Participation of cathepsin L in modori phenomenon in carp (Cyprinus carpio) surimi gel

Cathepsin L (Cat L) in carp (Cyprinus carpio) dorsal muscles was purified and its molecular weight determined by SDS polyacrylamide gel electrophoresis (SDS–PAGE) was 36 kDa. Its optimal temperature and pH were 50 °C and 5.5, respectively. The results of the effects of specific substrates, activators and inhibitors on the enzymatic activity showed that Cat L belongs to the family of cysteine proteinases containing thiol. Compared to the control, the gel strength of surimi with the addition of purified Cat L decreased significantly by 24.33% while that of surimi with both purified Cat L and inhibitors increased by 13.7% and 21.6%, respectively, suggesting the participation of Cat L in the modori phenomenon occurring in carp surimi. Both the SDS–PAGE electrophoretic pattern and microstructure figure revealed that Cat L could hydrolyse the main protein in carp surimi and was one of the enzymes involved in the modori phenomenon.     

Behead and live long or the tale of cathepsin L

In recent decades Saccharomyces cerevisiae has proven to be one of the most valuable model organisms of aging research. Pathways such as autophagy or the effect of substances like resveratrol and spermidine that prolong the replicative as well as chronological lifespan of cells were described for the first time in S. cerevisiae. In this study we describe the establishment of an aging reporter that allows a reliable and relative quick screening of substances and genes that have an impact on the replicative lifespan. A cDNA library of the flatworm Dugesia tigrina that can be immortalized by beheading was screened using this aging reporter. Of all the flatworm genes, only one could be identified that significantly increased the replicative lifespan of S.cerevisiae. This gene is the cysteine protease cathepsin L that was sequenced for the first time in this study. We were able to show that this protease has the capability to degrade such proteins as the yeast Sup35 protein or the human α‐synuclein protein in yeast cells that are both capable of forming cytosolic toxic aggregates. The degradation of these proteins by cathepsin L prevents the formation of these unfolded protein aggregates and this seems to be responsible for the increase in replicative lifespan.     

杂色鲍组织蛋白酶L的分离纯化与性质研究

通过硫酸铵分级沉淀、SP-Sepharose阳离子交换层析、Sephacryl S-200HR凝胶过滤和Hydroxyapatite羟基磷灰石层析,从杂色鲍（Haliotis diversicolor）内脏中分离纯化了组织蛋白酶L。SDS-PAGE结果显示,纯化蛋白的分子量约为28ku。该酶最适温度37℃,最适pH5.5。当温度低于40℃,pH在5.0～6.5范围内该酶较稳定。底物特异性实验与抑制剂实验结果表明,该酶属于半胱氨酸蛋白酶。金属离子Mn2＋、Ba2＋和Mg2＋对该酶有不同程度的激活作用,而Co2＋、Cu2＋、Zn2＋、Fe2＋和Ca2＋等对该酶起抑制作用。     

Investigations on the conversion of onion aroma precursors S‐alk(en)yl‐L‐cysteine sulphoxides in onion juice production

Onion flavour develops when the cells are disrupted, allowing the enzyme alliinase (EC 4.4.1.4) to act upon the aroma precursors S‐alk(en)yl‐L ‐cysteine sulphoxides (ACSOs). For maximum flavour release, optimal cell disintegration and complete enzymatic conversion of ACSOs are desired. To assess an industrial scale onion juice extraction in regard to the extent of cell disintegration and the rate of enzymatic ACSO conversion, samples from all mass flows (ie onion, macerate, juice and pomace) were subjected to ACSO analysis. Applying a modified HPLC method, which allows baseline separation of the aroma precursors, three ACSOs, namely methyl‐, propyl‐ and 1‐propenyl‐L ‐cysteine sulphoxides (MCSO, PCSO and 1‐PeCSO), were determined. Initial ACSO ratios changed in the course of increasing degree of comminution from 17:83 to 100:0 (MCSO/1‐PeCSO). Only negligible amounts of PCSO were detected (<9.2 nmol g^?1 fresh weight (fw)), contributing less than 0.2% to the total ACSO content. For evaluation of the disintegration efficiency a chart was established consisting of an abscissa representing a percentage scale of ruptured cells and ordinates for the development of the marker parameters ACSOs and reaction product pyruvate relative to their initial contents. From this correlation a cell disintegration percentage after grinding of about 70% was deduced according to the determined ACSO contents. On a molar basis, macerate, juice and pomace showed 15, 0.6–2.4 and 0.7–1.0% of initial ACSO contents respectively. Only trace amounts of 1‐PeCSO were found in the juice (<16 nmol g^?1 fw). MCSO was converted to almost 90%, indicating that enzymatic transformation, in a practical sense, goes to completion. Copyright © 2004 Society of Chemical Industry     

