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Seasonal variation of acid-soluble collagens extracted from the swim bladders and skins of bighead carp (Hypophthalmichthys nobilis) and grass carp (Ctenopharyngodon idella)
Food Bioscience 15 (2016) 27–33
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Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Seasonal variation of acid-soluble collagens extracted from the swim bladders and skins of bighead carp (Hypophthalmichthys nobilis) and grass carp (Ctenopharyngodon idella) Jinhua Hu a,b, Tiancheng Li a, Xiaoyong Liu c, Dasong Liu a,n a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China Synergetic Innovation Center of Food Safety and Nutrition, Wuxi, Jiangsu Province 214122, China c Shandong Haizhibao Ocean Science and Technology Co., Ltd., Weihai, Rongcheng, Shandong Province 264300, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 March 2016 Received in revised form 19 April 2016 Accepted 29 April 2016 Available online 29 April 2016
This study investigated the seasonal variation in the physicochemical properties of acid-soluble collagens (ASC) from the scales and skins of bighead carp and grass carp. The electrophoresis patterns of protein fraction and peptide hydrolysis treated by V8 protease were characterized using gel electrophoresis. Changes in thermostability were measured using differential scanning calorimetry, and collagen fibrils in vitro were observed using transmission electron microscopy. The ASC were mainly determined as type I collagens with triple helical structures. For collagens from the same tissue of the same fish species, similar protein fraction patterns and peptide hydrolysis patterns were observed, and the amino acid contents also showed no clear pattern of change with season. The thermal transition temperature of ASC showed a maximum seasonal variation of 0.5 °C, with no systematic pattern. The collagens could assemble into fibrils in vitro, and the D-periodicities of the fibrils showed no significant seasonal variation. The results suggested that the skins and swim bladders of two major freshwater species were reasonably stable as an alternative collagen source for year-round production. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Collagen Seasonal variation Grass carp Bighead carp Thermostability
1. Introduction Collagens are a group of major structural proteins with a unique right-handed triple helical structure, which are assembled from three left-handed helical polypeptide chains (Gelse, Pöschl, & Aigner, 2003). Collagens are divided into different types based on their variation in structure, function and tissue distribution, and up to now, the identification of 29 collagen types has been reported (McCormick, 2009). Among them, type I and type III collagens are the most abundant types, and found in most connective tissues of the vertebrae, such as bones, tendons, skins and intramuscular connective tissue (Gelse et al., 2003; Nagai, Suzuki, & Nagashima, 2008). Collagens are traditionally obtained from the bones and skins of cows and pigs and used extensively in the food, biomedical, pharmaceutical and cosmetic industries (Lee, Singla, & Lee, 2001; Tzaphlidon, 2004). Recently, for the safety concerns (Binsi, Shamasundar, Dileep, Badii, & Howell, 2009) and the dietary restriction due to religious or other reasons (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005; Regenstein, Chaudry, & n
Corresponding author. E-mail address: [email protected] (D. Liu).
http://dx.doi.org/10.1016/j.fbio.2016.04.006 2212-4292/& 2016 Elsevier Ltd. All rights reserved.
Regenstein, 2003), the by-products, such as bones, scales, skins and swim bladders have been studied as alternative sources for collagens (Duan, Zhang, Du, Yao, & Konno, 2009). Previous studies have suggested that the physicochemical properties of fish collagens were important for their applications (Huang, Shiau, Chen, & Huang, 2011; Pati, Adhikari, & Dhara, 2010; Singh, Benjakua, Maqsood, & Kishimura, 2011), and could be influenced by many factors such as fish species. The collagens from warm-water fish, in general, have the higher thermostability than those from cold/ice-water fish (Eastoe, & Leach, 1977). The physicochemical properties may also differ in collagens prepared from different tissues and/or with various manufacture methods (Regenstein, & Zhou, 2007). In addition, because fish is cold-blooded, the properties of fish proteins including collagen could vary according to the temperature of their living environments, which mostly would result from the difference of location and/or the seasonal variation (Duan et al., 2012; Duan, Konno, Zhang, Wang, & Yuan, 2010; Nakaya, Watabe, & Ooi, 1995; Touhata et al., 2001; Touhata, Tanaka, Toyohara, Tanaka, & Sakaguchi, 2000; Yuan et al., 2006). Bighead carp and grass carp are two of the most abundant freshwater fishes cultivated in China, and the seasonal consistency of collagens obtained from their processing by-products are important for their use as alternative sources of collagen. Thus, the
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objective of present study was to discuss the seasonal variation in the physicochemical properties of acid soluble collagens (ASC) from the scales and skins of bighead carp and grass carp, and to determine their suitability for using as the alternative sources on a year-round basis for collagen production.
2. Materials and methods 2.1.. Material Staphylococcus aureus V8 protease (EC 3.4.21.19) was from Sigma-Aldrich Co. (St. Louis, Mo., U. S. A.). The protein marker of ASC electrophoretic analysis (New England Biolabs, Inc., Ipswich, Ma., U. S. A.) was composed by serum albumin from bovine (66.4 kDa), phosphorylase B from rabbit muscle (97.2 kDa), MBPβ-galactosidase and β-galactosidase from E. coli (158 kDa and 116 kDa), and myosin from rabbit muscle (212 kDa). Besides all of the above 5 markers, the protein marker of hydrolysis peptide analysis (New England Biolabs, Inc.) also contained aprotinin from bovine lung (6.5 kDa), glutamic dehydrogenase from bovine liver (55.6 kDa); lysozyme from chicken egg white (14.3 kDa); trypsin inhibitor from soybean (20.1 kDa); and thiosephosphate isomerase, thioredoxin reductase and MBP2 from E. coli (27.0 kDa, 34.6 kDa and 42.7 kDa). All other chemicals were used in analytical grade.
