Investigation of the Chemical Composition and Functional Proteins of Chicken Gizzard Inner Lining

Abstract

Chicken gizzard inner lining (GIL) is a by-product of animal meat products. This study examines the chemical characteristics and functional proteins of GIL. Our results demonstrate that GIL is protein-rich and contains essential amino acids for humans. In addition, we analyzed soluble GIL proteins in GIL and categorized the identified proteins into 12 functional groups or processes. Moreover, biochemical analysis revealed that the 18-kDa antrum mucosa protein (Gastrokine-1 protein, GKN-1), which is known to support gastroprotective and physiological activities, was the most abundant protein of GIL. In conclusion, GIL is an important source of proteins and bioactive compounds, such as GKN-1. Therefore, GIL may have some capabilities as important applications in food processing.

Introduction

Meat is generally recognized as a good source of proteins with high biological value, group B vitamins, minerals, and other trace elements. In addition, meat has been reported to potentially possess many bioactive compounds, including prebiotics, immunomodulatory regulators, mineral-binding compounds, hypotensive chemicals, cholesterol-lowering compounds, opioids, antimicrobial ingredients, and antioxidants (Bauchart et al., 2006; Cheng et al., 2009; Ahhmed and Muguruma, 2010; Toldrá et al., 2012; Udenigwe and Howard, 2013; Lafarga and Hayes, 2014). On the other hand, animal by-products are materials of animal origin that are generally not consumed by the humans and thereby often wasted in the food industry. However, some animal by-products have antimicrobial and antidiabetic properties as well as hypolipidemic and anti-inflammatory effects (Chernukha et al., 2018; Toldrá et al., 2016). In addition, these by-products have recently been shown to be enriched in bioactive and proteinaceous products that can provide a potential of source of additional nutrition to consumers (Cheng et al., 2009, 2016; Jayathilakan et al., 2012). Moreover, specific proteins and tissues are accumulated and expressed in each cell and some of them have specific activities (Fagerberg et al., 2014). Currently, some by-products derived from various animal parts are available such as supplemental foods, and these may exert different bioactive effects; thus, the extraction of beneficial substances of such by-products should be investigated for their applications in human health.

Chicken (Gallus gallus) gizzard is a popular food, but the gizzard inner lining (GIL) is not currently processed for direct human consumption, and approximately 22.5 million kg of GIL is wasted annually in the slaughter industry (i). Thus, useful conversion of wasted GIL into industrially beneficial products can provide some economic merits. In reality, the GIL has been used as food supplements and extensively studied since ancient times in China. According to the book of Compendium of Materia Medica, a classical Chinese medicine book, GIL is called “endothelium corneum gigeriae galli,” which is purported to help maintain the integrity of the stomach lining and is believed to produce effects that improve indigestion, diarrhea, and hematuria and reduce gallstones and urethral stones. Chicken gizzard is covered by a solid layer of a carbohydrate-protein complex (called the “koilin” layer) (Hofmann and Pregl, 1907), while mammals lack koilin (Hodges, 1974). Akester (1986) found that the gizzard in birds is well developed (with powerful muscles and a thick abrasive inner membrane), which enables birds to digest hard materials (e.g., grain, insects, etc.). Therefore, although GIL is thought to be a good source of protein and could potentially be used for human consumption, research has been rarely conducted to analyze the components of GIL as food supplements and to investigate their functions.

The aims of this study were to analyze the chemical components of GIL and investigate their functional roles. This study analyzed the proximate components of GIL, its pH, color, and amino acid composition. This study also conducted a proteomic analysis via biochemical and instrumental analyses to identify and quantify the major and bioactive proteins from broiler GIL.

Materials and Methods

Sampling and pretreatment    GIL used in this study were obtained from 5-week-old chicken broilers purchased from a local slaughterhouse. The obtained GIL was washed with 0.01 mol/L of phosphate buffered saline (pH 7.2), wiped dry with a paper towel, wrapped it in aluminum foil, and stored in liquid nitrogen. The GIL samples were divided into three sections. The first section was used to determine pH, color, and to conduct a proximate analysis. The second section was used to analyze amino acid composition. Each GIL was powdered with table-type pulverizing machine RT-34 (Mill Powder Tech, Tainan, Taiwan) at 3,450 rpm for 1 min. Further, it was lyophilized with freeze dryer FD-5N (EYELA, Tokyo, Japan), vacuum packed with table-type vacuum packaging machine YU-601A (Yeou Yuan Industrial, Taichung, Taiwan), and stored at −80 °C for later analysis. The last section was used to conduct a proteomic analysis. To prepare for this, 5 g samples were cut from the GIL and vacuum packed 0.003-inch standard barrier pouches, 6 × 8 inches in size (Prime Source Vacuum, Nylon/PE, Kansas, MO, USA).

