Extraction and Assay of Acetic Acid Esterase From Malted Barley*

The extraction and the assay conditions for acetic acid esterase from barley malt have been optimised. An assay method was developed using diacetin as a substrate. The presence of reduced glutathione and the detergent Triton‐X‐100 in the extraction medium improved the yield of enzyme. The optimal pH for extraction was 8.0. When malt extracts were held at various temperatures acetic acid esterase was denatured at temperatures above 30°C. The optimum pH when measuring activity was 7.0. The crude enzyme extract released acetic acid from both soluble and insoluble cell wall materials. An insoluble, active form of the enzyme remained in the residual grain solids after the soluble form had been extracted.     

Partial Purification of Ferulic Acid Esterase from Malted Barley

A partial purification of ferulic acid esterase, which degrades feruloyl glycerol, has been achieved from barley malt in small yields. Coloured and viscous materials were removed from the malt extract using batch‐elution anion exchange chromatography. Further steps included gradient elution anion exchange chromatography and gel filtration chromatography. Estimations of the molecular weight varied greatly from 22KDa to 158 KDa, possibly because the protein interacted with the matrix of the gel exclusion chromatography column and because multiple forms of the enzyme were present. The partially purified feruloyl esterase had an apparent Km of 0.46% feruloyl glycerol. However more than one enzyme may be present and the substrate contains two isomers and so Michelis‐Menten kinetics may not be appropriate.     

Hydrolysis of Brewers' Spent Grain by Carbohydrate Degrading Enzymes

In this work four commercial cellulase‐hemicellulase mixtures with different activity profiles were used for solubilization of carbohydrates from brewers' spent grain (BSG). After the enzyme treatment, both the solubilised fraction and the unhydrolysed residue were characterized. Treatment with 5,000 nkat/g xylanase for 5 h at 50°C resulted in the solubilisation of 13–14% of the BSG dry weight as monosaccharides. This corresponded to the solubilisation of 26–28% of the original carbohydrates and 30–34% of original arabinoxylans, depending on the enzyme cocktail used. The relatively low hydrolysis level indicates that the majority of the BSG biomass is rather recalcitrant towards the cellulose‐hemicellulase enzyme mixtures applied in this study. The enzyme activity profile had a crucial impact on the chemistry of the oligosaccharides produced through the solubilisation of BSG. The presence of feruloyl esterase (FAE) activity in the enzyme cocktail resulted in the production of free ferulic acid, arabinoxylo‐oligosaccharides and their corresponding monomers. However, when the enzyme mixture was devoid of FAE activity, ferulic acid was still bound to the oligosaccharides. The unhydrolysed fraction was still found to contain over 40% of carbohydrates after enzymatic treatment despite the extensive enzyme dosages used. The protein fraction remained largely unaffected (i.e. insoluble) by the carbohydrate‐disrupting enzyme treatments. In addition to the recalcitrant carbohydrates, the residue was enriched with lignin and lipid type structures.     

产阿魏酸酯酶菌株的筛选及其酚酸释放研究

本文采用平板筛菌和牛津杯透明圈试验法筛选具有产阿魏酸酯酶的菌株,将其接种于麸皮中进行固态发酵,采用分光光度法和HPLC法分析发酵麸皮其阿魏酸酯酶活力、总酚酸以及阿魏酸的释放量,并进行个体形态、菌落特征和TIS测序鉴定,结果表明:筛选出一株菌株的阿魏酸酯酶酶活优于其他菌株,在发酵第4 d该菌株的阿魏酸酯酶酶活达最高为163.5 m U/g;而且发酵麸皮释放总酚酸的能力较高,将麸皮的总酚酸量提高6.6倍,其中阿魏酸的量达到0.697%,比没有发酵的麸皮阿魏酸的含量增加了0.22%。对其进行分子鉴定,该菌株与烟曲霉(Aspergillus clavatus)同源性达100%,确认为烟曲霉命名为烟青。将该菌应用于麸皮发酵提高阿魏酸含量,是农副产品增值利用以及药用功能开发的主要研究方向。     

The Use of an Extracellular Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids During Mashing

