Functional properties of oat globulin modified by a calcium-independent microbial transglutaminase

Oat globulin was modified by a calcium-independent microbial transglutaminase (TG). The TG-polymerized protein had higher solubility than the control at acidic pH and had improved water- and fat-binding properties. Incubation of 10% (w/v) oat globulin dispersions in the presence of TG at 37 degrees C led to the formation of a well-developed viscoelastic gel network with a microstructure characterized by thick strands and large clusters. The TG-induced gels had higher modulus values, lower loss tangent values, and lower frequency dependency than the heat-induced gels. The TG-induced gel system has the characteristics of classical polymer gel with permanent "chemical" cross-links, whereas the heat-denatured system has the characteristics of a temporary "physical" gel with breakable cross-links. Fourier transform infrared spectroscopy showed marked shift and intensity changes in several major bands, suggesting pronounced changes in protein conformation during TG-induced gelation. Aggregation of protein molecules was also indicated by the progressive increases in two infrared bands (1679-1682 and 1622-1625 cm(-)(1)) associated with the formation of intermolecular beta-sheets and strands. Results suggest that new food polymers with unique functionality can be produced from oat globulin treated with TG and that elastic gels can be formed near neutral pH, instead of the alkaline pH required for thermally induced oat globulin gels.     

Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein

Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.     

Effects of microfluidization treatment and transglutaminase cross-linking on physicochemical, functional, and conformational properties of peanut protein isolate

Peanut protein isolate (PPI) was treated by high-pressure microfluidization (40, 80, 120, and 160 MPa) and/or transglutaminase (TGase) cross-linking. It was found that individual microfluidization at 120 MPa was more effective in improving the solubility, emulsifying properties, and surface hydrophobicity of PPI than at other pressures (e.g., 40, 80, or 160 MPa). Individual TGase cross-linking also effectively changed the physicochemical and functional properties of PPI. Microfluidization (120 MPa) or TGase cross-linking caused the unfolding of PPI structure, resulting in the decrease of α-helix and β-turns levels and the increase of β-sheet and random coil levels, as proved by Fourier transform infrared (FTIR) and circular dichroism (CD) spectra. Compared with individual treatments, microfluidization followed by TGase cross-linking significantly (p < 0.05) improved the emulsion stability during long-term storage (20 days). Moreover, the combined treatments led to looser structure of PPI and resulted in more obvious changes in physicochemical properties.     

Physicochemical and structural properties of oat globulin polymers formed by a microbial transglutaminase

Oat globulin was polymerized by a microbial transglutaminase (TG), and some physicochemical and functional properties of polymers were studied. Reversed-phase HPLC revealed that the number of epsilon-(gamma-glutamyl) lysine isopeptide bonds formed after 4 h of enzyme incubation was 2.21 micromol/g of protein. SDS-PAGE showed that the oat globulin acidic polypeptides (AP) were more susceptible to polymerization than the basic polypeptides (BP), and the reactivities of both AP and BP were enhanced by the addition of other substrate proteins. Differential scanning calorimetry showed that both the denaturation temperature and denaturation enthalpy were decreased after TG treatment. Fourier transform infrared spectroscopy revealed marked increases in the intensity of two intermolecular beta-sheet bands associated with aggregate formation but little conformational changes in the polymerized protein. TG incubation led to progressive changes in flow properties of oat globulin dispersions, indicating enhanced pseudoplasticity and increased viscosity and yield stress.     

Modification of the rheological properties of whey protein isolate through the use of an immobilized microbial transglutaminase

A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using <10 mg of the same enzyme, illustrating the capacity of immobilized enzymes to be used more frequently in applications of this nature.     

Biochemical changes in myofibrillar protein isolates exposed to three oxidizing systems

The objective of the study was to compare three different oxidizing systems commonly present in muscle foods for their influence on the biochemical properties of muscle proteins. Myofibrillar protein isolate (MPI) prepared from pork serratus ventralis muscle was suspended (30 mg protein/mL) in 15 mM piperazine-N,N-bis(2-ethane sulfonic acid) buffer (pH 6.0). Oxidation was induced by incubating the protein suspension at 4 degrees C for 24 h with (i) a hydroxyl radical-generating system (HRGS: 10 microM FeCl3, 0.1 mM ascorbic acid, and 0.05-5.0 mM H2O2), (ii) a lipid-oxidizing system (LOS: 0.05-5.0 mM linoleic acid and 3750 units of lipoxidase/mL), or (iii) a metmyoglobin-oxidizing system (MOS: 0.05-0.5 mM metmyoglobin). Changes in oxidized MPI were measured as Ca- and K-ATPase activities, formation of protein carbonyls and 2-thiobarbituric acid-reactive substances (TBARS), loss of protein thermal stability, and protein aggregation. The three oxidizing matrixes induced complex MPI changes; for example, the Ca- and K-ATPase activities were altered mainly by low-concentration oxidants, but the changes were unique for each oxidizing system. The carbonyl content in MOS-treated MPI was the highest, while the TBARS production, changes in thermal properties, and loss of the myosin heavy chain were the greatest in HRGS-treated MPIs. Overall, the hydroxyl radical-producing medium appeared to be the most oxidative to myofibrillar proteins under the experimental conditions employed in the study.     

