Glycoprotein derived from the hot water extract of mint plant, Perilla frutescens britton

Glycoprotein showing inhibitory activity against mast cell degranulation and hyaluronidase activity was purified from the hot water extract of mint plant (Perilla frutescens Britton). The purified inhibitor gave a single band detected with Coomassie brilliant blue staining and periodic acid-Schiff staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The molecular mass was estimated to be 6.0 kDa on SDS-PAGE. The inhibitor did not become inactivated when boiled for 30 min or digested with trypsin, V8 protease, or proteinase K but was inactivated by NaIO(4) oxidation. The inhibitor prevented mast cell degranulation and hyaluronidase activity (IC(50) = 0.42 mg/mL) in a dose-dependent manner. The inhibitor also inhibited the protein kinase C activity. It is possible to purify and characterize a glycoprotein with putative pharmacological properties from mint plants.     

Effect of low and high pH treatment on the functional properties of cod muscle proteins

The functional properties of cod myosin and washed cod mince (myofibrillar protein fraction) treated at high (11) and low (2.5) pH were investigated after pH readjustment to 7.5. The solubility of refolded myosin was essentially the same as the native myosin. The pH-treated myofibrillar proteins had increased solubility over the whole ionic strength range studied. Acid and alkali treatment gave myosin and myofibrillar proteins improved emulsification properties, which were correlated with an increase in surface hydrophobicity and surface/interfacial activity. Enhanced gel strength was observed with acid- and alkali-treated myosin compared to native myosin, while the same treatment did not significantly improve the gel strength of acid- and alkali-treated myofibrillar proteins. The acid- and alkali-treated protein samples unfolded and gelled at a lower temperature than did the native proteins, suggesting a less conformationally stable structure of the refolded proteins. Functional studies show that acid and alkali treatment, which leads to partial unfolding of myosin may improve functional properties of cod myosin and myofibrillar proteins, with the greatest improvement being from the alkali treatment. The results also show that improvements in functionality were directly linked to the extent of partial unfolding of myosin on acid and alkali unfolding and refolding.     

In vitro proteolysis of myofibrillar proteins from beef skeletal muscle by caspase-3 and caspase-6

The objective of the study was to investigate in vitro degradation of myofibrils by caspase-3 or -6. Myofibrillar proteins prepared from beef skeletal muscle were incubated with caspase-3 or -6 at 30 °C for 2 or 12 h, and subsequently, protein degradation was detected. Results showed that caspase-3 and -6 reproduced the degradation patterns of titin and nebulin observed during normal postmortem (PM) aging; however, they only reproduced the 28 kDa fragment derived from troponin-T. Caspase-3 induced only minor degradation of desmin. However, caspase-6 caused increasing degradation of desmin with extended incubation time and produced three degradation fragments (45, 29, and 27 kDa) of which only the 45 kDa fragment has been reported in aged beef. Therefore, caspase-3 or -6 could only reproduce a part of myofibrillar protein degradation or degradation fragments observed in naturally aged meat and may be involved in PM proteolysis of muscle proteins together with other endogenous proteases.     

Perspectives into factors limiting in vivo digestion of legume proteins: antinutritional compounds or storage proteins?

The in vivo protein digestibility of raw and cooked common bean (Phaseolus vulgaris L.) and faba bean (Vicia faba L.) and of protein fractions extracted from them was determined with growing rats. Overnight-fasted rats were intubated with a protein suspension or fed the same amount of protein added to a basal diet. The rats were killed 1 h later, the contents of stomach and small intestine were washed out, and their protein contents were measured. The in vivo digestibility of proteins of raw common bean flour was 72.4% and not significantly improved after cooking. In contrast, the digestibility of faba bean proteins was decreased from 86.5 to 60.6% by the thermal treatment. Globulins from either species had similar digestibilities (approximately 70%). Proteins in the soluble fraction of cooked beans were more digestible than those in the insoluble fraction, which contained the bulk of the proteins. Hemagglutination assay and trypsin inhibitor determination indicated that after the thermal treatment only very low, nonharmful, levels of both lectin and inhibitor remained. Faba bean contained more polyphenols than common bean samples, with most of the polyphenols being bound to globulins. However, protein-bound polyphenols were markedly decreased after cooking. SDS-PAGE characterization of the gastrointestinal digesta of globulins and amino acid analysis of undigested proteins of whole cooked common bean and faba bean suggested that it is mainly the structural properties of the storage proteins and not their binding of polyphenols, which determines the extent of protein aggregation on autoclaving and may therefore be responsible for their low digestibility.     

