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Introduction
Meat and meat products play an important role in the human diet as they provide proteins and essential amino acids that are difficult to obtain from vegetables and fruits (Kushi et al., 2006). In particular, poultry meat is preferred by consumers owing to its nutritional value as well as cost (Stanciu, 2020). The average poultry meat consumption of the Organization for Economic Cooperation and Development (OECD) member countries was 31.7 kg/capita in 2020, which reflects an increase of approximately 5 kg from 26.57 kg/capita in 2010. In contrast, the world’s average poultry meat consumption was 14.88 kg/capita in 2020, compared to approximately 12.73 kg/capita in 2010. The consumption of poultry meat in the United States was 50.1 kg/capita in 2020, whereas it was 14 kg/capita in China, and 18.7 kg/capita in Korea (OECD, 2020). Poultry production and consumption have increased significantly over the past 40–50 years, and are expected to increase further, especially in developing countries, making chicken the most valuable source of meat protein for the growing world population (Baldi et al., 2020; OECD and FAO, 2020). In a survey of 1,100 adult men and women aged between 20 and 69 years old in Korea, the annual consumption of chicken was found to have increased due to easy delivery and online purchases, despite the challenges introduced by COVID-19. When purchasing chicken meat at home, the first criterion was freshness (63.6%), followed by price (39.9%), meat quality (36.9%), and expiration date (29.1%). In addition, the most common improvement for promoting chicken consumption was providing grade-based information (83.7%) and soft-textured meat (93.5%) (Rural Development Administration, 2020). The livestock industry focuses on health-oriented livestock products. In particular, meat processing products were developed to meet the requirements of modern consumers who prioritize improved living standards, diet, and food safety (Hafez and Attia, 2020). Therefore, chicken products have been improved in various ways, such as changing the cooking methods and developing partial meat products (Kook et al., 2020).
Most waste in the meat industry is generated during slaughtering, which raises concerns for environmental protection and sustainability (Russ and Meyer-Pittroff, 2004). The slaughter by-products include bones, tendons, skin, gastrointestinal contents, blood, and internal organs (Grosse, 1984). These can be generally utilized as food or reprocessed as secondary by-products in agriculture and industry (Liu, 2002). Many studies have been conducted to utilize the slaughter by-products (Jayathilakan et al., 2012; Min et al., 2017; Mirzapour-Kouhdasht et al., 2020). Collagen is an animal protein that accounts for approximately 30% of the animal protein and can be easily obtained from livestock, including fish, pigs, and chicken (Aberoumand, 2012; Cheng et al., 2009; Huo and Zheng, 2009; Muyonga et al., 2004; Pataridis et al., 2008). It provides strength and support to the skin, bone, cartilage, tendon, and blood vessels, and plays a major role in the extracellular matrix (Erdmann et al., 2008; Mendis et al., 2005; Ngo et al., 2011). Collagen forms a scaffold for tissue treatment and wound healing and is used in applications related to drug delivery systems in the urology, biotechnology, and medical fields (Nune et al., 2017). In the food industry, collagen is used in protein supplements, fragrances, gelling agents, emulsifiers, and additives aimed at improving texture (Nazeer and Kumar, 2012). The ingredients in poultry feed are high in methionine and lysine and should be a source of energy and minerals. Collagen is high in methionine (6%) and lysine (19%), so it plays an important role as a feed supplement in the poultry industry (Nazeer et al., 2011). Proper protein feed is important for broiler growth and meat quality (Beski et al., 2015). Many studies have focused on improving the meat quality of chicken through feed supplementation using natural products and by-products, such as collagen (Choi, 2005; Park et al., 2005; Woo et al., 2007). Supplementation with hydrolyzed collagen is known to have a positive impact on tibial dimensions, strength, and the mineral content of broilers (Güz et al., 2019). Collagen feeding can play an important role in increasing the muscle content of the broiler, and the collagen component proline can provide a higher initial growth rate than other amino acids, and thereby is a necessary ingredient. Therefore, collagen-related studies have focused on extracting collagen from by-products produced by the livestock and fish industries and using it as an effective supplement for alternative feed (Nurubhasha et al., 2019). The current study aimed to improve the availability of pig skin, which is a by-product of the pig industry, to enhance the quality of broiler breast meat.
