Biodegradation of cell wall components of maize stover colonized by white-rot fungi and resulting impact on in-vitro digestibility

Treatment of crop residues with some species of white-rot fungi can enhance digestibility. This study was conducted to investigate changes in in-vitro dry matter disappearance (IVDMD) and degradation of cell wall constituents in maize (Zea maize L) stover treated with three white-rot fungi: Cyathus stercoreus, Phlebia brevispora and Phanerochaete chrysosporium. Solid fermentation of maize stover for 28 days at 27°C improved IVDMD from 409 g kg^?1 (control) to 514 g kg^?1 for P brevispora and 523 g kg^?1 for C stercoreus. In contrast, growth of P chrysosporium reduced IVDMD from 409 to 298 g kg^?1. All fungi degraded cell wall p-coumaric acid (PCA) and ferulic acid (FA), but P chrysosporium was the least effective in degrading PCA and FA. Conversely, P chrysosporium degraded lignin 1·6 times more effectively than C stercoreus and 1·4 times more than P brevispora, indicating that lignin degradation alone cannot account for the IVDMD enhancement and that degradation of PCA and FA may be important. Hemicelluloses were preferentially and highly utilized by all the fungi. Cellulose was extensively degraded only by P chrysosporium (69% lost after 28 days of incubation), while substrate colonized by the other two fungi retained more than 84% original cellulose. Incubation of C stercoreus and P brevispora decreased the concentrations of both xylose and arabinose, but increased glucose concentration, whereas P chrysosporium removed less xylose and decreased glucose concentration. Preferential removal of arabinose over xylose by the fungi caused an increase in the xylose to arabinose ratio of the treated residues. Enhanced digestibility may have resulted from cleavage of lignin-carbohydrate bonds. Results of this study suggest that digestibility enhancement of maize stover colonized by white-rot fungi is regulated by a complex combination of various factors, including the degradation of structural carbohydrates, cell wall phenolic acids and lignin.     

Relationship between Phenolic Acids Formed During Rumen Degradation of Maize Samples andin vitroDigestibility

The organic matter (OMD) and cell wall (CWD) digestibility of stalks of 25 different maize samples were determined in an in vitro assay using rumen fluid. The stalk OMD and CWD varied between 62–80% and 44–62%, respectively. The free phenolic acids formed in the liquid phase during fermentation in in vitro incubations were assessed by HPLC and ¹H NMR. The following nine phenolic acids could be demonstrated to be present: p -hydroxybenzoic (HBA), p -hydroxyphenylacetic (HPAA), vanillic (VA), syringic (SYRA), 3-(4-hydroxyphenyl)propionic (HPPA), p -coumaric (PCA), ferulic (FA), phenylacetic (PAA) and 3-phenylpropionic (PPA) acids. Large variation was observed among the maize samples for all phenolic acids identified. The most abundant phenolic acids, PPA, PAA and HPAA were present in concentrations ranging from 0·57 to 1·41 mM, 0·32 to 0·46 mM, and 71 to 105 μM, respectively, and were present as free monomeric molecules. HBA concentrations ranged from 6·9 to 21·1 μM and this acid was also present as free molecules. The other phenolic acids could only be observed after NaOH treatment of the liquid phase and were detected in minor amounts (0 to 12 μM). Correlation studies revealed a negative relation between digestibility and NaOH labile esters in the medium (ie PCA, FA, VA and SYRA esters) and a positive relationship between digestibility and PPA and PAA. When path analysis was applied, an inverse effect of PCA esters in the medium on both OMD and CWD ( r =-0·71 and −0·56, respectively) was observed and a high positive effect of PPA acid on CWD ( r =+0·67). Principal component analysis revealed close associations between released phenolic acid esters, neutral detergent fibre, and cell wall esters of PCA and FA.     

