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The fractionation of serine protease inhibitor (SPI) from fish roe extracts was carried out using polyethylene glycol-4000 (PEG4000) precipitation. The protease inhibitory activity of extracts and PEG fractions from Alaska pollock (AP), bastard halibut (BH), skipjack tuna (ST), and yellowfin tuna (YT) roes were determined against target proteases. All of the roe extracts showed inhibitory activity toward bromelain (BR), chymotrypsin (CH), trypsin (TR), papain-EDTA (PED), and alcalase (AL) as target proteases. PEG fractions, which have positive inhibitory activity and high recovery (%), were the PEG1 fraction (0–5%, w/v) against cysteine proteases (BR and PA) and the PEG4 fraction (20–40 %, w/v) against serine proteases (CH and TR). The strongest specific inhibitory activity toward CH and TR of PEG4 fractions was AP (9278 and 1170 U/mg) followed by ST (6687 and 2064 U/mg), YT (3951 and 1536 U/mg), and BH (538 and 98 U/mg). The inhibitory activity of serine protease in extracts and PEG fractions from fish roe was stronger than that of cysteine protease toward common casein substrate. Therefore, SPI is mainly distributed in fish roe and PEG fractionation effectively isolated the SPI from fish roes. Keywords: Polyethylene glycol, Roe, Serine protease inhibitor, Recovery Background hake skeletal (Martone et al. 1991), and the roe of Alaska Protease inhibitors commonly accumulate in high quan- pollock, bastard halibut, skipjack tuna, yellowfin tuna tities in plant and animal tissues (Sangorrin et al. 2001), (Kim et al. 2015; Ji et al. 2011), herring (Oda et al. 1998), plant seeds, bird eggs, and various body fluids. Protease and carp (Tsai et al. 1996). inhibitors are also found in poultry (Lopuska et al. 1999), In industries of surimi-based product, commercial blood plasma (Rawdkuen et al. 2005; Rawdkuen et al. protease inhibitors are used to prevent the modori (gel 2007), fish roe (Kim et al. 2013a,b; Choi et al. 2002; softening) phenomenon and to maximize the gel strength Klomklao et al. 2014), and viscera (Kishimura et al. 2001). of surimi. The most commonly used inhibitors are bovine These inhibitors play a significant role in the regulation plasma protein (BPP), chicken egg white, potato powder, of proteolysis, whether the target enzymes are of exogen- and whey protein concentrate (Hamann et al. 1990; ous or endogenous origin. Protease inhibitors permit the Weerasinghe et al. 1996; Kim et al. 2015). However, regulation of the rate of proteolysis in the presence of the the use of BPP has been prohibited, due to the occurrence active enzyme (Barret 1986; Knight 1986; Cherqui et al. of mad cow disease. Egg white is expensive and has an 2001). The presence of protease inhibitors has been undesirable egg-like odor, while off-color problems may demonstrated in the blood and muscle of rainbow be encountered when potato powder is used (Akazawa trout (Clereszko et al. 2000), chum salmon (Yamashita et al. 1993). Therefore, alternative food-grade proteinase and Konagaya 1991), white croaker (Sangorrin et al. 2001), inhibitors from marine resources for surimi production are still needed. Fish roe, a byproduct generated from fish processing * Correspondence: heu1837@dreamwiz.com Department of Food and Nutrition/Institute of Marine Industry, Gyeongsang (3.0–30.0 % depend on fish species), is a highly nutritious National University, Jinju 52828, South Korea material rich in essential fatty acids and amino acids Full list of author information is available at the end of the article © 2016 The Author(s) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lee et al. Fisheries and Aquatic Sciences (2016) 19:25 Page 2 of 8 (Narsing Rao et al. 2012). Protease inhibitors in fish roe Fish roes were stored at −70 °C in sealed polyethylene can have a major impact on nutritional value as they bags until needed for inhibitor extraction. inhibit pancreatic serine proteases, thus impairing protein digestion. However, fish roe can be used as a potential Chemicals source of proteinase inhibitor and can be for a variety of Polyethylene glycol-4000 (PEG4000), which is a chemical applications such as medicine, agriculture, and food tech- used for fractionation, was obtained from the Yakuri Pure nology (Klomklao et al. 2014). ChemicalsCo.,Ltd.(Kyoto, Japan).Trypsin,chymotrypsin, Protein fractionation methods may be divided into those bromelain, and papain were from Sigma-Aldrich Chemical based on differential solubility, differential interaction with Co. (St. Louis, MO, USA). Alcalase 2.5 type DX, Neutrase solid media, and differential interaction with physical 0.8 L, Flavourzyme 500 MG, and Protamex were purchased parameters (Rawdkuen et al. 2005). In our previous study from Novozymes (Bagsvaerd, Denmark). Aroase AP-10 and (Kim et al. 2013a), the protease inhibitor was fractionated Pancidase NP-2 were from Yakult Pharmaceutical Co., Ltd. from fish eggs using methods based on protein solubility (Tokyo, Japan). Protease-NP was purchased from Amore- using organic solvent and ammonium sulfate (AS). AS pacific Co., Ltd. (Seoul, Korea). Casein and Nα-benzoyl- fractionation in isolating the protease inhibitor was more DL-arginine-2-naphthylamide hydrochloride (BANA) as effective than organic solvent precipitation (Kim et al. substrates were purchased from Sigma-Aldrich Chemical 2013a). However, AS fractionation methods have the Co.(St.Louis,MO, USA).The buffersolutions (0.1 M disadvantage of either requiring a high concentration or sodium phosphate buffer, pH 6.0; 0.1 M Tris-HCl buffer, cooling to avoid denaturation (Rawdkuen et al. 2007). In pH 9.0) for enzyme reaction were prepared according to the case of organic solvent fractionation, the component the method of Dawson et al. (1986). Sodium dodecyl sulfate obtained by fractionation has a notable capacity for use, as (SDS) and glycine were purchased from Bio Basic Inc. a result of the denaturation of the protein during the (Ontario, Canada). Coomassie brilliant blue R-250 was process (Kim et al. 2014; Rawdkuen et al. 2007). purchased from Bio-Rad Laboratories, Inc. (Hercules, In order to avoid the disadvantages of these techniques, CA, USA). Glycerol and β-mercaptoethanol were pur- polyethylene glycol (PEG) is an alternative precipitating chased from Sigma-Aldrich Chemical Co. (St. Louis, agent for protein fractionation. Chicken plasma was frac- MO, USA). Bromophenol blue was purchased from tionated into the protease inhibitor by PEG precipitation Junsei Chemical Co., Ltd. (Tokyo, Japan). (Rawdkuen et al. 2005; Rawdkuen et al. 2007). PEG has sev- All chemicals used were analytical grade. eral advantages over other precipitants, including the least denaturation of proteins at ambient temperatures, negli- Preparation of the CE gible temperature control required in the range 4–30 °C, Crude extracts (CEs) were prepared according to the relatively small amount of precipitant required compared modified method of Kim et al. (2013a). For extraction of with AS or organic solvents, and low residual PEG concen- CE from fish roes, the frozen roes were partially thawed tration in the precipitate since most of the PEG is retained and homogenized with 3 volumes (w/v)ofdeionized in the supernatant (Sharma and Kalonia 2004). distilled water. The homogenates were incubated at 20 °C The objectives of this study were