Stabilizing effects of sucrose–polymer formulations on the activities of freeze-dried enzyme mixtures of alkaline phosphatase,nucleoside phosphorylase and xanthine oxidase

To stabilize two freeze-dried enzyme mixtures, consisting of alkaline phosphatase, nucleoside phosphorylase and xanthine oxidase, the effects of sucrose–polymer (bovine serum albumin, gelatin, dextran, polyethylene glycol and polyvinylpyrrolidone) formulations on the remaining activity of the enzyme mixtures were investigated. The enzyme mixtures were freeze-dried with the additives, and then stored at 25, 40 and 55 °C. The glass transition temperatures (T_g) of the freeze-dried samples were assessed in order to determine their physical stability. The T_g values of sucrose–polymer formulations, with the exception of sucrose–polyethylene glycol, were higher than that of sucrose alone. Comparison of the remaining activities of freeze-dried samples showed that sucrose–bovine serum albumin and/or –gelatin prevented activity loss more effectively than did sucrose. Sucrose–polyethylene glycol showed protective ability equivalent to that of sucrose. On the other hand, sucrose–dextran and/or –polyvinylpyrrolidone diminished the stabilizing effect of sucrose. During storage, sucrose–gelatin prevented gradual activity loss to a much greater degree than did sucrose alone.     

马鲛鱼黄嘌呤氧化酶抑制肽的制备 工艺优化及抗氧化活性研究

目的 探究马鲛鱼黄嘌呤氧化酶抑制肽制备的最佳条件及其抗氧化活性。方法 以马鲛鱼为原料,黄嘌呤氧化酶抑制肽采用超声辅助酶法制备,并筛选最佳反应用酶;以单因素实验为基础,通过Box-Behnken响应面分析法,以黄嘌呤氧化酶抑制活性为响应值,优化得到酶解最优条件。通过1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除等方法测定膜分离物的抗氧化活性;肌肽和鹅肌肽的含量测定采用了高效液相色谱法。结果 最佳反应用酶为复配的中性蛋白酶与复合蛋白酶,总加酶量0.3%（中性蛋白酶∶复合蛋白酶=1:1, m:m）。优化所得的最佳酶解条件为：酶解温度50℃,加酶量为0.2%,pH为7.0。在此条件下黄嘌呤氧化酶抑制率37.49%,与回归理论模型基本符合。3个组分的膜分离物中,相对分子量小于1 kDa的酶解产物的抗氧化性能力最强。马鲛鱼中鹅肌肽的含量为1.558 mg/g,而肌肽含量则为0.10 mg/g。结论 马鲛鱼制备及工艺优化所得的黄嘌呤氧化酶抑制肽对黄嘌呤氧化酶有较好的抑制作用,为工业化制备马鲛鱼黄嘌呤氧化酶抑制肽提供了数据基础和理论依据,肌肽和鹅肌肽的含量测定为分离纯化马鲛鱼黄嘌呤氧化酶抑制肽试验奠定了基础。。     

Effect of maltodextrin on the stability of freeze-dried borojó (Borojoa patinoi Cuatrec.) powder

The adsorption isotherms (20 °C) and the relationship between water content and glass transition temperatures were modeled in freeze-dried borojó powder studying the effect of the addition of two maltodextrins with different dextrose equivalent (DE). Both compositional and physicochemical analyses of borojó pulp were performed. At room temperature (20 °C), the critical water content that ensures the glassy state of the product during storage increased from 5.9 g water/100 g product to 8.5–9.1 g water/100 g product when maltodextrins were added, without there being any significant differences related to the different DE. The critical water activity also increased from 0.285 to 0.504–0.510, being the borojó powder with maltodextrins added more stable.     

Effect of luteolin on xanthine oxidase: Inhibition kinetics and interaction mechanism merging with docking simulation

Xanthine oxidase (XO) catalyses hypoxanthine and xanthine to uric acid in human metabolism. Overproduction of uric acid will lead to hyperuricemia and finally cause gout and other diseases. Luteolin is one of the major components of celery and green peppers, its inhibitory activity on XO and their interaction mechanism were evaluated by multispectroscopic methods, coupled with molecular simulation. It was found that luteolin reversibly inhibited XO in a competitive manner with inhibition constant (K_i) value of (2.38 ± 0.05) × 10⁻⁶ mol l⁻¹. Luteolin could bind to XO at a single binding site and the binding was driven mainly by hydrophobic interactions. Analysis of synchronous fluorescence and circular dichroism spectra demonstrated that the microenvironment and secondary structure of XO were altered upon interaction with luteolin. The molecular docking results revealed luteolin actually interacted with the primary amino acid residues located within the active site pocket of XO.     