化学机械法制浆废液中污染物质的白腐菌降解——第1部分 CTMP废液的污染负荷与白腐菌生化降解的探索研究

研究了马尾松-尾叶桉热磨化学机械法制浆废液的污染特性及其生化可降解性,探索了利用白腐菌降解废液中污染物质的可行性,分析了污染物质生化降解的过程及其影响因素。研究结果表明白腐菌能够适应热磨化学机械法制浆废液的水质条件,有效地降解导致耗氧量的污染物质,同时去除悬浮物并脱除色度。     

化学机械法制浆废液中污染物质的白腐菌降解 第2部分 无外加营养盐条件下污染物质的白腐菌生化降解

进行了白腐菌处理化学机械法制浆废液的工艺实验研究 ,包括利用白腐菌降解酶的作用去除污染物质与脱色的可行性、生化处理过程影响因素的分析。研究结果表明白腐菌具有降解废液中污染物质的能力。在无外加营养盐的条件下 ,白腐菌可以直接利用废液中存在的碳水化合物降解产物作为碳源生长 ,进而开始降解其它污染物质的生化过程     

果汁中9种酚酸类化合物的RP-HPLC-PAD分析

研究了同时测定5种果汁中没食子酸、儿茶酸、绿原酸、原儿茶酸、咖啡酸、表儿茶酸、阿魏酸、莽草酸和丁香酸等9种生物活性酚酸的反相高效液相色谱-光电二极管阵列检测新方法。采用ZorbaxEclipseXDB-C18色谱柱(5μm,250mm×4.6mmi.d.),流动相为甲醇-0.5%乙酸,梯度洗脱,流速1.0mL/min,以保留时间和特征光谱对分离出的组分予以定性确证,用峰面积进行定量。9种组分的质量浓度与其峰面积在一定的范围内有良好的线性关系,相关系数为0.9987～0.9999。9种组分的平均回收率为86.5%～98.8%(相对标准偏差为3.1%～5.8%)。检测限为0.2～1.0mg/L。结果表明,该方法也适用于其他复杂体系中没食子酸等9种生物活性成分的分析。     

Monitoring Phenolic Composition of Maturing Maize Stover by High Performance Liquid Chromatography and Pyrolysis/Gas Chromatography/Mass Spectrometry*

Maize stovers collected every 14 days over an 84-day growth period were subjected to high-performance liquid chromatography with electrochemical detector (HPLC-ED) and pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) in order to monitor changes in the phenolic composition. Prior to HPLC-ED analyses, ground samples were sequentially extracted with (i) methanol, (ii) 0·1M sodium hydroxide and (iii) 2M sodium hydroxide in the presence of nitrobenzene to separate, respectively, free phenolic monomers, alkali-labile phenolic monomers and alkali-resistant lignin. In turn, solution (ii) was treated with alkaline nitrobenzene to obtain (iv) alkali-labile lignin. Pyrolysis was carried out on ground native samples by using a platinum heated filament pyrolyser. Increases in the absolute phenolic concentrations in the residues of 0·1M sodium hydroxide extraction and in the ratio of alkali-resistant lignin vs total lignin were observed by HPLC-ED during the first 28–42 days of maturation, reaching a steady level in the remaining maturation period. A linear increase of syringyl units vs guaiacyl units was for found the alkali-resistant lignin fraction over the entire period of maturation. Similar trends were showed by PY/GC/MS with regard to relative lignin content and syringyl/guaiacyl ratio. Both techniques showed their usefulness to gauge changes in the phenolic composition during the lignification process.     

Structural changes in wheat straw components during decay by lignin-degrading white-rot fungi in relation to improvement of digestibility for ruminants

Structural changes and resulting in-vitro dry matter digestibility (IVDMD) were determined during the course of solid-state fermentation of wheat straw using the lignin-degrading white-rot fungi Sporotrichum pulverulentum, Pycnoporus cinnabarinus and Cyathus stercoreus. The first fungus grew very rapidly on straw but degraded hemicelluloses and cellulose non-selectively resulting in very low IVDMD increases (50% after 35% weight loss). P. cinnabarinus and C. stercoreus preferentially degraded hemicelluloses achieving high improvements in IVDMD (maximum increase of 63 and 94%, respectively) with limited dry weight losses (12 and 18% after 7 and 13 days, respectively). The three fungi exhibited some selectivity among the individual hemicellulose components: O-acetyls were removed essentially at the same rate as xylan, while uronic acids accumulated as incubation proceeded. Conversely, the arabinose content decreased rapidly, especially with C. stercoreus and P. cinnabarinus, suggesting that removal of this pentose was partly responsible for digestibility improvement. Esterified phenolic acids were rapidly degraded during the first stages of decay by all three fungi although P. cinnabarinus and C. stercoreus degraded ferulic acid faster than p-coumaric acid. Lignin was preferentially degraded compared to polysaccharides by all three fungi. The amount of lignin removed, as determined by Klason, correlated well with IVDMD improvement (r=?0.97), while acid detergent lignin (ADL) showed a lower correlation (r=-0.86). Acidolysis yields of decayed lignin pointed to preferential degradation of β-O-4 ether linked units by the fungi. Syringyl units were removed faster than guaiacyl units only after 5 to 10% weight loss was obtained.     

