Assessment on the fermentability of xylooligosaccharides from rice husks by probiotic bacteria

Liquors from rice husk autohydrolysis, containing xylooligosaccharides (XOS), other saccharides, and nonsaccharide compounds, were refined by membrane processing to increase the proportion of substituted XOS in refined liquors. XOS were assayed for composition and degree of polymerization (DP) distribution and hydrolyzed with commercial enzymes for obtaining XOS with DP in the range of 2-6. Nanofiltered, hydrolyzed liquors were subjected to ion exchange processing to yield a final product containing monosaccharides, XOS (accounting for 55.6% of the nonvolatile solutes), and other nonvolatile compounds. The solution obtained after enzymatic hydrolysis with commercial xylanases (in which 82.8% of XOS were in the DP range of 2-6) was examined as a medium for promoting the growth of Bifidobacterium adolescentis CECT 5781, B. longum CECT 4503, B. infantis CECT 4551, and B. breve CECT 4839. The growth rate of B. adolescentis (0.58 h(-1)) was higher than the ones determined for B. longum, B. infantis, and B. breve (0.37, 0.30, and 0.40 h(-1), respectively). The percentage of total XOS consumption by B. adolescentis was 77% after 24 h, the highest percentage of utilization corresponding to xylotriose (90%), followed by xylobiose (84%), xylotetraose (83%), and xylopentaose (71%).     

Production and characterization of films from cotton stalk xylan

Composite film production based on cotton stalk xylan was studied, and the mechanical and physical properties of the films formed were investigated. Xylan and lignin were separated from cellulose by alkali extraction and, then, lignin was removed using ethanol washing. Self-supporting continuous films could not be produced using pure cotton stalk xylan. However, film formation was achieved using 8-14% (w/w) xylan without complete removal of lignin during xylan isolation. Keeping about 1% lignin in xylan (w/w) was determined to be sufficient for film formation. Films were produced by casting the film-forming solutions, followed by solvent evaporation in a temperature (20 degrees C) and relative humidity (40%) controlled environment. The elastic modulus and hypothetical coating strength of the films obtained by using 8% xylan were significantly different from the ones containing 10-14% xylan. The water vapor transfer rates (WVTR) decreased with increasing xylan concentration, which made the films thicker. The glycerol addition as an additional plasticizer resulting in more stretchable films having higher WVTR and lower water solubility values. As a result, film production was successfully achieved from xylan, which was extracted from an agricultural waste (cotton stalk), and the film-forming effect of lignin on pure xylan has been demonstrated.     

Solid-state fermentation of soybean and corn processing coproducts for potential feed improvement

Two agro-industrial coproducts, soybean cotyledon fiber and distiller's dried grains with solubles (DDGS), were used as substrates to evaluate the effect of coculturing three different fungi, Aspergillus oryzae , Trichoderma reesei , and Phanerochaete chrysosporium , on enzyme production by solid-state fermentation (SSF). When soybean fiber was used as the substrate, a maximum xylanase activity of 757.4 IU/g and a cellulase activity of 3.2 IU/g were achieved with the inoculation and incubation of T. reesei and P. chrysosporium for 36 h, followed by A. oryzae for an additional 108 h. This inoculation scheme also resulted in the highest xylanase activity of 399.2 IU/g compared to other fungi combinations in the SSF of DDGS. A large-scale SSF by this fungus combination produced fermented products that had xylanase and cellulase activities of 35.9-57.0 and 0.4-1.2 IU/g, respectively. These products also had 3.5-15.1% lower fiber and 1.3-4.2% higher protein contents, suggesting a potential feed quality improvement.     

In vitro fermentability of differently substituted xylo-oligosaccharides

Xylo-oligosaccharides (XOS) with various substituents were fermented in vitro by fecal inocula (FI) from four human volunteers to study the influence of substitution on the ability and rate of fermentation and on the production of short-chain fatty acids (SCFA) and lactate. By all FI used nonsubstituted XOS (nXOS) and arabino-XOS (AXOS) were fermented more quickly than the more complex structures of acetylated XOS (AcXOS) and XOS containing a 4-O-methylglucuronic acid group (GlcA(me)XOS). In the first stage (0-40 h) of the fermentations of nXOS and AXOS mainly acetate and lactate were formed. The fermentations of AcXOS and GlcA(me)XOS resulted in a lower lactate production, whereas the concentration of propionate and butyrate increased. These results put emphasis on the detailed elucidation of the structural features of nondigestible oligosaccharides in general to understand their fermentation mechanisms more precisely.     