Proximate analysis    An analysis of moisture content, protein, fat, and ash from GIL was conducted following methods outlined by the Association of Official Analytical Chemists International (2002). Determination of pH was based on a method by Ockerman (1985). In this study, 5 g of the GIL-lyophilized powder, which was prepared as described in the amino acid analysis method, were placed into stomacher bags (B01065WA, Nasco WHIRL-PAK, USA), to which 45 mL of distilled water was added; the GIL samples were processed in a Stomacher 400 (Seward, UK) at high speed for 1 min. The pH of the homogenate samples was measured with a gel-type pH electrode (InLab^® Solids, Mettler-Toledo, International Inc., Swiss) and pH meter (PHM201, Radiometer Analytical SAS, France).

Color value    A color value was for each sample was determined using a method developed by Lyon et al. (1980). A colorimeter (DR. Landge, Model LMG-051, Germany) was used to detect color space (CIE L*, a*, b*). Brightness was determined by L*, which ranges between 100 (brightest white color) to zero (darkest black color). The red/green opponent colors are represented along an a* axis, with red characterized by positive a* values and green characterized by negative a* values. The yellow/blue opponent colors are represented along the b* axis, with yellow characterized by positive b* values and blue characterized by negative b* values.

Analysis of amino acid composition    Amino acid composition was analyzed following a method outlined by Simpson et al. (1976); this analysis was entrusted to National Animal Industry Foundation (Taipei, Taiwan). Each 50 mg lyophilized sample was hydrolyzed with 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole under a vacuum in sealed tubes at 115 °C for 24 h. The analysis of amino acid composition was executed by high-performance liquid chromatography (Agilent 1100, Santa Clara, CA, USA) as a one-time analysis method following the manufacturer's instructions.

Proteome isolation of GIL    Fresh GIL-lyophilized powder (200 g), which were prepared under the same condition as that used in amino acid analysis, were mixed in 1 L of homogenization buffer (5 mmol/L Tris-HCl at pH 6.8, 0.004% pefabloc, a protease inhibitor cocktail tablet, and 10% sucrose) (Roche Applied Science, Mannheim, German) with a blender (Waring 7009G, USA) at 22,000 rpm for 5 min. The homogenization was performed thrice. The mixture was then centrifuged at 15,000 × g for 5 min at 4 °C. The resulting supernatant was collected and its protein concentration determined by the Bradford method (Kruger, 2002).

Proteomic analys is us ing two-dimensional gel electrophoresis (2-DE)    Protein concentrations for GIL proteome extract was determined using the Bradford method (Bio-Rad, Hercules, California). After the proteins were quantified, each lyophilized protein sample was dissolved in 175 µL lysis buffer (9.5 mol/L urea, 2% NP-40, 2% v/v ampholyte 3-10, and 65 mmol/L dithiothreitol [DTT]); then, added to 175 µL of rehydration buffer (0.5% v/v Pharmalyte 3-10, 2% CHAPS, and 8 mol/L urea) for 30 min at 20 °C. After being uniformly mixed, the sample was centrifuged at 17,530 × g for 30 min at 4 °C. The supernatant (containing 500 µg soluble proteins) was applied onto immobilized, pH-gradient (IPG) gel strips (18 cm in length, pH 3–10) and stored for 12 h to stabilize the contents. Next, a dimensional isoelectric focusing (IEF) electrophoresis was conducted using a Protean IEF system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) to achieve a final total voltage of 64,000 Vh (Görg et al., 2000). The IPG strips were equilibrated twice, once for 25 min in 15 mL of equilibration buffer 1 (2.0% SDS, 2.0% DTT, 30% glycerol, 6 mol/L urea, 50 mmol/L Tris-HCl at pH 8, and 0.002% bromophenol blue) and once for 25 min in 15 mL of equilibration buffer 2 (2.0% SDS, 30% glycerol, 6 mol/L urea, 50 mmol/L Tris-HCl, 0.001% bromophenol blue, and 2.5% iodoacetamide). Proteins were resolved in a second dimension on 12.5% SDS-PAGE gel using a Daltsix vertical electrophoresis system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). For whole proteome analysis, 18 GIL samples were analyzed (six samples with three replicates each). Gels were stained with Coomassie Blue (Gelcode, IL, USA) for 24 h. The stain was removed, and 350 mL of neutralization solution (0.1 mol/L Tris-phosphoric acid buffer at pH 6.5) was added for 3 min and then the samples were de-stained with 350 mL of 25% methanol until the background stain was completely removed (Neuhoff et al., 1988).

Proteomic gel-image analysis    Gel images were recorded using an image scanner (Image scanner III, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) prior to analysis with Melanie 7 software (GeneBio, Geneva, Switzerland). Protein spots from gel images of each GIL were exposed, matched with landmarks to help locate, and each spot was quantitated and then the percentage of each spot to the total amount was calculated.