Thiol-quinone adduct formation in myofibrillar proteins detected by LC-MS

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol-quinone adducts (RS-QH(2)) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H(2)O(2). GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine-quinone adduct, Cys-QH(2) (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol-quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H(2)O(2). Direct reaction with 4MBQ resulted in each case in increased Cys-QH(2) formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H(2)O(2). The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.     

Are molecular weights of proteins determined by superose 12 column chromatography correct?

Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.     

蚕豆发芽过程中蛋白质和抗氧化能力的变化

为更好地开发利用蚕豆资源,研究蚕豆发芽过程中蛋白质及蛋白抗氧化能力的变化规律,采用凯氏定氮法测定发芽蚕豆的蛋白质含量,采用氨基酸分析仪测定氨基酸组分含量,运用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术观察发芽蚕豆蛋白组成的变化,通过测定发芽蚕豆蛋白的1,1-二苯基-2-三硝基苯肼(DPPH)自由基和2,2'-联氮双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)自由基的清除能力分析其抗氧化能力。结果表明,蚕豆发芽过程中蛋白和氨基酸含量升高,促进部分蚕豆蛋白由大分子量转化分解成小分子量,提高了蚕豆蛋白的抗氧化能力,在蚕豆发芽第9天时蛋白质含量达到34.1 g·100g^-1、氨基酸含量为29.16 g·100g^-1,均达到最大值,蚕豆蛋白的自由基清除能力达到最强。综上,蚕豆的发芽过程可以增加蚕豆的营养物质成分含量,增强蚕豆的抗氧化能力。本研究结果为蚕豆蛋白、蚕豆芽等功能食品的开发利用提供了参考依据。     

Determination of the N-glycosylation patterns of seed proteins: applications to determine the authenticity and substantial equivalence of genetically modified (GM) crops

Methods have been developed to determine the N-glycosylation pattern of proteins at the single-seed level in two different biological systems. These were the well-characterized and widely consumed storage protein phaseolin from several species of Phaseolus (bean) and the α-amylase inhibitor from the same Phaseolus species expressed transgenically in pea. The N-glycosylation pattern of the α-amylase inhibitor expressed transgenically in pea was different from that of the inhibitor present in common bean (P. vulgaris), the species of origin of the gene. However, multivariate analysis showed that the differences in N-glycan patterns between the α-amylase inhibitors from common bean and pea were less than those between the inhibitors from common bean and two related bean species, lima bean (Phaseolus lunatus) and tepary bean (Phaseolus acutifolius).     

Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening

The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS. A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E. coli transformant. As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein. Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography. The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa). The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins. The inhibitor could inhibit the gel softening of mackerel surimi.     

Hydrolytic action of Lactobacillus casei CRL 705 on pork muscle sarcoplasmic and myofibrillar proteins.

Lactobacillus casei CRL 705 was screened, among other meat isolates, for its proteinase and aminopeptidase activities toward synthetic substrates and, according to that, selected for specific assays on muscle proteins. The hydrolytic effects of whole cells, cell free extracts (CFE), and the combination of both on muscle sarcoplasmic and myofibrillar protein extracts was evaluated by SDS-PAGE and reverse phase HPLC analyses. The proteinase activity of whole cells caused the degradation of a great number of sarcoplasmic protein bands. A partial hydrolysis was also associated with CFE that when combined with whole cells showed an important additional degradation. Peptide profiles from sarcoplasmic protein extracts were greatly modified regardless of the addition of whole cells or CFE, although their combination intensified these changes. The generation of free amino acids was remarkable when whole cells and CFE were incorporated together to sarcoplasmic protein extracts.     

Heat-induced aggregation of Phaseolus vulgaris L. Proteins: an electron spin resonance study

The mechanism of heat-induced aggregation of Phaseolus vulgaris L. proteins and of subunit interactions of importance for susceptibility of proteins to proteolysis was studied by electron spin resonance (ESR) spectroscopy. The mobility of a spin label bound to lysine residues was monitored at two different pH-induced (neutral and alkaline) association states of proteins extracted from raw and cooked common bean. The molecular weight of the protein complexes was assessed by size exclusion-high performance liquid chromatography (SE-HPLC) of labeled proteins. Upon alkaline dissociation, both native and denatured protein subunits underwent a reassociation process to form soluble complexes of molecular weight higher than the species originally present at neutral pH. However, unlike native proteins, impaired mobility of the spin label was observed in the aggregates that are formed after dissociation of subunits of denatured proteins, indicating a reduced accessibility of lysine residues. Trapping of lysine residues inside protein aggregates may explain limited digestibility in the small intestine of proteins in cooked legumes.     