Enzymatic synthesis of aroma compound xylosides using transfer reaction by Trichoderma longibrachiatum xylanase

Enzymatic synthesis of aroma compound xylosides was performed by Trichoderma longibrachiatum xylanase. Information concerning the nature of xylosides present in the reaction medium was obtained by GC-EI-MS, by GC-NCI-MS of TFA derivatives, and by positive FAB-MS of the reaction mixtures. Moreover, the structures of isolated benzyl beta-D-xylopyranoside and 4-O-beta-xylopyranosyl-beta-D-xylopyranoside were established by (1)H and (13)C NMR and heteronuclear two-dimensional ((1)H-(13)C) chemical shift correlation. The results obtained for hexyl and benzyl alcohol xylosides indicated that a reaction implying a transfer of one to two or three xylose units from xylan was involved. The enzyme was able to recognize xylobiose, xylotriose, and xylan as xylose donors. Benzyl xyloside, produced independently of xylobioside and xylotrioside, was found as the major kinetic product of the reaction. Benzyl xyloside was produced in higher quantities and at a higher rate than that obtained for the di- and trixyloside derivatives. The maximum production for benzyl xyloside, 1.29 g/L, was obtained in the presence of hexane (50%) used as cosolvent. Xylosides and xylobiosides of several aroma compounds, (Z)-hex-3-en-1-ol, heptan-2-ol, geraniol, nerol, and citronellol, were synthesized in different amounts, from 850 mg/L for (Z)-hex-3-en-1-yl xylosides to 1.5 mg/L for citronellyl xylosides. No synthesis occurred when menthol, linalool, and eugenol were used as acceptors.     

Production of xylooligosaccharides from xylans by extracellular xylanases from Thermobifida fusca

Xylooligosaccharides are produced for use as a valuable food sweetener or additive. They have many beneficial biomedical and health effects. In this study, a process for producing xylooligosaccharides from lignocellulolytic agricultural waste was developed. Bagasse, corncob, wheat bran, and peanut shell were used as carbon sources for production of xylanolytic enzymes from Thermobifida fusca NTU22. When using bagasse as the carbon source, the xylanolytic enzymes that simultaneously accumulated in the broth in a 500 mL Hinton flask after 72 h of cultivation at 50 degrees C were measured as xylanase (14.0 U/mL), beta-xylosidase (74.1 mU/mL), and acetyl esterase (29.1 mU/mL). The optimum pH and temperature for xylanases were 6.0-8.0 and 70 degrees C, respectively. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gel. This was followed by heat treatment at 70 degrees C for 30 min that eliminated 90% of the beta-xylosidase activity. The xylanase and acetyl esterase activities were still 100%. Two percent of xylan extracted from the bagasse was then hydrolyzed by heat-treated crude xylanase preparation at 60 degrees C, pH 7.0, for 10 h. The xylooligosaccharides that accumulated in the broth were about 23.7%. After the purification process by activated charcoal chromatography, the purity of xylooligosaccharides was 71.4%.     

Synthesis of morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) by dipeptidyl aminopeptidase IV derived from Aspergillus oryzae

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)), tetrapeptide, was synthesized using dipeptidyl aminopeptidase IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was incubated with Phe-Pro-NH(2) in the presence of DP IV under various conditions of temperature, concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40% yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt.HCl and 20 mM Phe-Pro-NH(2).HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4 degrees C for 24 h. Amino group protection was unnecessary for synthesis of morphiceptin by DP IV.     

Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose

The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.     

