Alternative Techniques for Producing a Quality Surimi and Kamaboko from Common Carp (Cyprinus carpio)

ABSTRACT: The demand for surimi and kamaboko is increasing in the world at the same time as the supply of the fish traditionally used has declined. In an effort to increase the range and hence supply of fish used, factors increasing the quality of surimi and kamaboko from common carp were investigated. The best surimi and kamaboko characteristics were produced by a modified conventional method (MCM) rather than traditional method (TM), alkaline‐aided method (AAM), and pH modified method (PMM). MCM processing used centrifugation instead of decanting and filtering to optimize dewatering and remove the sarcoplasmic proteins (Sp‐P). The temperature sweep test, at the end of sol–gel transition stage (at 75 °C), showed significantly (P < 0.05) greater G′ for the kamaboko from MCM than that from other methods tested. Furthermore, the greatest and the least gel strengths were obtained with MCM and TM kamaboko, respectively. The protein recovery was about 67%, 74%, 87%, and 92% for TM, AAM, MCM, and PMM, respectively. TM and MCM resulted in the removal of Sp‐P as determined by SDS‐PAGE. The superiority of MCM kamaboko gel characteristics was supported by scanning electron micrographs (SEM) of the gel, which showed a significantly (P < 0.05) greater number of polygonal structures than for the TM kamaboko, which had the fewest and largest polygonal structures. The pH‐shifting methods improved the textural quality of the resultant kamaboko compared with TM. However, a simple modification (centrifugation compared with decanting) by MCM in the surimi process can further improve the quality of the surimi and kamaboko gels. Furthermore, because it removed Sp‐P and still preserved gel strength, it suggests that Sp‐P are not required for gel strength.     

Contribution of sarcoplasmic proteins to myofibrillar proteins gelation

Surimi, a refined protein extract, is produced by solubilizing myofibrillar proteins during the comminuting and salting stages of manufacturing. The resulting paste gels on heating to produce kamaboko or a range of analog shellfish such as crab claw, filament sticks, fish mushroom, and so on. The myosin molecule is the major myofibrillar protein in gelation. It is believed that washing steps during the traditional surimi process play an important role in enhancing the gel properties of the resultant kamaboko by removing water-soluble (sarcoplasmic, Sp-P) proteins. By contrast, some researchers claim that retaining Sp-P or adding it into the surimi gel network not only does not interfere with the action of myofibrillar proteins during the sol-gel transition step but also improves the gel characteristics of the resultant kamaboko. It seems that retention of Sp-P or their addition into raw surimi does enhance the textural properties of kamaboko gel perhaps by functioning as a proteinase inhibitor, particularly against trypsin and trypsin-like proteinases but this depends on the type of applied surimi process. Among different types of Sp-P, it has been claimed that some proteins such as endogenous transglutaminase (TGase) play a more important role than other Sp-P in bond formation, by catalyzing the cross-linking of myosin heavy chain (MHC) molecules during low-temperature setting of surimi, resulting a more elastic kamaboko gel.     

葡萄糖氧化酶对鲢鱼糜凝胶特性的影响

为了研究葡萄糖氧化酶(GOD)诱导的氧化对鲢鱼糜凝胶特性的影响,以冷冻鲢鱼糜为原料,分析在不同的GOD添加量(0、0.1‰、0.3‰、0.5‰、0.7‰、0.9‰)下,鱼糜的弹性模量(G′)、损耗模量(G′′)以及鱼糜凝胶的凝胶强度、白度、微观结构、持水性、水分分布状态和蛋白交联等指标的变化情况.结果表明,随着GOD添...     