Materials and Methods
Six liters of distilled water and 3 kg of pig skin were put into an electronic pressure extractor (KS 220S, Kyungseo E&P, Incheon, Korea) at 80°C for 5 h. After heating, the insoluble collagen was strained through the gauze. The collagen extract was hydrolyzed at 60°C for 5 h using protease (Love Me Tender, H GROUP USA LLC, Atlanta, GA, USA) and concentrated at 80°C for 12 h. The collagen extract was cooled at room temperature for 20 min and filtered to 9,450 daltons using a 10,000-dalton filter (Multi-Angle Light Scattering, Korea Basic Science Institute, Daejeon, Korea). The final collagen extracted from pig skin was stored at 4°C for 24 h and used for feed additive.
Fifty Ross 308 broilers, two weeks old with an average weight of 322±0.3 g, were housed for two weeks before the experiment. The experiment was conducted with five random, completely blocked treatments, classified into the following groups: the negative control (basal diet, NC), positive control (basal diet+0.1% fish collagen (fish collagen premium power, Graviola House, Korea), PC), T1 (basal diet+0.1% pig skin collagen), T2 (basal diet+0.5% pig skin collagen), and T3 (basal diet+1.0% pig skin collagen) groups. The basal diet met or exceeded the National Research Council (NRC, 1994) requirements. All broilers were allowed to consume both feed and water ad libitum. After completing the two-week feeding regime, the tibial bone size, weight and skin thickness of the broilers were measured and the left breast was harvested for meat quality analysis.
In this study, five replicates of the same treatment were prepared and analyzed. The average value was considered the result.
Moisture, fat, protein, and ash (%) were measured in accordance with the Association of Official Agricultural Chemists method (AOAC, 2012).
pH was measured after adding 50 mL of distilled water to 5 g of breast meat sample. All samples were homogenized for 30 s using a homogenizer (Stomacher®400 Circulator, Seward, UK) and the pH was measured using a pH meter (Mettler Toledo Delta 340, Mettler-Toledo, Leicester, UK).
The water-holding capacity was estimated by the centrifugation method as reported by Laakkonen et al. (1970). The crushed sample (0.5±0.05 g) was placed in the upper filter tube of the centrifuge tube, heated in an 80°C water bath for 20 min, and cooled thereafter for 10 min. The centrifuge tube centrifuged for 10 min at 3,360×g. It is displayed as the ratio of the remaining sample weight / the sample weight before heating.
To measure the texture characteristics of the sample, the mastication test, shear, and cutting tests were performed using a rheometer (COMPAC-100, Sun Scientific, Tokyo, Japan), and the sample was placed at right angles to the direction of the muscle fibers in a 3cm-thick steak shape. The muscle was sheared and heated to an internal temperature of 70°C, and then allowed to cool under running water for 30 min. From the cooled sample, a 1-cm-diameter core was drilled in a cylindrical shape along the muscle fiber direction to collect the sample and then cut in the direction perpendicular to the muscle fiber using a rheometer (Compac-100, Sun Scientific) to measure the shear force. The measurement was repeated three or more times.
After shaping a 2-cm-thick sample into a circle (100±5 g), it was put in a polypropylene bag and vacuum packed. The amount of drip loss generated during storage in a refrigerator at 4°C for 24 h was measured as the weight ratio (%) of the initial sample.
After shaping a 3-cm-thick sample into a circle (150±5 g), it was put into a polypropylene bag, vacuum packed, and heated in a 70°C water bath for 40 min, followed by cooling for 30 min. The weight lost after heating was measured as the weight ratio (%) of the first sample.
The surface color of the breast meat was measured using a spectrophotometer (Model JX-777, Color Techno. System, Tokyo, Japan) standardized with a white plate (CIE L*, 94.04; CIE a*, 0.13; CIE b*, –0.51), with the CIE L* value on a HunterLab color system representing lightness, the CIE a* value representing redness, and the CIE b* value representing yellowness using a white fluorescent lamp (D65).
