Post mortem muscle protein degradation during ice‐storage of Arctic (Pandalus borealis) and tropical (Penaeus japonicus and Penaeus monodon) shrimps: a comparative electrophoretic and immunological study

Water‐, low‐salt‐ and high‐salt‐soluble protein fractions from the abdominal muscles of Pandalus borealis, Penaeus japonicus and Penaeus monodon extracted immediately after death and after 5, 16, 24, 48, 72, 96 and 120 h (P borealis) or 16, 22, 43, 71 and 92 h (Penaeus spp) of ice‐storage were analysed by one‐ and two‐dimensional electrophoresis and immunological techniques. The most evident effect in P borealis was the decrease in the relative amount of myosin heavy chain (MHC) and a concomitant increase in the number and intensity of bands of molecular size about 100 kDa cross‐reacting with anti‐MHC antiserum. MHC degradation of P borealis was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) of partially isolated native myosin. Other prominent features were the disappearance of bands of about 67 and 50 kDa after 24 h and the appearance of a band of slightly less than 50 kDa after 5 h of ice‐storage. These last bands showed the potential to be used as freshness markers. One spot tentatively identified as desmin did not suffer significant changes in any of the three species. Two bands (about 100 and 96 kDa) gave a positive reaction with the α‐actinin antibody in the zero‐time extract of P borealis, but after 24 h only one faint 96 kDa band was detected. In contrast, the extracts of P japonicus and P monodon did not suffer significant alterations during the examined period, and even after 92 h of ice‐storage only the 100 kDa anti‐α‐actinin cross‐reacting band was clearly visible in the high‐salt extract of P japonicus. © 2001 Society of Chemical Industry     

Preparation and characterisation of isoflavone aglycone‐rich calcium‐binding soy protein hydrolysates

Isoflavone aglycone‐rich calcium‐binding soy protein hydrolysates were prepared by subcritical water treatment and subsequent protease hydrolysis. Contaminated β‐glucosidase in the Protease M preparation could effectively convert glycosides into aglycones. Compared with Alcalase hydrolysates, Protease M hydrolysates exhibited higher molecular weight (>5000 Da) and more hydrophobic characteristics because of its weaker proteolytic activity. The antioxidant activity of Protease M hydrolysates was obviously improved. Initial increased DPPH and ABTS radical scavenging rate of Protease M hydrolysates may be ascribed to the conversion of isoflavones (<30 min) and a gradual release of antioxidant peptides. In the later hydrolysis, a gradual exposure of isoflavones involved in the interior of heat‐induced protein aggregates was mainly responsible for further improved antioxidant activities. Higher calcium‐binding capacity (up to 7.86%) with lower yield of peptide–calcium complex was observed for Protease M hydrolysates. These results could help researchers to develop a feasible protocol for producing nutrient‐enhanced soy protein hydrolysates.     

Gel‐enhancing effect and protein cross‐ linking ability of tilapia sarcoplasmic proteins

BACKGROUND: Tilapia (Oreochromis niloticus) sarcoplasmic proteins contain substantial transglutaminase (TGase) activity. The enzyme catalyzes the protein cross‐linking reaction, resulting in a more elastic gel. The objective was to investigate the gel‐enhancing effect of sarcoplasmic proteins from tilapia as related to TGase activity. RESULTS: Total TGase activity of sarcoplasmic proteins concentrate (SpC) increased about 3.6‐fold after ultrafiltration using 30 kDa membrane, but specific activity remained unchanged, indicating minimal TGase purification by ultrafiltration. Addition of 1 mg mL^?1 SpC containing 40 units TGase activity induced cross‐linking of tilapia actomyosin, and the extent of cross‐linking increased with added level of SpC. Myosin heavy chain (MHC) and troponin were preferably cross‐linked by tilapia SpC, while actin and tropomyosin were not affected. Higher retention of MHC was observed concomitantly with greater content of cross‐linked protein when SpC was added to lizardfish surimi. Lizardfish surimi with 10 g kg^?1 SpC added and pre‐incubated at 37 °C for 1 h exhibited 91.6% and 26.7% increase in breaking force and deformation, respectively, when compared to the control. CONCLUSIONS: Residual TGase activity in SpC played an important role in catalyzing the protein cross‐linking and enhancing actomyosin gelation. SpC could be a potential ingredient for improving textural properties of fish protein gel. Copyright © 2007 Society of Chemical Industry     