Danvers, Ma., U. S. A.). The precipitate was re-extracted for 48 h and centrifuged under the same condition. Both of the collected supernatants were combined and salted out using NaCl (2.0 M) for 12 h. The consequent precipitates were collected by 20 min centrifugation ( 4 °C) at 10,000g and re-dissolved using acetic acid (0.5 M). This solution were then dialyzed against acetic acid (0.1 M) first and then against DI water in a membrane bag (7 kDa cut-off according to the manufacturer, Shanghai Green Bird Science and Technology Development Co., Shanghai, China). The final products (ASC samples) after dialysis were lyophilized using a Labconco freeze dryer (Labconco Corp., Kansas, Mo., U. S. A.). 2.4. Amino acid analysis The ASC samples ( 100 mg) were sealed in an evacuated vial and hydrolyzed using 8 mL HCl (6 M) at 110 °C for 24 h. The precolumn derivatization was first conducted using o-phthalaldehyde and fluorenylmethyl chloroformate. Then, the amino acids were analyzed using a C18 ODS HYPERSIL column (5 mm particle size; 250 mm 4.6 mm i. d.; Agilent Technologies, Inc.) and an Agilent 1100 Series high performance liquid chromatography system (Agilent Technologies, Inc., Santa Clara, Ca., U. S. A.). The relative content of each amino acid was calculated by the corresponding peak area of the standard and sample (Sigma-Aldrich Co.). The results were recorded as residues per 1000 total residues recovered and served as an initial estimate of the amino acid composition.
2.2. Fish samples The live bighead carp of 3–4 kg and live grass carp of 4–5 kg were purchased in January, March, May, July, September and November of 2012 from the same local market (Wuxi, Jiangsu province, China). All of the fishes were shifted in water to the laboratory within 1 h. In a walk-in chill room ( 4 °C), the fish were stunned to the head by a sharp blow with a wooden stick. The skins and swim bladders were first removed using a scalpel and then washed in cold deionization water (DI water). The clean swim bladders and skins were further treated using a scissor and cut into small pieces (0.5 0.5 cm2), and then immediately stored in a refrigerator ( 20 °C) until collagen extraction. Three different lots of bighead carp and grass carp were used in seasonal variation study and the extraction was performed once for each lot. In addition, the environmental temperatures outside the room were recorded twice every day for the year of 2012, in the early morning at 1 am and in the afternoon at 1 pm, and the average of these two temperatures was recorded as the daily environmental temperature. The average monthly temperature was then calculated based on these average daily temperatures recorded in the month for fish sampling (Table 2). 2.3. Preparation of collagens from the skins and swim bladders The ASC extraction was done referring to the methods slightly modified from reference (Kittiphattanabawon et al., 2005; Nagai, & Suzuki, 2000). All of the experiments were processed in a walk-in chill room ( 4 °C). The removal of non-collagenous proteins and pigments was done by soaking the swim bladders and skins in 20-fold 0.1 M NaOH for 12 h which was repeated for 3 times and then washed by DI water. The removal of fat was done by suspending the alkaline treated swim bladders and skins in 20-fold butyl alcohol (10% v/v) for 12 h and repeated 3 times. The resulting residues were washed thoroughly using cold DI water, and then extracted with 40-fold acetic acid (0.5 M) for 72 h. The supernatants were collected by 20 min centrifugation ( 4 °C) at 10,000g using an Avanti J-E centrifuge (Beckman Coulter, Inc.,
2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was conducted following Laemmli's method (Laemmli, 1970) of with a stacking gel (4%) and a resolving gel (5%). A 2 mg/mL ASC in 1% SDS was mixed with Tris–HCl buffer (pH 6.8, 62.5 mM) containing SDS (2% w/v) and glycerol (25% v/v) with a ratio of 1:1 (v/v). Each well was loaded with 3 mL sample solution. The electrophoresis was done using a Mini-PROTEAN Tetra Cell system (Bio-Rad laboratories, Inc., Hercules, Ca., U. S. A.). The gel after electrophoresis was stained with Coomassie Brilliant Blue R-250 (0.1% w/v) in acetic acid (6.8% v/v) and methanol (50% v/v) for 4 h, followed by destaining using methanol (5% v/v) and acetic acid (7.5% v/v). 2.6. Peptide hydrolysis patterns The ACS peptide hydrolysis patterns were prepared and then studied referring to the methods slightly modified from Satio, Kunisaki, Urano, and Kimura (2002). The solution A was 2 mg/mL ASC dissolved in sodium phosphate buffer (pH 7.2, 0.1 M) containing 0.5% w/v SDS. The solution B was Staphylococcus aureus V8 protease dissolved in 2000 volumes of the same buffer. Solution A (100 mL) and solution B (10 mL) were incubated together at 37 °C for 30 min The termination of reaction was done by 3 min heat treatment in boiling water. The resulting peptides were analyzed using SDS-PAGE with a stacking gel (4%) and resolving gel (7.5%). 