Changes of trypsin in activity and secondary structure induced by complex with trypsin inhibitors and tea polyphenol

To reveal the relationships between the activity of trypsin and its structural change, changes of trypsin in biological activity induced by complex with Bowman-Birk trypsin inhibitor (BBTI), Kunitz soybean trypsin inhibitor (KSTI, type I-S) and tea polyphenol (TP) were detected and their relationship with the secondary structure changes were studied by far-UV circular dichroism (CD) spectra measurement. BBTI and KSTI were also irradiated by ultrasonic to compare the effects on trypsin. The rank was found as KSTI > BBTI > TP according to their inhibitory activities against trypsin. Yet BBTI exhibited much stronger resistance against ultrasonic irradiation than KSTI. BBTI, KSTI and TP were found inactivate trypsin by modifying the secondary structures and far-UV spectrum of trypsin. Complex of trypsin with ultrasonic-treated BBTI and native BBTI and KSTI exhibited the similar modified effects in secondary structures, decrease of α-helix and β-turn content, increase of β-sheet content and unchanged random coil content basically. But complex of trypsin with ultrasonic-treated KSTI exhibited less modified effects because of inactivation by ultrasonic irradiation. The changes of trypsin in secondary structure induced by complex with TP showed different from those induced by complex with BBTI and KSTI, increase of α-helix content, decrease of random coil content and unchanged β-sheet and β-turn content basically.     

Studies on the interaction of ‐epigallocatechin‐3‐gallate from green tea with bovine β‐lactoglobulin by spectroscopic methods and docking

The binding interaction between‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐lactoglobulin (βLG) was thoroughly studied by fluorescence, circular dichroism (CD) and protein–ligand docking. Fluorescence data revealed that the fluorescence quenching of βLG by EGCG was the result of the formation of a complex of βLG–EGCG. The binding constants and thermodynamic parameters at two different temperatures and the binding force were determined. The binding interaction between EGCG and βLG was mainly hydrophobic and the complex was stabilised by hydrogen bonding. The results suggested that βLG in complex with EGCG changes its native conformation. Furthermore, preheat treatment (90 °C, 120 °C) and emulsifier (sucrose fatty acid ester) all boosted the binding constants (K_a) and the binding site values (n) of the βLG‐EGCG complex. This study provided important insight into the mechanism of binding interactions of green tea flavonoids with milk protein.     

Inactivation of carboxypeptidase A and trypsin by UV–visible light

In the present work, the effect of UV–visible irradiation on the activity of carboxypeptidase A, and trypsin enzymes is shown. The irradiation of the above-mentioned enzymes inhibits their activity, in such a way that sufficiently high irradiation times annul their catalytic action. For carboxypeptidase A a total inactivation after 20 min of irradiation is observed, while trypsin is inactivated completely after 12 min of irradiation. Fitting the data to the Lineweaver–Burk graphs shows that, in the case of CPA enzyme, the inhibition caused by irradiation is similar to that of uncompetitive type. For trypsin, the irradiation acts similarly to a mixed inhibition-type.

Industrial relevance

UV irradiation is a technology used in food treatment, since it has been shown to be effective in the destruction of microorganisms. It can also be applied in the sterilization of enzymatic preparations used in the food industry, but it can have harmful effects, since it can go so far as to inactivate some of the enzymes. In some cases it interests to inactivate enzymes, and it is for it that this treatment type can be effective. In other cases it interests that the enzymes remain active. This way, it will be necessary to avoid that they are exposed to the light.     

Quantification of zinc-porphyrin in dry-cured ham products by spectroscopic methods Comparison of absorption, fluorescence and X-ray fluorescence spectroscopy