Proteomic analysis and protein identification    After 2-DE, the target spot was excised on gel surfaces using a clean scalpel. The excised gel was incubated with 100 mmol/L ammonium bicarbonate solution included 10 mmol/L DTT and 55 mmol/L iodoacetamide for 30 mins to allow reduction and alkylation. Then, the samples were digested with trypsin solution (10 ng/µL trypsin in 50 mmol/L ammonium bicarbonate with 10% acetonitrile) on ice for 30 mins, and each enzymatic reaction was stopped with 1.0% formic acid. To extract the digestives, the samples were treated with 50% acetonitrile/0.2% trifluoroacetic acid for 45 min and sonicated for 5 min to collect the supernatants; this process was repeated four times. Lastly, the samples were treated with 90% acetonitrile with 0.2% trifluoroacetic acid for 5 min and sonicated for 5 min, and the supernatants were collected. The collected supernatants were vacuum-concentrated with a SpeedVac evaporator (Tokyo Rikakikai Co. Ltd., Tokyo, Japan) and identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) (Perkins et al., 1999). To identify proteins and peptide mass fingerprints, the MALDI-TOF-MS data were submitted to the Bruker algorithm (Daltonics Biotools software) via the Mascot search engine (www.matrixscience.com) to access the NCBI database of chicken. The NCBInr 20110922 version has 15,173,690 sequences and 5,201,003,429 residues. The search parameters were: Taxonomy (Chordata), enzyme (trypsin), fixed modification (carbamidomethyl), variable modification (oxidation), miss cleavages max (1), mass tolerance (±100 ppm), and mass values (MH+).

The extraction of GKN-1 protein in GIL    The purification of GKN-1 (Gastrokine 1) was referenced and adjusted using the method of Hnia et al., (2008). Fresh GIL was mixed in a blender (thrice for 5 min each) with 1 L of extracted buffer 3 (1 mmol/L EDTA, 300 mmol/L KCl, 5 mg/L of supplemented soybean trypsin inhibitor, 0.5 mmol/L MgCl₂, 2 mg/L leupeptin, 50 mmol/L imidazole, and 5 mmol/L of phenylmethylsulfonyl fluoride at pH 6.9) (Roche Applied Science, Mannheim, Germany). The buffer was freshly prepared for every use. The mixture was heated to 85 °C for 4 min to denature the proteins, cooled on ice, and then centrifuged for 30 min at 4,800 × g. The collected supernatant was adjusted to 50% with ammonium sulfate by moderately mixing with a magnetic agitator. After centrifugation, the resulting protein pellet was resuspended in 25–30 mL buffer 4 (0.5 mmol/L DTT, 20 mmol/L Tris-HCl, 1 mmol/L ethylene glycol tetraacetic acid, pH 7.5). The homogenate was dialyzed against the buffer 4 in a cold room overnight with a moderate magnetic agitation (the A part of the extraction phase). After centrifugation, the dialyzed homogenate was equilibrated with the buffer 4 and subjected to Trisacryl DEAE-Cellufine A-200 chromatography (JNC corporation, Tokyo, Japan). The first eluted fractions were gathered, equilibrated with the buffer 4, and then subjected to Trisacryl CM-Cellufine C-500 chromatography (JNC corporation). After washing with the buffer 4 (which contains 10 mmol/L NaCl), the reserved proteins were specifically eluted with the buffer 4, which contains 100 mmol/L NaCl. Then, all 20 kDa enriched fractions were concentrated and collected using Amicon cells with a PM-10 membrane. Lastly, the protein mixture was chromatographed on a Sephacryl S-200 HR column (GE Healthcare, United Kingdom) (the B part of the extraction phase) and fractions containing only the 20 kDa protein were gathered and analyzed using SDS-PAGE. The preparation of samples for SDS-PAGE followed the method provided by Hnia et al. (2008). In addition, the crude extractions (only the completed A part) were prepared for a GKN-1 protein analysis and then subjected to the protein concentration analysis procedure using the chicken GKN-1 ELISA kit (MBS2500022, MyBioSource Inc., USA).

Statistical analysis    SAS (Statistical Analysis System, Ver. 8.02 for Windows, 2001) software was used to analyze data. Color value data were analyzed with a Student's t-test. Proteomic analysis data were compared using one-way ANOVA, and the post hoc analysis was conducted using the Tukey–Kramer method. A probability level of p < 0.05 was considered to be statistically significant in this study.