不同食用蛋白的添加对鲤鱼鱼糜流变和凝胶特性的影响

目的:研究大豆分离蛋白(soybean protein isolated,SPI)、乳清分离蛋白(whey protein isolate,WPI)、花生分离蛋白(peanut protein isolate,PPI)的添加对鲤鱼鱼糜流变和凝胶性质的影响。方法:利用流变仪、质构仪、色差计等对添加不同蛋白鱼糜的弹性模量、黏性模量、凝胶强度、破断强度、凹陷深度、持水性以及白度进行测定,并采用相关性分析法研究各指标之间的相互关系。结果:不同添加量的SPI、WPI和PPI均能有效地改善鱼糜的弹性模量、黏性模量、破断强度、凝胶强度和持水性,但会降低破断深度和白度,但各测定指标间存在显著相关(p<0.05)。SPI和PPI的添加对鱼糜的流变性、破断强度、凝胶强度的提高效果更好,添加量为8%时,鱼糜的凝胶强度均达到最大值,其中SPI组可达3806.70 g·mm,比对照组增加了34.63%;WPI对鱼糜的保水性效果最好,添加量为8%时,失水率仅为12.6%;白度随着蛋白添加量的增加而降低,其中PPI组与WPI组引起的白度降低较少,且差异不显著(p>0.05)。结论:在实际鱼糜制品的生产中,应根据产品的特征选择适合的蛋白种类和合理的添加量,来提高鱼糜制品的品质。     

Effects of CaCl<Subscript>2</Subscript> on chemical interactions and gel properties of surimi gels from two species of carps

Effects of CaCl₂ on chemical interactions, textural properties and expressible moisture content of suwari and kamaboko gels from yellowcheek carp and grass carp were investigated. And the correlations between the contents of chemical interactions and physical properties of surimi gels were analyzed. The contents of chemical interactions, especially non-disulfide covalent bonds, disulfide bonds and hydrophobic interactions of suwari and kamaboko gels, varied with addition concentration of CaCl₂ and fish species. Suwari and kamaboko gels from yellowcheek carp exhibited higher breaking force, deformation and gel strength than these from grass carp. Surimi gels (suwari and kamaboko gels) from yellowcheek carp and grass carp exhibited their maximum gel strength when 40 mmol/kg and 100 mmol/kg CaCl₂ was added, respectively. Addition of CaCl₂ at high concentration resulted in low water holding capacity of surimi gels. Correlation analysis indicated that the contents of nonspecific associations, ionic bonds, hydrophobic interactions and sulfhydryl groups exhibited significant correlation with breaking force of surimi gels from yellowcheek carp and grass carp. Additionally, the content of non-disulfide covalent bonds had significant positive correlations with breaking force and expressible moisture of surimi gel from yellowcheek carp.     

超高压对鲤鱼肉糜-MgCl2凝胶特性的影响

为研究超高压对低盐鲤鱼肉糜凝胶品质的影响,本文在低盐条件下（2% NaCl）,探讨了不同压力超高压处理（100~400 MPa）对含有0.3%氯化镁（MgCl₂）鲤鱼肉糜凝胶的蒸煮得率、保水性、色泽、质构、凝胶强度和流变学特性的改善作用。结果表明:适当的压力处理结合MgCl₂可以显著提高鲤鱼肉糜的凝胶品质。当处理压力为200 MPa时,鲤鱼肉糜凝胶的蒸煮得率、保水性、硬度、凝胶强度均达到最大值,分别为93.14%、95.24%、3327.25 g、3277 g。与对照组相比,适当的压力处理可以显著改善鲤鱼肉糜-MgCl₂凝胶的储能模量（G'）,同时对产品的色泽影响较小。因此,超高压结合氯化镁可以改善低盐鲤鱼鱼糜凝胶的品质,为低盐淡水鱼糜制品的开发提供理论依据。     

变性淀粉对白鲢鱼鱼糜凝胶特性和蛋白构象的影响

为了研究原木薯淀粉、羟丙基淀粉、乙酰化二淀粉磷酸酯和醋酸酯淀粉对白鲢鱼鱼糜凝胶特性和蛋白构象的影响,分析不同淀粉添加量条件下,白鲢鱼鱼糜白度、凝胶强度、持水性、质构特性、储能模量(G')、化学作用力、蛋白二级结构和微观结构的变化规律.结果表明,添加淀粉可以显著提高鱼糜凝胶的持水性.当其添加量为1％时,可提高鱼糜凝胶的白...     