Identification of the epsilon-(gamma-glutamyl)lysine cross-linking sites in alpha-lactalbumin polymerized by mammalian and microbial transglutaminases

To investigate the site specificity of two transglutaminases (TGases), that is, the enzymes from guinea pig liver (GTGase) and Streptoverticillium (MTGase), the acyl acceptor and donor sites in alpha-lactalbumin were determined. Alpha-lactalbumin was cross-linked in the presence of dithiothreitol by GTGase and MTGase for 15 and 30 min, respectively. Cross-linked alpha-lactalbumins by GTGase and MTGase were digested with lysylendopeptidase followed by the separation of the resulting peptides using reverse-phase HPLC. By the sequence analysis of the peptide fragments containing two N termini, which indicates the presence of cross-linked peptide, the lysine residues targeted by TGases were identified as follows: for GTGase, Lys16, Lys93, and Lys122; for MTGase, Lys5. These peptide fragments were further digested by V8 protease. Separation and sequence analyses of the resultant peptides were performed to identify glutamine residue involved in cross-linking. It was found that Gln54 was cross-linked to lysine residues by GTGase and MTGase in common. It is suggested that the difference in the numbers of lysine residues targeted by GTGase and MTGase may be responsible for the difference in the polymerization process of alpha-lactalbumin between GTGase- and MTGase-catalyzed systems.     

Modification of glutamine and lysine residues in holo and apo alpha-lactalbumin with microbial transglutaminase

The molecular structures determine the physical properties of milk proteins and are important for the texture of many dairy-based foods. Bovine alpha-lactalbumin (alpha-LA) is a globular 123 amino acid Ca(2+) binding milk protein. Modification with microbial Ca(2+) independent transglutaminase (MTGase) was used to modify lysines and glutamines in holo and apo alpha-LA. At 30 degrees C no lysines or glutamines are modified in holo alpha-LA, whereas in apo alpha-LA lysines 13, 16, 108, and 114, and glutamines 39 and 43, are modified. At 50 degrees C lysines 13, 16, 108, and 114, but no glutamines, are modified in holo alpha-LA, whereas in apo alpha-LA lysines 5, 13, 16, 108, and 114, and glutamines 39, 43, 54, 65, and 117, are modified. The methods presented here offer the possibility to manipulate the availabilities of residues in alpha-LA to the MTGase reaction and enable the preparation of alpha-LA species with different degrees of modification and hence with different physical properties.     

羟自由基导致肉类肌原纤维蛋白氧化和凝胶性降低

为研究羟自由基（·OH）氧化体系中肌原纤维蛋白（myofibrillar protein,MP）氧化及其凝胶特性的变化,试验分析了羟自由基氧化体系中不同H2O2浓度对蛋白氧化程度及MP凝胶白度、持水力、质构特性（texture profiles analysis,TPA）与弹性模量等特征指标的影响。结果表明：随H2O2浓度的增加,MP中羰基值上升,蛋白氧化程度加剧,凝胶白度、保水性、硬度、咀嚼性及弹性模量则与H2O2浓度呈显著负相关。与对照组相比,当H2O2浓度增加至20 mmol/L时,羰基含量增加至2.82 nmol/mg蛋白（p＜0.05）,凝胶白度、持水性及硬度则分别下降了2.83%、14.65%及52.77%（p＜0.05）。扫描电镜（scanning electron micrograph,SEM）观察表明,MP氧化导致凝胶微观结构破坏,形成空隙较大且分布不均的网络;低场核磁共振分析（nuclear magnetic resonance,NMR）结果显示,随 H2O2浓度的增加,MP 凝胶中的一部分不易流动水“态变”为自由水,凝胶持水力降低。综上所述,·OH氧化体系中肌原纤维蛋白氧化会影响其凝胶形成,破坏蛋白凝胶结构,降低凝胶功能,这为肉类生产加工过程中蛋白氧化控制提供理论参考。     

Stability and emulsion-forming ability of water-soluble fish myofibrillar protein prepared by conjugation with alginate oligosaccharide

Carp myofibrillar protein (Mf) was conjugated with alginate oligosaccharide (AO) through the Maillard reaction under low relative humidity, and the functional properties of the Mf-AO conjugate were investigated under different NaCl concentrations and pH levels. Mf became highly solubilized at lower NaCl concentrations by conjugation with AO, with a slight loss of available lysine. The thermal stability of Mf was effectively improved by conjugation with AO. Heat treatment at 80 degrees C for 2 h had no effect on the solubility of the Mf-AO conjugate attached to 227 microg/mg of AO regardless of the NaCl concentration and pH. Furthermore, the Mf-AO conjugate showed excellent emulsion-forming ability regardless of NaCl concentration. The improved functionalities of Mf by conjugation with AO remained even at a nearly isoelectric point. These results indicate that conjugation with AO through the Maillard reaction is an effective way to prepare high-functional food material from fish muscle protein.     