Results and Discussion

Proximate analysis and color values    GIL was mostly composed of moisture and protein; other components were fat and ash (Table 1). GIL was acidic (pH 3.4). The most distinguishing feature of GIL was that it was high in protein (42.3 g/100 g) and low in fat (0.74 g/100 g), in comparison to reference data (Arafa, 1977; Chen et al., 2016). The amount of protein in raw gizzard and broiler meat have been reported to be lower than that in GIL, whereas the fat content in raw gizzard and broiler meat have been reported to be higher than that in GIL (Arafa, 1977; Chen et al., 2016). This indicates that GIL is a better source of protein and nutrients than meat or gizzard without lining (Table 1). Also, the pH of GIL was found to be acidic (pH 3.4). We presumed that the low pH is due to the fact that GIL is part of the stomach tissue, which is normally in continual contact with stomach acid.

Table 1. Proximate analysis of Gizzard inner lining, raw gizzard, and broiler meat

Items	Gizzard inner lining	Raw gizzard †	Broiler meat ‡
Moisture (g/100g)	57.01 ± 1.27	76.77	74.93 ± 0.20
Crude protein (g/100g)	42.30 ± 1.58	19.20	20.46 ± 0.44
Crude fat (g/100g)	0.74 ± 0.19	2.01	2.03 ± 0.09
Ash (g/100g)	0.34 ± 0.07	1.21	1.17 ± 0.07
pH value	3.40 ± 0.04	-	-

Reference data from the proximate analysis of raw gizzard (Arafa, 1977)^† and the proximate analysis of broiler meat (Chen et al., 2016)^‡. Details information of these references is described in the references list of text.

GIL is composed of a rough (an inner luminal surface) and smooth surface (a surface attached to the gizzard muscle), as shown in Figure 1. In addition, the colors of the rough and smooth surfaces differ from one another (Table 2). The L* value of the rough surface of the samples was significantly lower than that of the smooth surface, and the a* and b* values were significantly higher (p < 0.05, Student's t-test) than those of the smooth surface. Akester (1984) indicated that GIL is quite complex, consisting of vertical rodlets of hard koilin packed in tight clusters that create an abrasive surface like coarse sandpaper. The rough surfaces are dyed dark orange due to the carotenoid in the chicken's feed (such as corn), while the smooth surfaces (not in contact with feed) are bright yellow. Akester (1986) also found that when GIL is separated from the gizzard muscle, the hard koilin breaks, revealing a smooth, bright yellow surface. Our study found that the histological characteristics of GIL conformed to the reported histological characteristics.

Fig. 1.

Rough and smooth surfaces of gizzard inner lining are shown. The smooth surface is responsible for the attached gizzard muscle (bright yellow), whereas the rough surface is responsible for assisting in digestion (dark orange).

Table 2. Color values of rough and smooth surfaces of gizzard inner lining

Items	Color value
	Rough surface	Smooth surface
L*	63.1 ± 0.12a	73.0 ± 1.11b
a*	8.7 ± 0.23a	5.2 ± 0.12b
b*	54.4 ± 1.51a	31.7 ± 0.24b

Different letters ^a-b indicate significant differences between values in each item (p < 0.05).

Amino acid composition    In this study, we found that GIL was rich in glutamic acid (13.16%), aspartic acid (11.41%), leucine (8.27%), valine (8.00%), and arginine (7.86%) (Table 3). These results were similar to values obtained by Li et al. (2002), who reported that glutamic acid (13.07%) and aspartic acid (11.46%) were the principal amino acids in GIL. Moreover, the nine essential amino acids (EAA) for humans were also present in GIL.

Table 3. Amino acid composition of chicken gizzard inner lining

Amino acid (AA)	Concentration (mg/100g)	Total AA (%)
Glutamic acid (Glu)‡	11905	13.16
Aspartic acid (Asp)‡	10318	11.41
Leucine (Leu)†	7475	8.27
Valine (Val)†	7233	8.00
Arginine (Arg)‡	7109	7.86
Tyrosine (Tyr)	6901	7.63
Isoleucine (Ile)†	5563	6.15
Phenylalanine (Phe)†	5305	5.87
Glycine (Gly)‡	5201	5.75
Threonine (Thr)†	4451	4.92
Alanine (Ala)‡	4383	4.85
Serine (Ser)	3981	4.40
Proline (Pro)	3194	3.53
Lysine (Lys)†	2333	2.58
Cysteine (Cys)	2114	2.34
Tryptophan (Trp)†	1299	1.44
Methionine (Met)†	1061	1.17
Histidine (His)†	614	0.68
Total AA	90440	100.00
Total EAA/total AA	35334†	39.07

^†  EAA= Essential amino acids,

^‡  amino acids of umami.

