Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

ENZYMATIC PROCESSING OF CRUDE XYLOOLIGOMER SOLUTIONS OBTAINED BY AUTOHYDROLYSIS OF EUCALYPTUS WOOD

Liquors from the aqueous processing of Eucalyptus wood, containing soluble xylan-degradation products (mainly substituted xylooligomers) were subjected to the action of commercial enzyme complexes (Celluclast, Bioxilanasa-AXC, Econase^R HC 400, Cellulase T 4 and Hemicellulase 90) containing endo-xylanase, xylobiase and acetyl-xylan-esterase activities to assess their suitability to obtain either xylose-containing solutions (useful as fermentation media) or low-DP xylooligosaccharides (useful as food additives). Under selected conditions, the Celluclast, Bioxilanasa-AXC, Econase^R HC 400 and Cellulase T 4 complexes were able to convert into xylose up to 90% of the substituted xylooligosaccharides present in the autohydrolysis liquors. The enzyme Hemicellulase 90 allowed the conversion of up to 67% of the initial substituted xylooligosaccharides into low-DP oligosaccharides. The most remarkable finding was that a significant concentration of DP2 oligomers (that could be incorporated into functional foods as prebiotic ingredients) was achieved at a short reaction time.     

ENZYMATIC PROCESSING OF CRUDE XYLOOLIGOMER SOLUTIONS OBTAINED BY AUTOHYDROLYSIS OF EUCALYPTUS WOOD

ABSTRACT

Liquors from the aqueous processing of Eucalyptus wood, containing soluble xylan-degradation products (mainly substituted xylooligomers) were subjected to the action of commercial enzyme complexes (Celluclast, Bioxilanasa-AXC, Econase^R HC 400, Cellulase T 4 and Hemicellulase 90) containing endo-xylanase, xylobiase and acetyl-xylan-esterase activities to assess their suitability to obtain either xylose-containing solutions (useful as fermentation media) or low-DP xylooligosaccharides (useful as food additives). Under selected conditions, the Celluclast, Bioxilanasa-AXC, Econase^R HC 400 and Cellulase T 4 complexes were able to convert into xylose up to 90% of the substituted xylooligosaccharides present in the autohydrolysis liquors. The enzyme Hemicellulase 90 allowed the conversion of up to 67% of the initial substituted xylooligosaccharides into low-DP oligosaccharides. The most remarkable finding was that a significant concentration of DP2 oligomers (that could be incorporated into functional foods as prebiotic ingredients) was achieved at a short reaction time.     

Arabinose-Containing Oligosaccharides from Tamarind Xyloglucan

Oligosaccharide mixtures were obtained by enzymatic degradation of tamarind xyloglucan from the seeds of Tamarindus indica by commercial Aspergillus niger endo-(1→4)-β-D -glucanase, and their composition analyzed by HPAE-PAD liquid chromatography revealing two major groups of oligosaccharides. The first group contained oligomers of DP 7, 8 and 9, while the second group consisted of a large variety of oligomers of DP 12 to 18. After purification by SEC and HPLC in semi-preparative scale, single oligomers were characterized by glycosyl-residue composition, glycosyl-linkage analysis and plasma desorption mass spectrometry. Beside β-D -Glucosyl, β-D -galactosyl and α-D -xylosyl units, some higher oligomers contained β-D -galactosyl-(1→5)-α-L -arabinosyl units, linked to O-6 in the glucosyl units of the (1→4)-β-D -glucan backbone. Other L -arabinosyl residues were located in terminal positions of α-D -xylosyl side chains. The variety of isolated oligomers reflected a complicated random distribution of side chains in tamarind xyloglucan. Based on the characteristics of the oligomers, four structure forming units for the polysaccharide are proposed.     