Antioxidative and xanthine oxidase inhibitory activities and phytochemical screening of the hydro-alcoholic extract of mace,aril of Myristica fragrans: Implication as an adjuvant therapy in gout

Myristica fragrans derivatives are used as spices in cuisines and also serve as an important source of traditional medicine worldwide. The present study has evaluated the antioxidative potential of the hydro-methanolic extract of the fruit aril of M. fragrans, which is also referred to as mace. This study also includes a quantitative phytochemical assessment of the extract and focuses on the ability of the extract to inhibit xanthine oxidase, a molecular target involved in nucleic acid metabolism and the enzyme responsible for the development of gout disease. The extract was found to contain different classes of secondary metabolites, including flavonoids, phenolics, tannins, alkaloids, and saponins. Furthermore, the antioxidative potential of the extract was evaluated against the radicals 2,2-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt and 2,2-diphenyl-1-pycrylhydrazyl, and there was a progressive reduction of the radicals with increasing doses of the extract. Moreover, a ferric reducing power assay also showed the antioxidant activity of the crude extract as compared to ascorbic acid, a well-known antioxidant. Another significant finding of the study is that the mace extract also considerably inhibits the enzyme xanthine oxidase in a concentration-dependent manner. Our results partially support the use of mace in dietary regimens as a nutraceutical that could serve as a prophylactic strategy or an adjuvant for the prevention and therapy of gout disease.     

Albumin causes a synergistic increase in the antioxidant activity of green tea catechins in oil-in-water emulsions

Model oil-in-water emulsions containing epicatechin (EC) and epigallocatechin gallate (EGCG) showed a synergistic increase in stability in emulsions containing added albumin. EGCG showed a stronger synergy (35%) with ovalbumin than did EC. Oxidation of the oil was monitored by determining peroxide values and hexanal contents. The effect of bovine serum albumin (BSA) on model oil-in-water emulsions containing each of the green tea catechins [epicatechin gallate (ECG), EGCG, EC and epigallocatechin (EGC)] was studied during storage at 30 °C. The green tea catechins showed moderate antioxidant activity in the emulsions with the order of activity being ECG ≈ EGCG > EC > EGC. Although BSA had very little antioxidant activity in the absence of phenolic antioxidants, the combination of BSA with each of the catechins showed strong antioxidant activity. BSA, in combination with EC, EGCG or EGC, showing the strongest antioxidant activity with good stability after 45 days storage. Model experiments with the catechins stored with BSA in aqueous solutions confirmed that protein–catechin adducts with antioxidant activity were formed between the catechins and protein. The antioxidant activity of the separated protein–catechin adducts increased strongly with storage time and was stronger for EGCG and ECG than for EC or EGC.     

Robust size control of bovine serum albumin (BSA) nanoparticles by intermittent addition of a desolvating agent and the particle formation mechanism

In polymeric nanoparticle preparation, despite similar conditions, large fluctuations in particle size distributions are usually observed. Herein, we demonstrate that the intermittent addition of a desolvating agent can improve reproducibility in the preparation of polymeric bovine serum albumin (BSA) nanoparticles. Using this modification, BSA nanoparticles of controlled size can be manufactured with narrow particle size distributions. In our study, ethanol as a desolvating agent was added intermittently to 1% BSA solutions at different pHs with stirring at 700 rpm. The effect of the preparation parameters on size and optical density of the fabricated nanoparticles were studied. The average particles sizes of BSA nanoparticles prepared at pH values of 6, 7 and 9 were approximately 100, 200 and 300 nm, respectively. As ethanol addition increased, desolvation of BSA molecules resulted in formation of loose-structured particles with pH-dependent size. Beyond that, only particle density increased, but size remained unchanged with further addition of ethanol. Consistently uniform particle size distribution was achieved by adding ethanol intermittently.     