The two primary amino acids (glutamic acid and aspartic acid) occur in a variety of foods and are known as constituents in umami (Stapleton et al., 1999). In particular, glutamic acid is often used as a food additive and flavor enhancer in the form of its sodium salt (monosodium glutamate), and it could be used as a raw material for monosodium glutamate in the food processing industry (Ikeda, 2002). Chicken and its by-products are often used in the preparation of bouillon in cooking, and the level of free glutamic acid in the heated soup of chicken muscles is remarkably higher than that of beef and pork (Nishimura et al., 1988). Moreover, the free glutamic acid level in consommé prepared with chicken by-products (stock prepared from wasted tissue components from bone, skin, and meat) was very high, and the products were suggested to be useful food ingredients for soup stock (Shibata et al., 2002). Since the GIL include high concentrations of umami amino acids, the heated soup of GIL also might yield a high level of umami taste in a food cooking and processing, suggesting its usefulness as a food ingredient and as an umami source. In addition, GIL is also rich in other EAA (for humans), such as leucine, valine, isoleucine, phenylalanine, etc. The ratio of EAA to total amino acids in GIL was 39.07% (mg/100 g protein). These rations for pork, beef, and chicken are 39%, 41%, and 41%, respectively (Qian et al., 2010). Hence, the amino acids content of GIL is complete, with abundant EAAs. Further, total EAA/total AA was nearly 40% (the FAO/WHO standard regulations for energy and protein requirements). Furthermore, Pivnenko et al. (1998) reported that chicken stomach includes these EAA as well as bioactive ingredients and that the peptide constituents isolated from the cuticle of the chicken stomach exhibit antiproteinase and immunostimulatory effects. Therefore, the digestive products from GIL are believed to include some important amino acids, such as EAA, and bioactive compounds.

Characteristics of GIL from proteomic analysis    GIL were homogenized and then soluble proteins were stained and assayed by 2-DE. A total of 90 differentially-expressed spots was observed (Fig. 2). The percent contents of all soluble protein from GIL are presented in Table 4 (Intensity percentage > 1.00) and supplemental data (Intensity percentage < 1.00). The proteomic analysis showed that the major proteins in GIL were 18-kDa antrum mucosa protein, zinc finger protein, and serum albumin precursor, with the sums of their intensity percentages being 21.94%, 17.62%, and 14.34%, respectively. The minor proteins were identified as alpha-enolase, glucose-regulated protein, secretogranin II, recombination activating gene 1, Zw10 protein, aconitate hydratase, and mitochondrial (supplemental data). In addition, all proteins were categorized into 12 kinds of groups or processes: positive regulation of cell proliferation (sum of intensity percentage, 22.8%), cell metabolic process (9.8%), immune response (7.6%), endopeptidase inhibitor activity (7.6%), transportation (5.4%), cell component (4.3%), potassium channel inhibitor activity (3.3%), carbonate binding (2.2%), cellular response to glucose starvation (1.1%), cellular response to growth factor stimulus (1.1%), glycerol ether metabolic process (1.1%), glycolysis (1.1%); proteins that could not be categorized into any of the above 12 groups or processes were regarded as no significant match (32.6%).

Fig. 2.

2-DE PAGE results of gizzard inner lining. Proteins were visualized using Coomassie Blue staining. Spot numbers refer to the number in Table 4.

Table 4. Functional roles of major differentially abundant proteins in gizzard inner lining, identified using MALDI-TOF-MS

Spot No.	Intensity (%)†	Protein	Accession number	Theoretical Mr/pI	Classification of bioactivity
1	14.06	NF-X1-type zinc finger protein NFXL1	gi|334331279	108580/10.40	Cell component
110	10.24	serum albumin precursor	gi|45383974	71868/5.40	Transportation
105	5.88	similar to 18-kDa antrum mucosa protein	gi|118101298	25995/9.40	Positive regulation of cell proliferation
71	5.77	Ig light chain	gi|212072	22769/5.20	Immune response
111	4.48	liprin-beta-1	gi|71896847	No significant match	Cell metabolic process
3	4.33	similar to 18-kDa antrum mucosa protein	gi|118101298	25995/9.40	Positive regulation of cell proliferation
2	4.09	cystatin precursor	gi|319655747	16682/9.04	Endopeptidase inhibitor activity
312	3.55	NF-X1-type zinc finger protein NFXL1	gi|334331279	52539/6.25	Cell component
5	3.44	serum albumin precursor	gi|45383974	71868/5.40	Transportation
17	2.96	similar to 18-kDa antrum mucosa protein	gi|118101298	25995/9.40	Positive regulation of cell proliferation
72	2.36	similar to 18-kDa antrum mucosa protein	gi|118101298	25995/9.40	Positive regulation of cell proliferation
97	1.80	Unidentifiable	No significant match	No significant match	No significant match
60	1.77	marker protein	gi|211503	20204/5.20	Cell metabolic process
87	1.63	similar to complement component C	gi|149062562	No significant match	Immune response
86	1.51	transthyretin precursor	gi|45384444	16356/4.95	Transportation
57	1.44	cystatin precursor	gi|319655747	16682/9.04	Endopeptidase inhibitor activity
214	1.41	Peptidyl-prolyl cis-trans isomerase FKBP1A	gi|45383498	12022/9.10	Cell metabolic process
260	1.35	hypothetical protein	gi|297465811	40733/12.60	No significant match
185	1.32	transthyretin precursor	gi|45384444	16356/4.95	Transportation
52	1.28	beta-galactoside-binding lectin	gi|45382785	15225/6.71	Carbonate binding
264	1.22	MHC class I antigen	gi|32400553	19045/7.60	Immune response
8	1.12	similar to 18-kDa antrum mucosa protein	gi|118101298	25995/9.40	Positive regulation of cell proliferation
10	1.10	similar to 18-kDa antrum mucosa protein	gi|118101298	25995/9.40	Positive regulation of cell proliferation
98	1.09	PIAS-NY protein	gi|13899014	45671/10.4	Immune response