A purified extracellular ferulic acid esterase from Lactobacillus acidophilus K1 was successfully used during mashing for the release of free phenolic acids into sweet wort. The enzyme was produced in bioreactors and partially purified to obtain the monoenzyme preparation. Release of free ferulic and vanillic acid into the wort at 52°C (with the use of 4.09–14.60 enzyme activity units/L of the mash) and ferulic acid at 62°C (14.60 units/L) was observed. Free p‐OH‐benzoic and syringic acids were effectively released at 26°C at each enzyme concentration used. Free p‐OH‐benzoic acid was also released by the enzyme (14.60 U/L) at 52°C‐74°C. Free protocatechuic acid was effectively hydrolyzed by the enzyme preparation (8.75 U/L and 14.60 U/L) at 26°C‐52°C. Free caffeic acid (effectively released at 26°C‐62°C) originated from chlorogenic acid. No p‐coumaric acid was released due to the action of bacterial esterase during mashing. Ferulic acid esterase from L. acidophilus K1 exhibited no ability to release free phenolic acids during mashing at 62°C or at 74°C due to its low thermostability. In conclusion, L. acidophilus K1 is an attractive source for the production of ferulic acid esterase, which can be useful for the release of antioxidant phenolic acids in the early stages of mashing.     

Use of Xylose Dehydrogenase from Trichoderma viride in an Enzymic Method for the Measurement of Pentosan in Barley

A xylose dehydrogenase has been isolated from Trichoderma viride. It has been used as part of an enzyme‐based assay to measure pentosan in barley. Pentosan is extracted with perchloric acid and then hydrolyzed by a cocktail of enzymes including xylanase, arabinofuranosidase, feruloyl esterase and acetyl esterase. Some 92% conversion to xylose was obtained, and this is measured in turn using a xylose dehydrogenase‐based assay mixture involving NAD‐dependent reduction of phenazine methosulphate monitored from absorbance increase at 585 nm. The xylose dehydrogenase is somewhat unstable and this will need to be enhanced for the assay to be functional.     

阿魏酸酯酶的制备

阿魏酸酯酶是一种新型酶制剂,在食品、饲料和造纸工业中都有着广阔的应用前景,但目前还没有工业化产品。文中研究了超滤和冷冻干燥制备阿魏酸酯酶的工艺条件,结果表明,酶液pH值为8.06时有利于酶蛋白截留;装液厚度为6mm时,酶粉剂冷冻干燥时间为24h;添加1%～2%的聚乙二醇可以保护酶液在冷冻干燥过程中不失活;阿魏酸酯酶的液体和固体产品在保存过程中均表现出了较好的稳定性。     

The Use of a Novel Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids from Brewer's Spent Grain

In this work, an extracellular ferulic acid esterase was produced in bioreactor cultivations of Lactobacillus acidophilus K1 strain. The enzyme was partially purified using ultrafiltration (10 kDa), dialysis (4–6 kDa) and Fast Protein Liquid Chromatography (Sepharose CM, Sephacryl S‐300). A considerable increment of enzyme activity (31‐fold) in the final preparation was achieved. Two distinct bands (approx. 21.5 kDa and 39 kDa) were obtained after SDS‐PAGE. A high similarity of the purified enzyme (LC‐MS/MS analysis) to tannase and ferulic acid esterase from Burkholderia ambifaria MEX‐5 was obtained. The optimal pH and temperature for the enzyme activity were 6.3 and 37°C, respectively. The enzyme preparation effectively released phenolic acids (mainly ferulic and p‐coumaric acid) from brewer's spent grain. This novel enzyme preparation can be used for the utilisation of a valuable and inexpensive by‐product of the brewing industry.     

Exposure or release of ferulic acid from wheat aleurone: Impact on its antioxidant capacity

The relationship between the aleurone cell integrity and the exposure or release of bioavailable ferulic acid (FA) with the antioxidant capacity of aleurone in in vitro and under simulated gastric conditions was explored. The antioxidant capacity of aleurone was increased by around 2-fold when its median particle size was reduced to under 50 μm. The opening of aleurone cells increased the physical exposure of FA bound to the insoluble polysaccharides, which seemed to be responsible of the increased antioxidant capacity. Synergistic combination of xylanase and feruloyl esterase was found to be the most efficient enzymatic treatment releasing up to 86% of total FA in bioaccessible forms. This enzymatic treatment significantly enhanced the radical scavenging activity of aleurone by up to 4-fold, which overlapped the overall antioxidant potential estimated from the total content of FA in aleurone. The improvement in the antioxidant capacity of aleurone was also observed in the simulated gastric digestion by inhibition of lipid oxidation.     