Enrichment of cheeses manufactured from cow's and sheep's milk blends with sheep-like species-related alkylphenols

Enhancement of concentrations of species-related sheep-like alkylphenols, p- and m-cresols and 3- and 4-ethylphenols, in experimental Manchego-type cheeses manufactured from cow's and sheep's milk blends (80:20) by using arylsulfatases was investigated. A food-grade arylsulfatase from Aspergillus oryzae (ATCC 20719) was produced using a stimulatory medium, and crude dried cells were used as the enzyme source. Exogenous arylsulfatases from Helix pomatia and A. oryzae were added to cheese curd, and the amounts of species-related alkylphenols were measured. Arylsulfatase from H. pomatia released limited amounts of alkylphenols in the cheese only when used at a high level. Arylsulfatase from A. oryzae released substantial amounts of alkylphenols during 2 months of ripening. The concentrations of alkylphenols in A. oryzae arylsulfatase-treated cheese were comparable to the previously reported levels present in aged Manchego-type cheeses manufactured from pure sheep's milk.     

selective production and characterization of levan by Bacillus subtilis (Natto) Takahashi

To meet the industrial need of an efficient microbial method for increased levan production, Bacillus subtilis (natto) Takahashi, a commercial natto starter for preparing fermented soybeans (natto), was used to produce levan. After cultivation for 21 h, 40-50 mg of levan mL(-1) was produced in medium containing 20% (w/w) sucrose, which was approximately 50% yield on available fructose. The product consisted of two fractions with different molecular masses (1794 and 11 kDa), which were easily separated by fractionation using an ethanol gradient. The products were well characterized by GPC, 13C NMR, and 1H NMR. The various sugars and concentrations, initial pH, fermentation temperature, and agitation speed affected the levan production by B. subtilis (natto) Takahashi. Takahashi strain is the most efficient levan-producing strain among all of the B. subtilis strains tested and, as previously reported, it produced the highest yield of levan in the least time (21 h) under the common cultivation condition.     

Determination of glucosamine and N-acetyl glucosamine in fungal cell walls

A new method was developed to determine glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) in materials containing chitin and chitosan, such as fungal cell walls. It is based on two steps of hydrolysis with (i) concentrated sulfuric acid at low temperature and (ii) dilute sulfuric acid at high temperature, followed by one-step degradation with nitrous acid. In this process, chitin and chitosan are converted into anhydromannose and acetic acid. Anhydromannose represents the sum of GlcN and GlcNAc, whereas acetic acid is a marker for GlcNAc only. The method showed recovery of 90.1% of chitin and 85.7-92.4% of chitosan from commercial preparations. Furthermore, alkali insoluble material (AIM) from biomass of three strains of zygomycetes, Rhizopus oryzae, Mucor indicus, and Rhizomucor pusillus, was analyzed by this method. The glucosamine contents of AIM from R. oryzae and M. indicus were almost constant (41.7 +/- 2.2% and 42.0 +/- 1.7%, respectively), while in R. pusillus, it decreased from 40.0 to 30.0% during cultivation from 1 to 6 days. The GlcNAc content of AIM from R. oryzae and R. pusillus increased from 24.9 to 31.0% and from 36.3 to 50.8%, respectively, in 6 days, while it remained almost constant during the cultivation of M. indicus (23.5 +/- 0.8%).     

Enzymatic production of xylooligosaccharides from cotton stalks

Xylooligosaccharide (XO) production was performed from xylan, which was obtained by alkali extraction from cotton stalk, a major agricultural waste in Turkey. Enzymatic hydrolysis was selected to prevent byproduct formation such as xylose and furfural. Xylan was hydrolyzed using a commercial xylanase preparation, and the effects of pH, temperature, hydrolysis period, and substrate and enzyme concentrations on the XO yield and degree of polymerization (DP) were investigated. Cotton stalk contains about 21% xylan, the composition of which was determined as 84% xylose, 7% glucose, and 9% uronic acid after complete acid hydrolysis. XOs in the DP range of 2-7 (X6 approximately X5>X2>X3) were obtained with minor quantities of xylose in all of the hydrolysis conditions used. Although after 24 h of hydrolysis at 40 degrees C, the yield was about 53%, the XO production rate leveled off after 8-24 h of hydrolysis. XO yield was affected by all of the parameters investigated; however, none of them affected the DP of the end product significantly, except the hydrolysis period. Enzyme hydrolysis was maintained by the addition of fresh substrate after 72 h of hydrolysis, indicating the persistence of enzyme activity. The optimal hydrolysis conditions were determined as 40 degrees C, pH 5.4, and 2% xylan. The obtained product was fractionated via ultrafiltration by using 10, 3, and 1 kDa membranes. Complete removal of xylanase and unhydrolyzed xylan was achieved without losing any oligosaccharides having DP 5 or smaller by 10 kDa membrane. After a two-step membrane processing, a permeate containing mostly oligosaccharides was obtained.     

