected from the loin and belly cuts. We observed that the dietary supplementations showed a greater effect on the aroma volatiles composition of loins rather than on those of the belly cut. With regards to aldehydes, the concentration of 4 aldehydes (hexanal, heptanal, E,2-hetenal and E,E,2,4-decadienal) as well as total aldehydes content in the loins were significantly influenced by the feeding diets while, only an aldehyde (benzaldehyde) in belly cut was affected. Aldehydes are mainly produced from the thermal oxidation of UFAs [48,49], and some of them are produced from the Maillard reaction [46]. Hexanal, heptanal and E,2-heptenal, are known to be the oxidation products of C18:2n6 [49]. These aldehydes, with a low odor detection threshold (0.003–0.005 ppm) and associated with green, grassy and harsh odors, are considered as the unpleasant compounds in cooked meat [47]. Interestingly, compared with the control group, the dietary supplementations decreased (p–< 0.05) the concentrations of these unpleasant aldehydes. This phenomenon may be explained by the decrease of PUFAs (e.g., C18:2n6) content of the meat (in case of loin cut) as the result of the dietary supplementations (Table 5). Similar to the current findings, Elmore et al. [50] stated that a small change in fatty acids of meat could result in an alteration in aroma volatiles of cooked meat. For the oleic acid-derived aldehydes (e.g., octanal, nonanal and decanal) associated with desirable odors (e.g., fatty note), no effects of the dietary supplementations were observed. This is probably because of the C18:1n9 content that was similar in both the control and supplementation groups (Table 5).

With regards to alcohols, the dietary supplementations did not affect this volatile class in the belly cut, and but reduced (p < 0.05) the amount of 1-pentanol, 1-heptanol and total alcohols content in the loin. Alcohols are formed as a result of the fatty acids oxidation, and are not important contributors of cooked meat flavor because of their high odor threshold (0.5–4 ppm) [4]. Therefore, the lower alcohols content in the loin of pigs received the dietary supplementation could be related to its lower PUFAs (e.g., C18:2n6 and C18:3n3) content compared to the control group (Table 5).

Sulfur-and nitrogen-containing compounds, and pyrazines, as the Maillard reaction-derived products with desirable odor notes (meaty and roasty odor notes), are the most important contributors of cooked meat flavor [45,51]. Interestingly, the dietary supplementations led to an increase of total sulfur-and nitrogen-containing compounds as well as pyrazines content in both the cuts. The increases of sulfur-and nitrogen-containing compounds as well as pyrazines contents in the cooked meat of pigs received the dietary supplementations may be related to the fact that: (i), higher availability of amino acids (from supplemented amino acids) in fresh meat, produced a higher amount of intermediated products (e.g., ammonia formed in the Strecker reaction during cooking) which interact with lipid-derived aldehydes in the later stages of Maillard reaction to yield these compounds [47], and (ii), the lower amount of unpleasant aldehydes (hexanal and heptanal produced from C18:2n6) elevated the formation of pyrazines, and sulfur-and nitrogen-containing compounds [49]. Regarding this, Elmore et al. [48] also noted that many of Maillard reaction-derived flavor volatiles are not formed or are formed at lower level when a higher PUFA content is present. Frank et al. [52] found that number and quantity of Maillard compounds (e.g., pyrazines) in meat increased with increasing IMF content. In the present study, the pigs received the dietary supplementations had a higher IMF content as aforementioned (Table 2).

Overall, although the WHD is known as a novel pig breed with a slow growth rate compared to the other commercial pig breeds [15], the slaughter weight of WHD pigs was similar to that of the commercial pig breeds when finished at a similar age [33]. As earlier mentioned, a significant proportion of worldwide consumers has a strong preference for highly-marbled pork [5], the marbling degree or IMF level, therefore, has become a major interest to the meat industry [1]. In the present study, WHD meat presented a considerably higher IMF content compared to that of other commercial breeds [19,53]. This suggests that WHD pig, with a good potential of growth and IMF accumulation, could be considered as an outstanding breed for production of high-quality meat to fulfil the consumer’s preference. On the other hand, an excessive fat level may result in a more trimming loss and high risk of rejection by consumers [54]. In the present study, the dietary supplementation significantly reduced the fat deposition in the belly cut. This implies that dietary amino acids and chromium supplementation emerged as an effective nutritional intervention for improving IMF and lessening the undesirable fat (e.g., subcutaneous fat) deposition in pork carcasses.