非肌肉蛋白对未漂洗革胡子鲶鱼鱼糜凝胶特性的影响

为进一步提高未经漂洗革胡子鲶鱼(Clarias gariepinus,CG)鱼糜的凝胶特性,本试验在CG鱼糜中添加0.4%转谷氨酰胺酶和20 mmol/kg CaCl₂基础上(CK组),再依次添加不同浓度的大豆分离蛋白(soybean protein isolation,SPI)、蛋清蛋白(egg white protein,EWP)和浓缩乳清蛋白(whey protein concentrate,WPC),SPI添加量分别为CG鱼糜肉重的2%(SPIL)、5%(SPIM)和7%(SPIH),EWP添加量分别记为EWPL(0.1%)、EWPM(0.3%)和EWPH(0.5%),WPC记为WPCL(0.2%)、WPCM(0.4%)和WPCH(0.6%),共制备出10组CG鱼糜凝胶。研究非肌肉蛋白对CG鱼糜的凝胶特性、色泽、持水力、动态流变学性质和微观结构等品质特性的影响。结果表明:整体上非肌肉蛋白的添加会提高CG鱼糜的凝胶特性和TGase活力。其中,SPIL、EWPH和WPCL三组的CG鱼糜凝胶表现出较好的凝胶强度,依次为3322.46、3555.90和4149.31 g·mm,并且网络结构较其他组更紧密、均匀;EWP和WPC的三种添加浓度均会提高CG鱼糜凝胶的白度值;SPI均可使CG鱼糜凝胶的T₂₂向弛豫时间短的方向迁移,其中SPIH组的凝胶持水性(86.64%)最高。动态流变结果表明,三种非肌肉蛋白的添加对CG鱼糜的弹性模量G'和损耗模量G″均产生显著影响,SPIH组、EWPM组和WPCL组为组内最高值。综上分析,在CG鱼糜凝胶中分别添加鱼糜总量的2% SPI、0.5% EWP、0.2% WPC,可显著提高CG鱼糜的凝胶特性。     

Participation of cysteine protease cathepsin L in the gel disintegration of red bulleye (Priacanthus macracanthus) surimi gel paste

BACKGROUND: Endogenous proteases, among them cysteine‐type proteases, are reported to contribute to gel disintegration, resulting in kamaboko of poor quality. Severe gel disintegration occurs in red bulleye surimi gel paste. The objective of this study was to clarify the participation of cysteine protease cathepsin L in the gel disintegration of red bulleye surimi. The surimi was made into kamaboko with and without cathepsin L inhibitors. To confirm its hydrolysis action, crude cathepsin L was also extracted and added to the surimi to make kamaboko. RESULTS: The gel strength of kamaboko obtained by both one‐step (50 °C, 2 h) and two‐step (50 °C, 2 h + 80 °C, 20 min) heating was very low in the absence of inhibitors. Protease inhibitors E‐64 and leupeptin were found to enhance the gel strength considerably. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the hydrolysis of kamaboko was promoted by crude cathepsin L and inhibited by E‐64 and leupeptin. The gel strength of two‐step heated kamaboko was increased from 12 to 110 and 130 g cm^?2 by E‐64 and leupeptin respectively at a concentration of 0.2 g kg^?1 surimi. CONCLUSION: Endogenous cathepsin L of red bulleye surimi participates in gel disintegration during kamaboko processing. It does so by degrading the myosin heavy chain of actomyosin and consequently hindering the gelation of red bulleye surimi. Copyright © 2009 Society of Chemical Industry     

不同盐对鲤鱼肌原纤维蛋白结构和凝胶特性的影响

肌原纤维蛋白是鱼肉中的主要蛋白质,对鱼糜类制品的质量起到关键的作用。文中研究了NaCl、CaCl2和Na4P2O73种盐类及其浓度对鲤鱼肌原纤维蛋白结构特性(表面疏水性、蛋白聚合程度)和凝胶特性(质构、微观结构、蛋白浊度)的影响。结果表明:鲤鱼肌原纤维蛋白的等电点为5.5;3种盐加入肌原纤维蛋白后会引起肌原纤维蛋白发生一定程度的聚集,表面疏水性降低;随着盐浓度的增大,肌原纤维蛋白凝胶硬度、弹性、浊度降低;添加CaCl2比其它盐类更明显降低鱼糜的凝胶硬度、弹性和浊度,且差异性显著(P<0.05);NaCl和Na4P2O7使鱼糜凝胶微观结构更致密、CaCl2使鱼糜凝胶微观结构变得松散。因此,在鲤鱼肌原纤维蛋白中添加NaCl、CaCl和NaPO对鱼糜肌原纤维蛋白结构和凝胶特性均有不同程度的影响。     

