Heat-induced changes in myofibrillar protein structures and myowater of two pork qualities. A combined FT-IR spectroscopy and low-field NMR relaxometry study

Low-field NMR T(2) and Fourier transform infrared (FT-IR) measurements were performed on meat samples of two qualities (normal and high ultimate pH) during cooking from 28 degrees C to 81 degrees C. Pronounced changes in both T(2) relaxation data and FT-IR spectroscopic data were observed during cooking, revealing severe changes in the water properties and structural organization of proteins. The FT-IR data revealed major changes in bands in the amide I region (1700-1600 cm(-)(1)), and a tentative assignment of these is discussed. Distributed NMR T(2) relaxation data and FT-IR spectra were compared by partial least-squares regression. This revealed a correlation between the FT-IR peaks reflecting beta-sheet and alpha-helix structures and the NMR relaxation populations reflecting hydration water (T(2B) approximately 0-10 ms), myofibrillar water (T(21) approximately 35-50 ms), and also expelled "bulk" water (T(2) relaxation times >1000 ms). Accordingly, the present study demonstrates that definite structural changes in proteins during cooking of meat are associated with simultaneous alterations in the chemical-physical properties of the water within the meat.     

Influence of aging and salting on protein secondary structures and water distribution in uncooked and cooked pork. A combined FT-IR microspectroscopy and 1H NMR relaxometry study

Fourier transform infrared (FT-IR) microspectroscopy and low-field (LF) proton NMR transverse relaxation measurements were used to study the changes in protein secondary structure and water distribution as a consequence of aging (1 day and 14 days) followed by salting (3%, 6%, and 9% NaCl) and cooking (65 degrees C). An enhanced water uptake and increased proton NMR relaxation times after salting were observed in aged meat (14 days) compared with nonaged meat (1 day). FT-IR bands revealed that salting induced an increase in native beta-sheet structure while aging triggered an increase in native alpha-helical structure before cooking, which could explain the effects of aging and salting on water distribution and water uptake. Moreover, the decrease in T2 relaxation times and loss of water upon cooking were attributed to an increase in aggregated beta-sheet structures and a simultaneous decrease in native protein structures. Finally, aging increased the cooking loss and subsequently decreased the final yield, which corresponded to a further decrease in T2 relaxation times in aged meat upon cooking. However, salting weakened the effect of aging on the final yield, which is consistent with the increased T2 relaxation times upon salting for aged meat after cooking and the weaker effect of aging on protein secondary structural changes for samples treated with high salt concentration. The present study reveals that changes in water distribution during aging, salting, and cooking are not only due to the accepted causal connection, i.e., proteolytic degradation of myofibrillar structures, change in electrostatic repulsion, and dissolution and denaturation of proteins, but also dynamic changes in specific protein secondary structures.     

Monitoring protein structural changes and hydration in bovine meat tissue due to salt substitutes by Fourier transform infrared (FTIR) microspectroscopy

The objective of this study was to investigate the influence of NaCl and two salt substitutes, MgSO4 and KCl, in different concentrations (1.5, 6.0, and 9.0%) on meat proteins by using Fourier transform infrared (FTIR) microspectroscopy. Hydration properties and secondary structural properties of proteins were investigated by studying the amide I, amide II, and water regions (3500-3000 cm(-1)) in FTIR spectra. By applying multivariate analysis (PCA and PLSR), differences between samples according to salt concentration and salt type were found and correlated to spectral bands. The most distinctive differences related to salt type were obtained by using the water region. It was found that samples salted with MgSO4 exhibited hydration and subsequent denaturation of proteins at lower concentrations than those salted with NaCl. Samples salted with KCl brines showed less denaturation even at the 9.0% concentration. The FTIR results were further supported by water-binding capacity (WBC) measurements.     

Infrared imaging of sunflower and maize root anatomy

Synchrotron radiation infrared microspectroscopy (SR-IMS) permits the direct analysis of plant cell-wall architecture at the cellular level in situ, combining spatially localized information and chemical information from IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers, such as cellulose, lignin, and proteins, in the root tissue of hydroponically grown sunflower and maize plants. Principal components analysis (PCA) was employed to reveal the major spectral variance between maize and sunflower plant tissues. The use of PCA showed distinct separation of maize and sunflower samples using the IR spectra of the epidermis and xylem. The infrared band at 1635 cm(-1), representing hydrocinnamic acid in (H type) lignin, provided a conclusive means of distinguishing between maize and sunflower plant tissues.     