Purification and Characteristics of Feruloyl Esterase from Aspergillus awamori G-2 Strain

ABSTRACT:  For food industry production processes and other uses, a mold that produces high levels of feruloyl esterase was obtained from laboratory mold collections and other sources. It was Aspergillus awamori G-2 that produces high levels of feruloyl esterase. The feruloyl esterase was purified using ion-exchange chromatography, size-exclusion chromatography, and HPLC chromatography. The enzyme was identified as a monomer protein using size-exclusion chromatography. Its optimum temperature and pH were, respectively, 40 °C and pH 5. Its activity was stable at pH 3 to 5. The enzyme was combined with xylan and starch, but it was absorbed by cellulose. The km of the feruloyl esterase was 0.0019% (0.01 mM). The enzyme showed stable activity at pH 3 and 50 °C, making this enzyme useful for food production.     

核桃内种皮不同形态酚酸类化合物的溶剂提取率比较

该文研究溶剂对核桃内种皮中不同形态酚酸类化合物的提取率,建立了适用于核桃内种皮中酚酸类化合物的提取方法.以新疆核桃为原料,比较不同比例的甲醇、乙醇溶液对核桃内种皮中可溶性游离态、可溶性酯化态和不溶性结合态酚酸的提取率.结果表明,50％(体积分数,下同)甲醇适合提取可溶性游离态绿原酸、没食子酸;50％(体积分数,下同)乙...     

HPLC–DAD–ESI–MS Analysis of Phenolic Compounds During Ripening in Exocarp and Mesocarp of Tomato Fruit

Identification of phenolic compounds was done by means of liquid chromatography (HPLC) coupled to mass spectrometry (MS) using the electrospray ionization interface (ESI). Quantification of phenolic compounds was carried out by using HPLC with diode array detector (DAD) in exocarp and mesocarp of tomato fruit at 6 different ripeness stages (mature‐green, breakers, turning, pink, light‐red, and red). Several phenolic compounds were identified including chlorogenic acid, caffeic acid, p‐coumaric acid, ferulic acid, and rutin and some combined phenolic acids were tentatively identified, mainly glycosides, such as caffeoyl hexose I, caffeoyl hexose II, caffeoylquinic acid isomer, dicaffeoylquinic acid, p‐coumaroyl hexose I, p‐coumaroyl hexose II, feruloyl hexose I, feruloyl hexose II, siringyl hexose, and caffeoyl deoxyhexose hexose. Fruit exocarp had higher quantities of total soluble phenolics (TSP) compared to mesocarp. During ripening, TSP increased in both exocarp and mesocarp, mainly in exocarp. While rutin increased, chlorogenic acid decreased in both tissues: exocarp and mesocarp.     

小麦与小麦芽阿魏酸酯酶酶活力的测定及其酶学性质研究

以阿魏酸乙酯作为底物,确定了阿魏酸酯酶的酶解工艺条件:酶解时间100 min;酶液与底物的体积比为0.75;提取液pH值6.2。针对阿魏酸酯酶酶学性质的研究表明:最适温度60℃,30～35℃范围内热稳定性较强,保温60 min后其活力可保持在80%以上;最适反应pH值5.0,在pH值5.0～5.6范围内对阿魏酸酯酶的破坏力相对较小,保温60 min其活力仍可保持在80%以上;Cu2+和Zn2+对阿魏酸酯酶有强烈的抑制作用,而ED-TA则对其有明显的促进作用。对5种小麦及其麦芽的阿魏酸酯酶酶活力进行测定:麦芽中的阿魏酸酯酶酶活均高于小麦,其中以烟2415、鲁麦21、郑麦004变化较大。     