2.7. Differential scanning calorimetry (DSC) The DSC characterization was carried out using the Q2000 Series DSC (TA Instruments, Inc., New Castle, De., U. S. A.). The samples were prepared according to the method of Kittiphattanabawon et al. (2005) with slight modification. An ASC acetic acid solution (0.05 M) of a concentration of 25 mg/mL was prepared at 4 °C for 24 h. The samples (9.5–10.5 mg) were sealed in aluminum pans and scanned at 20–50 °C with a rate of 1 °C/min, and an empty sealed pan was used as the reference. The peak temperature
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Table 1 Seasonal variation in the Pro and Hyp content of ASC. Species
Tissue
Amino acid
Month for sampling
Bighead carp
Skin
Pro Hyp Imino Pro Hyp Imino Pro Hyp Imino Pro Hyp Imino
Jan. 1177 4a 61 70ab 1787 4a 1127 1a 627 3a 1747 4a 1127 5b 71 70a 1837 5a 1097 2a 64 71a 1737 3a
Swim bladder
Grass carp
Skin
Swim bladder
a–c
acids
acids
acid
acid
Mar. 1197 2a 617 1ab 1807 3a 1097 8a 637 3a 1727 11a 1147 2b 64 74a 178 76a 1137 1a 577 7a 1707 6a
May 1207 1a 527 1c 1727 0a 106 7 3a 677 1a 1737 2a 1177 1ab 627 10a 1797 9a 1137 6a 66 70a 1797 6a
Jul. 124 7 2a 577 6bc 1817 4a 117 73a 577 13a 1747 10a 1217 3a 64 7 5a 1857 2a 1107 2a 64 7 9a 1747 7a
Sept. 1157 15a 657 1a 1807 16a 1127 2a 64 73a 176 75a 1177 2ab 597 0a 176 71a 1127 8a 68 73a 1807 5a
Nov. 115 71a 637 0ab 1787 0a 116 79a 607 6a 1767 15a 115 71ab 657 5a 180 76a 108 7 4a 717 1a 1797 5a
Means in a row followed by different lower-case letters differ significantly (p o 0.05).
Table 2 Seasonal variation in the D-periodicities of collagen fibrils assembled from ASC of grass carp skins. Month of sampling
Average monthly temperature of 2012 in Wuxi (ºC)
D- periodicity (nm)
Jan. Mar. May Jul. Sept. Nov.
4.0 72.1 10.0 74.9 22.3 72.1 30.4 71.8 23.6 74.7 11.0 72.5
66.7 70.4a 67.1 71.0a 66.8 70.5a 67.2 71.0a 66.5 70.5a 66.6 70.4a
a Means in a column followed by different lower-case letters differ significantly (po 0.05).
of each endothermic peak was recorded by the maximum value of thermal transition temperature (Td). 2.8. Electron microscopy observation of collagen fibrils ASC from skins of grass carp with a concentration 1.1 mg/mL was obtained in HCl (5 mM) at 4 °C and then mixed with phosphate buffer (pH 7.2) containing 15 mM KH2PO4, 27 mM KCl, 80 mM Na2HPO4 and 1370 mM NaCl at a ratio of 9:1. The collagen fibrils were formed after 3 h at 30 °C. The solution (20 mL) was dropped onto a 200 mesh copper grid and stained using phosphotungstic acid (1% w/v). The collagen fibrils were observed using a JEM-2100 electron microscopy (JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 200 kV (Li, Asadi, Monroe, &
Fig. 1. SDS-PAGE patterns of ASC from the skins and scales of bighead carp and grass carp: (a) and (b) show those from the skins and swim bladders of grass carp, and (c) and (d) show those from the skins and swim bladders of bighead carp. The number above each line refers to the month of 2012, and the first lane is the protein standards (SD).
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Fig. 2. Peptide hydrolysis patterns of ASC from the skins and scales of bighead carp and grass carp after V8 protease digestion: (a) and (b) show those from the skins and swim bladders of grass carp, and (c) and (d) show those from the skins and swim bladders of bighead carp. The number above each line refers to the month of 2012, and the left lane is the protein standards (SD).
Douglas, 2009). The transmission electron microscopy (TEM) images were analyzed using NIH ImageJ 1.41 software (National Institutes of Health, Bethesda, Md., U. S. A.). Ten measurements of the TEM images were used to calculate the average D-periodicity for each sample and each measurement covered 5 periodicities on a fibril. 2.9. Statistical analysis Statistical analysis was analyzed by SAS version 8.0 (1999, SAS Institute, Inc., Cary, N. C., U. S. A.). Variance analysis was done using the General Linear Model procedure. The means difference were determined using the Duncan test at an α level of 0.05.
3. Results and discussion 3.1. Amino acid compositions Fig. 3. The thermal transition temperature (Td, °C) of ASC from the skins and scales of bighead carp and grass carp rehydrated in 0.05 M acetic acid. ●, ASC from the skins of grass carp; ○, ACS from the swim bladders of grass carp; ■, ASC from the skins of bighead carp; □, ASC from the swim bladders of bighead carp.