Dynamic MRI and thermal simulation to interpret deformation and water transfer in meat during heating

Understanding and controlling structural and physical changes in meat during cooking is of prime importance. Nuclear magnetic resonance imaging (MRI) is a noninvasive, nondestructive tool that can be used to characterize certain properties and structures both locally and dynamically. Here we show the possibilities offered by MRI for the in situ dynamic imaging of the connective network during the cooking of meat to monitor deformations between 20 and 75 °C. A novel device was used to heat the sample in an MR imager. An MRI sequence was developed to contrast the connective tissue and the muscle fibers during heating. The temperature distribution in the sample was numerically simulated to link structural modifications and water transfer to temperature values. The contraction of myofibrillar and collagen networks was observed at 42 °C, and water began to migrate toward the interfascicular space at 40 °C. These observations are consistent with literature results obtained using destructive and/or nonlocalized methods. This new approach allows the simultaneous monitoring of local deformation and water transfer, changes in muscle structure and thermal history.     

Myowater dynamics and protein secondary structural changes as affected by heating rate in three pork qualities: a combined FT-IR microspectroscopic and 1H NMR relaxometry study

The objective of this study was to investigate the influence of heating rate on myowater dynamics and protein secondary structures in three pork qualities by proton NMR T2 relaxation and Fourier transform infrared (FT-IR) microspectroscopy measurements. Two oven temperatures at 100 degrees C and 200 degrees C corresponding to slow and fast heating rates were applied on three pork qualities (DFD, PSE, and normal) to an internal center temperature of 65 degrees C. The fast heating induced a higher cooking loss, particularly for PSE meat. The water proton T21 distribution representing water entrapped within the myofibrillar network was influenced by heating rate and meat quality. Fast heating broadened the T21 distribution and decreased the relaxation times of the T21 peak position for three meat qualities. The changes in T21 relaxation times in meat can be interpreted in terms of chemical and diffusive exchange. FT-IR showed that fast heating caused a higher gain of random structures and aggregated beta-sheets at the expense of native alpha-helixes, and these changes dominate the fast-heating-induced broadening of T21 distribution and reduction in T21 times. Furthermore, of the three meat qualities, PSE meat had the broadest T21 distribution and the lowest T21 times for both heating rates, reflecting that the protein aggregation of PSE caused by heating is more extensive than those of DFD and normal, which is consistent with the IR data. The present study demonstrated that the changes in T2 relaxation times of water protons affected by heating rate and raw meat quality are well related to the protein secondary structural changes as probed by FT-IR microspectroscopy.     

蛋白质组学在生鲜肉肉色变化机制研究中的应用

肉色是影响消费者购买欲望的最直观因素。宰后生鲜肉肉色的变化机制和肉色改善一直是肉类科学领域研究的热点。肉色体系复杂,其稳定性受动物宰前因素、从肌肉向可食用肉转化过程和宰后商业流通链条件等因素的影响。目前,仍有许多影响途径和作用机制不够清楚。研究者已采用蛋白质组学技术发现了许多与肉色相关的蛋白标志物和可能影响途径。该文基于近年的研究,综述了不同处理条件下与肉色相关的差异蛋白,主要包括结构性蛋白中的肌动蛋白、肌球蛋白和肌联蛋白,以及肌浆蛋白中的伴侣蛋白、热休克蛋白、代谢酶类和氧化还原酶类等,分析了差异蛋白表达对肉色的影响及其表达调控肉色体系的可能机制,为今后肉色变化机制的研究和肉色稳定性的调控提供了思路。     

Determination of nitrogen and protein content of meat and meat products

Chemical and instrumental methods for determination of nitrogen and protein are reviewed for their mode of action and utility in analysis of meat proteins and products. Although the Kjeldahl digestion method is satisfactory for determining total nitrogen, it is imprecise for determining total protein content. Presence of variable amounts of nonprotein nitrogenous components and of connective tissue proteins such as collagen and elastin produces error if the formula (N X 6.25) is used to calculate crude protein. Such fibrous proteins have higher nitrogen levels (over 18%) than other muscle proteins (about 16%), and a higher than actual protein value will be determined unless a lower conversion factor is used to correct for their content. To determine meat protein content more accurately, a combination of Kjeldahl determination with one or more additional tests to correct for nonprotein and fibrous protein content is recommended. The choice of the additional method(s) is based on the user's requirement for protein characterization, available time, type of meat product, and sample size.     

Probing the binding of the flavonoid diosmetin to human serum albumin by multispectroscopic techniques

The binding mechanism of molecular interaction between diosmetin and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using atomic force microscopy (AFM) and various spectroscopic techniques including fluorescence, resonance light scattering (RLS), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by diosmetin was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The RLS spectra and AFM images showed that the dimension of the individual HSA molecules were larger after interaction with diosmetin. The thermodynamic parameters, ΔH° and ΔS° were calculated to be -24.56 kJ mol(-1) and 14.67 J mol(-1) K(-1), respectively, suggesting that the binding of diosmtin to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies and denaturation experiments in the presence of urea indicated site I as the main binding site for diosmetin on HSA. The binding distance between diosmetin and HSA was determined to be 3.54 nm based on the F?rster theory. Analysis of CD and FT-IR spectra demonstrated that HSA conformation was slightly altered in the presence of diosmetin.     