玉米芯功能性低聚糖分级纯化及抗氧化活性研究

探究玉米芯功能性低聚糖分级纯化前后不同组分的组成、理化性质和抗氧化活性。以玉米芯为原料,采用活性炭柱层析法对玉米芯功能性低聚糖进行分级纯化,对纯化前后组分的聚合度、相对分子质量、阿魏酸基团以及红外光谱特性进行分析,并对酶解低聚糖粗提物(EH)及其纯化组分的自由基清除能力和抑制脂质过氧化能力进行比较分析。结果表明：活性炭水洗脱低聚糖组分(WO)、醇洗脱低聚糖组分(EO)均主要由木糖和阿拉伯糖组成,其中WO含有结合态阿魏酸,为阿魏酰阿拉伯低聚木糖;EO不含结合态阿魏酸,为阿拉伯低聚木糖。EH及其纯化组分WO、EO均具有一定的清除自由基(DPPH·、·OH、ABTS⁺·)能力和抑制脂质过氧化能力,且随浓度的增加而增加,WO抗氧化活性最强,EH次之,EO抗氧化活性较弱。     

深绿木霉嗜酸性阿魏酸酯酶酶学性质及生物质转化分析

为实现生物质的高效降解,制备具有重要生理功能的阿魏酸,本实验从深绿木霉XS6发酵液中分离纯化深绿木霉阿魏酸酯酶（Trichoderma atroatroviride feruloyl esterase,TaFAE）,并研究酶学特性。发酵液经(NH4)2SO4沉淀、DEAE-Cellulose DE52阴离子交换层析和Sephadex G-200凝胶过滤层析后,得到电泳纯的TaFAE,比活力134.3 U/mg,回收率28.9%,纯化倍数31.52。经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得酶分子质量约为66.4 kDa。TaFAE对阿魏酸甲酯亲和力最高,以其为底物,酶Km值和Vmax值分别为0.53 mmol/L和16.74 μmol/（min·mg）。以芥子酸甲酯为底物,酶促反应速率最大,达到32.21 μmol/（min·mg）,是阿魏酸甲酯的2 倍,对咖啡酸甲酯无活性,说明该酶具有严格的底物特异性。TaFAE最适pH值和温度分别为pH 4.0和40 ℃。在pH 2.0～9.0的范围内,30～40 ℃温度下都表现出良好的稳定性。金属离子Ca2＋和Mg2＋对TaFAE活性有显著激活作用,重金属离子Hg2＋和Pb2＋几乎完全抑制酶活性;β-巯基乙醇、二硫苏糖醇、十二烷基硫酸钠、Trition X-100增强酶活性,苯甲基磺酰氟有较强的抑制作用。木聚糖酶降解生物质的体系中加入TaFAE可显著增加阿魏酸和还原糖产量,表明TaFAE和木聚糖酶有良好协同作用。综上所述,TaFAE优良的耐酸性及其他酶学性质说明其在食品和饲料行业有较好的应用潜力。     

Solubilization and Activity Detection in Polyacrylamide Gels of a Membrane‐Bound Esterase from an Oenological Strain of Saccharomyces cerevisiae

Due to the importance of esters in determining wine aroma, the presence of different esterases in soluble and insoluble cell fractions of an oenological strain of Saccharomyces cerevisiae was studied. Cells of an oenological yeast strain were separated into three fractions corresponding to the cytoplasm, periplasm and plasma membrane, all showing esterase activity. The conditions for optimal solubilization of the membrane‐bound esterase were found, as well as those allowing its electrophoretic analysis, using the detergent disodium‐n‐lauryl‐β‐iminodipropionate (Deriphat) in the cathodic buffer. Comparison of the activity bands detected on gels revealed the presence of different esterase iso‐forms in the three sub‐cellular fractions. The method can also be used as the first electrophoretic step in two‐dimensional PAGE. Therefore, the procedures described are also a promising tool for the study of the yeast enzymes in relation to their effects during winemaking.     