Partial isolation and characterization of a cysteine proteinase inhibitor from Lima bean (Phaseolus lunatus)

Lima beans (Phaseolus lunatus) have been shown to contain cysteine proteinase inhibitor (CPI) activity, but the CPI has not been isolated or characterized. Accordingly, our objective was to isolate and partially characterize a CPI from lima bean. The isolation scheme included water extraction of lima bean flour followed by a chromatography series using DEAE Sepharose, Phenyl Sepharose, hydroxyapatite, and reversed-phase high performance liquid chromatography. This scheme resulted in the partial purification of a approximately 20 000-dalton protein with high inhibitory activity against papain. This isolated lima bean CPI had an N-terminal sequence homologous with other members of the cystatin class of CPIs. The protein was relatively heat labile; suggesting it could be inactivated with normal cooking, which is favorable for its use in transforming plants to create insect resistance.     

Tyrosinase-aided protein cross-linking: effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase. Keywords: Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.     

Intense degradation of myosin light chain isoforms in Spanish dry-cured ham

One of the main biochemical changes that take place during the processing of dry-cured ham is the degradation of the muscle protein fraction, mainly due to the action of muscle enzymes. In the present study, the isolation and tentative identification of 137 fragments from myosin light chain 1 (MLC 1), together with 88 fragments originated from myosin light chain 2 (MLC 2), have been achieved for the first time in Spanish dry-cured ham, proving the intense proteolysis experienced by myofibrillar proteins after dry-cured processing. This study was carried out by use of proteomic technology for peptide identification, and the possible enzymes contributing to the degradation of these proteins were also further discussed.     

Study the N Turnover of Legume Seed Meals for Designing a Slow-Release Nitrogen Fertilizer

To mitigate environmental problems and synchronize releasing nitrogen (N) with crop demand, slow-release N fertilizers can be a solution. In this research, the mechanism of the N immobilization in stable sources (not unstable sources such as microbial biomass and extractable organic N) for finding an appropriate compound in designing a slow-release N fertilizer was investigated. The experiments were carried out in a randomized complete block design using an incubation chamber to study the N mineralization in coarse and fine fractions of yellow lupin, blue lupin, and faba bean. The results showed that the major N immobilization occurred at 10 to 17 days after incubation. At this phase, only the polyphenols had a significant correlation coefficient with the N immobilization (r = 0.80). At 17 to 31 and 31 to 61 days after incubation, the N immobilization had significant relationship with cellulose (r = 0.96) and hemicellulose (r = 0.89), respectively. It seems that with advancing incubation time, cellulose and hemicellulose were released from cell walls, and similarly to polyphenol were bound to nitrate N (NO₃^?-N), ammonium N (NH₄⁺-N), or extractable organic N through different interactions. Although the main mechanisms of N immobilization in soil after adding plant materials with a high carbon (C)/N ratio are described in the literature, the available data do not yet present an appropriate composition of targeted, innovative, and slow-release N fertilizers. According to the obtained results, tests are suggested to find the optimum nitrification inhibitor using the powder of plant residues with different ratios of these compounds incorporated with inorganic fertilizers.     