to find the best condi- for 6 h, stirring every 1 h, and then centrifuged at tions for the polyethylene glycol fractionation of protein 12,000×g for 20 min at 4 °C. The supernatant was used as inhibitor and characterize the roe protease inhibitor from “crude extracts” for further study. Alaska pollock and bastard halibut as white-fleshed fish and skipjack tuna and yellowfin tuna as dark-fleshed fish Fractionation of protease inhibitor from CE with PEG roes. Four CEs from fish roes were continuously fractionated using PEG4000 in the range of 0–5 % (PEG1), 5–10 % (PEG2), 10–20 % (PEG3), and 20–40 % (w/v, PEG4), and Methods these fractions were collectedbycentrifugation(15,000×g, Materials for 30 min at 4 °C) and dissolved in a minimum quantity of Alaska pollock (AP, Theragra chalcogramma) roe was cold deionized water. The fractions were stored at −25 °C obtained from Blue Ocean Co. (Busan, Korea). Bastard until further analysis. halibut (BH, Paralichthys olivaceus) was purchased from the fish market (Tongyoung, Korea) and immediately brought to the laboratory. Roe was separated from BH Protein concentration and stored at −70 °C in sealed polyethylene bags. Skipjack The protein concentration of CE and PEG fractions tuna (ST, Katsuwonus pelamis) and yellowfin tuna (YT, from fish roes was determined according to the method Thunnus albacares) roes were obtained from Dongwon of Lowry et al. (1951) by bovine serum albumin as a F&B Co., Ltd. (Changwon, Gyungnam, Korea). standard protein. Lee et al. Fisheries and Aquatic Sciences (2016) 19:25 Page 3 of 8 Determination of inhibitory activity of CE and PEG SDS-PAGE and native PAGE gel electrophoresis fractions toward target proteases Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Enzyme activities against 0.1 % (w/v)chymotrypsin(CH) (SDS-PAGE) was carried out for the determination of the and trypsin (TR) as serine protease; 0.1 % (w/v)papain- purity and molecular weight of the samples, as described EDTA (PED) and bromelain (BM) as cysteine protein; and by Laemmli (1970), using a 10 % Mini-PROTEAN® TGX™ 1%(v/v) Alcalase (AL) and Neutrase (NE) and 1 % (w/v) Precast gel (Bio-Rad Laboratories, Inc., Hercules, CA, Protease-NP (PN), Pancidase NP-2 (NP), Protamex (PR), USA). Samples were prepared by mixing the CE and PEG Aroase AP-10 (AP-10), and Flavourzyme (FL) as commer- fractions at a 4:1 (v/v) ratio with the SDS-PAGE sample cial food-grade protease were measured using casein as a treatment buffer (62.5 mM Tris-HCl (pH 6.8), 2 % SDS substrate according to the methods of Ji et al. (2011). (w/v, pH 8.3), 10 % glycerol, 2 % β-mercaptoethanol, and The CE and PEG fractions were examined for inhibi- 0.002 % bromophenol blue). The samples were heated in a tory activity against commercial proteases as mentioned boiling water bath at 100 °C for 5 min and loaded (20 μg above. Protease inhibitory activity was measured using protein) on the SDS-polyacrylamide gel, and electrophor- casein and BANA as substrates. esis was performed at constant amperage (10 mA/gel) When casein was used as a substrate, 50 μL of the using a Mini-PROTEAN® Tetra cell (Bio-Rad Laboratories inhibitor solution (CE and PEG fractions) was mixed Inc., Hercules, CA, USA). After electrophoresis, the gel with enzymes (10–100 μL) in 1.5 mL of 0.1 M sodium was stained in a staining solution containing Coomassie phosphate buffer (pH 6.0) or 0.1 M Tris-HCl buffer (pH brilliant blue R-250. De-staining was carried out using a 9.0). After incubation for 10 min at room temperature, solution containing acetic acid, methanol, and water 0.5 mL of 2 % casein was added and mixed thoroughly. (1:2:7, v/v/v). The molecular weight of samples was esti- The