Effect of heat shock protein 27 on the in vitro degradation of myofibrils by caspase‐3 and μ‐calpain

This study was designed to investigate the effect of heat shock protein 27 (HSP27) on the in vitro degradation of myofibrils induced by caspase‐3 or μ‐calpain. Myofibrillar proteins were prepared from at‐death beef muscles and incubated with caspase‐3 or μ‐calpain with and without HSP27, or with HSP27 alone, at 30 °C for 2 h, and protein degradation was assessed. Results showed that caspase‐3 promoted the degradation of titin, nebulin and troponin‐T, and μ‐calpain promoted the degradation of nebulin, desmin and troponin‐T, observed during normal PM ageing. Moreover, the addition of HSP27 restricted the degradation of troponin‐T in μ‐calpain‐ and caspase‐3‐treated myofilaments, and restricted the degradation of desmin in μ‐calpain‐treated myofilaments. Therefore, HSP27 may indirectly or directly interact with caspase‐3 and μ‐calpain, reducing their activity and mediating PM proteolysis of muscle proteins to affect meat tenderisation.     

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     

Preliminary studies on the effect of processing on the IgE reactivity of tomato products

Tomatoes and tomato products, obtained from a market, from companies or prepared in research laboratories, were tested in order to investigate how different processes could modulate the IgE reactivity of these products. The protein fraction of the samples was extracted by using a low‐temperature method in order to avoid degradation, and then separated on SDS PAGE to evaluate the protein profile. All the separated samples were blotted onto nitrocellulose membranes and then individually tested with pooled human sera that had been obtained from patients allergic to tomato, in order to evaluate the potential IgE binding of the protein bands. Heat treatments conventionally employed for the production of tomato‐based products were found to strongly degrade proteins thus dramatically reducing their IgE reactivity; characteristic protein profiles were found for specific processes such as hot and cold break. Ultra‐high‐pressure treatments were found to generally preserve the integrity of tomato proteins and this was reflected by the unaltered IgE reactivity observed. Copyright © 2007 Society of Chemical Industry     

Influence of gender and spawning on thermal stability and proteolytic degradation of proteins in Australian red claw crayfish (Cherax quadricarinatus) muscle stored at 2 °C

Thermal stability and proteolytic degradation of male (M), nonspawning female (F) and spawning female (SF) red claw crayfish (Cherax quadricarinatus) muscle proteins during refrigerated storage (2 °C) were investigated. The thermal transition temperatures (T_max) of myosin and actin remained relatively constant during storage, but their enthalpies of denaturation (ΔH) increased, especially in SF samples. SF muscle proteins were more heat‐stable (greater T_max and ΔH values, P < 0.05) than M and F muscle proteins. Protein degradation occurred in all muscle groups, more rapidly in M and F muscles than in SF muscle. The diminishments of a 69‐kDa component and troponin‐I and the appearance of a 55‐kDa polypeptide represented the most salient proteolytic changes. The results suggested that the spawning status was a more significant factor than gender in affecting the quality of red claw muscle proteins and their changes during refrigerated storage.     

Barley Grain Non‐specific Lipid‐Transfer Proteins (ns‐LTPs) in Beer Production and Quality

Plant non‐specific lipid‐transfer proteins (ns‐LTPs) are known for their ability to transfer various lipids between membranes in vitro. These ubiquitous basic proteins, that all share alpha structure stabilized by four disulphide bridges, are characterized by the presence of a hydrophobic cavity able to accommodate lipid molecules. According to molecular mass, this multigene family is subdivided into two subfamilies, ns‐LTP1 (9 kDa) and ns‐LTP2 (7 kDa); both located in the aleurone layer of the cereal grain endosperm. Ns‐LTP1 is a prominent protein in barley grain, malt and beer. Numerous studies performed on its structure and function confirm its important role in grain protection, as well as brewing technology. As the major beer protein crucial for many aspects of brewing, ns‐LTP1 can affect beer production and quality. Comparatively, there is less data available on the less abundant ns‐LTP2. In this review, the focus is on recent progress on the structure, biological and technological function of barley grain ns‐LTP1 and ns‐LTP2, with an emphasis on their importance in brewing.     