白菜型冬油菜‘陇油7号’叶片响应低温胁迫的差异蛋白鉴定与分析

为了从蛋白质组学的角度探讨超强抗寒品种白菜型冬油菜‘陇油7号’的抗寒机理,本研究采用TCA(三氯乙酸)-丙酮沉淀法,提取低温胁迫(4℃,7 d)前后的叶片总蛋白,对蛋白提取方法、IPG(固定pH梯度)胶条种类等环节进行了优化,运用双向电泳和质谱分析技术,鉴定了低温胁迫下‘陇油7号’5叶期叶片总蛋白质组分的表达差异模式。结果表明:改进后的蛋白质提取液(含DTT,二硫苏糖醇)和PVPP(聚乙烯吡咯烷酮)得到的蛋白质平均浓度较改进前高3.42μg·μL-1,除盐时间较改进前短1.14 h;同时,含蛋白酶抑制剂苯甲基磺酰氟(PMSF)的蛋白提取液获得的蛋白质种类丰富,凝胶图谱中可检测到661个蛋白点,较改进前可测蛋白点数(587)提高11.2。采用17 cm p H 4~7的IPG胶条的电泳能更好地分离蛋白,得到重复性好、分辨率高的蛋白质组图谱。利用PDQuest 8.0软件分析比较了超强抗寒品种‘陇油7号’低温胁迫前后的蛋白组表达谱,发现低温胁迫前后共有15个质变的蛋白质点,推测这些差异蛋白点可能与低温胁迫的响应有关。进一步对质变的蛋白质点进行了质谱分析,鉴定出11个与低温胁迫相关的蛋白质点,这些蛋白包括光合作用相关的蛋白、糖代谢相关的蛋白、物质运输相关的蛋白和逆境响应相关的蛋白。而且,低温胁迫处理前后,‘陇油7号’叶片蛋白质的表达水平存在明显差异,这些差异蛋白可能在冬油菜抗寒响应中发挥重要作用。     

Proteomic identification of actin-derived oligopeptides in dry-cured ham

An intense proteolysis of muscle proteins, mainly due to the action of endogenous proteolytic enzymes, has been reported to occur during the processing of dry-cured ham. This gives rise to an important generation of free amino acids and peptides of small size that contribute directly or indirectly to flavor characteristics of the final product. The nature and properties of the free amino acids generated during postmortem proteolysis have been well established. However, little is known about the identity of peptides generated during the processing of dry-cured ham. In the present paper, we describe the isolation (by ethanol precipitation followed by size exclusion and reverse phase chromatographies) and identification (by matrix-assisted laser desorption/ionization time-of-flight MS and collision-induced dissociation MS/MS) in a Spanish dry-cured ham of the following four oligopeptides: (A) TKQEYDEAGPSIVHR, (B) ITKQEYDEAGPSIVHRK, (C) DSGDGVTHNVPIYE, and (D) DSGDGVTHNVPIYEG. This is the first time that these peptide fragments have been reported in dry-cured ham at the end of processing. Sequence homology analysis revealed that the four peptides originated from muscle actin, indicating an extensive hydrolysis of this protein during dry-curing. Some of the cleavage sites corresponding to these fragments in actin were reproduced by other authors through the incubation of this myofibrillar protein in the presence of cathepsin D (EC 3.4.23.5), thus supporting a relevant action of this enzyme during the processing of dry-cured ham.     

Phaseolin in vitro pepsin digestibility: role of acids and phenolic compounds

Great Northern bean (Phaseolus vulgaris L.) phaseolin proteolysis at 37 degrees C, varying HCl concentrations (10 mM to 1 M), phaseolin:pepsin ratios ranging from 5:1 to 100:1 (w/w), and incubation times up to 24 h was investigated. The results suggest that phaseolin is not resistant to in vitro pepsin hydrolysis. At a phaseolin-to-pepsin ratio of 100:1 (w/w), native phaseolin was completely digested in 24 h when incubated in 50 mM HCl, while heat-denatured phaseolin (30 min at 100 degrees C, boiling water bath) was digested in 1 h under similar conditions. When incubated at 37 degrees C for 24 h, acid alone, even at as low a concentration as 10 mM, caused a partial breakdown of native phaseolin. The degree of phaseolin hydrolysis by HCl was dependent on the acid concentration used. The rate of native phaseolin hydrolysis increased with increasing HCl concentration rather than pepsin concentration. Common food acids were able to partially hydrolyze phaseolin. Among the food acids tested, oxalic acid was the most effective in hydrolyzing phaseolin. Spectroscopic studies revealed a significant change in secondary and tertiary structures when native phaseolin was incubated in dilute HCl. None of the tested phenolic compounds adversely affected phaseolin hydrolysis by pepsin.     

Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates

Sunflower protein isolates and the proteases pepsin and pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Hydrolysates obtained after 3 h of incubation with pepsin and 3 h with pancreatin were studied. An ACE inhibitory peptide with the sequence Phe-Val-Asn-Pro-Gln-Ala-Gly-Ser was obtained by G-50 gel filtration chromatography and high-performance liquid chromatography C18 reverse phase chromatography. This peptide corresponds to a fragment of helianthinin, the 11S globulin from sunflower seeds, which is the main storage protein in sunflower. These results show that sunflower seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with pepsin and pancreatin.