鲢鱼与金线鱼混合鱼糜的凝胶特性

为研究鲢鱼与金线鱼混合鱼糜的凝胶特性,本实验对混合鱼糜凝胶的凝胶强度、持水性、蒸煮损失、白度值、横向弛豫时间、微观结构和肌原纤维蛋白进行分析。结果表明：混合鱼糜的白度值较纯金线鱼鱼糜显著提高（P＜0.05）,且鲢鱼鱼糜与金线鱼鱼糜质量比5∶1混合后的凝胶白度值高于纯鲢鱼鱼糜;此配比下的混合鱼糜凝胶性能最好,其中凝胶破断力、凹陷距离、凝胶强度和持水性较鲢鱼鱼糜分别提高了18.33%、27.29%、51.01%和5.65%,较金线鱼鱼糜分别提高了5.84%、19.50%、26.49%和3.45%;该配比下的混合鱼糜凝胶的蒸煮损失较鲢鱼鱼糜和金线鱼鱼糜分别降低了15.88%和7.48%。低场核磁共振分析显示鲢鱼鱼糜与金线鱼鱼糜质量比5∶1混合后的鱼糜凝胶的横向弛豫时间T22比鲢鱼鱼糜凝胶T22缩短了10.34 ms。通过扫描电镜观察发现鲢鱼鱼糜与金线鱼鱼糜质量比5∶1混合时可形成高度均匀、致密的空间凝胶网络结构。肌原纤维蛋白凝胶电泳图显示该配比下肌球蛋白重链（myosin heavy china,MHC）发生交联,大分子聚集体形成,进入凝胶中MHC更少,条带更细窄。     

动物肝脏均质液的添加对鱼糜凝胶特性的影响

为了研究畜禽动物肝脏的添加对鱼糜凝胶特性的影响，本研究选取猪肝和鸭肝制备均质液，分别以5%或10%的比例添加入鲢鱼鱼糜中，并测定分析混合鱼糜凝胶的质构特性、流变学性质、水分分布、分子间作用力等。结果显示，肝脏均质液的加入提高了鱼糜凝胶的硬度以及储能模量（G’）和损耗模量（G’’），降低了凝胶的白度值、回复性和内聚性，对凝胶体系中水分分布未见显著影响；随着肝脏均质液添加量由5%提高到10%，鱼糜凝胶的硬度未呈现显著变化，而白度、回复性和内聚性则进一步下降；鸭肝均质液的TG酶粗酶活显著高于猪肝组，5%添加量下，破坏体系中离子键、氢键、疏水键、二硫键之后鸭肝混合鱼糜凝胶中蛋白质溶解性高于猪肝组，SDS-PAGE分析显示添加鸭肝组的样品相比猪肝组其肌球蛋白重链条带变浅，可能是更多的肌球蛋白参与了TG酶介导的分子间共价交联所致；10%添加量的情况下，猪肝组鱼糜凝胶强度以及不易流动水的比例相比对照组显著下降，而鸭肝组未见差异。初步研究表明5%猪肝均质液的添加对冷冻鲢鱼鱼糜凝胶的性能有显著影响，可能与其中含有TG酶有关，需要进一步研究揭示。     

Characteristics of surimi and kamaboko from sardines

Sardines of varying freshness (1 to 3 days in ice) were manually or mechanically processed into fish mince and surimi using 1 to 3 washing steps. Standard kamaboko gels were prepared by grinding thawed surimi with 3% NaCl and 5% potato starch, stuffing into sausage casings, holding at 37°C for 30 min and cooking at 90°C for 50 min.
Three washing steps decreased the yield of washed fish mince (21-27 g per 100 g of whole fish), and the protein recovery (50-55% of that present in the unwashed mince), but caused efficient lipid removal (80%) leading to surimi containing only 0.2-1.4% lipid. The texture and colour of the final kamaboko were also improved.
The texture parameters (folding score, rigidity, elasticity index and gel strength) of kamaboko prepared from very fresh sardines were markedly enhanced by gel setting during incubation at 37°C for 30 min or 4°C for 24 hr. Incubation at 60°C for 30 min led to soft, coarse gels. Omitting potato starch or replacement by spray dried egg white had little effect on texture.
The texture of kamaboko prepared from less fresh sardines was less firm and elastic, did not improve when incubation was carried out at 37 or 4°C before cooking, and was softer and coarser after incubation at 60°C. Partial or total replacement of potato starch by egg white, soy protein isolate or bovine serum albumin markedly improved the texture. Egg white also increased the colour lightness.     