mixture was incubated for 1 h at 40 °C. The enzym- mated using Precision Plus Protein™ standards (10–250 K) atic reaction was terminated by adding 2 mL of 5 % from Bio-Rad Laboratories, Inc., (Hercules, CA, USA). TCA and then centrifuged at 1910×g for 15 min at 4 °C. Native PAGE was performed according to the procedure The liberated soluble peptides in the supernatant were of Kim et al. (2015), except that the sample was not heated estimated by measuring the absorbance at 280 nm to and the SDS and reducing agent were left out. determine the residual protease activity. Protease activities against 0.1 % TR and 0.1 % PED were Zymography measured using BANA as the substrate according to the Casein zymography was performed on native PAGE. methods of Rawdkuen et al. (2007) with a slight modifica- Briefly, after electrophoresis, the gel was flooded with tion. The 0.1 % TR (50 μL) and 0.1 % PED (100 μL) were 3 mL of 0.1 % chymotrypsin. The gel was incubated for added to 50 μg of inhibitor solution in 1.5 mL of 0.1 M 60 min at 40 °C to allow the protease to diffuse into the Tris-HCl buffer (pH 9.0) and 0.1 M sodium phosphate gel and then washed with distilled water. The gel was buffer (pH 6.0), respectively. The mixture was incubated immersed in 0.1 M Tris-HCl buffer, (pH 9.0) with 2 % for 10 min at room temperature. Then, 50 μLof10mM casein (v/v) for 2 h. The gel was then rinsed with distilled BANA was added and vortexed immediately to start the water, fixed, and stained with Coomassie brilliant blue R- enzyme reaction. After incubating for 1 h at 40 °C, 0.5 mL 250 to develop inhibitory zones detected as a dark band of 2 % HCl/ethanol was added to terminate the reaction. on a clear background. The reaction mixture was centrifuged at 1910×g for 15 min. The residual activity of enzymes was measured by Statistical analysis the absorbance at 540 nm (U-2900, UV-VIS spectropho- All experiments were conducted in triplicates. The aver- tometer, Hitachi, Tokyo, Japan). age and standard deviations were calculated. Data were One unit of enzyme activity was defined as an increase analyzed using the analysis of variance (ANOVA) proced- of 0.1 absorbance per 1 h. ure by means of the statistical software SPSS 12.0 KO One unit of inhibitory activity was defined as the (SPSS Inc., Chicago, IL, USA). The mean comparison was amount of an inhibitor that reduced 1 unit/mg of target made using Duncan’s multiple range test (P <0.05). protease activity for 1 h. Relative inhibitory activity (RIA) was calculated as Results and discussion follows: Inhibitory activity of CEs Commercial protease inhibitory activities of the crude extract (CE) from fish roes (AP, BH, ST, and YT) are RIAðÞ % ¼ 〔ðÞ C−A =C〕 100 shown in Fig. 1. Inhibitory activities against 11 commer- cial proteases were measured using casein as a substrate. C = enzyme activity of control (without inhibitor), A = The highest relative inhibitory activity (RIA, %) was enzyme activity of sample (with inhibitor) found in all CEs for CH as a serine protease. Of the CEs, Lee et al. Fisheries and Aquatic Sciences (2016) 19:25 Page 4 of 8 these results confirmed that the CE from fish roes belongs to the serine protease inhibitor family. Ji et al. (2011) confirmed the distribution of protease inhibitory activity in CEs from fish roes. ST (Choi et al. 2002) and YT (Klomklao et al. 2014) were reported to possess high trypsin inhibitory activity. The protease inhibitor from chum salmon egg (Kim et al. 2006), AP egg (Ustadi et al. 2005a), and glassfish egg (Ustadi et al. 2005b) inhibited the cysteine proteases such as papain and cathepsin L, but not trypsin, a serine protease. Protein content of PEG fractions The