Characterization of Acetylxylan Esterase from White-Rot Fungus Irpex lacteus

The carbohydrate esterase family 1 (CE1) in CAZy contains acetylxylan esterases (AXEs) and feruloyl esterases (FAEs). Here we cloned a gene coding for an AXE belonging to CE1 from Irpex lacteus (IlAXE1). IlAXE1 was heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. IlAXE1 hydrolyzed p-nitrophenyl acetate, α-naphthyl acetate and 4-methylumbelliferyl acetate, however, it did not show any activity on ethyl ferulate and methyl p-coumarate. We also examined the activity on partially acetylated and feruloylated xylan extracted from corncob by hydrothermal reaction. Similarly, ferulic and p-coumaric acids were not liberated, and acetic acid was only detected in the reaction mixture. The results indicated that IlAXE1 is an acetylxylan esterase actually reacted to acetyl xylan. However, since IlAXE1 was unable to completely release acetic acid esterifying xylopyranosyl residues, it is assumed that acetyl groups exhibiting resistance to deacetylation by IlAXE1 are present in corn cob xylan.     

Characterisation of black cumin,pomegranate and flaxseed meals as sources of phenolic acids

The article focuses on the extraction of ten phenolic acids from black cumin (Nigella sativa L.), pomegranate (Punica granatum L.) and flax (Linum usitatissimum L.) seed meals. The extracts have been fractionated as free, esterified and insoluble‐bound phenolic compounds and quantitatively determined by HPLC–PDA. The analysed meals can be utilised for obtaining valuable phenolic acids. However, the distribution of phenolic compounds varies depending on the meal source. The insoluble‐bound fraction has been the richest for the black cumin meal, both qualitatively and quantitatively, containing all ten analysed phenolics. In the pomegranate meal, the main phenolic has been gallic acid, accounting for nearly 48% in free form. The esterified form of the flaxseed meal has been abundant with ferulic (1025.44 ± 3.99 mg kg^?1 dry weight), caffeic and p‐coumaric acids. The total amount of phenolic acids would be underestimated if only free fractions would be taken into account, while neglecting esterified (for the pomegranate and flax meals) and insoluble‐bound fractions (for the black cumin and pomegranate meals).     

高产阿魏酸酯酶菌株的筛选鉴定及产酶条件优化

为提高白酒酿造过程中阿魏酸的含量,该研究从浓香型白酒大曲中分离筛选产阿魏酸酯酶的菌株,通过形态观察、生理生化试验及分子生物学技术对其进行菌种鉴定,并以阿魏酸酯酶活力为评价指标,采用单因素试验及响应面试验对其发酵条件进行优化。结果表明,筛选得到一株产阿魏酸酯酶的菌株,编号为M2。经鉴定,其为一株蜡样芽孢杆菌（Bacillus cereus）,该菌株产阿魏酸酯酶的最佳发酵条件为接种量5%,发酵温度30 ℃,初始pH值5.5,发酵时间72 h。在此优化条件下,阿魏酸酯酶活力为33.89 U/L,比优化前提高10.03%,具有较强的产阿魏酸酯酶能力。     

Antioxidant Properties of Free,Soluble Ester and Insoluble‐Bound Phenolic Compounds in Different Barley Varieties and Corresponding Malts

Ten different barley cultivars and their corresponding malts were used to obtain different fractions. Phenolics extracted belonged to free, soluble esters and insoluble‐bound fractions. Total phenolic content (TPC) of the free fraction, as measured according to the Folin‐Ciocalteu method, ranged from 37.7 to 167.2 mg gallic acid equiv/kg of dried material (GAE/kg_dw) for barley and between 34.1 and 72.3 mg GAE/kg_dw for malt. The bound phenolic content ranged from 210.3 to 320.5 and between 81.1 and 234.9 mg GAE/kg_dw for barley and malt, respectively. The contribution of bound phenolics to the TPC was significantly higher than that of free and esterified fractions. Catechin and ferulic acid, quantified by high performance liquid chromatography with diode array detector (HPLC‐DAD), were the most abundant phenolics in the free and bound fractions, respectively. The p‐coumaric acid content was lower in hulless genotypes, as compared to hulled genotypes, showing that it is mainly concentrated in the hull. The antioxidant activities of the phenolic fractions were investigated using the radical scavenging assay (DPPH) and ferricyanide reducing power. The bound phenolics demonstrated a significantly higher antioxidant capacity compared to the free and esterified phenolics. During the malting process, a significant decrease of the bound phenolics was observed with a corresponding increase of the esterified fraction.