The amino acid profiles of ASC from the swim bladders and skins showed that proline (Pro) and hydroxyproline (Hyp) are two of the most abundant amino acids in collagen. The characterization of amino acid sequence is done by the repeating sequence of Gly-X-Y
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Fig. 4. TEM images of collagen fibrils assembled from ASC of grass carp skins in vitro. (a) January, (b) March, (c) May, (d) July, (e) September, (f) November. The vertical bars represent the standard deviations (n¼ 3).
triplets, in which X is mostly composed by Pro and most of Y is Hyp. The content of Pro and Hyp is critical for the physicochemical properties of collagens, particularly for their thermostability (Regenstein et al., 2007). Table 1 shows the content of these two amino acids in ASC obtained from different seasons, and no clear patterns of change were observed. For the ASC from grass carp skins, the content of Pro was in the range from 112 to 121 residues/1000 residues, the content of Hyp was in the range from 59 to 71 residues/1000 residues, and the total content of the imino acid was in the range of 176–185 residues/1000 residues. While for the ACS from grass carp swim bladders, the content of Pro was in the range from 108 to 113 residues/1000 residues, the content of Hyp was in the range from 57 to 71 residues/1000 residues, and the total content of imino acid was in the range of 170–180 residues/1000 residues. For the ASC from bighead carp, the contents of Pro, Hyp, and the total imino acid were also in similar ranges as those of grass carp. Previous researches have suggested that the amino acid composition of fish collagen varied, particularly for the imino acid content, depending mainly on the sources (Regenstein et al., 2007). Generally, the imino acid content of collagens from warmwater fish is in the range around 170–180 residues/1000 residues, which is normally higher than that from cold-water fish and ice fish but lower than that from mammals (Regenstein et al., 2007). In addition, the amino acid composition may differ from collagens from different tissues and/or prepared by different methods. The maturation stage of fish source did not significantly influence the amino acid composition of collagen extracts as revealed in the studies of Nile perch, a fresh water fish (Muyonga, Cole, & Duodu, 2004a, 2004b). Although the local average temperature in this study changed from 4 °C in January to 30 °C in July (Table 2), we
did not observe a clear changing pattern with regarding to the seasonal variation of amino acid composition, particularly for the imino acid content. 3.2. Protein fraction patterns and peptide hydrolysis patterns by SDS-PAGE Fig. 1 shows the SDS-PAGE patterns of ASC extracted from swim bladders and skins of grass carp (Fig. 1(A) and (B)) and bighead carp (Fig. 1(C) and (D)). All of the ACS were typical type I collagen, and consisted of three different α chains, namely, α1(I), α2(I), and α3(I). Moreover, the β chains (dimmers of the α-chains) and some highly cross-linked components were also observed in all of the ASC. However, for the ACS from the same tissue of the same fish species, the SDS-PAGE patterns showed no significant seasonal variation in protein fraction distribution. In a previous study on scale collagens from carp and silver carp, it was suggested that the content of the α1 chain was lower for scale collagen from winter fish than those from the summer, while the content of α2 and α3 chains were higher for scale collagen from winter fish (Duan et al., 2012). However, the results with the collagens from the skins and swim bladders in the present study are inconsistent with their results. The seasonal consistency of ASC's subunit MW was further investigated by examining the peptide hydrolysis patterns (Fig. 2). Compared with the intact collagens (Fig. 1), the intensity of band for high molecular weight peptide fragments decreased while that of the low molecular weight components increased. The peptide hydrolysis patterns of ASC from grass carp were slightly different from those from bighead carp. However, for the ASC from the
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same fish and same tissue, no significant seasonal variations were observed suggesting the major primary structures remain unchanged.
also thank Shuying Wang, Hongyang Pan and Keyv Lu for their help with the amino acid analysis and TEM measurement.
3.3. Thermostability using DSC
References
The thermostability of ASC was determined using DSC, and the maximum thermal transition temperatures (Td) were recorded. For the ASC from skins of both bighead carp and grass carp, the Td values changed from 35.1 to 35.8 °C, while those of ASC from swim bladders changed from 36.5 to 37.8 °C (Fig. 3), suggesting that the thermostability of ASC from swim bladders (the internal tissues) was higher than that of ASC from skins (the external tissues), which was in agreement with previous research on pepsin-solubilized collagens from bighead carp (Liu et al., 2015; Liu, Liang, Regenstein, & Zhou, 2012) and acid-solubilized collagens from sea bass (Nikoo et al., 2014; Sinthusamran, Benjakul, & Kishimura, 2013; Zhang et al., 2016). Thus, the data in the present study did not suggest any seasonal variation on thermostability (Fig. 3).
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3.4. Formation of collagen fibrils In the fibril formation process, collagen molecules assembled to the parallel staggered hole areas with less than full protein coverage and the overlapping areas with full protein coverage. Their different electron density resulted in the D-periods formation when examined using TEM. The differences in collagen molecules could influence the length of these hole areas and overlapping areas, which would lead to changes in the TEM observation if there were seasonal variation. Fig. 4 shows the TEM observations of collagen fibrils assembled from ACS of grass carp skins at various times. All the skin ASC could form ordered structures of fibril in vitro with characteristic D-periodicity, which served as an indication for the reconstitution of native-like collagen fibrils (Li et al., 2009). In addition, the D-periodicities of fibrils formed from ASC were all between 66 and 67 nm (Table 2), suggesting that there was no significant variation in the fibrogenesis of collagens with season (Chen et al., 2010).
4. Conclusions Bighead carp and grass carp are two of the most abundant freshwater fishes in China. The ASC from the scales and skins of these two fish species were mainly type I collagens with the major molecular form of α1(I)α2(I)α3(I). For collagens from the same tissue of the same fish species, all of the tests that were run in this research showed that there was no sign of significant seasonal variation. This suggests that the skins and swim bladders of these two major freshwater species were suitable for using as the alternative sources for collagen production on a year-round basis without concern for having to modify the process or the end use because of seasonal variation.
Conflict of interest statement There are no conflicts of interest to report.