protein contents of CE and PEG fractions from fish roes are shown in Fig. 3. The protein contents of the CE Fig. 1 Commercial protease inhibitory activity of the crude extract from of AP, BH, ST, and YT were 5655.0, 4183.0, 2849.6, and fish roes toward casein as a substrate. RIA (%) relative inhibitor activity 3711.0 mg/100 g roe, respectively. The highest protein content of PEG fraction by PEG precipitation was found AP showed the highest RIA (52.2 %), followed by ST in PEG1 (0–5 % fraction) for AP and BH. The protein (29.7 %), BH (18.1 %), and YT (14.0 %). RIAs (0.1–3.1 %) recovered in the PEG1 fraction of AP and BH represented for TR as a serine protease were lower than those of CH. 55.1 and 46.8 % of the total protein content of PEG frac- RIAs of BR and PED as a cysteine protease were observed tions, respectively. Among the PEG fractions obtained for AP, ST, and YT except for BH. Among the commercial from the CE of ST, the PEG4 fraction had the highest food-grade proteases, RIAs in all CEs were observed for protein content (350.8 mg/100 g roe), which constituted AL. The other proteases, such as FL, PR, NE, AP-10, and approximately 38.8 % of the total protein content of PEG PN, showed no effect on the inhibitory activity. Therefore, fractions, followed by PEG4 (349.4 mg/100 g roe), PEG2 these results suggested that the CE from fish roes belongs (177.5 mg/100 g roe), and PEG1 fraction (26.3 mg/100 g to the serine protease inhibitor family and is also more roe). The protein content recovered from the PEG3 and sensitive to reaction with chymotrypsin than trypsin. PEG4 fractions of YT represented 42.3 and 40.5 % of the The protease inhibitory activities for trypsin (TR) and total protein content of PEC fractions. From the result, papain-EDTA (PED) of the CE from fish roes are shown greater protein in the PEG fraction suggested that a higher in Fig. 2. Inhibitory activities were measured using BANA amount of protease inhibitors was precipitated. Bovine as a specific substrate for trypsin and papain. RIA for tryp- blood plasma (Lee et al. 1987) and chicken plasma sin was the highest in AP (23.0 %), followed by ST (Rawdkuen et al. 2005; Rawdkuen et al. 2007) were (12.1 %), BH (8.4 %), and YT (8.0 %). Whereas, when PED fractionated into proteins and protease inhibitor by as a cysteine protease was used, the CEs of all fish roes PEG precipitation with high separation efficiencies. showed no effect on the inhibitory activity. Therefore, Fig. 2 Commercial protease inhibitory activity of the crude extracts from Fig. 3 Protein content (mg/100 g roe) of PEG fractions obtained fish roes by the polyethylene glycoltowardBANAasasubstrate. Means from the crude extracts of fish roes by the polyethylene glycol with different letters within the sample are significantly different at P < precipitation. Means with different letters within the sample are 0.05 by Duncan’s multiple range test. RIA (%) relative inhibitor activity significantly different at P < 0.05 by Duncan’s multiple range test Lee et al. Fisheries and Aquatic Sciences (2016) 19:25 Page 5 of 8 Inhibitory activity of PEG fractions the inhibitory activity for BR and PED. Of the PEG frac- Commercial protease inhibitory activity and the recovery tions, the PEG1 fraction of ST and YT showed the highest of the CE and PEG fractions from fish roes are shown in SIA for BR (72.6 and 45.7 U/mg, respectively) and PED Table 1. Inhibitory activities against 1 % AL, 0.1 % BR, (618.6 and 566.2 U/mg, respectively). From this result, it 0.1 % PED, 0.1 % CH, and 0.1 % TR were measured using can be stated that the cysteine inhibitor from the PEG casein as a substrate. fraction of AP, ST, and YT is more concentrated