Zinc–protoporphyrin (Zn–pp), which has been identified as the major pigment in certain dry-cured meat products, was extracted with acetone/water (75%) and isolated from the following meat products: Parma ham, Iberian ham and dry-cured ham with added nitrite. The quantification of Zn–pp by electron absorption, fluorescence and X-ray fluorescence (XRF) spectroscopy was compared (concentration range used [Zn–pp] = 0.8–9.7 μM). All three hams were found to contain Zn–pp, and the results show no significant difference among the content of Zn–pp quantified by fluorescence, absorbance and X-ray fluorescence spectroscopy for Parma ham and Iberian ham. All three methods can be used for quantification of Zn–pp in acetone/water extracts of different ham types if the content is higher than 1.0 ppm. For dry-cured ham with added nitrite, XRF was not applicable due to the low content of Zn–pp (<0.1 ppm). In addition, XRF spectroscopy provides further information regarding other trace elements and can therefore be advantageous in this aspect. This study also focused on XRF determination of Fe in the extracts and as no detectable Fe was found in the three types of ham extracts investigated (limit of detection; Fe ? 1.8 ppm), it allows the conclusion that iron containing pigments, e.g., heme, do not contribute to the noticeable red colour observed in some of the extracts.     

Investigation on the inactivation of trypsin by oenothein B: isothermal titration calorimetry and docking studies

The aim of this study was to investigate the inhibitory mechanism of oenothein B (OeB), a unique oligomer ellagitannin with a rigid structure, on porcine trypsin using fluorescence spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and molecular docking. Trypsin activity was strongly inhibited by OeB in a competitive way. Fluorescence quenching of trypsin by OeB was a static quenching. The CD spectra showed that binding of OeB to trypsin altered trypsin's conformation. The ITC and docking studies revealed that the inhibitory mechanism of OeB occurred via binding to the interior hydrophobic groups of trypsin and the formation of hydrogen bonds with trypsin through binding to the amino acid residues Asn97, His573, Ser195 and Gln192. This study provides a theoretical and computational basis for the precise control of trypsin in food industry. Based on the results, OeB may be used in food technology research as novel bioactive trypsin inhibitor.     

Interactions of different polyphenols with bovine serum albumin using fluorescence quenching and molecular docking

Polyphenols are responsible for the major organoleptic characteristics of plant-derived foods and beverages. Here, we investigated the binding of several polyphenols to bovine serum albumin (BSA) at pH 7.5 and 25 °C: catechins [(−)-epigallocatechin-3-gallate, (−)-epigallocatechin, (−)-epicatechin-3-gallate], flavones (kaempferol, kaempferol-3-glucoside, quercetin, naringenin) and hydroxycinnamic acids (rosmarinic acid, caffeic acid, p-coumaric acid). Fluorescence emission spectrometry and molecular docking were applied to compare experimentally determined binding parameters with molecular modelling. Among these polyphenols, (−)-epicatechin-3-gallate showed the highest Stern–Volmer modified quenching constant, followed by (−)-epigallocatechin-3-gallate. Similarly, (−)-epicatechin-3-gallate had the highest effect on the Circular Dichroic spectrum of BSA, while the changes induced by other polyphenols were negligible. Molecular docking predicted high binding energies for (−)-epicatechin-3-gallate and (−)-epigallocatechin-3-gallate for the binding site on BSA near Trp213. Our data reveal that the polyphenol structures significantly affect the binding process: the binding affinity generally decreases with glycosylation and reduced numbers of hydroxyl groups on the second aromatic ring.     

荧光光谱法结合分子对接探究pH对姜黄素与牛血清白蛋白结合的影响

目的 采用荧光光谱法结合分子对接探究姜黄素(curcumin,CUR)与牛血清白蛋白(bovine serum albumin,B SA)在不同pH条件下相互作用。方法 通过荧光光谱法计算结合常数和热力学参数分析CUR对BSA荧光猝灭作用和机制;采用位点Marker和分子对接分析结合位点。结果 CUR与BSA结合形成了复合物,产生内源性荧光猝灭作用,属于静态猝灭。不同pH条件下的结合作用力不同,但结合位点数目均为1。由同步荧光可知,色氨酸残基附近疏水性增强,说明与CUR结合后BSA构象出现了收缩,结合位点靠近色氨酸。位点Marker实验证明pH影响CUR与BSA结合位点,分子对接结果表明, pH 7.4时CUR与BSA的结合位点位于Sudlow’s site I附近。结论 本研究表明pH影响CUR与BSA的结合反应,为CUR的活性保护及蛋白基递送载体研究提供理论支持。     

Quality evaluation of fish and other seafood by traditional and nondestructive instrumental methods: Advantages and limitations