Electrophoresis to determine the molecular weight distribution in soluble proteins from various foods and their rumen‐resistant residues in cattle

Electrophoresis was used to visually identify and determine the molecular weight (MW) distribution of rumen‐degradable and rumen‐resistant or escape peptides in soluble proteins from 12 agricultural and distillers' food raw materials (RM) and their residues (RU) following 18 h of in sacco rumen incubation (dg₁₈) in cattle. Soluble proteins were extracted by using water, salt, acid and alkali in succession to represent albumins, globulins, glutalin 1 and glutalin 2 respectively. RM and RU differed substantially in the MW range, number and intensity of bands for various soluble proteins. The bands were mostly below the MW range of 66 kDa. Low‐MW (<25 kDa) peptides were greater in number than high‐MW (>25 kDa) peptides in almost all soluble proteins from RM. Individual peptides behaved differently during rumen incubation. Their resistance to or escape from rumen degradation varied with the class of food, type of soluble protein and their MW range. On average, low‐MW albumins in agricultural foods were more resistant to rumen degradation (0.41 RM vs 0.12 RU; 29%) than their high‐MW counterparts (0.12 RM vs 0.02 RU; 21%). In contrast, high‐MW glutalin 1 was more resistant (0.03 RM vs 0.22 RU) than low‐MW glutalin 1 (0.09 RM vs 0.26 RU) in most agricultural foods. Globulins contained the least and glutalins the most resistant peptides in distillers' foods. While this study reveals an association between dg₁₈ and protein type, structure and size, we do not recommend the immediate use of electrophoresis for routine food evaluation unless more studies are undertaken. It may, however, be suitable for further characterisation of the degradation of specific, selected peptides by specific micro‐organisms. © 2001 Society of Chemical Industry     

Expression of HIV-1 nef in yeast: The 27 kDa nef protein is myristylated and fractionates with the nucleus

The nef gene of human immunodeficiency virus type 1 (HIV-1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of M_r 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV-1 antisera. Under low-level steady-state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the nucleus.     

LeMAPK4 participated in cold‐induced ethylene production in tomato fruit

BACKGROUND: Mitogen‐activated protein kinase (MAPK, EC 2.7.11.24) cascade from several plant species has been shown to be activated during response to abiotic stress. Ethylene plays an important role in fruit tolerance to environmental stress. However, the mechanisms by which MAPK regulates defence systems in fruit and the relationship between MAPK and ethylene remain to be determined. RESULTS: MAPK inhibitor significantly decreased the chilling tolerance of tomato (Lycopersicon esculentum cv. Lichun) fruit during cold storage. Moreover, decreases in ethylene content, LeACS2 expression and activities of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and ACC oxidase (ACO, EC 1.14.17.4) due to MAPK inhibitor occurred within 24 h after cold treatment. Upon treatment with cold and ethephon, the ethylene content, activities of ACS and ACO and expression of LeACS2, LeACO1 and LeMAPK4 increased. CONCLUSION: The results showed the regulation of MAPK in ethylene biosynthesis to protect tomato fruit from cold stress. In addition, the participation of LeMAPK4 in cold‐induced ethylene biosynthesis in tomato fruit was indicated. © 2013 Society of Chemical Industry     

Evolution of cacao bean proteins during fermentation: a study by two‐dimensional electrophoresis

The protein content of cacao (Theobroma cacao) beans was studied by quantitative two‐dimensional electrophoresis (2‐DE) and by measuring total and protein nitrogen by the Kjeldahl method from the unfermented stage up to the seventh day of fermentation. The major trends in evolution of protein concentration were followed by measuring the intensities of some of the most abundant bean polypeptides. During fermentation a biphasic proteolytic process was observed. Protein degradation was detected after two days of fermentation, and was most pronounced during the third day. Following the initial phase of degradation until the end of fermentation, very little further protein degradation was observed, possibly due to the release of polyphenolic compounds and their subsequent complexing with the remaining proteins. Total protein estimated by the Kjeldahl method decreased to 57% of the initial value during the fermentation period. The process of degradation is selective, with some polypeptides resisting more than others. The evolution of total protein and non‐protein nitrogen content is also described. © 1999 Society of Chemical Industry     

What makes protein indigestible from tissue‐related,cellular, and molecular aspects?

This paper gives an insight into key factors, which impair enzymatic protein digestion. By nature, some proteins in raw products are already poorly digestible because of structural peculiarities, or due to their occurrence in plant cytoplasmic organelles or in cell membranes. In plant‐based protein, molecular and structural changes can be induced by genetic engineering, even if protein is not a target compound class of the genetic modification. Other proteins only become difficult to digest due to changes that occur during the processing of proteinaceous products, such as extruding, boiling, or acidic or alkaline treatment. The utilization of proteinaceous raw materials in industrial fermentations can also have negative impacts on protein digestibility, when reused as fermentation by‐products for animal nutrition, such as brewers’ grains. After consumption, protein digestion can be impeded in the intestine by the presence of antinutritional factors, which are ingested together with the food or feedstuff. It is concluded that the encircling matrix, but also molecular, chemical, and structural peculiarities or modifications to amino acids and proteins obstruct protein digestion by common proteolytic enzymes in humans and animals.     