in the All PEG fractions obtained from CE of AP and BH PEG1 fraction (0–5 %). The highest SIA for CH was showed no effect on the specific inhibitory activity (SIA) observed in the PEG4 fractions of AP, ST, and YT except for AL as a commercial food-grade protease. The SIA of for BH. The SIA of 9278.3, 6687.0, and 3951.1 U/mg with 210.3 and 209.3 U/mg with recovery of 0.2 and 3.2 % recoveries of 12.0, 49.1, and 68.7 % were obtained for AP, were obtained for the PEG1 fraction of ST and YT, ST, and YT, respectively. The SIA and recovery for TR respectively. Among the PEG fractions of AP, the highest were highest in the PEG4 fraction of the four fish species. SIA (17.9 U/mg) and recovery (18.4 %) was found in the The SIA and recovery for TR in the PEG4 fraction were PEG1 fraction for BR, while the PEG2 fraction gave the 1170.9 U/mg and 45.2 % for AP, 98.2 U/mg and 19.8 % for highest SAI (220.8 U/mg) and recovery (11.8 %) for PED. BH, 2064.2 U/mg and 312.4 % for ST, and 1536.2 U/mg However, all the PEG fractions of BH showed no effect on and 419.2 % for YT. From the result, the greater SIA and Table 1 Commercial protease inhibitory activities of PEG fractions obtained from the crude extracts of fish roes by the polyethylene glycol precipitation toward casein as a substrate Fraction AP BH ST YT SIA (U/mg) Recovery (%) SIA (U/mg) Recovery (%) SIA (U/mg) Recovery (%) SIA (U/mg) Recovery (%) AL CE 235.0 100.0 40.4 100.0 925.6 100.0 144.0 100.0 PEG1 −44.9 −3.3 −537.2 −347.3 210.3 0.2 209.3 3.2 PEG2 −65.9 −0.6 −42.7 −2.5 180.6 1.2 85.8 1.8 PEG3 −23.4 −0.9 −4.7 −1.4 −160.1 −2.1 37.2 3.3 PEG4 −65.3 −0.8 28.4 10.6 −26.7 −0.4 18.5 1.6 BR CE 16.8 100.0 −58.7 100.0 9.9 100.0 16.2 100.0 PEG1 17.9 18.4 −93.1 41.4 72.6 6.8 45.7 6.2 PEG2 5.9 0.7 −23.0 0.9 −32.1 −20.2 −23.3 −4.2 PEG3 17.3 9.5 −30.1 6.3 −28.6 −35.5 −26.4 −20.6 PEG4 −14.1 −2.4 −19.4 5.0 −50.2 −62.5 −21.1 −15.7 PED CE 38.4 100.0 −54.5 100.0 35.6 100.0 112.5 100.0 PEG1 0.9 0.4 −66.2 31.7 618.6 16.1 566.2 11.0 PEG2 220.8 11.8 20.3 −0.9 50.6 8.9 137.0 3.6 PEG3 55.0 13.2 −42.9 9.6 9.3 3.2 50.5 5.7 PEG4 137.0 10.1 −23.2 6.4 15.8 5.5 22.4 2.4 CH CE 2183.9 100.0 672.5 100.0 1678.6 100.0 695.3 100.0 PEG1 673.4 5.3 −1477.5 −57.4 5768.6 3.2 2475.6 7.8 PEG2 1776.3 1.7 668.7 2.4 903.2 3.4 322.8 1.4 PEG3 767.7 3.2 288.4 5.2 1311.0 9.6 511.2 9.3 PEG4 9278.3 12.0 537.5 12.0 6687.0 49.1 3951.1 68.7 TR CE 73.1 100.0 74.5 100.0 81.4 100.0 44.3 100.0 PEG1 23.7 5.6 −990.1 −347.2 1999.7 22.7 −298.7 −14.8 PEG2 −259.4 −7.3 −416.9 −13.3 −513.2 −39.3 −299.0 −19.9 PEG3 −255.4 −32.2 −190.1 −31.2 302.8 45.7 118.9 33.9 PEG4 1170.9 45.2 98.2 19.8 2064.2 312.4 1536.2 419.2 Minus (−) values are no protease inhibitory activity Recovery (%) = (total inhibitory activity of fraction/total inhibitory activity of CE) × 100 PEG1–PEG4, 0–5, 5–10, 10–20, 20–40 % fractions obtained from polyethylene glycol-4000 precipitation AL alcalase, BM bromelain, PED papain-EDTA, CH chymotrypsin, TR Trypsin, CE crude extract, SIA specific inhibitory activity, RIA (%) relative inhibitor activity Lee et al. Fisheries and Aquatic Sciences (2016) 19:25 Page 6 of 8 Whereas, it was observed that PEG precipitation for ST and YT gave maximum recovery of the inhibitor in a 20– 40 % fraction (PEG4). Approximately 61.4 and 77.1 % of the total inhibitory activity of all PEG fractions were recoveredinthe PEG4fractionofST and YT,respectively. From theresults, theserineproteaseinhibitor from four fish roes was more likely concentrated in the PEG1 (for AP and BH) and PEG4 fraction (for ST and YT). Fractionation was commonly selected as a first step of purification, because the fractionation significantly reduced the volume of the solution and effectively