Chemical composition and biodegradability of crop residues colonized by white-rot fungi

Two experiments were conducted to study the degradation of rice (Oriza sativa L) straw and maize (Zea maize L) stover by selected white-rot fungi, using loss of organic matter, disappearance of cell wall constituents, and in-vitro dry matter digestibility (IVDMD) as indices of rumen microbial degradation. Four white-rot fungi—Cyrathus stercoreus, Dichomitus squalens and two strains (cellulase-less mutant and a wild type) of Phanerochaete chrysosporium—were selected for the first experiment. After 30 days of solid state fermentation. C stercoreus increased IVDMD of both substrates from 373 g kg^?1 (rice) and 393 g kg^?1 (corn) to approximately 540 g kg^?1 by selectively degrading hemicellulose as opposed to cellulose. The highest IVDMD was obtained with a minimal loss of dry matter (33 g kg^?1). In contrast, both strains (wild and mutant) of P chrysosporium degraded cellulose and hemicellulose indiscriminately, and thus lowered the IVDMD to 276. 257 as opposed to 373 for rice straw and 301. 307 as opposed to 393 g kg^?1 for maize stover. All the fungi showed a preference for degrading maize stover compared with rice straw, although the materials were similar in cellulose and lignin concentration. No direct correlation was found between lignin degradation and the improvement of substrate IVDMD of either substrate. The objective of the second experiment was to determine the rate and extent of digestibility of rice straw colonized by C stercoreus during two incubation periods (15 and 30 days). Dry matter disappearance (DMD) was determined by ruminal incubation of the substrate contained in nylon bags. The 30-day incubation period was superior (1.3 ×) to that of the 15-day period in improving in-situ DMD. Treatment of rice straw for 30 days not only improved the extent of dry matter digestibility (616 as opposed to 389 g kg^?1), but also increased (1.5 ×) the rate of disappearance of substrate.     

化学机械法制浆废液中污染物质的白腐菌降解 第3部分 白腐菌生化降解前后废液的气相色谱/质谱分析

在采用白腐菌处理化学机械法制浆废液探索研究的基础上，利用气相色谱／质谱联用检测技术，分析了生化处理前后废液中的有机污染物的相变化。研究结果表明，CTMP制浆废液中的毒性有机污染物主要为含有不同取代基的芳香族酚类，醇类、醛类、醚类、酯类和有机酸类等，预处理段废液和磨浆段废液相比，主要污染成分的相对含量存在着差异，经过白腐菌生化处理之后，废液中检测出来的污染物种类和含是均发生了明显变化，多种芳香族化合物的结构及其联接链发生断裂破坏，废液有机污染物的毒性显著降低。     

枳椇果梗不同类型酚酸含量及抗氧化活性

通过酸碱水解,有机溶剂萃取的方法制备枳椇果梗4种不同类型酚酸———游离酚酸(SFPA)、酯型酚酸(SEPA)、糖苷型酚酸(SGPA)、不溶性结合型酚酸(ISPA)。对各部分酚酸进行紫外可见光谱扫描确定其紫外吸收特性;以Folin-Ciocalteu法测定总酚酸含量,采用清除DPPH、ABTS+自由基和Fe3+还原法对不同类型酚酸体外抗氧化活性进行评价。结果表明:枳椇果梗不同类型酚酸均有特征紫外吸收;不同类型酚酸的总酚含量大小依次为:不溶性结合型酚酸﹥酯型酚酸﹥游离酚酸﹥糖苷型酚酸,不溶性结合型酚酸含量显著高于其他三类酚酸;在3种抗氧化体系中,不溶性结合型酚酸也表现出最强的抗氧化活性。     

Characterization of Enzyme Released Antioxidant Phenolic Acids and Xylooligosaccharides from Different Graminaceae or Poaceae Members