Although being one of the most vulnerable and perishable products, fish and other seafoods provide a wide range of health-promoting compounds. Recently, the growing interest of consumers in food quality and safety issues has contributed to the increasing demand for sensitive and rapid analytical technologies. Several traditional physicochemical, textural, sensory, and electrical methods have been used to evaluate freshness and authentication of fish and other seafood products. Despite the importance of these standard methods, they are expensive and time-consuming, and often susceptible to large sources of variation. Recently, spectroscopic methods and other emerging techniques have shown great potential due to speed of analysis, minimal sample preparation, high repeatability, low cost, and, most of all, the fact that these techniques are noninvasive and nondestructive and, therefore, could be applied to any online monitoring system. This review describes firstly and briefly the basic principles of multivariate data analysis, followed by the most commonly traditional methods used for the determination of the freshness and authenticity of fish and other seafood products. A special focus is put on the use of rapid and nondestructive techniques (spectroscopic techniques and instrumental sensors) to address several issues related to the quality of these products. Moreover, the advantages and limitations of each technique are reviewed and some perspectives are also given.     

Phytochemical profiles of black and yellow soybeans as affected by roasting

The current research aimed to compare the changes of phytochemicals, antioxidants, and anti-nutritional factors of yellow and black soybeans upon roasting. Yellow soybean was roasted at 210°C for 35 min and 230°C for 25 min, and black soybean was roasted at 210°C for 30 min and 230°C for 25 min. Isoflavones were significantly increased after the roasting process, while trypsin inhibitor was significantly decreased. The roasting process combined with the soaking process led to decrease in anthocyanin, soyasaponin, and antioxidant capacities. Black soybeans had more apparent variations in compounds than the yellow soybeans. For black soybeans, the processing under 230°C for 25 min presented more decrease in trypsin inhibitor and anthocyanin content. The roasting at a high temperature and short time produced products with a light color. Roasting combined with the soaking process had negative impacts on both yellow and black soybeans, but there were slight differences in the resultant compounds between different roasting temperatures.     

动态高压微射流诱导的去折叠胰蛋白酶结构预测

采用差示扫描量热法和圆二色谱分析了动态高压微射流处理前后胰蛋白酶结构的变化,结合前期研究中动态高压微射流诱导的去折叠胰蛋白酶构象的表征,通过PyMOL、3DSMax等软件分别对天然胰蛋白酶和动态高压微射流诱导的去折叠胰蛋白酶的三维空间构象进行模拟和预测,结果表明:与天然胰蛋白酶相比,动态高压微射流处理后胰蛋白酶热焓降低,二级结构构象单元百分含量发生变化,去折叠胰蛋白酶结构更加松散。模拟的胰蛋白酶分子构象示意图更直观地显示了胰蛋白酶的活性中心、二硫键以及二级结构信息,对动态高压微射流诱导的去折叠胰蛋白酶三维结构模型变化做一初探。      

基于生物信息学稻米半胱氨酸蛋白酶抑制剂来源生物活性肽的虚拟筛选及分子对接研究

稻米半胱氨酸蛋白酶抑制剂(Oryzacystatin,OC)是除贮藏蛋白外一类重要的稻米蛋白,为筛选稻米OC来源的生物活性肽,以稻米OC蛋白质序列为对象进行生物信息学分析和计算机辅助酶解,建立OC蛋白虚拟模拟胃肠道消化产物的肽数据库,预测其生物活性肽序列.进一步结合分子对接虚拟筛选具有结合潜力的活性肽并探讨其与靶蛋白的...     

Identification of Lactobacillus from koumiss by conventional and molecular methods

Lactobacilli are considered to be one of the most important potential probiotics in the dairy industry. Twelve strains of Lactobacillus were isolated from home-made koumiss samples, a traditionally fermented mare milk in China. The isolates were identified based on physiological and biochemical characteristics and analysis of 16S RNA sequences. They were proven to be Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus casei and Lactobacillus plantarum. The results demonstrated that both methods were essential to identify an isolate accurately.     