Carotenoid–protein interaction as an approach for the formulation of functional food emulsions

Functional foods represent an emerging market of growing economic importance. The formulation of carotene fortified food emulsions involves the difficulty to transfer the carotenoids into the lipid phase. Usually, undesirable organic solvents are used to dissolve the carotenoid in the lipid phase, in particular astaxanthin and β-carotene. Here, we present a novel approach in which the carotenoid is first bound to bovine serum albumin (BSA) and then the carotenoid–protein complex is used to prepare an emulsion. Absorbance spectroscopy indicates the formation of the complex in the aqueous phase and provides first results of the carotenoid load, which is supported by the colour of the cream phase. The droplets in the emulsion are visualized by confocal laser scanning spectroscopy, indicating the protein layer. The laser diffraction spectroscopy and confocal laser scanning spectroscopy provide the particle size distribution and insight of the stability of emulsion interface.     

牛血清白蛋白/黄酮醇纳米核壳颗粒的制备及其抗氧化活性研究

利用牛血清白蛋白和疏水性黄酮醇合成牛血清白蛋白/黄酮醇纳米核壳颗粒,采用TEM确定纳米核壳的粒径,HPLC分析明确包覆率,2种抗氧化方法(DPPH自由基,ABTS自由基)对包覆前后的黄酮醇类化合物抗氧化活性进行评价。结果表明:牛血清白蛋白/黄酮醇纳米核壳粒径约为20 nm;牛血清白蛋白与黄酮醇化合物的结合关系影响它的包覆率;由于具有抗氧化活性的基团与BSA形成氢键,使得被包覆的黄酮醇抗氧化活性降低。合成牛血清白蛋白黄酮纳米核壳颗粒可以作为提高黄酮稳定性保护黄酮抗氧化活性实现黄酮较高生物利用度的一种新途径。      

小麦粉多酚氧化酶活性测定方法的改进

选取3个小麦粉作为试验材料,对国家标准征求意见稿《粮油检验小麦粉多酚氧化酶测定》的方法进行检验,发现该法因缺少PPO粗酶提取过程,参加反应的酶量较少,不能反映小麦粉真实的PPO活性;其次PPO与底物的反应温度不是最适宜的,导致酶反应不充分;第三酶反应后再离心,使得一部分酶反应产物被沉淀截留,导致检测数据小且不准确。文章在该基础上对其进行改进,考察浸提时间、最适反应温度、底物邻苯二酚添加时间对PPO活性检测的影响,优化该检验方法。结果表明:优化后的检验方法测得的结果较为理想,采用浸提4h、离心后添加邻苯二酚、反应温度70℃,测得的PPO活性数值大大提高,重复性好,稳定可靠。并采用改进后的方法检测了50种小麦粉,得出不同等级的小麦粉PPO的范围。     

Multispectroscopic studies on the interaction of maltol,a food additive,with bovine serum albumin

The interaction between maltol, a food additive, and bovine serum albumin (BSA) under simulated physiological conditions was investigated by fluorescence, UV–Vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The results suggested that the fluorescence quenching of BSA by maltol was a static procedure forming a maltol–BSA complex. The positive values of enthalpy change and entropy change indicated that hydrophobic interactions played a predominant role in the interaction of maltol with BSA. The competitive experiments of site markers revealed that the binding of maltol to BSA mainly took place in subdomain IIA (Sudlow site I). The binding distance between maltol and BSA was 3.01 nm based on the Förster theory of non-radioactive energy transfer. Moreover, the results of UV–Vis, synchronous fluorescence, CD and FT-IR spectra demonstrated that the microenvironment and the secondary structure of BSA were changed in the presence of maltol.     

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。     

An explanation for the combined effect of xylanase–glucose oxidase in dough systems