Approximately 4 g of sample was placed in an Erlenmeyer flask, and 30 mL of sulfuric acid solution was added to the sample. The flask was covered with a lid and heated in a dry oven at 105°C for 16 h. The contents of the flask were transferred to a 500 mL volumetric flask, diluted with tertiary distilled water, and homogenized. A Whatman No. 2 150 mm filter was used to filter the sample. The filtrate (5 mL) was diluted to 100 mL, and 2 mL of the diluted sample was added to a test tube, to which 1 mL of oxidant solution was added, shaken, and finally left at 20°C to 25°C for 20 min. Next, 1 mL of a color reagent was added to each test tube, mixed, and these samples were incubated in a 60°C water bath for 15 min, cooled under flowing water for 3 min or more, and finally, the absorbance was measured at a fixed wavelength of 558 nm using a spectrophotometer. For preparation of the standard curve, 2 mL of the working standard solution was taken and its absorbance measured based on color development. The collagen content (g/100 g) was analyzed by substituting the sample absorbance into the regression equation (Kolar, 1990).
The left tibia bones of each broiler were removed as drumsticks with the flesh intact. The drumsticks were immersed in boiling water (100°C) for 10 min. After cooling to room temperature, the drumsticks were defleshed by hand and the patella was removed. Then, they were dried for 24 h at room temperature. The tibial length and bone weight were measured. Skin thickness was measured immediately after slaughter.
The sample (50 g) was homogenized in 250 mL of chloroform:methanol (2:1) solution at 3,000 ppm according to the method of Folch et al. (1957). The lipids were extracted from the homogeneous solution and sodium anhydrous sulfate was used to remove moisture from the liquid from which the lipids were extracted and the filtrate was concentrated at 50°C–55°C. One milliliter of tricosanic acid was added first, followed by 1 mL of 0.5 N NaOH. The sample was heated at 100°C for 20 min, cooled for 30 min, and 2 mL of BF3 was added, heated for 20 min, and finally cooled for 30 min. After adding heptane and 4 mL of NaCl, the supernatant was removed and injected into a gas chromatograph to measure the fatty acid content.
Amino acid analysis was conducted according to the standard analysis method proposed by the Livestock Technology Research Institute in 2000 using an amino acid analyzer. Briefly, 100 mg of sample (approximately 30 mg of crude protein) was placed in a decomposition bottle, and 40 mL of 6 N HCl was added, followed by the injection of nitrogen gas. It was placed in an evaporating flask, connected to a rotating evaporator, and the hydrochloric acid was removed at 50°C. When the evaporation was completed, the distilled water bottle was washed with distilled water, the contents transferred to the evaporative flask, and evaporation was conducted thrice. A small amount of buffer solution (pH 2.2) or distilled water was added to the final evaporative flask to dissolve the amino acids, and the sample was then filtered through No. 5B filter paper, and the volume was made up to 50 mL. This sample was used as the amino acid analyzer specimen and its absorbance was measured at 570 nm.
Statistical analysis was conducted using the General Linear Model (GLM) procedure of the SAS program (Statistical Analysis System 2002, Cary, NC, USA), and the significance of the comparisons between the means of the treatment groups was verified (p<0.05) using Duncan’s multiple range test.