The effect of high pressure carbon dioxide on the inactivation kinetics and structural alteration of phenylalanine ammonia-lyase from Chinese water chestnut: An investigation using multi-spectroscopy and molecular docking methods

The effect of high pressure carbon dioxide (HPCD) on the inactivation kinetic and structure of phenylalanine ammonia-lyase (PAL) from Chinese water chestnut (CWC) was studied in this paper. The inactivation kinetic of PAL treated by HPCD (1–4 MPa, 35–55 °C, and 5–60 min) were determined and fitted to the first order kinetics model with calculating kinetic parameters. As revealed by the circular dichroism spectral, the α-helix and β-turn content in secondary structure increased and the β-sheet content decreased. And the intensity of the fluorescence spectra reflecting tertiary structure decreased, together with the λ_max blue-shifted with the increasing pressure. Fluorescence and circular dichroism spectral results indicated that the conformation of PAL was altered by HPCD. The findings of particle size distribution and ζ potential showed that HPCD could cause the aggregates of PAL particles. Moreover, molecular docking indicated the interactions between small molecules (CO₂, H₂CO₃, HCO₃⁻, and CO₃²⁻) and PAL might result in a decrease in PAL activity by forming steric hindrance, preventing substrate from binding. Finally, this paper proposed a potential mechanism for inactivation of PAL by HPCD treatment, where the loss in PAL activity was correlated to changes in secondary and tertiary structure of PAL, which was induced by aggregation effect of HPCD.     

Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexation

BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman–Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged K_M value of trypsin for benzoyl‐DL ‐arginine‐p‐nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non‐competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near‐UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry     

Control of lightness and firmness of cold and reheated frankfurter-type sausages using different spectroscopic methods applied to raw batter

Muscle types and collagen, fat, and muscle protein minus collagen were varied in cooked frankfurter-type sausages made from beef and pork meat as well as pork backfat. The content of collagen was fixed at preset levels with pork rind. The amount of total muscle protein in the sausages varied between 5.9% and 11.9% and the fat between 16.1% and 22.1%. The collagen content varied between 1.3% and 4%. Spectroscopic measurements (near-infrared reflectance spectra 1100 to 2500 nm; front-face autofluorescence emission spectra 360 to 640 nm) on raw batters were used to predict the amounts of total muscle protein minus collagen, collagen, myoglobin, and fat (biochemical components), L* values from a Minolta chromameter, and firmness of cold (22 degrees C) and reheated sausages (60 degrees C). Lightness of sausages was most accurately determined from the batter data with a Minolta chromameter or the autofluorescence measurement system. Firmness of cold sausages could be described by the amounts of biochemical components plus the type of muscle used in the sausage. The 2nd-best approach was to use the shape of the near-infrared spectra to determine firmness. This was possible as the shape of near-infrared spectra depended on total protein content, and total protein content largely determined the firmness of cold sausages. If the sausages were reheated to 60 degrees C, near-infrared spectroscopy alone determined firmness of the sausages with a lower accuracy than a combined solution of fluorescence and near-infrared spectroscopy. The 2 spectroscopic techniques could thus be used to estimate the amount of biochemical components in sausages. Once these components were known, firmness could be calculated from a model between the amounts of biochemical components and firmness. For reheated sausages, as opposed to cold ones, there was a need to differentiate between collagen and the other muscle proteins in order to determine firmness. This was optimally achieved by using both autofluorescence and near-infrared spectroscopy.     

Study of the interaction of deoxynivalenol with human serum albumin by spectroscopic technique and molecular modelling

The mechanism of interaction between deoxynivalenol (DON) and human serum albumin (HSA) was studied using spectroscopic methods including fluorescence spectra, UV-VIS, Fourier transform infrared (FT-IR) and circular dichroism (CD). The quenching mechanism was investigated in terms of the association constants, number of binding sites and basic thermodynamic parameters. The distance between the HSA donor and the acceptor DON was 2.80?nm as derived from fluorescence resonance energy transfer. The secondary structure compositions of free HSA and its DON complexes were estimated by the FT-IR spectra. Alteration of the secondary protein structure in the presence of DON was confirmed by UV-VIS and CD spectroscopy. Molecular modelling revealed that a DON–protein complex was stabilised by hydrophobic forces and hydrogen bonding. It was potentially useful for elucidating the toxigenicity of DON when combined with biomolecular function effect, transmembrane transport, toxicological testing and the other experiments.