过敏原肌质钙结合蛋白在毕赤酵母中的表达、优化及免疫反应性研究

目的 探究天然肌质钙结合蛋白(sarcoplasmic calcium binding protein, SCP)的可替代物,为蟹类过敏原的检测提供基础材料,本研究首次利用毕赤酵母(Pichia pastoris, P. pastoris)高效表达表达三疣梭子蟹(Portunus trituberculatus)重要过敏原SCP,并检验其免疫反应性。方法 根据毕赤酵母的密码子偏好性优化SCP基因并构建重组质粒。将其热激转化至P. pastoris GS115菌株后经遗传霉素(Geneticin, G418)筛选获得阳性高拷贝子。最后通过甲醇诱导表达重组SCP并结合免疫印记(Western blotting, WB)和间接酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)验证其免疫反应性。结果 SCP在P. pastoris GS115中实现了可溶性高效表达,其表观分子量约为28 kDa。在摇瓶水平下,最佳诱导条件为pH为6.0、每24 h添加1.0%(v/v)甲醇,于28℃发酵144 h,在此条件下,纯度为91.6%的SCP产量可达15 mg/L。WB和间接ELISA结果表明,重组SCP具有IgG结合能力。结论 毕赤酵母表达系统可以得到纯度较高且免疫反应性良好的重组SCP。本研究为SCP的理化研究及产业化应用奠定了基础,并有望促进特异性甲壳类过敏原检测的发展。     

Comparative study of in vitro digestibility of major allergen,tropomyosin and other proteins between Grass prawn (Penaeus monodon) and Pacific white shrimp (Litopenaeus vannamei)

BACKGROUND: Stability in simulated gastric fluid is supposed to be an important parameter for the estimation of food allergenicity. In the present study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from Grass prawn and Pacific white shrimp in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system was investigated and comparatively studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), western blotting, and inhibition enzyme‐linked immunosorbent assay (ELISA). RESULTS: In the SGF system, proteins such as actin and myosin heavy chain (MHC) were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In the SIF system, MHC was also easily decomposed, while TM and actin were resistant to digestion. Western blotting using a specific polyclonal antibody against TM indicated that the degradation pattern of shrimp TM by SGF and SIF was almost unaffected by the presence of other myofibrillar proteins. Further study by IgE immunoblotting and inhibition ELISA using sera from crustacean‐allergic patients indicated that IgE binding of TM was decreased. CONCLUSION: Proteinase digestion is effective in reducing IgE binding of shrimp TM. It is also of interest to notice that Pacific white shrimp TM had higher digestion stability than Grass prawn TM. However, Pacific white shrimp TM revealed enhanced IgE binding over that of TM from Grass prawn and thus it is possibly more allergenic. Copyright © 2010 Society of Chemical Industry     

In vitro proteolysis of myofibrillar and sarcoplasmic proteins of European sea bass (Dicentrarchus Labrax L) by an endogenous m‐calpain

The effects of m‐calpain isolated from the skeletal muscle of sea bass on sarcoplasmic and myofibrillar proteins isolated from the same tissue were examined in vitro. Incubation of sarcoplasmic proteins with m‐calpain resulted in only a slight decrease (0.7 kDa) in the molecular weight (MW) of a 26.5 kDa protein. Degradation of myofibrils, monitored by quantification of TCA‐soluble peptides generated, resulted in the maximum amount of peptides being generated after 1 h of incubation at 25 °C. Noticeable modifications in the SDS‐PAGE profile of digested myofibrils were observed, including partial denaturation of myosin heavy chain and the release of tropomyosin, ～69 and ～27 kDa doublet bands and a few polypeptides of MW lower than 20 kDa in the soluble fraction. Examination of the degradation patterns of myofibrillar proteins using Western blotting showed that α‐actinin was partially degraded, with release of native α‐actinin and its fragments from myofibrils, whereas desmin was highly degraded after 2 h of digestion. © 2002 Society of Chemical Industry     

Molecular cross‐talk between members of distinct families of selenium containing proteins

Dietary intake of selenium has been associated with reduced risk of several cancer types, and this is likely due to its role as a specific constituent of selenium containing proteins. One of these, selenium‐binding protein 1 (SBP1), is a protein of unknown function that has been shown to be reduced in tumors of diverse tissue types as compared to the corresponding normal tissue. More importantly, SBP1 has also been reported to be a predictor of clinical outcome. Levels of SBP1 are inversely associated with the levels of another protein representative of a different class of selenoproteins, glutathione peroxidase1 (GPx‐1). GPx‐1 is an anti‐oxidant, selenocysteine containing enzyme implicated in several diseases, including cancer, due to the association of specific alleles with disease risk. The relationship between SBP1 and GPx‐1 represents a unique example of a molecular interaction between selenium containing proteins with a likely significant impact on human health and disease.     