Acknowledgements This research was partly supported by the 111 project (B07029), the Collaborative Innovation Center for Food Safety and Quality Control and the Postdoctoral Science Foundation of Weihai. We
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Contents lists available at ScienceDirect
Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Seasonal variation of acid-soluble collagens extracted from the swim bladders and skins of bighead carp (Hypophthalmichthys nobilis) and grass carp (Ctenopharyngodon idella) Jinhua Hu a,b, Tiancheng Li a, Xiaoyong Liu c, Dasong Liu a,n a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China Synergetic Innovation Center of Food Safety and Nutrition, Wuxi, Jiangsu Province 214122, China c Shandong Haizhibao Ocean Science and Technology Co., Ltd., Weihai, Rongcheng, Shandong Province 264300, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 March 2016 Received in revised form 19 April 2016 Accepted 29 April 2016 Available online 29 April 2016
This study investigated the seasonal variation in the physicochemical properties of acid-soluble collagens (ASC) from the scales and skins of bighead carp and grass carp. The electrophoresis patterns of protein fraction and peptide hydrolysis treated by V8 protease were characterized using gel electrophoresis. Changes in thermostability were measured using differential scanning calorimetry, and collagen fibrils in vitro were observed using transmission electron microscopy. The ASC were mainly determined as type I collagens with triple helical structures. For collagens from the same tissue of the same fish species, similar protein fraction patterns and peptide hydrolysis patterns were observed, and the amino acid contents also showed no clear pattern of change with season. The thermal transition temperature of ASC showed a maximum seasonal variation of 0.5 °C, with no systematic pattern. The collagens could assemble into fibrils in vitro, and the D-periodicities of the fibrils showed no significant seasonal variation. The results suggested that the skins and swim bladders of two major freshwater species were reasonably stable as an alternative collagen source for year-round production. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Collagen Seasonal variation Grass carp Bighead carp Thermostability
1. Introduction Collagens are a group of major structural proteins with a unique right-handed triple helical structure, which are assembled from three left-handed helical polypeptide chains (Gelse, Pöschl, & Aigner, 2003). Collagens are divided into different types based on their variation in structure, function and tissue distribution, and up to now, the identification of 29 collagen types has been reported (McCormick, 2009). Among them, type I and type III collagens are the most abundant types, and found in most connective tissues of the vertebrae, such as bones, tendons, skins and intramuscular connective tissue (Gelse et al., 2003; Nagai, Suzuki, & Nagashima, 2008). Collagens are traditionally obtained from the bones and skins of cows and pigs and used extensively in the food, biomedical, pharmaceutical and cosmetic industries (Lee, Singla, & Lee, 2001; Tzaphlidon, 2004). Recently, for the safety concerns (Binsi, Shamasundar, Dileep, Badii, & Howell, 2009) and the dietary restriction due to religious or other reasons (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005; Regenstein, Chaudry, & n
Corresponding author. E-mail address: [email protected] (D. Liu).
http://dx.doi.org/10.1016/j.fbio.2016.04.006 2212-4292/& 2016 Elsevier Ltd. All rights reserved.
Regenstein, 2003), the by-products, such as bones, scales, skins and swim bladders have been studied as alternative sources for collagens (Duan, Zhang, Du, Yao, & Konno, 2009). Previous studies have suggested that the physicochemical properties of fish collagens were important for their applications (Huang, Shiau, Chen, & Huang, 2011; Pati, Adhikari, & Dhara, 2010; Singh, Benjakua, Maqsood, & Kishimura, 2011), and could be influenced by many factors such as fish species. The collagens from warm-water fish, in general, have the higher thermostability than those from cold/ice-water fish (Eastoe, & Leach, 1977). The physicochemical properties may also differ in collagens prepared from different tissues and/or with various manufacture methods (Regenstein, & Zhou, 2007). In addition, because fish is cold-blooded, the properties of fish proteins including collagen could vary according to the temperature of their living environments, which mostly would result from the difference of location and/or the seasonal variation (Duan et al., 2012; Duan, Konno, Zhang, Wang, & Yuan, 2010; Nakaya, Watabe, & Ooi, 1995; Touhata et al., 2001; Touhata, Tanaka, Toyohara, Tanaka, & Sakaguchi, 2000; Yuan et al., 2006). Bighead carp and grass carp are two of the most abundant freshwater fishes cultivated in China, and the seasonal consistency of collagens obtained from their processing by-products are important for their use as alternative sources of collagen. Thus, the
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objective of present study was to discuss the seasonal variation in the physicochemical properties of acid soluble collagens (ASC) from the scales and skins of bighead carp and grass carp, and to determine their suitability for using as the alternative sources on a year-round basis for collagen production.
2. Materials and methods 2.1.. Material Staphylococcus aureus V8 protease (EC 3.4.21.19) was from Sigma-Aldrich Co. (St. Louis, Mo., U. S. A.). The protein marker of ASC electrophoretic analysis (New England Biolabs, Inc., Ipswich, Ma., U. S. A.) was composed by serum albumin from bovine (66.4 kDa), phosphorylase B from rabbit muscle (97.2 kDa), MBPβ-galactosidase and β-galactosidase from E. coli (158 kDa and 116 kDa), and myosin from rabbit muscle (212 kDa). Besides all of the above 5 markers, the protein marker of hydrolysis peptide analysis (New England Biolabs, Inc.) also contained aprotinin from bovine lung (6.5 kDa), glutamic dehydrogenase from bovine liver (55.6 kDa); lysozyme from chicken egg white (14.3 kDa); trypsin inhibitor from soybean (20.1 kDa); and thiosephosphate isomerase, thioredoxin reductase and MBP2 from E. coli (27.0 kDa, 34.6 kDa and 42.7 kDa). All other chemicals were used in analytical grade.