The proteins (Intensity percentage > 1.00) are shown, and the spot numbers refer to the numbers in Figure 2.

^†  The protein expression was represented as the percentage of the level of that of all the quantified spots.

Our 2-DE and MALDI-TOF-MS analyses revealed that GIL contains bioactive ingredients, which is consistent with the results obtained by Pivnenko et al. (1998). In Table 4, 7 types of the bioactivities of the major identified proteins from GIL are summarized. Therefore, it is believed that GIL is probably rich in bioactive proteins. The 18-kDa antrum mucosal protein was the most abundant protein occurring in GIL (21.94%); this protein positively regulates the activities related to cell proliferation. According to the HUGO Gene Nomenclature Committee (HGNC), the 18-kDa antrum mucosal protein and/or related proteins (Martin et al., 2003; Toback et al., 2003; Walsh-Reitz et al., 2005) is now formally termed as gastrokine-1 protein (GKN-1) (HGNC no. 23217). The protein GKN-1 is expressed in gastric antrum mucosa cells of many mammalian species and has a high expression when the stomach is injured (Oien et al., 2004; Yoshihara et al., 2006). Also, the mechanism of the effects of GKN-1suggests that its release onto apical cell surfaces is regulated and that protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating recovery and proliferation after injuries (Toback et al., 2003). In order to prove whether the 18-kDa antrum mucosal protein in this study was, in fact, the GKN-1 protein, we investigated its properties using SDS-PAGE and ELISA approaches.

Analysis of GKN-1 protein in GIL    The SDS-PAGE result confirmed that the approximate 18-kDa band was the same molecular weight as the GKN-1 protein from the fraction (Fig. 3). Moreover, refined extracts determined that the GKN-1 concentration in GIL was 0.14 mg/mL and that the crude extract concentration was 8.0 ng/mL by ELISA. Therefore, it was strongly suggested that GIL contains the GKN-1 protein. Previous studies have revealed various bioactivities related to the GKN-1 protein. For example, the GKN-1 protects the intestinal mucosal barrier by acting on specific, tight-junction proteins and by stabilizing perijunctional actin (Walsh-Reitz et al., 2005). GKN-1 released into the gel covering the mucosal surface might speed recovery after an epithelial injury caused by a diverse array of pharmacological agents and after bacterial cytolysis and infiltration (Martin et al., 2003). Thus, GIL might have some health-promoting effects, including the potential of GKN-1, as reported in the report by Martin et al. (2003) and Walsh-Reitz et al. (2005).

Fig. 3.

Factions containing only the 18-kDa protein (GKN-1) in gizzard inner lining, pooled and analyzed using SDS-PAGE.

The level of GKN-1 was approximately 21.94% (93 mg/g) of all identified proteins based on the MALDI-TOF-MS analysis (Table 4); ELISA indicated a lower concentration than MALDI-TOF-MS. The difference between MALDI-TOF-MS and ELISA results might be due to the differences in preparation and analytical methods used to process samples. Further investigations using another method such as LC-MS/MS analysis are warranted to determine the precise level of GKN-1 in GIL.

In conclusion, chicken GIL is an animal by-product that contains high protein levels and important amino acids, such as EAA, similar to other animal food products. Moreover, GIL proteins have potentially various bioactive properties as per MALDI-TOF-MS analysis. Also, our results indicate that GKN-1 protein, which has been reported to exhibit gastroprotective and physiological activities, might be one of the most important components of GIL. To the best of our knowledge, there are few reports to demonstrate these effects of GKN-1 from GIL during its use as a food ingredient or supplement. Hence, to validate the protective effects of GIL, particularly the GKN-1 protein derived from GIL, in vitro and/or in vivo assays need to be conducted in future. In addition, research pertaining to various applications of GIL in food processing is imperative.

Conflict of interest

The authors declare that they have no conflicts of interests.

Acknowledgments    The authors would like to thank Enago (www.enago.tw) for the English language review.