Results and Discussion
Table 1 shows the general composition of the breast meat from pig skin collagen-fed broilers. While the moisture content was the highest in the PC (74.33%) group, it was the lowest in the T3 group (72.94%). There was no significant difference between the NC, T1, and T2 groups in terms of moisture content. The protein content was significantly lower in the T1 group than in the NC group, and the PC group showed significantly low protein content. Previous studies reported that the growth of salmon was reduced upon replacing much of the fish protein concentrate fed to the fish with gelatin (Mundheim et al., 2004; Opstvedt et al., 2003). Studies have also shown that increasing the supply of bovine skin gelatin reduced the rate of protein digested from the meat and that the protein source affected the flow of endogenous amino acids into the intestines (Rutherfurd et al., 2015). According to research on the performance of broilers—which were provided feed in which a portion of soymeal was replaced by cow skin gelatin—the weight of the broilers decreased as the supply of cow skin gelatin gradually increased (Rutherfurd et al., 2015). These studies suggested that collagen supplementation may reduce the amino acid digestion rate of broilers, thus reducing the protein content in broiler breast meat. As shown in Table 1, groups T1 and PC had significantly higher levels of fat than the groups given other treatments. The fatty acid biosynthesis of algae depends upon the supply of dietary carbohydrates for acetyl-CoA production, and when such carbohydrates are ingested, insulin stimulation results in the increased activity of fatty acid synthase (FAS) and malate dehydrogenase (MD) (Hillgartner et al., 1995). Most of the fat present in poultry comes from carbohydrates, and glycolysis and NADPH are essential for the synthesis of fatty acids in the cytoplasm. Therefore, fatty acid synthesis depends largely upon the supply of carbohydrates from feed and the activity of the glycolytic system (Moon, 2018). The significantly higher fat content in the PC group can be attributed to the carbohydrate content of the feed and is not thought to be affected by collagen. The PC group also has a relatively high fat content owing to its low protein content. Table 2 shows that the ash content significantly increased with increases in the collagen levels. Feeding calf bone-extracted gelatin to early breeding chicks, with high bone hemostatic capability (in 14 days) significantly improved the tibial ash, calcium, and phosphorus content in the chicks. Considering that gelatin can improve bone strength by promoting the mineralization of cartilage sheets (Kim et al., 2017), the feeding of pig skin collagen can be assumed to increase the ash content in broiler breast meat.
Table 2 shows the quality characteristics of the broiler breast meat. The NC group showed significantly higher drip loss than the other treatments, whereas T2 showed the lowest. There was no significant difference between cooking loss and the CIE L* of Hunter color. However, the CIE a* of the T2 and PC groups was significantly lower than that of the other treatments, whereas the CIE b* of the T1 group was the lowest. The collagen content of the skin was significantly higher in the T1 group than in other treatment groups and was the lowest in the T3 group. The drip loss, cooking loss, water-holding capacity, and color of the breast meat are known to be related to pH (Berri et al., 2008; Fletcher, 1995; Mir et al., 2017). However, since there was no significant difference in pH, an association between drip loss, cooking loss, water-holding capacity, and color due to the difference in pH could not be determined. Increases in the water-holding capacity were reported to improve tenderness, juiciness, firmness, and appearance (Offer and Knight, 1988). Therefore, since there was no significant difference in the water-holding capacity of the broiler breast meat, it is judged that there was no significant difference in the shear force. A previous study reported a positive correlation between drip loss and time in storage, suggesting that the oxidative processes occurring in both the lipid and protein fractions during storage may alter the water-holding capacity (Lonergan et al., 2001). However, since there was no significant difference in drip loss and water-holding capacity in this study, the relationship between drip loss and water-holding capacity could not be determined. In this study, the collagen content of the broiler breast meat was found to increase with the collagen content of the feed, and the T3 group showed a significantly higher content than the NC group. Preceding studies reported that the human consumption of collagen extracted from pig skin and chicken feet was likely to affect the proliferation of fibroblasts and the formation of collagen fibroblasts in collagen-specific ways (Iwai et al., 2005). The results of the previous study may be related to the difference in the collagen content of the breast meat of broilers fed pig skin collagen in this study.
Table 3 shows the texture characteristics of broiler breast meat. Springiness and cohesiveness were significantly higher in all treatment groups relative to those in the NC group, and chewiness was significantly higher in the T3 group than in the other groups. Hardness was significantly higher in the following order: T3, PC, T2, and T1. There was no significant difference in shear force between the treatments. In a previous study, pigs that orally ingested feed with collagen peptides were shown to have a higher fibroblast density in the dermis, larger diameter and density of collagen fibrils, and a larger area of collagen than those that ingested the basic diet and feed with lactalbumin (Matsuda et al., 2006). Small-diameter collagen fibrils are mechanically weaker than thicker ones (Parry et al., 1978), and collagen fibrils are denser due to the proliferation of fibroblasts in the dermis (Yamamoto et al., 2002). Collagen has been proven to play a key role in determining the meat strength in various livestock, including birds (Sirri et al., 2016). Broilers that ingested pig skin may be expected to have a greater diameter and higher density of collagen fibrils in the dermis than those in the NC group. As the pig skin-fed broilers had harder meat due to the high density of fibroblasts in the dermis, springiness, cohesiveness, and chewiness of the meat may also be affected.