Restoration of the red colour of photo-bleached carthamine in calcium-alginate gel membranes

The restoration of the red colour of carthamine (photo-bleached in polymeric heterosaccharide gels) was investigated using sodium salts, potassium salts, and alkali. Ca-alginate gel was stained with carthamine, and the red-dish membranes were exposed to fluorescent light for 2·5, 4·0 and 20 h (at 28°C). The resulting orange-yellow gel membranes were treated with Na- or K-citrate solution and the restoration of the red colouration was followed spectrophoto-metrically. The results indicate that the red colour of carthamine can be retained within the gel membranes, while yellow-coloured compounds were removed and/or transformed by the alkaline salt solutions.     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

Effect of microwave heating on the low-salt gel from silver carp (Hypophthalmichthys molitrix) surimi

Gels from silver carp (Hypophthalmichthys molitrix) surimi were obtained using microwave (MW) heating (15 W/g power intensity for 20–80 s) at different levels of salt (0 g/100 g, 1 g/100 g, or 2 g/100 g). And the gel heated by MW was compared with the gel obtained by conventional water-bath heating (85 °C for 30 min). The gel strength increased when the salt level was increased. The mechanical and functional properties of non-salted, low-salt and regular-salt products were improved by MW heating for 60 s and 80 s, significantly (p < 0.05), except for the cook loss. The content of TCA-soluble peptides indicated that the MW heating inhibited the autolysis of proteins significantly (p < 0.05) during gelling. The SDS-PAGE and total content of –SH group proved that MW enhanced the cross-linking of proteins effectively through disulphide bonds and non-disulphide covalent bonds. The microstructure of the samples revealed that a fine compact network, with particles of protein aggregates, was formed in the low-salt gels (1 g/100 g) heated by MW for 60 s. All of these properties might be responsible for the formation of a superior textural low-salt gel induced by MW.     

A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.)

BACKGROUND: Proteases hydrolyse storage proteins to provide precursors for perpetuating species. The aim of this study was to investigate and characterise different proteases in germinating brown rice. RESULTS: The protease activity of brown rice increased sevenfold during 7 days of germination. It was highest on day 6 when determined at pH 3.5. With casein as substrate the proteases showed two catalytic groups: acidic proteases with an optimal pH of 3.5 and alkaline proteases with an optimal pH of 8.0. The acidic protease activity was inhibited by Ba²⁺ and Pb²⁺ but stimulated by Zn²⁺, while the alkaline protease activity was inhibited by Ca²⁺ and Pb²⁺ but stimulated by Mg²⁺ and Zn²⁺. SDS‐gelatin‐PAGE assay showed two protease activity bands at pH 3.5, while two different bands with higher molecular weights were observed at pH 8.0. Inhibition assay revealed that pepstatin A and E‐64 inhibited 67.63 and 38.26% respectively of the protease activity at pH 3.5, indicating the presence of aspartic and cysteine proteases. Metalloproteases played a major role under alkaline conditions (88.37% inhibition with EDTA). CONCLUSION: Germinated brown rice proteases fall into different classes with different properties. This study is helpful for their further purification. Copyright © 2010 Society of Chemical Industry     

Isolation of cathepsin B from the muscle of silver carp (Hypophthalmichthys molitrix) and comparison of cathepsins B and L actions on surimi gel softening

Cathepsin B from silver carp muscle was purified to 263-fold by acid treatment, ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 29 kDa as determined by SDS-PAGE and immunoblotting. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64, suggesting the purified enzyme belongs to the cysteine proteinase family. Optimal pH and temperature were 5.5 and 35 °C, respectively. The enzyme catalyzed the hydrolysis of Z-Arg-Arg-MCA with a parameter of K_m (90 μM) and K_cat (20.3 s⁻¹), but hardly hydrolyzed Arg-MCA. Analysis of surimi gel strength and microstructure showed that cathepsins B and L were capable of destroying the network structure of silver carp surimi gels, consequently causing gel softening. Cathepsin L might play an important role in the modori effect.     