Proteome analysis of Pithecellobium dulce seeds using two‐dimensional gel electrophoresis and tandem mass spectrometry

BACKGROUND: Pithecellobium dulce Benth. belongs to the Leguminosae family, which contains several members that are important components of human diets owing to their high protein content and quality. In this study the seed proteins from P. dulce were separated and identified using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry respectively. RESULTS: The 2‐DE protein map revealed a total of 317 distinct protein spots, including a cluster of about 12 proteins located in the region of pI 5–6 with molecular masses of 55–97 kDa that accounted for more than 50% of the total proteins. Ninety‐six of the most abundant protein spots were analysed using nano liquid chromatography/tandem mass spectrometry (LC/MS/MS), from which 27 were successfully identified through the query of acquired tandem mass spectral data used in MASCOT searching against a custom legume protein database. A further four proteins from the highly abundant protein cluster were putatively identified using mass spectrometry‐driven BLAST (MS‐BLAST) homology searches. CONCLUSION: This research has generated a 2‐DE proteome reference map for P. dulce seeds and used LC/MS/MS to characterise the proteins. The identification of proteins from P. dulce was carried out using the sequence database successively for MASCOT and MS‐BLAST homology‐based searches. Copyright © 2009 Society of Chemical Industry     

Purification and characterisation of relevant natural and recombinant apple allergens

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purification.     

“Potential Health Benefits of Lunasin: A Multifaceted Soy‐Derived Bioactive Peptide”

Bioactive peptides are small protein fragments derived from enzymatic hydrolysis of food proteins, fermentation with proteolytic starter cultures, and gastrointestinal digestion. These peptides have positive impacts on a number of physiological functions in living beings. Lunasin, a soy‐derived bioactive peptide, is one of the most promising among them. Lunasin encoded within 2S albumin (GM2S‐1) gene, identified as a novel peptide extracted from soybean seed. It is composed of 43 amino acid residues with a molecular weight of 5.5 kDa. Extensive scientific studies have shown that lunasin possesses inherent antioxidative, anti‐inflammatory, anticancerous properties and could also play a vital role in regulating of cholesterol biosynthesis in the body. Its high bioavailability and heat stable nature allow its potential use as dietary supplement. The present review summarizes some of the potential health and therapeutic benefits of lunasin reported hitherto.     

Expression analysis of endo‐1,4‐β‐glucanase genes during aril breakdown of harvested longan fruit

BACKGROUND: The aim of the present study was to investigate the expression profiles of three endo‐1,4‐β‐glucanase (EGase, EC 3.2.1.4) genes during aril breakdown of longan fruit stored at room temperature (25 °C), low temperature (10 °C) or on transferring fruit stored at 10 °C for 20 days to 25 °C. RESULTS: Three longan full‐length cDNAs, designated Dl‐EGase1, Dl‐EGase2 and Dl‐EGase3, were isolated and characterized. EGase activity in aril tissues of longan fruit increased with the appearance of aril breakdown symptoms, while RNA gel blot analysis revealed that the accumulations of three Dl‐EGase genes exhibited differential characteristics with the occurrence of aril breakdown. Dl‐EGase2 may be involved in the aril breakdown in longan fruit at the later stage of storage at room temperature. Conversely, expression of Dl‐EGase3 could be mainly involved in aril breakdown of fruit stored at 10 °C. In addition, Dl‐EGase3 and Dl‐Egase2 were related to the aril breakdown of fruit transferred from low temperature to room temperature. CONCLUSION: The results obtained in this study indicate that Dl‐EGase genes are involved in the aril breakdown of longan fruit and that considerable variation exists between expression patterns of individual members of the EGase gene family. Copyright © 2009 Society of Chemical Industry     

Comparative post‐harvest behaviour of traditional and virus‐resistant Muchamiel tomatoes

BACKGROUND: Nowadays, organoleptic quality is the primary objective for almost all tomato breeding programmes. In this study, post‐harvest behaviour of a breeding line with genetic resistance to important viruses (tomato mosaic virus, tomato spotted wilt virus and tomato yellow leaf curl virus) has been compared with the original traditional landrace (Muchamiel). The breeding line has been obtained by backcrossing, introgressing three resistance genes but aiming to keep the quality characteristics of the traditional variety. Tomatoes were picked at random and stored at 10 °C for 13 days. Quality analyses were made in both tomato samples: weight loss, colour, respiration rate, ethylene production, maturity index, instrumental hardness and sensory evaluation with trained panel. RESULTS: Fruits of the breeding line were characterized by higher hardness even with a higher maturity index. Results of sensory tests were in agreement with instrumental measurements. Organoleptic quality of Muchamiel virus‐resistant tomatoes was at least as high as that of traditional tomatoes, reaching the best scores in odour and aroma at the 13th storage day. CONCLUSION: Although a long time has been required to develop the breeding line, results indicate that organoleptic fruit quality has been recovered through backcrossing, confirming the success of the breeding programme. Copyright © 2010 Society of Chemical Industry     