Production of inulooligosaccharides by endoinulinases from Aspergillus ficuum

Inulooligosaccharide (IOS) production from inulin was studied using a partially purified endoinulinase and a purified endoinulinase, which originated from Aspergillus ficuum. At the optimal conditions, including 50 g/L inulin, an enzyme concentration of 10 U/g substrate, 45 °C, and pH 6.0, the inulin-degrading degree by partially purified endoinulinase was 74% and an IOS yield over 50% were observed after 72 h. The major products were identified as DP2 to DP4. The purified Endo-I was used for inulo-oligosaccharide production at the optimal conditions obtained with orthogonal experiments, including pH 5.0, 45 °C, 50 g/L inulin, and an enzyme concentration of 10 U/g substrate. With pure inulin as substrate, the maximum inulin-hydrolyzing degree was 75% and the total IOS yield was 70% after 72 h. The hydrolysis products consisted of DP2 to DP8 with HPLC, and DP3 and DP4 were relatively high. With Jerusalem artichoke juice as substrate, the inulin-hydrolyzing extent reached 89% and the maximum IOS production was up to 80% after 72 h. Various IOS with different DP (mainly DP2, DP3, DP4, DP5, DP7, DP8) were evenly distributed in the final reaction products.     

Optimization of Transglutaminase Cross-linking of Pumpkin Oil Cake Globulin; Improvement of the Solubility and Gelation Properties

Enzymatic cross-linking of hull-less pumpkin oil cake globulin was studied using microbial transglutaminase. Response surface methodology was employed to study the effect of enzyme/substrate ratio, temperature, and reaction time on protein cross-linking reaction, measured by degree of polymerization (DP) as a response. The second-order polynomial model showed good fit with the experimental data since the coefficient of determination was R ²?=?0.9297. Reaction time of 39.2 min, E/S ratio of 1/4.9 (w/w) and temperature of 28 °C were found to be optimal conditions to achieve the highest value of DP (69%). The solubility and gelation properties of the cross-linked proteins with different values of DP were assessed for improvement. The solubility of polymerized proteins was increased over the pH range 5.0–8.0 and affected with increase of DP, at each pH value studied. The highest solubility was achieved at DP of 60%, at pH 7.0 and was 24-fold higher than solubility of unmodified protein. Gelation properties of cross-linked protein were also improved and the highest decrease of least gelation concentration was achieved at pH 9.0.     

SUBSTRATE SPECIFICITY AND NATURE OF ACTION OF BARLEY β-GLUCAN SOLUBILASE*

Barley β/glucan solubilase was shown to be active, to differing extends, towards hot water (65°C) soluble β-glucan, CM-cellulose and cellodextrins (DP 2–8). However, the enzyme did not affect the viscosity of CM-pachyman or appear to solubilise cotton cellulose. When β/glucan was treated with lichenase a mixture of small molecular weight products was obtained including a DP 9 dextrin. This dextrin was not obtained when the β-glucan was treated with β-glucan solubilase prior to hydrolysis by lichenase. It has been concluded, therefore, that this β-glucan solubilase is an endo-type glucanase, which appears to attack the small proportion of long blocks of (1→4)-β-linked glucosyl residues reputed to be present in barley β-glucan.     

Aspergillus ficuum 内切菊粉酶的酶解条件优化及酶解动力学研究

采用Aspergillus ficuum 内切型菊粉酶Endo-Ⅰ 酶解商品菊粉制备低聚果糖,经正交试验确定其最适酶解条件为：pH5.0、温度45℃、底物浓度50g/L、加酶量10U/g,在此酶解条件下,低聚果糖得率可达70.37%,酶解产物以DP3 和DP4 为主,同时含有一定量的DP2、DP5、DP6、DP7、DP8;在此条件下酶解自制菊芋干粉时,低聚果糖得率为41.72%,酶解产物以DP3～DP6 的低聚果糖为主,DP2 含量很少,同时酶解液中还含有大量的果糖;在此条件下酶解自制菊芋提取液时,低聚果糖得率高达79.80%,酶解产物中除DP6 含量较低外,其他各聚合度的低聚糖分布比较平均。在此相同酶解条件下酶解72h 时,3 种底物中,以菊芋提取液酶解后的低聚果糖得率最高。因此,应用Aspergillus ficuum 内切型菊粉酶Endo-Ⅰ酶解菊芋制备低聚果糖宜选择菊芋提取液作底物。     

In vitro amylolytic degradation of natural and graft copolymerised cassava and potato starches