References

•  Ahhmed,  A.M. and  Muguruma,  M. (2010). A review of meat protein hydrolysates and hypertension. Meat Sci., 86, 110-118.
•  Akester,  A.R. (1984). The glandular layer and koilin membrane of the gizzard in the domestic fowl. J. Anat., 121, 730.
•  Akester,  A.R. (1986). Structure of the glandular layer and koilin membrane in the gizzard of the adult domestic fowl (Gallus gallus domesticus). J. Anat., 147, 1-25.
•  Arafa,  A.S. (1977). Pickled chicken gizzards: 1. acceptability and proximate analysis. Poult. Sci., 56, 1014-1017.
• Association of Official Analytical Chemists (AOAC) International. (2002). Official methods of analysis of AOAC international 17th edn. Arlington, Virginia, USA.
•  Bauchart,  C.,  Rémond,  D.,  Chambon,  C.,  Patureau Mirand,  P.,  Savary-Auzeloux,  I.,  Reynès,  C., and  Morzel,  M. (2006). Small peptides (<5 kDa) found in ready-to-eat beef meat. Meat Sci., 74, 658-666.
•  Chernukha,  I.M.,  Fedulova,  L.V.,  Kotenkova,  E.A.,  Takeda,  S., and  Sakata,  R. (2018). Hypolipidemic and anti-inflammatory effects of aorta and heart tissues of cattle and pigs in the atherosclerosis rat model. Anim. Sci. J., 89, 784-793.
•  Chen,  Y.,  Qiao,  Y.,  Xiao,  Y.,  Chen,  H.,  Zhao,  L.,  Huang,  M., and  Zhou,  G. (2016). Differences in physicochemical and nutritional properties of breast and thigh meat from crossbred chickens, commercial broilers, and spent hens. Asian-Australas J. Anim. Sci., 29, 855-864.
•  Cheng,  F.Y.,  Wan,  T.C.,  Liu,  Y.T.,  Chen,  C.M.,  Lin,  L.C., and  Sakata,  R. (2009). Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase. Anim. Sci. J., 80, 91-97.
•  Cheng,  F.Y.,  Lai,  I.C.,  Lin,  L.C., and  Sakata,  R. (2016). The in vitro antioxidant properties of alcalase hydrolysate prepared from silkie fowl (Gallus gallus) blood protein. Anim. Sci. J., 87, 921-928.
•  Fagerberg,  L.,  Hallström,  B.M.,  Oksvold,  P.,  Kampf,  C.,  Djureinovic,  D.,  Odeberg,  J.,  Habuka,  M.,  Tahmasebpoor,  S.,  Danielsson,  A.,  Edlund,  K.,  Asplund,  A.,  Sjöstedt,  E.,  Lundberg,  E.,  Szigyarto,  C.A.,  Skogs,  M.,  Takanen,  J.O.,  Berling,  H.,  Tegel,  H.,  Mulder,  J.,  Nilsson,  P.,  Schwenk,  J.M.,  Lindskog,  C.,  Danielsson,  F.,  Mardinoglu,  A.,  Sivertsson,  A.,  von Feilitzen,  K.,  Forsberg,  M.,  Zwahlen,  M.,  Olsson,  I.,  Navani,  S.,  Huss,  M.,  Nielsen,  J.,  Ponten,  F., and  Uhlén,  M. (2014). Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol. Cell Proteomics, 13, 397-406.
•  Görg,  A.,  Obermaier,  C.,  Boguth,  G.,  Harder,  A.,  Scheibe,  B.,  Wildgruber,  R., and  Weiss,  W. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 21, 1037-1053.
•  Hnia,  K.,  Notarnicola,  C.,  de Santa Barbara,  P.,  Hugon,  G.,  Rivier,  F.,  Laoudj-Chenibvesse,  D., and  Mornet,  D. (2008). Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle. PLoS ONE, 3, e3854.
•  Hodges,  R.D. (1974). The Histology of the Fowl. Academic Press, London.
•  Hofmann,  K.B. and  Pregl  F. (1907). Über Koilin. Hoppe Seylers Z. Physiol. Chem., 52, 448-471.
•  Ikeda,  K. (2002). New seasonings. Chem. Senses, 27, 847-849.
•  Jayathilakan,  K.,  Sultana,  K.,  Radhakrishna,  K., and  Bawa,  A.S. (2012). Utilization of byproducts and waste materials from meat, poultry and fish processing industries: a review. J. Food Sci. Technol., 49, 278-293.
•  Kruger,  N.J. (2002). The Bradford method for protein quantification. In: The protein protocols handbook 2nd edn, Humana press. Totowa, N. J., pp. 15-22.
•  Lafarga,  T. and  Hayes,  M. (2014). Bioactive peptides from meat muscle and by-products: generation, functionality and application as functional ingredients. Meat Sci., 98, 227-239.
•  Li,  Z.H.,  Chen,  D., and  Li,  Z.H. (2002). Determination of nutrient elements and amino acids in Gallus gallus domesticus. Amino Acids & Biotic Resources, 24, 20-21.