In the bakery industry, glucose oxidase is usually used in combination with xylanase. Although many theories exist on the mechanism of action of each enzyme, the positive effect of combining the two is as yet unexplained. In this paper we studied a possible basis for this synergy by focusing on the involvement of arabinoxylans. Water‐extractable arabinoxylans and water‐unextractable solids were studied in dough with or without glucose oxidase. Addition of glucose oxidase led to a stiffer and less extensible dough. Addition of arabinoxylans caused a further decrease in dough extensibility. Addition of xylanase could correct for this decrease. The role of arabinoxylans was further investigated by studying modified water‐extractable arabinoxylans and the effect of agents affecting arabinoxylan crosslinking (ferulic acid). Water‐extractable arabinoxylan was modified with xylanase to generate a low‐molecular‐weight fraction (WEAX_XYL). Alternatively, it was modified with NaOH to prepare a fraction with a decreased ferulic acid content (WEAX_OH). Addition of modified arabinoxylans diminished the effect of glucose oxidase. Glutenin macropolymer and water‐extractable arabinoxylan viscosity experiments were performed to obtain further detail on the role of arabinoxylans (AX). These results give rise to a new theory explaining the contribution of xylanase in its synergy with glucose oxidase. In this theory glucose oxidase not only catalyses the formation of protein disulfide bonds, but also of AX‐AX crosslinks. The latter negatively affect bread quality. Xylanase corrects for this latter effect by cleaving arabinoxylan complexes and generating small ferulic acid‐containing arabinoxylan fragments interfering with the crosslinking of high‐molecular weight arabinoxylans. Copyright © 2005 Society of Chemical Industry     

Inactivation kinetics of apple polyphenol oxidase in different pressure–temperature domains

The impact of high hydrostatic pressure and temperature on the stability of polyphenol oxidase (PPO) was studied in cloudy apple juice. Application of 200–500 MPa near room temperature or heat treatment at 45–55 °C at ambient pressure caused an increase of PPO activity of up to 65% in freshly squeezed apple juice. Combined pressure–temperature inactivation experiments with fully activated PPO (5 min treatment at 400 MPa and 20 °C) were carried out in the range of 0.1–700 MPa and 20–80 °C. Enzyme inactivation kinetics followed a 2.2 order reaction scheme at all pressure–temperature conditions tested. A polynomial model was successfully applied to describe the rate of PPO inactivation as a function of pressure and temperature and was used to construct a pressure–temperature isokinetic diagram. This diagram clearly showed synergistic effects of pressure and temperature on the inactivation of apple PPO at pressures above 300 MPa and antagonistic effects at lower pressures. Compared to ambient pressure conditions, temperatures required to inactivate PPO in apple juice were increased 10–15 °C at 100–300 MPa.

Industrial relevance

High pressure processing of fresh fruits is gaining popularity in the food industry because of its ability to inactivate microorganisms and some enzymes near room temperature with little impact on flavour or nutritional attributes of the food. However, quantitative data regarding the impact of process parameters on the target reaction are required to economically utilise this technology. This paper provides a mathematical model describing the combined effect of pressure, temperature and treatment time on the inactivation of PPO in cloudy apple juice.     

Investigation on the phase behaviour of gelatin/agarose mixture in an environment of reduced solvent quality

Investigation on the phase behaviour of a biopolymer mixture has been performed using 7.5% (w/w) gelatin and 1.5% (w/w) agarose in the presence of variable amounts of polydextrose as the co-solute from low to high levels of total solids. Mechanical observation of the system was performed using small deformation dynamic oscillation in shear along with thermal studies using modulated differential scanning calorimetry. Micrographs provided images of the changing morphology of the network with the addition of co-solute. Agarose and gelatin form non-interactive bicontinuous phases in the aqueous environment. Systematic increase in the concentration of polydextrose prevents the formation of a stable agarose network, with the polysaccharide chains dispersing in the high solids environment. Gelatin, on the other hand, retains its conformational stability even at a saturating co-solute environment through enhanced protein structuring. Vitrification studies on the high solids system at subzero temperatures provides information on the structural and molecular relaxation identified as a glass transition phenomenon. Fourier transform infrared spectroscopy was used to analyse potential direct interaction between polymers and co-solute. The extent of amorphicity in the system was confirmed using wide angle X-ray diffraction.     