Effects of rice residue on physicochemical properties of silver carp surimi gels

The objective of this study was to determine the effects of rice residue on the physicochemical properties of silver carp surimi gels. The whiteness of gels was slightly decreased when rice residue was added. Breaking force and deformation of gels were significantly decreased with more than 1% rice residue addition (p < 0.05). The trends of storage modulus, tan δ, and interactions demonstrated a negative effect of rice residue (more than 1%) on gel network. The addition of rice residue reduced the interactions in surimi gel network, such as hydrophobic interaction. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis indicated that there was little interaction between rice proteins and myofibrillar proteins. Rice residue deteriorated the freeze-thaw stability of surimi gels. Therefore, rice residue could be an inactive filler in the gel network unless it was less than 1%.     

Myofibril-Bound Serine Proteinase (MBP) and its Degradation of Myofibrillar Proteins

Proteolysis of a myofibril-bound serine proteinase (MBP) from carp Cyprinus carpio on myofibrillar proteins and their gel formation ability were investigated. MBP readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBP. The optimum degradation temperatures of MBP to myosin heavy chain in myofibril and kamaboko gel were 55°C and 60°C, respectively. The degradation effects of MBP on actin, α-actinin and tropomyosin were studied by the immunoblotting method. Because of its myofibril-bound and myofibrillar protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.     

外源添加物对鲢鱼-鳕鱼复合鱼糜凝胶性能的影响

以鲢鱼与鳕鱼为主要原料,选择外源添加物马铃薯淀粉、谷氨酰胺转氨酶（Glutamine transaminase, TGase）以及蛋清蛋白的添加量为单因素,以复合鱼糜凝胶强度为响应值,采用Box-Behnken响应面试验优化复合鱼糜凝胶性能,并用质构性能分析和扫描电镜对最优复合鱼糜凝胶性能进行验证。结果表明:鲢鱼-鳕鱼复合鱼糜在马铃薯淀粉添加量为16%,谷氨酰胺转氨酶（TGase）添加量为0.4%,蛋清蛋白添加量为6.0%,复合鱼糜凝胶强度最大。根据此配方（优化工艺组）所得复合鱼糜质构特性参数中的咀嚼性、粘结性、破断力与破断距离均显著高于未添加外源物普通工艺组（P<0.05）,同时优化工艺组的复合鱼糜的扫描电镜图显示其凝胶结构网状结构较为致密,凝胶孔洞较小,证明了外源添加物有助于改善复合鱼糜凝胶强度。     

Mechanisms of gelation of sardine proteins: influence of thermal processing and of various additives on the texture and protein solubility of kamaboko gels

Surimi prepared from freshly caught sardines was mixed with NaCl and other additives and used to prepare kamaboko gels. Protein-protein interactions involved in the setting (at 4 or 37°C) and/or the cooking (at 90°C) gelation steps were investigated (i) by assessment of kamaboko texture as a result of the type and concentration of additive added; (ii) by partial solubilization of kamaboko gels in buffers containing mercaptoethanol, sodium dodecyl sulphate (SDS) and/or urea, followed by determination of the soluble protein constituents by polyacrylamide gel electrophoresis. Cooked gels of high elasticity and of varying rigidity and gel strength were obtained in the 73–80% water range. Adequate gel texture required a NaCl content of 1.7–3.5% and a pH range of 6.4–8.4. Low concentrations of reducing agents (mercaptoethanol, dithiothreitol, cysteine) or of divalent cations (Ca²⁺, Mg²⁺) improved the texture of gels obtained by setting at 37°C with and without subsequent cooking at 90°C. On the other hand, the addition of N -ethyl maleimide or of ethylene diamine tetra-acetate led to texture deterioration after cooking. These data demonstrate the involvement of disulphide bonds and of electrostatic interactions in surimi gelation. Gel solubilization experiments indicate that the aggregation of myosin heavy chains through various types of protein-protein interactions may be responsible for the elastic gel network formed during setting at 37°C (30 min) or 4°C (24h). Strengthening of the gel network after cooking appears to be due to additional disulphide and hydrophobic interactions.     

