46 3 Vol.46, No.3 2015 5 OCEANOLOGIA ET LIMNOLOGIA SINICA May, 2015 鲀 (Navodon septentrionalis) * 1 1 1 2 1 (1. 316022; 2. 316022) 鲀 (ASC) (PSC), ASC PSC SDS-PAGE, : 鲀 ASC PSC 0.94% ± 0.07% 3.91% ± 0.14% Gly(265.0 251.8 /1000 ) Ala(111.0 107.6 /1000 ) Pro(87.0 83.1 /1000 ), His Tyr, Cys ASC PSC (Pro Hyp) 169.3 160.6 /1000 SDS-PAGE (FTIR) ASC PSC I, 17.6 C 16.5 C ph, ASC PSC ; NaCl 2%, ASC PSC,, 鲀 ASC PSC,,, 鲀 ; ; ; R285 doi: 10.11693/hyhz20150100016, 30,,,, (Duan et al, 2009;, 2011; Liu et al, 2012),,, (BSE) (TSE) (Jongjareonrak et al, 2005a, b; Chi et al, 2014) H1N1,,,,,,,, (Jongjareonrak et al, 2005a, b;, 2006;, 2009; Li et al, 2013a; Liu et al, 2012; Li et al, 2013b) 鲀 (Navodon septentrionalis) 鲀 鲀 鲀, (, 2013) *, 2014C33034 ;, 2011C02003 ;, Y201225031,, E-mail: zhaoy@hotmail.com :,,, E-mail: wangbin4159@hotmail.com : 2015-01-06, : 2015-02-18
704 46, 鲀,,, 鲀,,, 鲀 1 1.1 鲀 (N. septentrionalis),, 20 C SDS-PAGE Bio-Rad, (CSC) (Pepsin, 160 000 U/g) Sigma, 1.2 1.2.1 鲀 鲀,, 1..15(W/V) 0.1 mol/l NaOH 4 C 24 h (NaOH 6 h ),,,,, 1..20(V/V) 15% 48 h( 12 h ) 1.. 10(W/V) 0.5 mol/l EDTA-2Na(pH 7.4) 24 h(edta-2na 12 h ),, 1.2.2 1.2.2.1 (ASC) ASC Yu (2014) 鲀 1.. 15(W/V) 0.5 mol/l, 24 h, 4 C 20 000 g 30 min, Tris-HCl (0.05 mol/l, ph 7.5), NaCl 2.6 mol/l,, 4 C 20000 r/min 20 min 0.5 mol/l 0.1 mol /L 24 h 36 h, (ASC) ASC : ASC (%)= (g)/ (g) 100 1.2.2.2 (PSC) PSC Yu (2014) (ASC) 1..15 (W/V) 0.5 mol/l, 3% 4 C 48 h, 4 C 20000 g 30 min, Tris-HCl (0.05 mol/l, ph 7.5), NaCl 2.6 mol/l,, 4 C 20000 r/min 20 min 0.5 mol /L 0.1 mol /L 24 h 36 h, (PSC), PSC : PSC (%)= (g)/ (g) 100 1.2.3 ASC PSC ASC PSC AOAC (2003) 950.46B 920.153 960.39(a) 928.08 1.2.4 (AA) (AA) (2010) (2009) 1 mg ASC PSC, 2 ml HCl(6 mol/l)( KOH ),,, 110 C 24 h,,, 70 C,, 3 ph 2.20, 0.22 μm, HITACH L8800, 2% 1.2.5 SDS-PAGE SDS- PAGE Chi (2013a) 7.5%, 4%, 200 V 30 min, R-250 ( 0.5 g, 225 ml, 225 ml, 50 ml), 15 min, ( 100 ml, 100 ml, 800 ml), 1.2.6 (FTIR) FTIR (2011) ASC PSC 1 2mg 1..100 KBr,
3 : 鲀 (Navodon septentrionalis) 705, 450 4000 cm 1 1.2.7 ASC PSC ASC PSC Kittiphattanabawon (2005) (0.6%) 4 C /min 4 C 40 C 4 C T d 50% : =( )/ ( ) 1.2.8 ASC PSC ASC PSC Montero (1991) ASC PSC 3 mg/ml 0.5 mol/l, 4 C 24 h, 4 C 15 000 rmp 30 min 1.2.8.1 ph ASC PSC 8 ml 50 ml, 6 mol/l NaOH/HCl ph 1-11 ph NaOH/HCl 10 ml, 4 C 30 min, 4 C 15 000 rmp 60 min,, ph ph 1.2.8.2 NaCl ASC PSC (6 mol/l, 5 ml) 0.5 mol/l 5 ml NaCl (W/V ) NaCl 0 %, 1 %, 2 %, 3 %, 4 %, 5 % 6 % 4 C 30min, 4 C 15000 r/min 60 min,, NaCl NaCl 2 2.1 ASC PSC 鲀 ASC PSC 0.94% ± 0.07% 3.91% ±0.14%, PSC ASC 4.16,, 鲀 ASC PSC 1 :, ASC PSC, 表 1 马 面 鲀 鱼 头 ASC 和 PSC 中 营 养 成 分 分 析 (n=3) Tab.1 Composition of head of bluefin leatherjacket, ASC and PSC (n=3) 鲀 ASC PSC 63.70 ± 2.05 0.16 ± 0.03 0.13 ± 0.01 18.41 ± 0.30 1.22 ± 0.11 1.38 ± 0.29 1.01 ± 0.09 0.59 ± 0.12 0.43 ± 0.08 16.89± 0.86 98.03 ± 0.54 98.08 ± 0.47,, 2.2 ASC PSC ASC PSC 2 : ASC PSC, Gly(265.0 251.8 /1000 ), Ala(111.0 107.6 /1000 ) Pro(87.0 83.1 /1000 ) His Tyr, Cys ASC PSC (Pro+Hyp) 169.3 160.6 /1000 表 2 ASC PSC 和 CSC 的 氨 基 酸 组 成 及 含 量 ( 残 基 /1000 残 基 )(n=3) Tab.2 Amino acids compositions of ASC, PSC and CSC (residues/1000 residues) (n=3) ASC PSC CSC Hyp 82.0 77.5 95.8 Asp 50.0 51.3 46.1 Thr 26.9 29.8 18.5 Ser 40.6 43.2 33.6 Glu 75.6 78.9 76.5 Pro 87.0 83.1 122.0 Gly 265.0 251.8 333.0 Ala 111.0 107.6 121.0 Cys 0.0 0.0 0.0 Val 36.4 38.5 21.7 Met 18.5 19.4 6.15 Ile 25.2 28.4 11.5 Leu 42.2 45.1 23.6 Tyr 12.4 14.4 3.73 Phe 22.0 24.2 3.33 Lys 49.2 50.8 26.7 His 10.6 10.7 5.39 Arg 45.4 45.3 51.4 Total 1000.0 1000.0 1000.0 169.3 160.6 217.8 Gly,
706 46,, (Gly-X-Y) n, Gly,, X Y, Gly-X-Y X Pro, Y Hyp (Balti et al, 2011),, Sionkowska (1999) (2005) : Hyp, (Pro Hyp) 鲀 ASC PSC (CSC)(217.8 /1000 ), 鲀 ASC PSC, 2, ASC PSC Cys, 鲀 2.3 ASC PSC SDS-PAGE ASC PSC SDS-PAGE 1 : ASC PSC, α (α1 α2),, α1 130 kda,, α2 110 kda, ; α, ASC PSC β (α ), 200 kda, α1, β, γ (α ), 300 kda ASC PSC SDS-PAGE,, 鲀 I (Montero et al, 1991; Jongjareonrak et al, 2005a, b; Kittiphattanabawon et al, 2005; Balti et al, 2011; Liu et al, 2012; Chi et al, 2013; Li et al, 2013b), ASC PSC I 2.4 (FTIR) ASC PSC (4000 450 cm 1 ) 2 : ASC PSC I 3400 cm 1 N-H, 2900 cm 1 B C-H, 1650 cm 1 C O, 1540 cm 1 N-H, 1240 cm 1 N-H, 1450 1230cm 1 3 (Li et al, 2013b) ASC PSC, 2 鲀 ASC PSC (FTIR) Fig.2 FTIR spectra of ASC and PSC from the head of bluefin leatherjacket 1 鲀 ASC PSC SDS-PAGE Fig.1 SDS-PAGE pattern of ASC and PSC from the head of bluefin leatherjacket 1: ; 2: ASC; 3: PSC 2.5 ASC PSC (Viscosity) (T d ),,,,,, 3,,,,
3 : 鲀 (Navodon septentrionalis) 707,,,, (Usha et al, 2004), (thermal denaturation temperature, T d ) (T d ), (ηsp/c-t), T d ASC PSC 3 : ASC PSC 4 C 24 C,, 24 C 40 C, ASC PSC 17.6 C 16.5 C, T d (37 C) (Nagai et al, 2002; Huang et al, 2011) ASC PSC, 3 鲀 ASC PSC (n=3) Fig.3 Thermal behaviours of ASC and PSC from the head of bluefin leatherjacket measured by viscosity change in deionized water (n=3) 2.6 2.6.1 ph ASC PSC 4, ph 1 3, ASC, ph 4 7, ASC ph 7, ASC (22.3% ± 3.6%) ph 7 11, ASC, ph 1 6 PSC ph 1 4, ph 8 (39.1% ± 3.8%) ph 1 11, ASC PSC ph, 4 ph 鲀 ASC PSC (n=3) Fig.4 Solubility of ASC and PSC from the head of bluefin leatherjacket at different ph (n=3),,,, 鲀 ASC PSC ph 7 8 6 9 (Nagai et al, 2002; Kittiphattanabawon et al, 2005; Huang et al, 2011; Chi et al, 2013) 2.6.2 NaCl ASC PSC 5, NaCl 0% 2% (W/V), ASC PSC (>90%); NaCl 2% 5% (W/V), ASC PSC, NaCl 5% 6% (W/V), ASC PSC NaCl 0% 6% (W/V), ASC PSC, PSC ASC NaCl ASC PSC Bae (2008), Na,,,,, ;,,, 5 NaCl 鲀 ASC PSC (n=3) Fig.5 Solubility of ASC and PSC from the head of bluefin leatherjacket at different NaCl concentrations (n=3)