Occurrence of ochratoxin A- and aflatoxin B1-producing fungi in Lebanese grapes and ochratoxin a content in musts and finished wines during 2004

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 microg/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 microg/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 microg OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 microg OTA L(-1)).     

蒸汽爆破预处理和微生物发酵对玉米秸秆降解率的影响

为了提高玉米秸秆的利用效率,首先对玉米秸秆进行蒸汽爆破预处理（压力2.5 Mpa,维压200 s）,然后再进行米曲霉发酵,研究物理和生物学处理对秸秆成分及相关酶活变化的影响。结果表明,蒸汽爆破使秸秆中纤维素、半纤维素和木质素的降解率分别达到8.47%、50.45% 和36.65% （p＜0.05）。爆破预处理的秸秆再经米曲霉发酵6 d后,秸秆中纤维素和半纤维素的降解率分别为27.89%和64.80% （p＜0.05）,发酵秸秆中的滤纸酶、羧甲基纤维素酶、淀粉酶和蛋白酶活力分别达到335.10、1138.92、1954.20和201.99 U/g。爆破预处理后进行米曲霉发酵,对于提高玉米秸秆的降解率具有非常重要的意义。     

Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus

Several isoflavonoid phytoalexins produced by soybeans are known to be estrogenic, with potential beneficial health effects in humans. Increased production of phytoalexins by the soybean plant will facilitate research efforts in this area. In this study, phytoalexin induction and accumulation in soybean cotyledon tissue was observed using four species of Aspergillus: A. sojae, A. oryzae, A. niger, and A. flavus. All four Aspergillus species tested elicited phytoalexin accumulation in living soybean cotyledons. Results from a time course study indicated that maximum concentrations of the phytoalexin glyceollin, 955 microg/g fresh weight (fw), occurred at day 3 in soybean cotyledon tissue inoculated with A. sojae. Other Aspergillus species caused an accumulation of glyceollin at significantly lower levels. A maximum concentration of coumestrol of 27.2 microg/g fw was obtained from soybean cotyledons inoculated with A. niger. Soybean phytoalexins induced by food-grade A. sojae and A. oryzae allowed the collection of higher concentrations of phytoalexins for further examination in several in vitro and in vivo biological studies conducted to determine potential estrogenic activities.     

不同粮仓中稻谷优势霉菌的鉴定及差异性分析

为了研究稻谷中优势霉菌的种类、数量以及变化趋势,本试验以湖南省3个不同地区稻谷为研究对象,通过初步判断各粮仓优势霉菌,结合分子生物学方法对其ITS 序列进行分析,并对稻谷霉菌进行带菌量测定。结果表明,3个粮仓中,金牛仓稻谷中优势菌株分别为根霉、米曲霉、毛霉、黑曲霉、烟曲霉、黄曲霉、白曲霉、桔青霉;金山仓稻谷中优势菌株分别为根霉、米曲霉、毛霉、黑曲霉、黄曲霉、白曲霉、桔青霉;银光仓稻谷中优势菌株分别为根霉、米曲霉、毛霉、黑曲霉、黄曲霉。3个粮仓中根霉数量分布均表现为上层>下层>中层,米曲霉为中层>上层>下层,毛霉、黑曲霉、黄曲霉的分布为上层>中层>下层,同一霉菌在同一粮仓不同粮层间数量存在显著差异(P<0.05);随着储藏时间的延长,米曲霉、黑曲霉、黄曲霉数量逐渐减少,根霉、毛霉数量增加,同一霉菌在不同储藏时间霉菌数量存在差异显著(P<0.05)。本研究结果为稻谷安全储藏与霉菌防控提供了一定的理论依据。     