Investigation of the activity of cathepsin B in red shrimp (Solenocera crassicornis) and its relation to the quality of muscle proteins during chilled and frozen storage

Cathepsin B is a cysteine protease that has important effects on the quality of muscle products. In this study, the changes of cathepsin B activity and its relation to muscle proteins were investigated in intact and beheaded shrimp during chilled and frozen storage. The obtained results indicated that the water holding capacity (WHC), shear force, hardness, and myofibrillar protein (MP) content all significantly decreased in both the intact and beheaded shrimp samples with increasing storage period (p < 0.05). Specifically, beheading shrimp exhibited much more stable characteristics than intact shrimp samples during both chilled and frozen storage. The enzyme activity results suggested that cold temperature and storage induced the release of cathepsin B from the lysosomes to the mitochondria, sarcoplasm, and myofibrils in the muscle tissues. Furthermore, SDS-PAGE and transmission electron microscopy (TEM) analysis revealed that beheading the shrimp greatly inhibited the dissociation of shrimp muscle proteins during storage. The current findings suggest that cathepsin B located in the head of shrimp was likely transferred to the muscle through the first abdominal segment during storage, accelerating the dissociation of the muscle proteins. Therefore, beheading the shrimp was conducive to prolonging the shelf-life of stored shrimp products.     

Stability of betacyanin from red pitahaya (Hylocereus polyrhizus) and its potential application as a natural colourant in milk

The effects of temperature (30, 50, 85 and 100 °C) treatment and 10 weeks of refrigerated storage (4 °C) on the betacyanin content and colour stability of betacyanins from red pitahaya (Hylocereus polyrhizus) were investigated and compared to red beet (Beta vulgaris; E‐162) which was used as a control. Temperature treatment at 50 and 85 °C caused a greater reduction in betacyanin content (BC) in E‐162 compared to red pitahaya by inducing changes in betacyanin profile. The 10 weeks of refrigerated storage at 4 °C induced colour changes in betacyanins from red pitahaya and E‐162. In milk, betacyanins from red pitahaya presented a lower loss of BC and total colour changes (ΔE*) compared to E‐162. The microbial onset in milk containing betacyanins from red pitahaya and E‐162 was delayed up to day 5 of refrigerated storage at 4 °C compared to day 3 in plain milk. Milk containing betacyanins from red pitahaya showed better colour acceptability (3.89 ± 1.89) compared to E‐162 (6.10 ± 1.71). Hence, betacyanins from red pitahaya may be a potential natural functional colourant to simulate strawberry colour in milk.     

Effect and synergy of different exogenous additives on gel properties of the mixed shrimp surimi (Antarctic krill and white shrimp)

The object of this study was a high-protein Antarctic krill ball which was made of mixed shrimp surimi (Antarctic krill and white shrimp). The effects of four different exogenous additives on gel properties and spatial structure of mixed shrimp surimi systems during processing were investigated by texture profile analysis, low-field nuclear magnetic resonance, magnetic resonance imaging, Fourier-transform infrared spectroscopy, scanning electron microscopy and rheology analysis, according to product formulation and processing conditions. Different exogenous additives improved distinctly the mixed shrimp surimi system. The use of the four exogenous additives combined had a synergistic promotional effect. Acetylated distarch adipate, egg white powder and soy protein isolate played a vital role in producing high-protein Antarctic krill balls. The relatively limited improvement of transglutaminase could be attributed to processing temperature and the presence of autolytic enzymes originated intrinsically from Antarctic krill surimi.     

Partial properties of an aspartic protease in bitter gourd (Momordica charantia L.) fruit and its activation by heating

Bitter gourd (BG fruit) is usually heated in hot water to reduce bitterness and improve flavour before being served. Protein extract from BG was analyzed for protease activity by gelatin-gel electrophoresis. The study showed that the proteolytic activity in BG flesh was enhanced by heat-treatment at temperatures ranging from 50 °C to 75 °C. An aspartic protease (AP) was characterized by gel electrophoresis. The optimal AP activity was at pH 7; the pI of the AP was demonstrated to be 4.8; the protein molecular weight of the BG–AP was estimated to be 60 KD by SDS–PAGE. The AP was implicated in the proteolysis of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase.     