removed contam- inated proteins (Burnouf 1995). Rawdkuen et al. (2007) reported that PEG fractionation was more effective than AS fractionation. PEG might induce the conformational Fig. 4 Total inhibitory activity of PEG fractions obtained from the crude extracts of fish roes by the polyethylene glycol precipitation changes in the way which favored the inhibition of protease toward BANA as a substrate. Means with different letters within the (Rawdkuen et al. 2005). Hao et al. (1980) reported that a sample are significantly different at P < 0.05 by Duncan’s multiple variety of protease inhibitors were found in the 0–20 % range test. TIA (U/100 g of roe) total inhibitory activity PEG4000 fraction of plasma. recovery of the PEG4 fraction suggested that a higher Native PAGE and SDS-PAGE amount of serine protease inhibitor was precipitated in The native PAGE of the PEG fractions is shown in the PEG concentration range of 20–40 %. Fig. 5a. The PEG1, PEG2, and PEG3 fractions from AP Total inhibitory activity (TIA, U/100 g roe) of PEG frac- contained protein bands similar to those of CE. A weakly tions for trypsin using BANA as a specific substrate is cationic protein band which appeared in the PEG4 frac- shown in Fig. 4. Among all precipitates obtained from AP tion of AP was rarely found in other fractions. In the CE and BH, the PEG1 fraction had the highest inhibitory of BH, protein bands with cationic proteins, weakly cat- activity, followed by PEG4, PEG3, and PEG2 fraction. TIAs ionic protein, and weakly anionic protein were observed. of 151,206.6 and 170,464.7 U/100 g roe were recovered in After fractionation, increase in the weakly cationic protein the PEG1 fraction toward AP and BH, respectively. bands was observed with increasing PEG concentration. Fig. 5 Native PAGE (a) and SDS-PAGE (b) of PEG fractions obtained from the extracts of fish roes by the polyethylene glycol precipitation. Lane 1, CE; lane 2, PEG1; lane 3, PEG2; lane 4, PEG3; lane 5, PEG4. S standard maker Lee et al. Fisheries and Aquatic Sciences (2016) 19:25 Page 7 of 8 Fig. 6 Native PAGE (a) and inhibitory activity staining (b) for chymotrypsin of serine protease inhibitor fractions obtained from the extracts of skipjack tuna and yellowfin tuna roe by the polyethylene glycol precipitation. Lane 1, CE; lane 2, PEG1; lane 3, PEG2; lane 4, PEG3; lane 5, PEG4 The CE from ST and YT showed a similar protein pattern predominant proteins in PEG4. From the result, using with cationic protein, weakly cationic protein, and weakly 20–40 % PG fractionation was found to be an effective anionic protein bands. The PEG4 fraction from ST and YT method to fractionate the serine protease inhibitor from consisted of bands with weakly cationic protein and weakly ST and YT roes. anionic protein as the major components. The molecular weight distributions of the PEG fractions Conclusions estimated from the mobility in SDS-PAGE are shown in The protease inhibitor from fish roes was successfully frac- Fig. 5b. The CE of AP contained a variety of proteins with tionated by using 200–400 g PEG/L. The PEG fractions different high and low molecular weights. Protein bands from fish roes obtained showed high inhibitory activity in the ranges of 150–75, 50, 25–20, and 15–10 K were against trypsin and chymotrypsin as serine protease. PEG is observed. The PEG1, PEG2, and PEG3 fractions also had commonly exploited in large-scale protease inhibitor prep- a pattern similar to that of CE from AP. Whereas, the aration or purification from fish roes for both seafood and PEG4 fraction showed only a low molecular band in the surimi industry use. range of 15–10 K. Similar protein patterns were observed among the