Danvers, Ma., U. S. A.). The precipitate was re-extracted for 48 h and centrifuged under the same condition. Both of the collected supernatants were combined and salted out using NaCl (2.0 M) for 12 h. The consequent precipitates were collected by 20 min centrifugation ( 4 °C) at 10,000g and re-dissolved using acetic acid (0.5 M). This solution were then dialyzed against acetic acid (0.1 M) first and then against DI water in a membrane bag (7 kDa cut-off according to the manufacturer, Shanghai Green Bird Science and Technology Development Co., Shanghai, China). The final products (ASC samples) after dialysis were lyophilized using a Labconco freeze dryer (Labconco Corp., Kansas, Mo., U. S. A.). 2.4. Amino acid analysis The ASC samples ( 100 mg) were sealed in an evacuated vial and hydrolyzed using 8 mL HCl (6 M) at 110 °C for 24 h. The precolumn derivatization was first conducted using o-phthalaldehyde and fluorenylmethyl chloroformate. Then, the amino acids were analyzed using a C18 ODS HYPERSIL column (5 mm particle size; 250 mm 4.6 mm i. d.; Agilent Technologies, Inc.) and an Agilent 1100 Series high performance liquid chromatography system (Agilent Technologies, Inc., Santa Clara, Ca., U. S. A.). The relative content of each amino acid was calculated by the corresponding peak area of the standard and sample (Sigma-Aldrich Co.). The results were recorded as residues per 1000 total residues recovered and served as an initial estimate of the amino acid composition.
2.2. Fish samples The live bighead carp of 3–4 kg and live grass carp of 4–5 kg were purchased in January, March, May, July, September and November of 2012 from the same local market (Wuxi, Jiangsu province, China). All of the fishes were shifted in water to the laboratory within 1 h. In a walk-in chill room ( 4 °C), the fish were stunned to the head by a sharp blow with a wooden stick. The skins and swim bladders were first removed using a scalpel and then washed in cold deionization water (DI water). The clean swim bladders and skins were further treated using a scissor and cut into small pieces (0.5 0.5 cm2), and then immediately stored in a refrigerator ( 20 °C) until collagen extraction. Three different lots of bighead carp and grass carp were used in seasonal variation study and the extraction was performed once for each lot. In addition, the environmental temperatures outside the room were recorded twice every day for the year of 2012, in the early morning at 1 am and in the afternoon at 1 pm, and the average of these two temperatures was recorded as the daily environmental temperature. The average monthly temperature was then calculated based on these average daily temperatures recorded in the month for fish sampling (Table 2). 2.3. Preparation of collagens from the skins and swim bladders The ASC extraction was done referring to the methods slightly modified from reference (Kittiphattanabawon et al., 2005; Nagai, & Suzuki, 2000). All of the experiments were processed in a walk-in chill room ( 4 °C). The removal of non-collagenous proteins and pigments was done by soaking the swim bladders and skins in 20-fold 0.1 M NaOH for 12 h which was repeated for 3 times and then washed by DI water. The removal of fat was done by suspending the alkaline treated swim bladders and skins in 20-fold butyl alcohol (10% v/v) for 12 h and repeated 3 times. The resulting residues were washed thoroughly using cold DI water, and then extracted with 40-fold acetic acid (0.5 M) for 72 h. The supernatants were collected by 20 min centrifugation ( 4 °C) at 10,000g using an Avanti J-E centrifuge (Beckman Coulter, Inc.,
2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was conducted following Laemmli's method (Laemmli, 1970) of with a stacking gel (4%) and a resolving gel (5%). A 2 mg/mL ASC in 1% SDS was mixed with Tris–HCl buffer (pH 6.8, 62.5 mM) containing SDS (2% w/v) and glycerol (25% v/v) with a ratio of 1:1 (v/v). Each well was loaded with 3 mL sample solution. The electrophoresis was done using a Mini-PROTEAN Tetra Cell system (Bio-Rad laboratories, Inc., Hercules, Ca., U. S. A.). The gel after electrophoresis was stained with Coomassie Brilliant Blue R-250 (0.1% w/v) in acetic acid (6.8% v/v) and methanol (50% v/v) for 4 h, followed by destaining using methanol (5% v/v) and acetic acid (7.5% v/v). 2.6. Peptide hydrolysis patterns The ACS peptide hydrolysis patterns were prepared and then studied referring to the methods slightly modified from Satio, Kunisaki, Urano, and Kimura (2002). The solution A was 2 mg/mL ASC dissolved in sodium phosphate buffer (pH 7.2, 0.1 M) containing 0.5% w/v SDS. The solution B was Staphylococcus aureus V8 protease dissolved in 2000 volumes of the same buffer. Solution A (100 mL) and solution B (10 mL) were incubated together at 37 °C for 30 min The termination of reaction was done by 3 min heat treatment in boiling water. The resulting peptides were analyzed using SDS-PAGE with a stacking gel (4%) and resolving gel (7.5%). 