 Various enzymatic hydrolyses to maximise degradation of the starch moiety of grafted starch (starch–g-polyacrylonitrile) were tried. The percentage of α- and β-amylolyses of grafted starch were 55 and 50 compared to 80 and 70 respectively for natural starches. Sequential degradation with α-amylase and glucoamylase of grafted starch showed 70% hydrolysis. The maltooligosaccharide profile by HPLC of the hydrolysates of grafted starch showed oligomers up to a degree of polymerisation (DP) 3, whereas the natural starch hydrolysates showed up to DP 7. The percentage hydrolysis, as well as the enzyme degradation profile, remained similar for both potato and cassava starches. Further treatment of the grafted starch hydrolysates with Bacillus cereus cells showed the presence of very low molecular weight polyacrylonitrile chains grafted onto maltooligosaccharides. The size exclusion chromatography analysis of grafted starch indicated the amylose component of starch that undergoes graft copolymerisation. Received: 30 December 1999     

柱前衍生-高效液相色谱-质谱联用法鉴定糖浆 掺假蜂蜜

目的建立蜂蜜中掺假的快速鉴定方法。方法将蜂蜜样品溶解后进行真空干燥,然后对样品中的低聚糖进行荧光标记,以氨基柱为分析柱,采用乙腈和甲酸铵为流动相,对标记低聚糖进行高效液相色谱分离,采用ESI-MS的SIR模式对标记低聚糖进行定性测定。结果基于荧光标记试剂2-氨基苯甲酰胺(2-AB)和毒性较低的还原剂2-甲基吡啶-N-甲硼烷(2-PB)的还原氨化反应能有效地对蜂蜜中的带还原端的低聚糖进行荧光标记。天然蜂蜜含有单糖(DP1)、双糖(DP2)和少量三糖(DP3),掺假蜂蜜除此以外还含有少量四糖(DP4)、五糖(DP5)和六糖(DP6)。结论采用此法对蜂蜜样品进行蜂蜜掺假判定与国标方法测试结果基本吻合,而且不需对样品进行净化处理即可上机分析,为建立蜂蜜掺假的快速筛查方法提供参考。     

纤维素酶水解纤维低聚糖的研究

对纤维素酶催化水解纤维三糖、纤维四糖和纤维五糖的规律进行研究。结果表明：相对水解率随低聚糖聚合度增大而降低;中间产物低聚糖和底物低聚糖竞争性与酶作用,产物中低聚合度低聚糖优先被酶催化水解;过大的纤维素酶用量并不能显著增大水解率;相对水解率均随初始低聚糖浓度增大而减小;pH5.0 时,纤维素酶最适温度为55℃。     

Basic subsite theory assumptions may not be applicable to hydrolysis of cellooligosaccharides by almond beta-glucosidase

Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond beta-glucosidase were evaluated at pH 5.0 and 25 degrees C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1-6, 1970). The value of k0/Km decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2-6). Also, the Km and k0 values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.     

Fabrication,characterization, and cytotoxicity evaluation of cranberry procyanidins-zein nanoparticles

Cranberry procyanidins (CPs)-zein nanoparticles were fabricated using a modified liquid–liquid dispersion method. The CPs were purified using chromatographic methods and analyzed using HPLC equipped with a fluorescence detector (FLD) and mass spectrometer (MS). The purified CPs had a purity of 64.7% (w/w) and contained procyanidin oligomers (from dimers to decamers) and polymers, with polymers being the predominant component (38.7%, w/w). The particle size of the CPs-zein nanoparticles increased from 392 nm to 447 nm with the increase of the CPs-to-zein mass ratios from 1:8 to 1:2. Morphologically, the CPs-zein nanoparticles were spherical as observed by scanning electron microscopy (SEM). The loading efficiency of CPs in the CPs-zein nanoparticles decreased with an increase of CPs-to-zein mass ratios from 1:8 to 1:2, and ranged from 10% to 86%. The oligomers with higher degree of polymerization (DP) showed higher loading efficiency than the oligomers with lower DP, suggesting a greater binding affinity on zein proteins. The loading capacity of the CPs-zein nanoparticles fabricated using a high CPs-to-zein mass ratio (1:2) was significantly higher than those using a low mass ratio (1:8). Fourier transform infrared spectroscopy (FTIR) suggested that the primary interactions between the CPs and zein were hydrogen bond and hydrophobic interactions. Cell culture studies using human promyelocytic leukemia HL-60 cells showed that the CPs encapsulated in nanoparticles had decreased cytotoxicity compared to the CPs.     