Xylooligosacchrides (XOS) and phenolic acids were simultaneously produced from hemicelluloses using crude enzyme mixture synthesized by a novel Bacillus subtilis KCX006. The strain synthesized XOS-forming endo-xylanase and de-branching α-L-arabinofuranosidase and esterase but not β-xylosidase. This enzyme mixture can improve the yield of XOS and phenolic acids from xylan due to the absence of hydrolysis of XOS by β-xylosidase and release of phenolic acids by esterase. Hence, the enzyme mixture was tested for simultaneous production of XOS and phenolic acids from xylan (28–35%)-rich Graminaceae or Poaceae plant biomass, such as wheat bran, sugarcane bagasse, bamboo, and rise husk. Hemicellulose fractions of the biomasses were extracted by alkaline treatment and hydrolyzed using crude xylanase mixture. The profiles of XOS and phenolic acids formed were analyzed by HPLC. The lyophilized hydrolytic products were further analysed by ¹H NMR to identify the substitutions in XOS. The observed profile of XOS and phenolic acids varied with the biomass sources. Maximum amounts of XOS (665.2 mg/g dwt) and phenolic acids (89.69 μg/g dwt) were produced from hemicellulose A fractions of sugarcane bagasse and bamboo bagasse, respectively. HPLC and ¹H NMR analysis of XOS revealed the formation of free- and arabino-XOS. Phenolic acids consisted of hydroxycinnamic and hydroxybenzoic acids and the antioxidant activity correlated well with hydroxycinnamic acids content. The crude enzyme of B. subtilis is useful to produce mixture of XOS and phenolic acids from biomass.     

苹果酒中酚酸、黄烷-3-醇的检测

以小国光(Ralls)和富士(Fuji)及其所酿制的新鲜苹果酒为试验材料,采用反相高效液相色谱法测定分析苹果原汁、发酵中酒样和苹果成品酒中11种酚酸、5种黄烷-3-醇的含量。结果表明:苹果和苹果酒中存在4种酚酸(原儿茶酸、绿原酸、咖啡酸、对-香豆酸)、2种黄烷-3-醇(儿茶素和表儿茶素);不同品种的苹果原汁和苹果成品酒中酚类物质的含量都存在显著差异。其中,小国光苹果和小国光苹果酒中的酚类物质总含量较高;对于每个品种,绿原酸都是最主要的酚酸类物质,含量最高的黄烷-3-醇类物质都是表儿茶素。随着发酵过程的进行,苹果酒中酚类物质的含量有不同程度的增加。其增加趋势为S型曲线,即先平缓再较快再平缓。     

Free Phenolic Acids in Shanxi Aged Vinegar: Changes During Aging and Synergistic Antioxidant Activities

Shanxi aged vinegar (SAV) is the most famous one among the four typical famous vinegars in China, which is produced by spontaneous solid-state fermentation using sorghum, bran, and millet chaff and Daqu. In this study, free phenolic acids (FPAs) species and changes during SAV aging, and the synergistic antioxidant activities between FPAs was investigated. The results revealed that eight FPAs, including protocatechuic acid, p-hydroxybenzoic acid, salicylic acid, dihydro sinapic acid, p-coumaric acid, sinapic acid, dihydro ferulic acid, and ferulic acid, stably existed during SAV 0–5 year aging periods, whereas during the aging time evaporation of water and FPAs combining with melanoidins made prediction of precise FPAs contents difficult. With regard to FPAs synergistic antioxidant activities, it was observed that the FPAs antioxidative interrelations were antagonism in antiradical activity experiments. And, only a few groups of FPAs mixtures showed antagonism in reduction experiments.     

Inhibition of Horse Bean and Germinated Barley Lipoxygenases by Some Phenolic Compounds

Phenolic compounds have been repeatedly implicated as potent antioxidants. Efficiency has been frequently estimated by radical scavenging activity and few reports have considered lipoxygenase (LOX) inhibition. Horse bean LOX was inhibited by a range of phenolic acids, gallates and flavonoids. All compounds tested were uncompetitive inhibitors with the exception of flavonol aglycons which were non competitive type. In each class of compounds, inhibition constants were strongly affected by structures. Inhibition patterns of (-)-epicatechin on germinated barley LOXs were detailed: (-)-epicatechin acted as an uncompetitive inhibitor while (-)-epicatechin reduced hydroperoxide formation by its radical scavenging activity and thus limited enzyme activation.     