拟穴青蟹新型过敏原肌质钙结合蛋白的纯化、鉴定及分子克隆

为进一步认识蟹类的过敏原,采用免疫印迹方法,分析发现甲壳类过敏患者血清能与拟穴青蟹肌浆蛋白中分子质量约为21 kD的蛋白质产生特异的IgE结合反应,结果显示该蛋白可能是蟹类新型过敏原。通过硫酸铵盐析、阴离子交换和凝胶过滤柱层析等方法对21 kD-蛋白进行分离纯化,采用Western blotting和基质辅助激光解析电离飞行时间质谱（matrix assisted laser desorption ionization-time of flight-mass spectrometry,MALDI-TOF-MS）确认纯化的21 kD-蛋白为肌质钙结合蛋白（sarcoplasmic calcium binding protein,SCP）。采用SMART-RACE（SwitchingMechanism At RNA Termini-Rapid Amplification of cDNA Ends）的方法获得SCP的cDNA序列,该序列全长986 bp,开放阅读框为579 bp,编码193 个氨基酸,其理论分子质量21.94 kD,等电点4.44。拟穴青蟹SCP与甲壳类动物SCP具有较高的同源性,与昆虫SCP的同源性较差;三级结构模拟分析显示,SCP含有5 个螺旋-转角-螺旋结构区,即EF-手型结构区,并在其中两个手型结构区形成2 个钙离子结合位点;进一步预测得到SCP的4 个线性抗原表位和3 个构象性抗原表位。     

Angiotensin I‐converting enzyme inhibitory and Ca‐binding activities of peptides prepared from tuna cooking juice and spleen proteases

Tuna cooking juice is a by‐product from the tuna canning industry. In this study, tuna cooking juice was hydrolysed by proteases extracted from the spleen. Tuna cooking juice showed the highest ACE inhibitory and Ca‐binding activities after hydrolysis for 270 and 180 min, respectively. The hydrolysate was further fractionated by ultrafiltration. The permeate exhibited highest ACE inhibitory and Ca‐binding activities when passed through 1 and 5 kDa cut‐off membranes, respectively. Gel filtration chromatography was used to determine the MW of bioactive peptides that exhibited highest ACE inhibitory and Ca‐binding activities. Those peptides that exhibited highest ACE inhibitory and Ca‐binding activities were the MW range of 238–829 Da and 1355–1880 Da, respectively. These results suggest that the tuna cooking juice and the spleen protease extract are a potential source of bioactive peptides that can be utilised as bioactive ingredients in functional food and nutraceuticals.     

Molecular structure characteristics,functional parameters and in vitro protein digestion of pressure‐cooked soya bean flours with different amounts of water

When a food product is cooked at high temperature with different proportions of water, the differential degradation that occurs in the molecular structure promotes changes in their functional characteristics. In this study, water and soya bean flour (dry base) were mixed in different ratios (3:1, 2:1, 1:1, 0.25:1 and 0:1) and pressure cooked at 130 °C, 1.5 kg cm^?2 for 30 min, cooled down to 40 °C and air dried for 24 h. Protein changes due to thermal process were determined with the ATR‐FTIR, as well as some functional parameters and in vitro protein digestibility. At higher water:soya bean flour ratios (3:1 and 2:1), the protein digestibility increased due to denaturation of tertiary structures, while urease activity (UA) and the functional characteristics of water absorption index (WAI) and water solubility index (WSI) decreased. Pearson's correlation analysis revealed that molecular changes on amide I, II and in α‐helix: β‐sheet ratios were directly related with the amount of added water.     

Preparation and characterisation of selected physicochemical and functional properties of β‐chitosans from squid pen

Squid pen β‐chitosans prepared under various deacetylation conditions (30%, 35%, 40% and/or 45% NaOH for 15, 30 and/or 60 min) were characterised. β‐Chitosans (deacetylated with 35–45% NaOH for 15–60 min) had 87.1–96.2% degree of deacetylation (DD), 93.5–96.7% solubility and 120.5–654.9 mPa s viscosity. Treatment with 30% NaOH for 15–60 min yielded inadequately deacetylated β‐chitosans (DD = 51.9–80.2%). Two chitosans prepared under 35% NaOH for 15 min and 45% NaOH for 30 min (designated as 35%–15 and 45%–30, respectively) were further compared. Drying (sun‐drying vs. oven‐drying) methods did not affect DD. 35%–15 chitosan exhibited lower nitrogen, DD and bulk density, but higher viscosity compared with 45%–30 chitosan. Higher water‐ and fat‐binding capacity but lower DPPH radical scavenging activity were observed for 35%–15 chitosan compared with 45%–30 chitosan. Compared with 45%–30 chitosan, 35%–15 chitosan exhibited higher antibacterial activity against Salmonella Enteritidis and Listeria monocytogenes, but lower antibacterial activity against Escherichia coli.