708 46, 3 鲀 (ASC) (PSC), : (1) SDS-PAGE 鲀 (ASC) (PSC) (2) SDS-PAGE (FTIR) ASC PSC I (3) ASC PSC,,,, (4) ph ASC PSC, NaCl, ASC PSC,,, 2009.., 9(1): 77 83,,, 2005.., 34(23): 14 16,, 旸, 2011.., 34(2): 19 23,,, 2011. (Ommastrephes bartrami)., 42(6): 839 843,, 焜, 2013. 鲀 (Navodon septentrionalis)cyp19a., 44(4): 1086 1094,,, 2010.., 34(6): 801 808,,, 2006.., 6(1): 173 178,,, 2009. (Trichiurus lepturus)., 40(3): 307 312 Bae I, Osatomi K, Yoshida A et al, 2008. Biochemical properties of acid-soluble collagens extracted from the skins of underutilised fishes. Food Chem, 108(1): 49 54 Balti R, Jridi M, Sila A et al, 2011. Extraction and functional properties of gelatin from the skin of cuttlefish (Sepia officinalis) using smooth hound crude acid protease-aided process. Food Hydrocolloid, 25(5): 943 950 Chi C F, Wang B, Li Z R et al, 2013. Characterization of acid-soluble collagens from the cartilages of scalloped hammerhead (Sphyrna lewini), red stingray (Dasyatis akajei), and skate (Raja porosa). Food Sci Biotechnol, 22(4): 909 916 Chi C F, Wang B, Li Z R et al, 2014. Characterization of acid-soluble collagen from the skin of hammerhead shark (Sphyrna lewini). J Food Biochem, 38(2): 236 247 Duan R, Zhang J J, Du X Q et al, 2009. Properties of collagen from skin, scale and bone of carp (Cyprinus carpio). Food Chem, 112(3): 702 706 Huang Y R, Shiau C Y, Chen H H et al, 2011. Isolation and characterization of acid and pepsin-solubilized collagens from the skin of balloon fish (Diodon holocanthus). Food Hydrocolloid, 25(6): 1507 1513 Jongjareonrak A, Benjakul S, Visessanguan W et al, 2005a. Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of brownstripe red snapper (Lutjanus vitta). Food Chem, 93(3): 475 484 Jongjareonrak A, Benjakul S, Visessanguan W et al, 2005b. Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin. J Sci Food Agr, 85(7): 1203 1210 Kittiphattanabawon P, Benjakul S, Visessanguan W et al, 2005. Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chem, 89(3): 363 372 Li Z R, Wang B, Chi C F et al, 2013a. Influence of average molecular weight on antioxidant and functional properties of cartilage collagen hydrolysates from Sphyrna lewini, Dasyatis akjei and Raja porosa. Food Res Int, 51(1): 283 293 Li Z R, Wang B, Chi C F et al, 2013b. Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius). Food Hydrocolloid, 31(1): 103 113 Liu D S, Liang L, Regenstein J M et al, 2012. Extraction and characterisation of pepsin-solubilised collagen from Fins, scales, skins, bones and swim bladders of bighead carp (Hypophthalmichthys nobilis). Food Chem, 133(4): 1441 1448 Montero P, Jiménnez-Colmenero F, Borderìas J, 1991. Effect of ph and the presence of NaCl on some hydration properties of collagenous material from trout (Salmo