Study on the relationship between the degradation degrees of glutinous rice starch extruded with different α-amylases and the qualities of Chinese rice wine

In view that enzymatic extrusion can change the degradation degree of raw materials, this paper studied the regulation rules of four indexes of degradation degree of enzymatically extruded glutinous rice flour and the correlation between these indexes and the quality indexes of brewed Chinese rice wine. The results showed that at appropriate water content, dextrose equivalent (DE) and water solubility index (WSI) were linearly related to the enzyme content in extrusion with thermostable α-amylase (TS-αA) and mesophilic α-amylase (MS-αA), respectively. But water absorption index (WAI) was not significantly affected by enzyme content. The percentages of each degree of polymerisation (DP) (1–7) and that of the sum of the rest DPs called other DP were the fundamental reasons for different DE values, WSI, WAI and quality indexes of Chinese rice wine. DP1 was significantly positively correlated with alcohol degree, and negatively correlated with total acid. DP2–DP7 (except DP4) was significantly positively correlated with amino acid nitrogen. Besides, other DP was significantly positively correlated with total sugar and total acid. The percentages of DP1, DP2–DP7 and other DP could be respectively adjusted by changing enzyme content in extrusion with MS-αA, MS-αA/TS-αA and TS-αA to control the related quality indexes of Chinese rice wine.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

可德兰寡糖的制备及其组分分析

目的：可德兰多糖是目前发现的唯一不含分支的线性β-1,3- 葡聚糖,由于难溶于水而限制其生物活性的研究,为此对可德兰寡糖的制备及其组分进行研究。方法：采用化学法酸解可德兰多糖。结果：通过红外光谱与基质辅助激光解析/ 电离飞行时间质谱(MALDI-TOF MS)的检测,结果表明：可德兰多糖降解后结构未发生变 化,降解产物的聚合度为2～19,其中聚合度为5 的成分含量最高;高效阴离子交换色谱脉冲安培检测法分析结果表明：反应温度105℃、反应时间150～180min 是降解可德兰寡糖的合理反应条件,降解效果最佳。结论：本研究建立的方法可简单、高效的降解可德兰多糖,制备其寡糖。     

Untersuchungen zum Abbau von Maillard-Reaktionsprodukten durch amylolytische Enzyme

The enzymatic degradation of thermal treated α-glucans with amylolytic enzymes depends on the reaction environment (T, pH, moisture), the degree of polymerisation (DP) and the branch of the substrates as well as on the presence of amino compounds. The chemical changes of the α-glucans due to thermolysis at 180 °C are characterized by means of the amount of reducing substances and the amount of maltooligosaccharides (HPLC). In general the enzymatic degradability of the thermal treated α-glucans is decreased with increasing time of thermolysis, temperature and moisture content. The enzyme activity with the thermal treated α-glucans is diminished in the same way. The addition of amino compounds reduces the enzymatic degradability only at the beginning of the reaction. With increasing time of thermolysis the thermolysates without glycine addition are hardly degradated. As reason for these differences in the enzymatic degradation transglycosylation and non-enzymatic browning reactions (caramalisation/ Maillard-reaction) are assumed.     

Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii

In the genome data base of the hyperthermophilic archaeon Pyrococcus horikoshii, an open reading frame with sequence homology to a gene encoding alcohol dehydrogenase was found. It was demonstrated that the encoded enzyme was a thermostable L-threonine dehydrogenase which can oxidize the hydroxy alkyl residue of L-threonine associated with the reduction of NAD+ or NADP+. This enzyme is a member of the zinc-containing L-threonine dehydrogenase family. One enzyme molecule contained one zinc atom, and this metal was considered to contribute to the hyperthermostablility of the enzyme. The reaction of the enzyme proceeded via a sequential mechanism. The Michaelis constants (Km) for L-threonine and NAD+ were 0.013 and 0.010 mM, respectively, and the maximum reaction rate (Vmax) was 1.75 mmol NADH formed/min/mg-protein at 65 degrees C. The Km values for both L-threonine and NADP+ were larger than those for L-threonine and NAD+ with a similar Vmax value. These results indicate that the enzyme has lower affinity to NADP+ than to NAD+, and the binding affinity for L-threonine depends on the coenzymes.     

Texture properties of rice cakes made of rice flours treated with 4-α-glucanotransferase and their relationship with structural characteristics

Rice flours were treated using a thermostable 4-α-glucanotransferase (GTase) from Thermus aquaticus for 1, 3, and 48 h. Molecular weight of modified rice starch rapidly decreased within 1 h of reaction, then slowed down. As the reaction proceeded, the proportions of short (<DP 11) and long (>DP 30) branched chains of modified starch increased, whereas the proportion of medium chains decreased. Rice cakes were prepared with native and GTase-treated rice flours (substitution at 5%) and kept for 3 and 21 h. Texture profile analysis indicated significantly increased hardness, adhesiveness, chewiness, and resilience in rice cakes containing treated rice flours. Sensory analysis revealed that the rice cakes containing 48 h-treated flour had significantly increased springiness, hardness, toughness, and adhesiveness during both storage periods. Meanwhile, it displayed the lowest starch-like attribute and crumbliness. These results suggested that the substitution for 48 h GTase-treated rice flour could retard the retrogradation of rice cakes.     

Effects of dry period length and dietary energy source on inflammatory biomarkers and oxidative stress in dairy cows

Negative energy balance in dairy cows in early lactation has been associated with increased inflammation and oxidative stress in these cows. The objective of this study was to evaluate the effects of dry period (DP) length and dietary energy source on inflammatory biomarkers and oxidative stress in dairy cows. Holstein-Friesian dairy cows (60 primiparous and 107 multiparous) were assigned randomly to a 3 × 2 factorial design with 3 DP length (0, 30, or 60 d) and 2 early lactation rations (glucogenic or lipogenic). Cows were fed a glucogenic or lipogenic ration from 10 d before the expected calving date. Blood was collected in wk ?3, ?2, ?1, 1, 2, and 4 relative to calving. Dry period length affected inflammatory biomarkers and oxidative stress, especially in wk 1 and 2 after calving. Cows with a 0-d DP had higher levels of ceruloplasmin, cholesterol, and reactive oxygen metabolites, and they tended to have higher haptoglobin levels compared with cows with a 30- or 60-d DP. Cows with a 0-d DP had a lower plasma paraoxonase and bilirubin in the first 2 wk after calving and a lower liver functionality index compared with cows with a 60-d DP. Cows of parity >3 fed a glucogenic ration had higher cholesterol levels compared with cows of parity >3 fed a lipogenic ration. No interaction between DP length and ration was present for inflammatory biomarkers or oxidative stress variables. Plasma bilirubin levels for cows with a 0-d DP were negatively related to energy balance and metabolic status in these cows. Moreover, occurrence of clinical health problems (fever, mastitis, metritis, and retained placenta) was 41, 27, and 30% for cows with 0-, 30-, and 60-d DP, respectively. High levels of ceruloplasmin, cholesterol, and reactive oxygen metabolites in cows with 0-d DP were related to the occurrence of health problems in these cows. In conclusion, omitting the DP increased levels of ceruloplasmin, cholesterol, and reactive oxygen metabolites, and decreased levels of bilirubin and paraoxonase in plasma, independent of ration, compared with cows with a 60-d DP. These contrasting effects of DP length on inflammatory status could be explained in part by the improved energy balance and occurrence of health problems in these cows, but was not related to increased somatic cell count in cows with a 0-d DP. Cows with a 0-d DP had better energy balance, but also had higher levels of oxidative stress compared with cows with a 60-d DP. Moreover, occurrence of health problems did not differ between cows with different DP lengths.