(+)-Catechin-aldehyde condensations: competition between acetaldehyde and glyoxylic acid

(+)-Catechin reaction with two aldehydes (acetaldehyde and glyoxylic acid) was studied in winelike model solution. The two aldehydes were reacted either individually or together with (+)-catechin and in molar excess. The reactions were followed by HLPC-UV and HPLC-ESI/MS to monitor (+)-catechin disappearance as well as dimer and polymer appearance. In all reactions a reaction order of close to 1 for (+)-catechin disappearance was observed. (+)-Catechin disappearance was slower in the presence of acetaldehyde (t(1/2) = 6.7 +/- 0.2 h) compared to glyoxylic acid (t(1/2) = 2.3 +/- 0.2 h). When the two aldehydes were reacted together, (+)-catechin disappearance was faster (t(1/2) = 2.2 +/- 0.5 h). When aldehydes were reacted separately, the dimer appearance was independent of the type of aldehyde used but the ethyl-bridged dimer disappearance was slower with acetaldehyde. When aldehydes were reacted together, the dimer appearance changed. Ethyl-bridged dimers appeared before carboxymethine-bridged dimers, and their disappearance occurred earlier. Copolymers containing both ethyl and carboxymethine bridges were also observed.     

Studies on the starch and hemicelluloses fractionated by graded ethanol precipitation from bamboo Phyllostachys bambusoides f. shouzhu Yi

Starch from bamboo Phyllostachys bambusoides f. shouzhu Yi evaluated by means of solid-state 13C CP/MAS NMR and X-ray diffraction showed a typical B-type pattern with a very low degree of crystallinity (10.9%). In addition to starch, alkali-soluble hemicelluloses were further fractionated by graded precipitation at ethanol concentrations of 0 (HA), 15, 30, 45, 60, and 75% (v/v). Chemical composition and structural features of the six hemicellulosic subfractions were investigated by a combination of sugar analysis, GPC, FT-IR, GC-MS, 1D (1H and 13C) and 2D (HSQC) NMR spectra, and thermal analysis. The results showed that the bamboo hemicelluloses were O-acetylated 4-O-methyl-glucuronoarabinoxylans (GAX) consisting of a linear (1→4)-β-D-xylopyranosyl backbone decorated with branches at O-3 of α-L-arabinofuranosyl (5-12 mol%) or at O-2 of 4-O-methylglucuronic acid units and acetyl groups (0.8-11 mol%). The molecular weights of these polysaccharides ranged between 13400 and 67500 g/mol, and the molar ratios of A/X and G/X increased with ascending ethanol concentrations. Moreover, xylo-oligosaccharides (XOS) with DP 1-6 were produced by enzymatic hydrolysis of hemicelluloses and the total yields of XOS were range of 21.5 to 40.6%. The structure-property relationships were also established in order to improve enzyme accessibility.     

碱浓度和温度对玉米秸秆木聚糖提取率和品质的影响

由于提取率和产品品质等方面的原因,作为玉米秸秆主要成分之一的木聚糖还未能得到充分有效的利用。为改善玉米秸秆木聚糖的提取率和品质,对影响木聚糖提取的碱浓度和温度2个关键因素进行了研究。使用组分分析和凝胶渗透色谱等手段对木聚糖得率、纯度和结构进行了研究,同时分离得到纤维素和醇溶木质素。结果表明,Na OH浓度对木聚糖得率和结构的影响比温度显著,较优提取条件为:Na OH浓度为10%(w/v),提取温度90℃,此条件下木聚糖的溶出率和回收率分别为86.37%和76.92%,木聚糖纯度为63.90%。凝胶渗透色谱结果显示,碱浓度的增加使木聚糖的分子量降低,而随温度的升高则呈先升后降的趋势。此外,提取剩余物中纤维素的含量随碱浓度和温度强度的增加而增加。研究证明组合适当的碱浓度和提取温度可以在保证品质的前提下得到较高的木聚糖提取率。