2.7. Differential scanning calorimetry (DSC) The DSC characterization was carried out using the Q2000 Series DSC (TA Instruments, Inc., New Castle, De., U. S. A.). The samples were prepared according to the method of Kittiphattanabawon et al. (2005) with slight modification. An ASC acetic acid solution (0.05 M) of a concentration of 25 mg/mL was prepared at 4 °C for 24 h. The samples (9.5–10.5 mg) were sealed in aluminum pans and scanned at 20–50 °C with a rate of 1 °C/min, and an empty sealed pan was used as the reference. The peak temperature
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Table 1 Seasonal variation in the Pro and Hyp content of ASC. Species
Tissue
Amino acid
Month for sampling
Bighead carp
Skin
Pro Hyp Imino Pro Hyp Imino Pro Hyp Imino Pro Hyp Imino
Jan. 1177 4a 61 70ab 1787 4a 1127 1a 627 3a 1747 4a 1127 5b 71 70a 1837 5a 1097 2a 64 71a 1737 3a
Swim bladder
Grass carp
Skin
Swim bladder
a–c
acids
acids
acid
acid
Mar. 1197 2a 617 1ab 1807 3a 1097 8a 637 3a 1727 11a 1147 2b 64 74a 178 76a 1137 1a 577 7a 1707 6a
May 1207 1a 527 1c 1727 0a 106 7 3a 677 1a 1737 2a 1177 1ab 627 10a 1797 9a 1137 6a 66 70a 1797 6a
Jul. 124 7 2a 577 6bc 1817 4a 117 73a 577 13a 1747 10a 1217 3a 64 7 5a 1857 2a 1107 2a 64 7 9a 1747 7a
Sept. 1157 15a 657 1a 1807 16a 1127 2a 64 73a 176 75a 1177 2ab 597 0a 176 71a 1127 8a 68 73a 1807 5a
Nov. 115 71a 637 0ab 1787 0a 116 79a 607 6a 1767 15a 115 71ab 657 5a 180 76a 108 7 4a 717 1a 1797 5a
Means in a row followed by different lower-case letters differ significantly (p o 0.05).
Table 2 Seasonal variation in the D-periodicities of collagen fibrils assembled from ASC of grass carp skins. Month of sampling
Average monthly temperature of 2012 in Wuxi (ºC)
D- periodicity (nm)
Jan. Mar. May Jul. Sept. Nov.
4.0 72.1 10.0 74.9 22.3 72.1 30.4 71.8 23.6 74.7 11.0 72.5
66.7 70.4a 67.1 71.0a 66.8 70.5a 67.2 71.0a 66.5 70.5a 66.6 70.4a
a Means in a column followed by different lower-case letters differ significantly (po 0.05).
of each endothermic peak was recorded by the maximum value of thermal transition temperature (Td). 2.8. Electron microscopy observation of collagen fibrils ASC from skins of grass carp with a concentration 1.1 mg/mL was obtained in HCl (5 mM) at 4 °C and then mixed with phosphate buffer (pH 7.2) containing 15 mM KH2PO4, 27 mM KCl, 80 mM Na2HPO4 and 1370 mM NaCl at a ratio of 9:1. The collagen fibrils were formed after 3 h at 30 °C. The solution (20 mL) was dropped onto a 200 mesh copper grid and stained using phosphotungstic acid (1% w/v). The collagen fibrils were observed using a JEM-2100 electron microscopy (JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 200 kV (Li, Asadi, Monroe, &
Fig. 1. SDS-PAGE patterns of ASC from the skins and scales of bighead carp and grass carp: (a) and (b) show those from the skins and swim bladders of grass carp, and (c) and (d) show those from the skins and swim bladders of bighead carp. The number above each line refers to the month of 2012, and the first lane is the protein standards (SD).
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Fig. 2. Peptide hydrolysis patterns of ASC from the skins and scales of bighead carp and grass carp after V8 protease digestion: (a) and (b) show those from the skins and swim bladders of grass carp, and (c) and (d) show those from the skins and swim bladders of bighead carp. The number above each line refers to the month of 2012, and the left lane is the protein standards (SD).
Douglas, 2009). The transmission electron microscopy (TEM) images were analyzed using NIH ImageJ 1.41 software (National Institutes of Health, Bethesda, Md., U. S. A.). Ten measurements of the TEM images were used to calculate the average D-periodicity for each sample and each measurement covered 5 periodicities on a fibril. 2.9. Statistical analysis Statistical analysis was analyzed by SAS version 8.0 (1999, SAS Institute, Inc., Cary, N. C., U. S. A.). Variance analysis was done using the General Linear Model procedure. The means difference were determined using the Duncan test at an α level of 0.05.
3. Results and discussion 3.1. Amino acid compositions Fig. 3. The thermal transition temperature (Td, °C) of ASC from the skins and scales of bighead carp and grass carp rehydrated in 0.05 M acetic acid. ●, ASC from the skins of grass carp; ○, ACS from the swim bladders of grass carp; ■, ASC from the skins of bighead carp; □, ASC from the swim bladders of bighead carp.