The variation of superoxide dismutase (SOD) and xanthine oxidase (XO) activities in milk using an improved method to quantitate SOD activity

An improved method for determination of superoxide dismutase (SOD) in milk has been developed. The interference of endogenously occurring xanthine oxidase (XO) has been subs'antially reduced by addition of an ultrafiltration step, which removed 61 ± 10 (mean ± SD) % of the XO activity. This method was used to determine the SOD activity in milk serum from 15 Swedish Red and White (SRW) cows and 18 Swedish Friesian (SF) cows at various stages of lactation. The XO activity in raw milk was also determined. The milk from these cows had a mean SOD activity of 0.83 ± 1.14 U ml^?1 milk serum and a mean XO activity of 49 ± 22 mU ml^?1 raw milk (n = 127). A difference between breeds could be noted in XO activity, ie milk from SF cows had significantly (P < 0.05) higher mean XO levels compared with milk from SRW cows, 54 ± 23 mU ml^?1 than 44 ± 21 mU ml^?1. A significantly increased level of XO activity in milk from SRW cows during the lactation was found. No significant effect of breed or stage of lactation on SOD activity could be detected. Individual cows with high and low milk SOD levels during the entire lactation have been identified.     

一株产高活性多酚氧化酶菌株筛选鉴定及其酶学性质

采用变色圈法从土壤样品中分离筛选产多酚氧化酶的菌株,对其进行形态学观察、分子生物学鉴定,并对其所产多酚氧化酶的酶学性质进行研究。结果表明,筛选得到1株产多酚氧化酶活性较高的菌株,命名为ZL-2,其被鉴定为竹黄菌属（Shiraia sp.）。菌株ZL-2发酵8 d产多酚氧化酶酶活最高,达到521 U/mL。菌株ZL-2产多酚氧化酶最适底物为邻苯二酚,其最适pH值为6、最适温度为30 ℃;酶活在温度20～40 ℃及pH 3～6范围内稳定。金属离子Cu2+、Mg2+、Mn2+、Fe3+、Zn2+对多酚氧化酶都有激活作用,其中Cu2+激活作用最强;Ca2+、Al3+对酶活有一定抑制作用;L-抗坏血酸和亚硫酸氢钠对该酶抑制作用较强。     

Structure-affinity relationship of bovine serum albumin with dietary flavonoids with different C-ring substituents in the presence of Fe ion

The relationship of four dietary flavonoids, quercein (QU), luteolin (LU), taxifolin (TA) and (+)-catechin (CA), with different C-ring substituents binding with bovine serum albumin (BSA) have been investigated in the absence and presence of Fe³⁺ by means of spectroscopic methods. The quenching constant (K_SV), binding constant (K_a), binding site number (n) and spatial-distance (r) of flavonoids with BSA were calculated. The results indicated that hydroxyl group at 3-position and C2–C3 double bond affected the binding affinities between flavonoids and BSA. The values of the binding affinities were in the order: QU > CA > TA > LU. The presence of Fe³⁺ affected the interactions between flavonoids and BSA significantly, and the influence was distinct for the flavonoids with different C-ring structures. The binding affinities of QU, LU and CA for BSA were increased about 0.85%, 41.3% and 56.8% in the presence of Fe³⁺, probably because of the formation of QU–Fe³⁺–BSA complex, Fe³⁺–LU complex and the conformational change of BSA induced by Fe³⁺, respectively. The binding affinities of TA for BSA were decreased about 20.7%, mainly because of the existence of competitive binding between TA and Fe³⁺ with BSA. However, the quenching mechanism for QU, LU, TA and CA to BSA were based on static quenching combined with non-radiative energy transfer irrespective of the absence or presence of Fe³⁺ ion.     

一种多金属氧酸盐对多酚氧化酶的抑制活性评价及在南美白对虾保鲜上的应用

目的:研究新型多酚氧化酶抑制剂对南美白对虾的保鲜效果。方法:通过紫外和红外光谱表征合成的多酚氧化酶抑制剂H₈[P₂Mo₁₇Ni(OH₂)O₆₁](P₂Mo₁₇Ni),研究其对蘑菇多酚氧化酶(PPO)相对酶活的影响及抑制机制和抑制类型;再测定经P₂Mo₁₇Ni处理后的南美白对虾在贮藏过程中的感官评分、色差(L^*)值、总色差(ΔE)值、菌落总数、酸碱度(pH)值和总挥发性盐基氮(TVB-N)值的变化情况,评价P₂Mo₁₇Ni在南美白对虾保鲜上的作用。结果:P₂Mo₁₇Ni对体外蘑菇PPO的活性具有抑制作用,其半抑制浓度(IC₅₀)为(0.377±0.004) mmol/L,且对PPO的抑制模式为竞争型可逆抑制,抑制常数K_I为0.185 mmol...     

Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate

ABSTRACT:  Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 °C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.