CE and PEG fractions from BH, in which low Acknowledgements This research was supported by the National Research Foundation of Korea molecular proteins were predominant. The CE from ST (NRF) funded by the Ministry of Education, Science and Technology had protein bands in the ranges of 25–20 and 15–10 K. (2010–0009921). The PEG1 and PEG2 fractions showed low molecular protein bands (25–10 K). The PEG3 and PEG4 fractions Authors’ contributions HJL, HJK, and SHP carried out the enzymatic inhibitory activity analysis, showed bands with higher molecular weight protein than participated in the PEG fractionation, and drafted the manuscript. ISY, GWL, those of PEG1 and PEG2 fractions. The CE from YT and YJK participated in searching and screening references and performed the contained protein bands with a different molecular weight. statistical analysis. JSK and MSH conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read After fractionation, the molecular band in range of 15– and approved the final manuscript. 10 K was retained in the PEG4 fraction. Competing interests Native PAGE and detection of protease inhibitory activity The authors declare that they have no competing interests. by zymography Author details Due to the high serine protease inhibitory activity, the 1 Department of Food and Nutrition/Institute of Marine Industry, Gyeongsang PEG fractions of ST and YT were selected. The native National University, Jinju 52828, South Korea. Department of Seafood Science and Technology/Institute of Marine Industry, Gyeongsang National PAGE patterns and inhibitory activity staining for chymo- University, Tongyeong 53064, South Korea. trypsin of PEG fractions are depicted in Fig. 6. For native PAGE (Fig. 6a), a similar protein pattern was observed in Received: 15 February 2016 Accepted: 19 May 2016 CE (lane 1) and PEG4 fraction (lane 5), in which bands with weakly cationic protein and weakly anionic protein References were dominant. The inhibitory activity staining of the Akazawa H, Miyauchi Y, Sakurada K, Wasson DH, Reppond KD. 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Functional Submit your next manuscript to BioMed Central properties and in vitro antioxidant activity of roe protein hydrolysates of Channa striatus and Labeo rohita. Food Chem. 2012. doi:10.1016/j.foodchem. and we will help you at every step: Oda S, Igarashi Y, Manaka KI, Koibuchi N, Sakai-Sawada M, Sakai M, et al. • We accept pre-submission inquiries Sperm-activating proteins obtained from the herring eggs are homologous to trypsin inhibitors and synthesized in follicle cells. Dev Biol. 1998;204:55–63. � Our selector tool helps you to find the most relevant journal Rawdkuen S, Benjakul S, Visessanguan W, Lanier TC. Fractionation and � We provide round the clock customer support characterization of cysteine proteinase inhibitor from chicken plasma. � Convenient online submission J Food Biochem. 2005. doi:10.1111/j.1745-4514.2005.00027.x. Rawdkuen S, Benjakul S, Visessanguan W, Lanier TC. Cysteine proteinase inhibitor � Thorough peer review from chicken plasma: fractionation, characterization and autolysis inhibition � Inclusion in PubMed and all major indexing services of fish myofibrillar proteins. Food Chem. 2007. doi:10.1016/j.foodchem. � Maximum visibility for your research Sangorrin MP, Folco EJ, Martone CM, Sanchez JJ. Purification and characterization of a protease inhibitor from white croaker skeletal muscle Submit your manuscript at (Micropogon opercularis). Intl J Biochem Cell Biol. 2001;33:691–9. www.biomedcentral.com/submit
Fisheries and Aquatic Sciences – Springer Journals
Published: Jul 26, 2016
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