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.     

Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions

Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.     

Effect of interfacial protein cross-linking on the in vitro digestibility of emulsified corn oil by pancreatic lipase

The objective of this study was to investigate the influence of globular protein interfacial cross-linking on the in vitro digestibility of emulsified lipids by pancreatic lipase. 3% (wt/wt) corn oil-in-water emulsions stabilized by either lecithin or beta-lactoglobulin were prepared (pH 7). A portion of the beta-lactoglobulin stabilized emulsions was subjected to a heat treatment known to cross-link the adsorbed globular proteins (85 degrees C, 20 min). Pancreatic lipase and bile extract were then added to each emulsion at 37 degrees C (pH 7) and the evolution of the particle charge, particle size, appearance and free fatty acids released were measured over a period of 2 h. The rate and extent of lipid digestion did not differ greatly between lecithin and beta-lactoglobulin stabilized emulsions, nor did it differ greatly for unheated (BLG-U) or heated (BLG-H) beta-lactoglobulin stabilized emulsions. For example, the initial rate of lipid digestion was found to be 3.1, 3.4, and 2.3 mM fatty acids s(-1) m(-2) of lipid surface for droplets stabilized by BLG-U, BLG-H, and lecithin, respectively. Pancreatic lipase was able to adsorb to the droplet surfaces and access the emulsified lipids, regardless of the initial interfacial composition and the fact that some of the original emulsifier appeared to remain at the oil-water interface during digestion. These results help to explain why the human body is so efficient at digesting dietary triacylglycerols.     

Cross-linking of hen egg white lysozyme by microbial transglutaminase under high hydrostatic pressure: localization of reactive amino acid side chains

After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.     

传统风干牦牛肉加工中肌原纤维蛋白氧化与体外消化性变化

为了评价传统风干牦牛肉在加工过程中肌原纤维蛋白消化率的变化,并探讨蛋白质氧化影响其消化性的潜在机制。在牦牛肉自然风干加工过程中（40 d）采集样本,测定肌原纤维蛋白羰基、巯基、二硫键、二聚酪氨酸、粒径分布、体外消化率及脂质氧化等指标,并分析脂质氧化和蛋白质氧化对蛋白质消化性的影响。结果显示,脂质初级氧化产物（Primary Oxidation Value, POV）、硫代巴比妥酸反应物（Thiobarbituric Acid Reaction Substrates, TBARS）和蛋白质羰基化合物随加工时间的延长而显著增加（P<0.05）,并且相互间存在极显著相关性（P<0.01）。此外,二硫键和二聚酪氨酸含量显著增加（P<0.05）,这是蛋白质交联的主要形式。蛋白质被胃蛋白酶和胰蛋白酶总的水解率在整个加工过程中降低了19.58%（P<0.05）,蛋白质的水解率与羰基、二硫键、二聚酪氨酸和蛋白质粒径呈现极显著负相关性（P<0.01）。综上所述,在风干牛肉加工期间,脂质氧化产物促进了蛋白质氧化,蛋白质的羰基化和交联导致蛋白酶对蛋白质的水解率降低。研究结果对了解风干牦牛肉传统加工方式和进一步改进加工工艺具有积极的促进作用。     

Chemical reactions in cottonseed protein cross-linking by formaldehyde, glutaraldehyde, and glyoxal for the formation of protein films with enhanced mechanical properties

Amino acids involved in cottonseed protein cross-linking by formaldehyde, glutaraldehyde, and glyoxal during protein film formation were identified by an original technique. The entire HPLC amino acid profile (after acid hydrolysis) was studied, along with variations in reactive lysine contents, in films cross-linked or not with increasing quantities of formaldehyde, glutaraldehyde, and glyoxal. This strategy highlighted the formation of acid-resistant lysine derivatives that a simple reactive lysine determination would not have detected. The results-which agree with previously published data-enhance the overall understanding of cross-linking activities that occur in aqueous alkaline solutions during the formation of protein films made with cottonseed flour. Lysine was found to have a key role in protein cross-linking by dialdehydes, with the involvement of tyrosine in the presence of formaldehyde and of arginine in the presence of glyoxal. These results could provide valuable chemical tools for adjusting the mechanical properties of cottonseed protein films.     