The amino acid profiles of ASC from the swim bladders and skins showed that proline (Pro) and hydroxyproline (Hyp) are two of the most abundant amino acids in collagen. The characterization of amino acid sequence is done by the repeating sequence of Gly-X-Y
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Fig. 4. TEM images of collagen fibrils assembled from ASC of grass carp skins in vitro. (a) January, (b) March, (c) May, (d) July, (e) September, (f) November. The vertical bars represent the standard deviations (n¼ 3).
triplets, in which X is mostly composed by Pro and most of Y is Hyp. The content of Pro and Hyp is critical for the physicochemical properties of collagens, particularly for their thermostability (Regenstein et al., 2007). Table 1 shows the content of these two amino acids in ASC obtained from different seasons, and no clear patterns of change were observed. For the ASC from grass carp skins, the content of Pro was in the range from 112 to 121 residues/1000 residues, the content of Hyp was in the range from 59 to 71 residues/1000 residues, and the total content of the imino acid was in the range of 176–185 residues/1000 residues. While for the ACS from grass carp swim bladders, the content of Pro was in the range from 108 to 113 residues/1000 residues, the content of Hyp was in the range from 57 to 71 residues/1000 residues, and the total content of imino acid was in the range of 170–180 residues/1000 residues. For the ASC from bighead carp, the contents of Pro, Hyp, and the total imino acid were also in similar ranges as those of grass carp. Previous researches have suggested that the amino acid composition of fish collagen varied, particularly for the imino acid content, depending mainly on the sources (Regenstein et al., 2007). Generally, the imino acid content of collagens from warmwater fish is in the range around 170–180 residues/1000 residues, which is normally higher than that from cold-water fish and ice fish but lower than that from mammals (Regenstein et al., 2007). In addition, the amino acid composition may differ from collagens from different tissues and/or prepared by different methods. The maturation stage of fish source did not significantly influence the amino acid composition of collagen extracts as revealed in the studies of Nile perch, a fresh water fish (Muyonga, Cole, & Duodu, 2004a, 2004b). Although the local average temperature in this study changed from 4 °C in January to 30 °C in July (Table 2), we
did not observe a clear changing pattern with regarding to the seasonal variation of amino acid composition, particularly for the imino acid content. 3.2. Protein fraction patterns and peptide hydrolysis patterns by SDS-PAGE Fig. 1 shows the SDS-PAGE patterns of ASC extracted from swim bladders and skins of grass carp (Fig. 1(A) and (B)) and bighead carp (Fig. 1(C) and (D)). All of the ACS were typical type I collagen, and consisted of three different α chains, namely, α1(I), α2(I), and α3(I). Moreover, the β chains (dimmers of the α-chains) and some highly cross-linked components were also observed in all of the ASC. However, for the ACS from the same tissue of the same fish species, the SDS-PAGE patterns showed no significant seasonal variation in protein fraction distribution. In a previous study on scale collagens from carp and silver carp, it was suggested that the content of the α1 chain was lower for scale collagen from winter fish than those from the summer, while the content of α2 and α3 chains were higher for scale collagen from winter fish (Duan et al., 2012). However, the results with the collagens from the skins and swim bladders in the present study are inconsistent with their results. The seasonal consistency of ASC's subunit MW was further investigated by examining the peptide hydrolysis patterns (Fig. 2). Compared with the intact collagens (Fig. 1), the intensity of band for high molecular weight peptide fragments decreased while that of the low molecular weight components increased. The peptide hydrolysis patterns of ASC from grass carp were slightly different from those from bighead carp. However, for the ASC from the
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same fish and same tissue, no significant seasonal variations were observed suggesting the major primary structures remain unchanged.
also thank Shuying Wang, Hongyang Pan and Keyv Lu for their help with the amino acid analysis and TEM measurement.
3.3. Thermostability using DSC
References
The thermostability of ASC was determined using DSC, and the maximum thermal transition temperatures (Td) were recorded. For the ASC from skins of both bighead carp and grass carp, the Td values changed from 35.1 to 35.8 °C, while those of ASC from swim bladders changed from 36.5 to 37.8 °C (Fig. 3), suggesting that the thermostability of ASC from swim bladders (the internal tissues) was higher than that of ASC from skins (the external tissues), which was in agreement with previous research on pepsin-solubilized collagens from bighead carp (Liu et al., 2015; Liu, Liang, Regenstein, & Zhou, 2012) and acid-solubilized collagens from sea bass (Nikoo et al., 2014; Sinthusamran, Benjakul, & Kishimura, 2013; Zhang et al., 2016). Thus, the data in the present study did not suggest any seasonal variation on thermostability (Fig. 3).
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3.4. Formation of collagen fibrils In the fibril formation process, collagen molecules assembled to the parallel staggered hole areas with less than full protein coverage and the overlapping areas with full protein coverage. Their different electron density resulted in the D-periods formation when examined using TEM. The differences in collagen molecules could influence the length of these hole areas and overlapping areas, which would lead to changes in the TEM observation if there were seasonal variation. Fig. 4 shows the TEM observations of collagen fibrils assembled from ACS of grass carp skins at various times. All the skin ASC could form ordered structures of fibril in vitro with characteristic D-periodicity, which served as an indication for the reconstitution of native-like collagen fibrils (Li et al., 2009). In addition, the D-periodicities of fibrils formed from ASC were all between 66 and 67 nm (Table 2), suggesting that there was no significant variation in the fibrogenesis of collagens with season (Chen et al., 2010).
4. Conclusions Bighead carp and grass carp are two of the most abundant freshwater fishes in China. The ASC from the scales and skins of these two fish species were mainly type I collagens with the major molecular form of α1(I)α2(I)α3(I). For collagens from the same tissue of the same fish species, all of the tests that were run in this research showed that there was no sign of significant seasonal variation. This suggests that the skins and swim bladders of these two major freshwater species were suitable for using as the alternative sources for collagen production on a year-round basis without concern for having to modify the process or the end use because of seasonal variation.
Conflict of interest statement There are no conflicts of interest to report.
Acknowledgements This research was partly supported by the 111 project (B07029), the Collaborative Innovation Center for Food Safety and Quality Control and the Postdoctoral Science Foundation of Weihai. We
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