Chemical imaging of microstructures of plant tissues within cellular dimension using synchrotron infrared microspectroscopy

Synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) is an advanced bioanalytical technique capable of exploring the chemistry within microstructures of plant and animal tissues with a high signal to noise ratio at high ultraspatial resolutions (3-10 microm) without destruction of the intrinsic structures of a tissue. This technique is able to provide information relating to the quantity, composition, structure, and distribution of chemical constituents and functional groups in a tissue. The objective of this study was to illustrate how the SR-FTIR technique can be used to image inherent structures of plant tissues on a cellular level (pixel size, approximately 10 microm x 10 microm). The results showed that with the extremely bright synchrotron light, spectra with high signal to noise ratios were obtained from areas as small as 10 microm x 10 microm in the plant tissue, which allowed us to "see" plant tissue in a chemical sense on a cellular level. The ultraspatial resolved imaging of plant tissues by stepping in pixel-sized increments was obtained. Chemical distributions of plant tissues such as lignin, cellulose, protein, lipid, and total carbohydrate could be mapped. These images revealed the chemical information of plant intrinsic structure. In conclusion, SR-FTIR can provide chemical and functional characteristics of plant tissue at high ultraspatial resolutions. The SR-FTIR microspectroscopic images can generate spatially localized functional group and chemical information within cellular dimensions.     

基于稳态空间分辨光谱的猪肉新鲜度检测方法

该文探索肉品光学参数与新鲜程度变化之间的关系,从而为无损、快速地评定猪肉新鲜度提供一种方法。基于生物光子学原理,应用近红外稳态空间分辨光谱技术,设计了可见-短波近红外多通道光学参数检测装置,在系统校准基础上获得猪肉在新鲜度变化过程中的有效衰减系数谱。研究了逐日获得的猪肉有效衰减系数与反映肉品新鲜程度的挥发性盐基氮（TVB-N）含量之间的关系,并辅以菌落总数检测与感官指标评价等作为参考依据。研究结果表明,当肉品发生腐败,胞膜发生溶解破裂,其组织结构的改变导致散射变化;有效衰减系数在940、960 nm波长处与TVB-N含量变化呈显著相关。由此得出：在短波近红外范围内可以建立近红外光谱与TVB-N的相关关系,从而达到评价肉类新鲜度的目的。     

Using Fourier transform infrared (FT-IR) absorbance spectroscopy and multivariate analysis to study the effect of chlorine-induced bacterial injury in water

The effect of chlorine-induced bacterial injury on spectral features using Fourier transform infrared (FT-IR) absorbance spectroscopy was studied using a mixed bacterial culture of (1:1) ca. 500 CFU/mL each Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 15442 in 0.9% saline. Bacterial cells were treated with 0, 0.3, or 1.0 ppm of initial free chlorine (21 degrees C, 1 h of contact time). Chlorine-injured and dead bacterial cells retained the ATR spectral properties of uninjured or live cells in the region of C-O-C stretching vibrations of polysaccharides, indicative of the cell wall peptidoglycan layer and lipopolysaccharide outer leaflet. This confirms the observations of others that extensive bacterial membrane damage is not a key factor in the inactivation of bacteria by chlorine. The bactericidal effect of chlorine caused changes in the spectral features of bacterial ester functional groups of lipids, structural proteins, and nucleic acids, with apparent denaturation reflected between 1800 and 1300 cm (-1) for injured bacterial cells. Three-dimensional principal component analysis (PCA) showed distinct segregation and clustering of chlorine-treated and untreated cells. Cells exposed to chlorine at 0.3 or 1.0 ppm could be distinguished from the untreated control 73 and 80% of the time, respectively, using soft independent modeling of class analogy (SIMCA) analysis. This study suggests that FT-IR spectroscopy may be applicable for detecting the presence of injured and viable but not culturable (VBNC) waterborne pathogens that are underestimated or not discernible using conventional microbial techniques.     

Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.     

Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high-pressure homogenization

The conformational changes of whey proteins upon adsorption at the soy oil/water interface were investigated using Fourier transform infrared (FT-IR) spectroscopy. Significant changes were observed in the bands assigned to beta-sheets and alpha-helix structures following the adsorption of proteins at the oil/water interface. The remaining interfacial proteins after Tween 20 desorption revealed small changes in beta-sheet and alpha-helical structures, whereas in the desorbed whey proteins the unordered structures largely increased, and beta-sheet structures almost disappeared. These FT-IR results provide important knowledge about the conformational modifications in whey proteins occurring upon adsorption at the oil/water interface. Finally, specific conformational changes are necessary to stabilize emulsions: adsorption-induced unfolding, increase in alpha-helical structures to establish interactions with the oil phase, and aggregation between adsorbed whey proteins to form protein membranes. Moreover, the structural changes in whey protein adsorbed at the oil/water interface under high-pressure homogenization are irreversible.     