irideus Gibb) muscle and skin. J Sci Food Agr, 54(1): 137 146 Nagai T, Araki Y, Suzuki N, 2002. Collagen of the skin of ocellate puffer fish (Takifugu rubripes). Food Chem, 78(2): 173 177 Sionkowska A, Kamińska A, 1999. Thermal helix-coil transition in UV irradiated collagen from rat tail tendon. Int J Biol Macromol, 24(4): 337 340 Usha R, Ramasami T, 2004. The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity. Thermochim Acta, 409(2): 201 206 Yu D, Chi C F, Wang B et al, 2014. Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna (Katsuwonus pelamis). Chin J Nat Med, 12(9): 712 720
3 : 鲀 (Navodon septentrionalis) 709 ISOLATION AND CHARACTERIZATION OF ACID AND PEPSIN SOLUBLE COLLAGENS FROM THE HEAD OF BLUEFIN LEATHERJACKET (NAVODON SEPTENTRIONALIS) ZHAO Yu-Qin 1, WANG Yu-Mei 1, WANG Bin 1, CHI Chang-Feng 2, DING Guo-Fang 1 (1. School of Food and Pharmacy, Zhejiang Provincial Key Engineering Technology Research Center of Marine Biomedical Products, Zhejiang Ocean University, Zhoushan 316022, China; 2. School of Marine Science and Technology, National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316022, China) Abstract Every year about 100000 tons of by-product are wasted in food industry in processing bluefin leatherjacket (Navodon septentrionalis) in China. To make more effective use of the wastes, acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the head of bluefin leatherjacket were extracted, and their properties were determined by using amino acid analysis, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel), and FTIR (Fourier transform infrared) technologies. The yields of ASC and PSC were 0.94% ± 0.07% and 3.91% ± 0.14% (on wet weight basis), respectively. ASC and PSC contained glycine (265.0 and 251.8 residues/1000 residues) as the major amino acid and the contents of imino acids were 169.3 and 160.6 residues/1000 residues, respectively. Amino acid composition, SDS-PAGE, and FTIR confirmed that ASC and PSC were mainly composed of type I collagen with slight differences in molecular structure. The denaturation temperatures (T d ) of ASC and PSC were 17.6 C and 16.5 C, much lower than those of mammalian and tropical fish species due to low imino acid contents. Therefore, the collagen helices of ASC and PSC might be less stable than those of mammalian because of lower imino acid contents and T d. All the collagens were soluble at acidic ph (1 4) and their solubility became low in NaCl concentration above 2% (W/V). Therefore, the isolated collagens from the bluefin leatherjacket head could be utilized as a substitute to mammalian collagen in the preparation of peptides. Key words bluefin leatherjacket (Navodon septentrionalis); fish head; acid soluble collagen (ASC); pepsin soluble collagen (PSC)