Stability and antioxidative properties of acylated anthocyanins in three cultivars of red cabbage (Brassica oleracea L. var. capitata L. f. rubra)

BACKGROUND: Red cabbage is rich in acylated anthocyanins. The aim of the experiment has been to determine the stability of anthocyanins in extracts of red cabbage cultivars and examine their antioxidative properties. The (high‐performance liquid chromatographic) HPLC profile of anthocyanins characteristic of each red cabbage cultivar has also been determined. RESULTS: We have used three red cabbage cultivars: cv. Koda, Haco POL and Kissendrup SWE. The HPLC assays enabled us to distinguish nine anthocyanin compounds, which were all classified as cyanidin derivatives. These compounds occurred in different proportions in the three culitvars of red cabbage. The differences were observed in the degradation index of anthocyanins in extracts from the red cabbage cultivars examined, both stored at + 2 °C and frozen. The highest trolox equivalent antioxidant capacity (TEAC) was attained for anthocyanins from cv. Haco POL red cabbage, whereas the lowest value was determined for cv. Koda. CONCLUSION: There were differences in the stability and antioxidative properties of anthocyanin compounds in red cabbage extracts among the cultivars evaluated (Koda, Haco POL and Kissendrup SWE). The highest stability and strongest antioxidative properties were assigned to anthocyanins in extracts from cv. Haco POL. The HPLC profiles imply differences in proportions of anthocyanin compounds present in the three red cabbage cultivars. Copyright © 2009 Society of Chemical Industry     

Role of precursors on greening in crushed garlic (Allium sativum) bulbs, and its control with freeze-dried onion powder

BACKGROUND: Lachrymatory factor (LF) synthase in onion bulbs reacts with S‐1‐propenyl‐L ‐cysteine sulfoxide (1‐PeCSO), a key compound in garlic greening. In this study, freeze‐dried onion powder containing LF synthase was used in treatments to control garlic greening. Prior to the use of freeze‐dried onion powder to treat greening garlic bulbs, model reactions were conducted to confirm the reactivity of 1‐PeCSO in onion bulbs to garlic greening. RESULTS: While pink pigments were generated from 1‐PeCSO, green pigments were produced from the combination of 1‐PeCSO and S‐2‐propenyl‐L ‐cysteine sulfoxide (2‐PeCSO). However, pigments were formed in the systems containing 1‐PeCSO, amino acid and alliinase. Even non‐greening garlic bulbs stored at 20 °C turned green with the reaction of 200 g L^?1 1‐PeCSO; therefore 1‐PeCSO isolated from onion bulbs had the same role as 1‐PeCSO in garlic bulbs in terms of greening. Onion bulbs turned green after the addition of 600 g L^?1 2‐PeCSO. The addition of freeze‐dried onion powder inhibited garlic greening, and treatment with 15 g kg^?1 onion powder gave the best storage stability of crushed garlic bulbs. CONCLUSION: The addition of freeze‐dried onion powder inhibited the greening in crushed garlic bulbs, and treatment with 15 g kg^?1 onion powder gave the best storage stability of crushed garlic bulbs. Copyright © 2011 Society of Chemical Industry     

Effect of fermentation on the antioxidant activity of red beans (Phaseolus radiatus L. var. Aurea) ethanolic extract

The antioxidant activities of 50% ethanol extracts from red bean non-fermented and fermented by Bacillus subtilis or Aspergillus oryzae were determined using Sprague–Dawley rats as a testing model. Oral administration of all the extracts decreased the malondialdehyde (MDA) level in the liver but not in the brain tissue. Bacillus subtilis fermented extract increased the levels of ascorbic acid, α-tocopherol and glutathione (GSH) and the superoxide dismutase (SOD) activity in the liver tissue, while it increased only the ascorbic acid level in the brain tissue. Aspergillus oryzae fermented extract increased the levels of GSH and the SOD activity in the liver tissue, and it also increased GSH and ascorbic acid in the brain tissue. In general, the extracts from fermented red bean were more effective than the non-fermented extract in raising the antioxidant levels in the liver tissue.