Surfactant protein of the Streptomyces subtilisin inhibitor family inhibits transglutaminase activation in Streptomyces hygroscopicus

Transglutaminase (TGase) is widely used in the food industry for improving protein properties by catalyzing the cross-linking of proteins. In Streptomyces, TGase is secreted as a zymogen, and an activation process has been observed in liquid culture. However, the activation mechanism remains unclear. In the present study, the TGase activation process in Streptomyces hygroscopicus was investigated by biochemical approaches. In a liquid culture, Pro-TGase was secreted and gradually was converted into active TGase during the growth period; however, in a cell-free system in which cells were removed from the liquid culture, TGase activation stalled unexpectedly. Subsequently, the TGase activation process was found to be inhibited by a TGase-activating protease inhibitor (TAPI). N-Terminal amino acid sequencing and a homology search of the purified TAPI revealed that it is a member of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found that TAPI (0.1 mg/mL) decreased the surface tension of water from 72 to 60 mJ/m2 within 5 min, suggesting that it possesses surface activity. This is the first report that an SSI member functions as a surfactant protein. On the basis of these findings, a model for TAPI-regulated TGase activation process was proposed. This study provides novel insights into the TGase activation process in Streptomyces.     

Maillard reaction and protein cross-linking in relation to the solubility of milk powders

Protein changes in relation to solubility, Maillard reaction (MR), and protein cross-linking in whole milk powder (WMP), skim milk powder (SMP), and whey protein concentrate (WPC) stored at different relative humidities (RHs) were investigated by chemical and electrophoretic methods. WMP and SMP reached minimum solubility rapidly, while WPC showed no change in solubility. The loss of solubility corresponded with development of high-molecular-weight protein complexes observed by two-dimensional electrophoresis. The maximal MR rate occurred at 66% RH for WMP and SMP (high lactose/protein ratios) and 84% RH for WPC (low lactose/protein ratios) based on the furosine and hydroxymethylfurfural contents. However, browning was greatest at 84% RH in all powders. The minimum solubility corresponded with the casein and fat contents. The retention of solubility and minimal protein cross-linking of WPC compared to casein-containing powders suggest that the casein content and cross-linking strongly influence the decrease in the solubility of milk powder.     

粗酶水解全脂豆粉提取油脂和蛋白

水酶法提取大豆油和蛋白是一项可替代溶剂浸提制油工艺的绿色环保技术,但是商品酶的价格较高且酶活易受外界环境影响,使水酶法制油技术的应用受到限制。该试验在优化过的培养基中接种枯草芽孢杆菌发酵培养42 h,所得发酵液经测定含有碱性和中性两种蛋白酶,所得粗酶经透析浓缩后,在碱性蛋白酶活为(2?000±200) U/mL,中性蛋白酶活为(1?500±200) U/mL时,在酶液中接入挤压膨化豆粉水解。通过对酶解条件的优化,试验证实在温度55℃,料液比1∶8 g/mL,起始pH值为10的条件下水解6 h,总油提取率有最大值,达到了94.2%,总蛋白提取率为90.1%,跟商品Alcalase碱性蛋白酶提取相比,总油提取率增加了1.9%,总蛋白提取率降低了2%。通过对粗酶和商品Alcalase酶水解豆粉过程产生的乳状液破乳后油的品质分析,发现二者所得油的性能指标没有明显区别,品质均优于浸提法制油,粗酶提取的水解蛋白比用Alcalase碱性蛋白酶水解的分子量更小,范围分布更广。     

Salt-induced changes in pork myofibrillar tissue investigated by FT-IR microspectroscopy and light microscopy

FT-IR microspectroscopy and light microscopy were used to investigate pork muscle musculus semitendinosus samples, taken from three animals, that were subjected to brine salting at different concentrations (0.9, 3, 6, and 9% NaCl). Differences in spectral characteristics and in microstructure were observed in meat from animals differing in initial pH and varying salt concentrations. The FT-IR data displayed changes in the amide I region from 1700 to 1600 cm(-1). This spectral range was analyzed by principal component analysis (PCA) and partial least-squares regression (PLSR). These methods revealed correlations between the spectral data and the different animals, salt content, moisture content, pH value, and myofiber diameter. A shrinking share of alpha-helical components was related to an increase in salt concentration in the muscle. At the same time, a greater share in nonhydrogenated C=O groups (1668 cm(-1)) was related to an increase in salt concentration in the meat. The share of aggregated beta-strands differed with respect to the different animals but was not influenced by salt concentration. The meat at higher pHs (>6) had less aggregated beta-strands than that at lower pHs (5.6-5.7). It could be demonstrated that simultaneous with changes in microstructure, pH value, salt, and moisture content were alterations in the protein amide I region as measured by FT-IR microspectroscopy, revealing a relationship between these biophysical and chemical parameters and secondary protein structure attributes.