•  Lyon,  C.E.,  Lyon,  B.G.,  Davis,  C.E., and  Townsend,  W.E. (1980). Texture profile analysis of patties made from mixed and flake-cut mechanically deboned poultry meat. Poult. Sci., 59, 69-76.
•  Martin,  T.E.,  Powell,  C.T.,  Wang,  Z.,  Bhattacharyya,  S.,  Walsh-Reitz,  M.M.,  Agarwal,  K., and  Toback,  F.G. (2003). A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa. Am. J. Physiol. Gastrointest. Liver Physiol., 285, 332-343.
•  Neuhoff,  V.,  Arold,  N.,  Taube,  D., and  Ehrhardt,  W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis, 9, 255-262.
•  Nishimura,  T.,  Rhue,  M.R.,  Okitani,  A., and  Kato,  H. (1988). Components contributing to the improvement of meat taste during storage. Agric. Biol. Chem., 52, 2323-2330.
•  Ockerman,  H.W. (1985). “Quality control of post-mortem muscle tissue”. Department of Animal Science, Ohio State University, Columbus, USA.
•  Oien,  K.A.,  McGregor,  F.,  Butler,  S.,  Ferrier,  R.K.,  Downie,  I.,  Bryce,  S., and  Keith,  W.N. (2004). Gastrokine 1 is abundantly and specifically expressed in superficial gastric epithelium, down-regulated in gastric carcinoma, and shows high evolutionary conservation. J. Pathol., 203, 789-797.
•  Perkins,  D.N.,  Pappin,  D.J.,  Creasy,  D.M., and  Cottrell,  J.S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20, 3551-3567.
•  Pivnenko,  T.N.,  Epstein,  L.M.,  Okladnikova,  S.V.,  Gazha,  A.K., and  Besednova,  N.N. (1998). Antiproteinase and immunostimulating effects of peptide constituents isolated from cuticle of chicken stomach. Prikl. Biokhim. Mikrobiol., 34, 455-459.
•  Qian,  A.P.,  Yan,  S.A.,  Lin,  X.X., and  Cheng,  W.W. (2010). The content of amino acid in the poultry meat and its nutritive evaluation. Chinese agricultural science bulletin, 26, 94-97.
•  Shibata,  K.,  Sainen,  S., and  Yasuhara,  Y. (2002). Effect of chicken body parts and additional meat on the taste and taste components of soup stock and consommé. J.Home Econ. Jpn. (Nippon Kasei Gakkaishi)., 53, 901-916 (in Japanese).
•  Simpson,  R.J.,  Neuberger,  M.R., and  Liu,  T.Y., (1976). Complete amino acid analysis of proteins from a single hydrolysate. J. Biol. Chem., 251, 1936-1940.
•  Stapleton,  J.R.,  Roper,  S.D., and  Delay,  E.R. (1999). The taste of monosodium glutamate (MSG), L-aspartic acid, and N-methyl-D-aspartate (NMDA) in rats: are NMDA receptors involved in MSG taste? Chem. Senses, 24, 449-457.
•  Toback,  F.G.,  Walsh-Reitz,  M.M.,  Musch,  M.W.,  Chang,  E.B.,  Del Valle,  J.,  Ren,  H.,  Huang,  E., and  Martin,  T.E. (2003). Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein, are mitogenic and motogenic. The Am. J. Physiol. Gastrointest. Liver Physiol., 285, G344-353.
•  Toldrá,  F.,  Aristoy,  M.C.,  Mora,  L., and  Reig,  M. (2012). Innovations in value-addition of edible meat by-products. Meat Sci., 92, 290-296.
•  Toldrá,  F.,  Mora,  L., and  Reig,  M. (2016). New insights into meat by-product utilization. Meat Sci., 120, 54-59.
•  Udenigwe,  C.C. and  Howard  A. (2013). Meat proteome as source of functional biopeptides. Food Res. Int., 54, 1021-1032.
•  Walsh-Reitz,  M.M.,  Huang,  E.F.,  Musch,  M.W.,  Chang,  E.B.,  Martin,  T.E.,  Kartha,  S., and  Toback,  F.G. (2005). AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins. The Am. J. Physiol. Gastrointest. Liver Physiol., 289, G163-171.
•  Yoshihara,  T.,  Kadota,  Y.,  Yoshimura,  Y.,  Tatano,  Y.,  Takeuchi,  N.,  Okitsu,  H.,  Umemoto,  A.,  Yamauchi,  T., and  Itoh  K. (2006). Proteomic alteration in gastic adenocarcinomas from Japanese patients. Mol. Cancer, 5, 75.
• i) http://usda.mannlib.cornell.edu/usda/nass/PoulProdVa//2010s/2017/PoulProdVa-04-28-2017.pdf (August 14, 2018)