Using synchrotron-based FTIR microspectroscopy to reveal chemical features of feather protein secondary structure: comparison with other feed protein sources

Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein. An understanding of the structure of the whole protein is often vital to understanding its digestive behavior in animals and nutritive quality. Usually protein secondary structures include alpha-helix and beta-sheet. The percentages of these two structures in protein secondary structures may influence feed protein quality and digestive behavior. Feathers are widely available as a potential protein supplement. They are very high in protein (84%), but the digestibility of the protein is very low (5%). The objective of this study was to use synchrotron-based Fourier transform infrared (FTIR) microspectroscopy to reveal chemical features of feather protein secondary structure within amide I at ultraspatial resolution (pixel size = 10 x 10 microm), in comparison with other protein sources from easily digested feeds such as barley, oat, and wheat tissue at endosperm regions (without destruction of their inherent structure). This experiment was performed at beamline U2B of the Albert Einstein Center for Synchrotron Biosciences at the National Synchrotron Light Source (NSLS) in Brookhaven National Laboratory (BNL), U.S. Dept of Energy (NSLS-BNL, Upton, NY). The results showed that ultraspatially resolved chemical imaging of feed protein secondary structure in terms of beta-sheet to alpha-helix peak height ratio by stepping in pixel-sized increments was obtained. Using synchrotron FTIR microspectroscopy can distinguish structures of protein amide I among the different feed protein sources. The results show that the secondary structure of feather protein differed from those of other feed protein sources in terms of the line-shape and position of amide I. The feather protein amide I peaked at approximately 1630 cm(-1). However, other feed protein sources showed a peak at approximately 1650 cm(-1). By using multicomponent peak modeling, the relatively quantitative amounts of alpha-helix and beta-sheet in protein secondary structure were obtained, which showed that feather contains 88% beta-sheet and 4% alpha-helix, barley contains 17% beta-sheet and 71% alpha-helix, oat contains 2% beta-sheet and 92% alpha-helix, and wheat contains 42% beta-sheet and 50% alpha-helix. The difference in percentage of protein secondary structure may be part of the reason for different feed protein digestive behaviors. These results demonstrate the potential of highly spatially resolved infrared microspectroscopy to reveal feed protein secondary structure. Information from this study by the infrared probing of feed protein secondary structure may be valuable as a guide for feed breeders to improve and maintain protein quality for animal use.     

Monitoring biochemical changes in bacterial spore during thermal and pressure-assisted thermal processing using FT-IR spectroscopy

Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (approximately 10(9) CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and TP (121 degrees C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm(-1)). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in alpha-helix and beta-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm(-1), respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm(-1)) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores.     

Determination of soy in meat

A number of methods may be used for determining soy flour in meat products. Highly purified soy products are more difficult to determine because the nonprotein components used to quantify the flour are reduced. Immunoassays have been used to directly measure protein content of soy products. Immunological methods for determination of soy proteins in meat are complicated by changes in the structure of the soy proteins during processing. These changes alter the available epitopes, changing the immunoreactivity of soy proteins. The epitopes available are dictated by the details of the processing. Other workers circumvented this problem by denaturing the soy protein with urea and mercaptoethanol, and then removing these agents by dialysis; whatever the initial protein conformation, all soy samples came to the same final conformation after the denaturing agents were removed. The assay used antibody made against the "renatured protein." These steps made the assay long and laborious. Attempts to develop a rapid assay were complicated by the same protein denaturation problems. Sodium dodecylsulfate gel electrophoresis coupled with immunoblotting may be the best quantitative approach.     

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.     

Cooking temperature is a key determinant of in vitro meat protein digestion rate: investigation of underlying mechanisms

The present study aimed to evaluate the digestion rate and nutritional quality of pig muscle proteins in relation to different meat processes (aging, mincing, and cooking). Under our experimental conditions, aging and mincing had little impact on protein digestion. Heat treatments had different temperature-dependent effects on the meat protein digestion rate and degradation potential. At 70 °C, the proteins underwent denaturation that enhanced the speed of pepsin digestion by increasing enzyme accessibility to protein cleavage sites. Above 100 °C, oxidation-related protein aggregation slowed pepsin digestion but improved meat protein overall digestibility. The digestion parameters defined here open new insights on the dynamics governing the in vitro digestion of meat protein. However, the effect of cooking temperature on protein digestion observed in vitro needs to be confirmed in vivo.