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two‐dimensional zymography and direct identification of zymographic spots by mass spectrometry

BACKGROUND: Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow‐up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes. RESULTS: Kiwi crude extracts were analysed by two‐dimensional zymography on gelatin‐containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI‐ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. CONCLUSIONS: The innovative achievements of the present study are the: (1) two‐dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI‐ToF MS analysis of the zymographic spots. Copyright © 2010 Society of Chemical Industry     

Starch Granule‐Associated Proteins and Polypeptides: A Review

This paper reviews current information regarding the (non‐gluten) proteins which are naturally located in and on starch granules, and which are termed starch granule associated proteins (SGAPs). At least ten major SGAPs can be extracted from most starches and have molecular weights in the range of approximately 5 to 149 kDa. A substantial number of these proteins are located at the starch granule surface, where their presence in association with that of other minor granule components (such as lipids), appears to significantly influence the overall properties of both starch granules and starchy products. The different extraction conditions which can be exploited to selectively remove SGAPs are detailed. Although some SGAPs may serve as supplementary storage proteins, the majority of SGAPs are believed to be starch biosynthetic enzymes, a proportion of which remain trapped within the growing granule structure. The identity and quantities of the biosynthetic enzymes varies with botanical source, genetic variety and even with time. Current knowledge regarding the identities, compositions, locations, properties and functions of several of the more common SGAPs (e.g. the ∼15 kDa friabilin/puroindoline proteins, the ∼30 kDa protein, and the ∼60 kDa starch granule‐bound starch synthase (SGBSS) protein) are reviewed in detail.     

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     

Purification and partial characterization of xyloglucan‐hydrolyzing enzymes from watermelon placental tissue

BACKGROUND: Watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai) fruit matrix glycans are comprised largely of xyloglucans (XGs). As in other fruits, these polymers show significant molecular mass downshifts during ripening. In the present study, we describe the purification and characteristics of a number of xyloglucanases (XGases) from the placental tissue of ripe watermelon. XGases were extracted from watermelon fruit placental tissue and purified by sequential ion‐exchange, gel‐permeation and concanavalin A chromatography. RESULTS: Five XGases (P1S2, P2S2, P3S1, P3S2, P3S3) were recovered with molecular masses ranging from 30.5 to 77.5 kDa on SDS‐PAGE. All XGases showed maximum activity at pH 5–5.5 and 35–40 °C against tamarind seed XG and were also active against XG‐rich matrix glycans from watermelon fruit. The enzymes were strongly inhibited by mercury and hydrolyzed XG without generation of oligomers or monomers. P3S3 had the highest activity against XG. The purified enzymes were active toward carboxymethylcellulose, indicating that they were not XG specific. CONCLUSION: The pattern of molecular mass downshifts during XG hydrolysis by the purified XGases and the absence of monomeric and oligomeric products are consistent with endo‐type catalysis for the XGases and with a role for these enzymes in the degradation of cell wall XG during ethylene‐induced watersoaking of watermelon fruit placental tissues. Copyright © 2009 Society of Chemical Industry     

Chemical,antioxidant and sensory properties of tomato‐watermelon‐pineapple blends,and changes in their total antioxidant capacity during storage

A conductometric analysis of the effect of condensate of peroxides generated during lipid oxidation in an accelerated stability test was adapted to test the hypothesis that total antioxidant capacity of tomato products would sometimes increase during processing and in storage. Tomato pulp blends made from a mixture of tomato (Lycopersicon esculentum var Roma VF), watermelon (Citrullus vulgaris var Babylack) and pineapple (Ananas comosus var Smooth cayennes) were analysed for basic quality profiles of dry matter, Brix, titratable acidity (TTA) and pH, total reducing sugars, Component antioxidants phytochemicals and total antioxidant capacity. The lowest sensory score (overall impression) of 4.80 ± 2.59 was recorded for tomato juice while, blend TWP 111p had highest score of 6.20 ± 1.99. There was significant difference (P < 0.05) in the basic quality profile of the pulp blends except for TTA values (0.37 ± 0.02 to 0.45 ± 0.05) and 2‐Furfurals (2.47 ± 0.03 to 2.71 ± 0.01). The fresh blend of 50% tomato, 25% watermelon and 25% pineapple had the highest total antioxidant capacity of 3.69 ± 0.52 mg 100 mL^?1 catechin equivalent. The total antioxidant capacity of the stored pulp increased from 2.95 ± 0.13 to 6.22 ± 0.32 mg 100 mL^?1 catechin equivalent in pasteurised TWP 211p blend by 60 days when stored at 40 °C. Total antioxidant status of tomato‐based fruit mix increased during the first 80 days.