典型加工条件下鱼糜肌原纤维蛋白品质变化规律及调控方法研究进展

鱼糜制品（鱼丸、鱼糕、蟹肉棒等）因为其健康营养、方便快捷等特点而深受消费者欢迎，具有广阔的市场前景。鱼糜制品是基于鱼糜肌原纤维蛋白的胶凝性能所形成的弹性凝胶体，其加工工序包括斩拌、加热、添加外源物等。因此，鱼糜肌原纤维蛋白在典型加工条件下发生结构以及功能特性的变化会影响鱼糜制品的品质。本文主要综述了鱼糜肌原纤维蛋白在物理场（加热、斩拌）、化学场（pH值、离子强度）、生物场（酶、微生物）等典型加工条件下的结构及功能特性变化规律及调控方法，以期为进一步发展鱼糜制品加工业提供理论依据。     

Optimal blending of differently refined fish proteins based on their functional properties

Two different mixtures (Alaska pollock surimi with grass carp fish protein isolate (FPI) and grass carp surimi with grass carp FPI) were investigated for their compatibility and functionalities. As the proportion of FPI increased, it was observed surface hydrophobicity and surface reactive sulfhydryl (SRSH) content increased significantly, indicating the degree of fish protein unfolding prior to gelation was much higher than surimi alone. Comparable results were shown as measured by storage modulus (G′) in oscillatory dynamic rheology, demonstrating the gelling temperature was reduced when the proportion of FPI increased. Effects of mixing surimi and FPI on gel functionality (hardness, cohesiveness, and whiteness) exhibited a linear pattern when the proportion of surimi was larger than or equal to that of FPI. However, there were no linear relationships when the proportion of FPI exceeded that of surimi.

Practical applications

Commercial surimi has been successfully used in the Western world over 30 years. Unlike surimi which is a refined fish myofibrillar protein composite, fish protein isolate (FPI) is a refined composite of myofibrillar protein and sarcoplasmic protein. The former is made by avoiding any chemical/physical denaturation, while the latter can be made by inducing chemical denaturation and renaturation with pH shift. Even though FPI is not currently available in a commercial scale, it has a great potential to replace all or a part of surimi for the manufacture of fish protein gel products. This study reveals how to optimally mixed these two differently refined fish proteins based on their functional properties. The results suggested that blending surimi and FPI may be feasible only when the proportion of FPI does not exceed 50%.     

Effect of Various Types of Egg White on Characteristics and Gelation of Fish Myofibrillar Proteins

ABSTRACT:  Three types of egg white protein (regular dried egg white [REW], special dried egg white [SEW], and liquid egg white [LEW]) were compared for their effect in surimi (CON), containing no egg white (EW). They were characterized for enzyme inhibition and time of EW addition, either with cryoprotectants prior to freezing or during gel preparation, using Pacific whiting surimi. In addition, the setting (suwari) effect and fish protein–EW protein interactions (dynamic rheological properties, total sulfhydryl groups, and fracture gel analysis) were evaluated using Alaska pollock surimi. After 12 mo frozen storage, adding 2% and 3% SEW to Pacific whiting surimi during chopping significantly ( P  < 0.05) increased the force and deformation values compared to adding the respective EW before freezing. The highest ( P  < 0.05) force (175.2 g) and deformation (9.0 mm) values after 12 mo were obtained when 3% SEW was added during chopping. Enzyme autolysis showed that addition of 2% EW protein was effective at inhibiting enzyme activity. During setting, adding 2% SEW maintained force (1047.4 g) and deformation (17.9 mm) values similar ( P  > 0.05) to CON (1055.1 g; 19.3 mm) and significantly ( P  < 0.05) better than 2% REW (666.1 g; 15.6 mm). Texture results corresponded well to other data where 2% SEW showed the lowest total sulfhydryl groups (48.3 mole/10⁵ g), and higher elastic modulus (G'), which suggests improved protein interaction for gel formation. Overall, the addition of 2% to 3% SEW improved gel textural properties of Pacific whiting and Alaska pollock fish protein.