高效液相色谱法测定大豆中游离氨基酸含量

以邻苯二甲醛和氯甲酸芴甲酯联合柱前衍生,反相C18短柱分离,二元梯度洗脱,二极管阵列检测器（用于检测胱氨酸和赖氨酸）和荧光检测器（用于检测其他氨基酸）联合检测,建立了反相高效液相色谱法测定大豆种质资源中20种游离氨基酸含量。各氨基酸浓度在5～900μmol／L范围内线性良好,相关系数为0．9991以上。检出限低,灵敏度高。以5％三氯乙酸为提取溶剂,游离氨基酸的提取效果好,标准添加回收率为96．32％～102．25％,相对标准偏差为1．56％～4．30％（n=6）。本方法可用于大豆及其它谷物中游离氨基酸含量的测定。     

苦荞中酚酸和原花色素的分析测定

采用了反相液相色谱法 (RP HPLC) ,结合二极管阵列检测器 (DAD)分析 ,测定了苦荞籽粒各部分 (壳、麸皮、外层粉、内层粉 )的酚酸和原花色素。测定结果表明 ,苦荞中的酚酸种类主要是苯甲酸类———原儿茶酸和对羟基苯甲酸 ;酚酸总量在 94.61～ 1745 .3 3mg/kg。原花色素含量在 0 .0 3 %～ 5 .0 3 %。     

苦丁茶中酚酸类化合物的HPCE分离测定

通过建立了高效毛细管电泳法同时分离测定苦丁茶中山奈酸、芦丁、没食子酸、原儿茶酸、咖啡酸、没食子酸、原儿茶酸等7种酚酸类物质含量的方法。对缓冲液离子浓度和pH、分离电压和毛细管温度等电泳分离条件进行了优化,结果发现在柱温25℃、电压20kV、20mmol/L pH7.5磷酸二氢钾-硼砂缓冲液的电泳条件下,可在13min内实现了7种酚酸类物质的分离检测。本方法具有较高的灵敏度,呈现较好的线性关系,r为0.9982~0.9996,迁移时间重现性为0.1557%~0.1664%,峰面积重现性为1.080%~3.466%,回收率为80.14%~93.24%。本研究采用建立好的方法测定了不同产区苦丁茶中7种酚酸类化合物的含量。     

Determination of phenolic acids in plant cell walls by microwave digestion

Microwave digestion (750 W for 90 s) with 4 M NaOH was used to release esterified and etherified hydroxycinnamic acids from cell walls of maize (Zea mays L), wheat (Triticum aestivum L), barley (Hordeum vulgare L) and oilseed rape (Brassica napus L) stems. Subtraction of values for saponifiable phenolic acids obtained after treatment with I M NaOH at room temperature from digest results provided a measure of β-ether linked units. These were exclusively (E + Z)-ferulic acid in the cereal straws. Only trace amounts of ether- and ester-linked hydroxycinnamic acids were released from the dicotyledon, ripe straw. Microwave digestion was shown to be an order of magnitude more effective than dioxane-HCl at liberating β-ether bound phenolic acids and as effective, but substantially quicker, than previously described high-temperature alkaline digestions.     

铜皂比色法测定稻谷中脂肪酸的研究

介绍一种简单快速的比色法测定稻谷中脂肪酸含量。采用异辛烷作为提取溶剂,醋酸铜-吡啶溶液作为显色指示剂。该法具有直观准确、易于操作的特点。     

GC-FID测定黄酒中17种游离氨基酸

建立以N-(特丁基二甲基硅烷)-N-甲基三氟乙酰胺(MTBSTFA)为衍生试剂测定黄酒中17种游离氨基酸的气相色谱(GC)方法。比较了衍生温度、时间、及辅助试剂等对衍生效率的影响,确定N,N-二甲基甲酰胺(DMF)为辅助衍生试剂,衍生温度80℃,衍生时间60 min,可以定量检测黄酒中主要的游离氨基酸。各氨基酸在50～500 mg/L内线性良好(相关系数均大于0.99)。对黄酒样品的测定结果与高效液相色谱测定结果相比皮尔逊相关系数为0.998,样品的平均加标回收率为87.11%～119.47%。结果表明:GC-FID方法操作简便、准确、重现性好,适用于黄酒中17种游离氨基酸的分离检测。该测定方法将为开发气相-燃烧-同位素比率质谱(GC-C-IRMS)测定黄酒中氨基酸13C稳定同位素丰度以及分析黄酒酿造过程中的代谢实验提供研究基础。