Table 4 shows the bone length, weight, and skin thickness of broiler breast meat. No significant difference between the treatments was seen (Table 4). The bone weight was the heaviest in the T1 group and tended to decrease with the addition of collagen extract. Although there was no significant difference in the four treatment groups, the bone length tended to increase in the T1 and T2 groups compared to the NC group and tended to decrease in the T3 group. The addition of gelatin extracted from calf bones to broiler feed is known to result in longer broiler tibiae than those in the NC group (Beyranvand et al., 2019). In animal experiments, the intake of hydrolyzed collagen by livestock was reported to improve bone density and bone mineral content (Wu et al., 2004). The amount of type I collagen in bone substrate increased when mice consumed hydrolyzed collagen (Nomura et al., 2005). Research has shown that the consumption of alkali and acid-treated bone gelatin enhanced the surface area of the small intestine, and consequently improved bone properties and the intestinal absorption of phosphorus (van Harn et al., 2017). There is a report that gelatin intake promotes bone-forming cell differentiation and bone regeneration in broilers and that gelatin can improve bone strength by promoting the mineralization of cartilage sheets (Kim et al., 2017). The results of previous studies show that collagen intake and bone are related, but no significant difference was observed in this research. Compared to the NC group, there was no significant difference in skin thickness between the T1 and T2 groups, although it was significantly lower in the T3 group. The results of a previous experiment showed that the thickness of the dermis in pigs fed collagen peptide was similar to that of pigs fed lactalbumin group and the NC group (Matsuda et al., 2006).
Table 5 shows the fatty acid analysis of the broiler breasts. No significant difference was observed overall. The γ-linoleic acid (C18:3n6) content was the highest in the NC group (at 16.38%) and tended to decrease as the extract content in the treatments increased. The γ-linoleic acid (C18:3n6) content in the PC group was 14.80% and it was the lowest level in the T3 group at 13.59%. Feeding pigs with diets containing conjugated linoleic acid has been reported to significantly increase the L* and a* values of pig tenderloin, although it did not increase the b* value (Dunshea et al., 2005). As shown in Table 5, the γ-linoleic acid (C18:3n6) content of NC and T1 was high, and as shown in Table 2, the L* and a* values of the NC and T1 groups were high. The results of this study are similar to those reported in previous studies. The L* and a* values were attributed to the γ-linoleic acid content (C18:3n6). Table 6 shows the amino acid analysis of broiler breast meat. While there was no significant difference in the overall amino acid content, lysine, histidine, and arginine showed lower levels in the T1, T2, and T3 groups than in the NC group.
Conclusion
This study was conducted to investigate the quality of broiler breast meat after feeding broilers pig skin collagen, a by-product of the pig farming industry, and to upgrade broiler breast meat to meet the consumer requirements. The protein content was highest in the NC and T3 groups (p<0.05). Drip loss was the highest in the negative control (p<0.05). As the pig skin collagen content increased in broiler feed, the collagen content, springiness, cohesiveness, chewiness, and hardness also gradually increased (p<0.05). In terms of Hunter color, CIE L* and the CIE a* value were high in the NC and T1 groups (p<0.05), and the CIE b* was low in the T1 group (p<0.05). Bone weight was the highest in the T1 group, bone length was the shortest in the T3 group, and the skin also was the thinnest in the T3 group (p<0.05). Therefore, the breast meat of pig skin collagen-fed broilers was of better quality than that of the NC group. Among them, T3 was considered to have the most improved broiler breast meat quality because it had the highest collagen content, best texture characteristics, and the lowest drip loss, despite the disadvantage of poor skin thickness. Research is needed to offset the disadvantages of poor skin thickness for higher meat quality.
