Production of high-content inulo-oligosaccharides from inulin by a purified endoinulinase

Inulo-oligosaccharides were produced from inulin with high yield by using a purified endoinulinase from a commercial inulinase preparation. The maximum yield of oligosaccharide achieved was around 96% irrespective of substrate concentrations ranged from 50 to 150 g inulin/l. A wide range of degradation products from inulin, varying in their DP (degree of polymerization) 2 to 6, were obtained where the major oligosaccharides were DP3 and 4. The reaction pH gave rise to a significant difference in yield and sugar composition of inulo-oligosaccharides.     

Enzymatic production of inulo-oligosaccharides from chicory juice

Batchwise production of inulo-oligosaccharide from chicory juice was carried out by an endoinulinase from Pseudo-monas sp. The maximum yield of oligosaccharides (OS) was about 80% in total sugar basis with substrate at 30–100 g/l. Compared with pure inulin of the same origin as a substrate, the same OS yield was obtained but it showed quite a different product distribution in degree of polymerization (DP) and sugar composition, where DP2, DP3 and DP4 were major components. © Rapid Science Ltd. 1998     

Bioconversion of inulin to 2,3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase

Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin.     

Production of inulooligosaccharides from chicory extract by endoinulinase from Xanthomonas oryzae No. 5

Inulooligosaccharides (IOS) production from chicory extract was carried out using endoinulinase obtained from a new isolate, Xanthomonas oryzae No. 5. The IOS production from chicory extract was maximum when 50 g/liter of chicory extract was utilized as the substrate. As the substrate concentration increased, the IOS production accordingly decreased probably due to substrate inhibition. For a comparative study, enzyme reactions were carried out from pure inulin as substrate. Though total IOS contents indicated higher IOS yield with pure inulin compared to that of chicory extract, the distribution of inulooligosaccharide components between pure inulin and chicory extract was not significantly different; i.e. DP5 and higher oligosaccharides are major products in case of both chicory extract and pure inulin as substrate. A considerable amount of oligofructose (about 30%, w/w), which were originally present in chicory extract, resulted in the change of the enzyme kinetics. A reaction pH 7 was found to be most suitable for enzyme reaction. The initial reaction rates increased with increasing enzyme dosage, although the relative composition of the IOS produced remain unchanged.     

Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE–PAD) was evaluated for the quantitation of polyglucose metabolites (DP2–DP7) in human plasma. The method was investigated for accuracy, precision, specificity, linearity, range and analyte stability. Samples were prepared by dilution into the standard range (0.1–10 μg/ml) followed by deproteinization using a 30?000 molecular mass cut-off filtration device. The limit of detection was 0.05 μg/ml for all metabolites. Method precision for DP2–DP7 varied from approximately 2% R.S.D. in the upper range to approximately 15% R.S.D. at the limit of quantitation. Samples were stable following one or two freeze–thaw cycles and, after preparation, they could be refrigerated for up to 72 h. Application of this method to clinical plasma samples from continuous ambulatory peritoneal dialysis (CAPD) patients administered one daily night-time intraperitoneal exchange of 2 l of 7.5% polyglucose solution for four weeks indicated that plasma levels of DP2, DP3 and DP4 increased from baseline levels of <0.01 g/l to steady-state levels of 1.2±0.3, 1.2±0.3 and 0.4±0.1 g/l (mean±S.D.), respectively. These steady state plasma levels for DP2 and DP3 are comparable to previously reported levels in patients administered daily overnight 7.5% polyglucose dialysis solution.     

Inulin-type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects

AIMS: We assessed to what extent fructans of different degrees of polymerization (DP) differ in their prebiotic effectiveness towards in vitro microbial communities from the proximal and distal colon. METHODS AND RESULTS: Two short chain fructans - oligofructose (DP 2-20) and inulin (DP 3-60) - were administered to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) at 2.5 g day(-1). The influence of fructan addition towards fermentation activity and microbial community composition from the different SHIME colon compartments were evaluated. Both fructans exerted prebiotic effects with significantly higher butyrate and propionate production and stimulation of lactic acid-producing bacteria. Compared with oligofructose, it was noted that it took more time before significant effects from inulin addition were observed. Yet, the higher short-chain fatty acid production and lower proteolytic activity showed that the prebiotic effects from inulin were more pronounced than oligofructose. Also, the bifidogenic effects from inulin vs oligofructose were higher in the distal colon compartments and this effect was prolonged in the distal colon once the addition was stopped. CONCLUSIONS: Inulin has more pronounced prebiotic effects than oligofructose towards both fermentation activity and bacterial community composition in the SHIME model. SIGNIFICANCE AND IMPACT OF THE STUDY: Its slower fermentation rate and higher prebiotic potency makes inulin a more interesting compound than oligofructose to beneficially influence the microbial community from both the proximal and distal colon regions.     

Enhanced production ofd(−)-lactic acid by mutants ofLactobacillus delbrueckii ATCC 9649

Summary Chemical mutagenesis with ethyl methanesulfonate (EMS) was used to develop strains ofLactobacillus delbrueckii (ATCC 9649) that tolerated increased lactic acid concentrations while continuously producing the acid. Three mutants (DP2, DP3 and DP4) were compared with wild-typeL. delbrueckii by standing fermentations with different glucose concentrations. All three mutants produced higher levels of lactic acid than the wild-type. In pH-controlled (pH 6.0) stirred-tank-batch fermentations, mutant DP3 in 12% glucose, 1% yeast extract/mineral salt/oleic acid medium produced lactic acid at a rate that was more than 2-times faster than the wild-type. Mutant DP3 also produced 77 g/l lactic acid compared with 58 g/l for the wild-type. Overall, compated with wild-type, the mutants DP2 and DP3 exhibited faster specific growth rates, shorter lag phases, greater lactic acid yields, tolerated higher lactic acid concentrations, and produced as much as 12% lactic acid in 12% glucose, 3% yeast extract/mineral salt/oleic acid medium which required an additional 9% glucose when the residual glucose concentration decreased to 3%. Mutant DP3 was stable for over 1.5 years (stored freeze dried). The strain development procedure was very successful; mutants with enhanced lactic acid-producing capacity were obtained each time the procedure was employed.Journal Paper No. J-14087 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Projects No. 2889 and 0178.     

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Optimization of simultaneously enzymatic fructo- and inulo-oligosaccharide production using co-substrates of sucrose and inulin from Jerusalem artichoke

Prebiotic substances are extracted from various plant materials or enzymatic hydrolysis of different substrates. The production of fructo-oligosaccharide (FOS) and inulo-oligosaccharide (IOS) was performed by applying two substrates, sucrose and inulin; oligosaccharide yields were maximized using central composite design to evaluate the parameters influencing oligosaccharide production. Inulin from Jerusalem artichoke (5–15% w/v), sucrose (50–70% w/v), and inulinase from Aspergillus niger (2–7 U/g) were used as variable parameters for optimization. Based on our results, the application of sucrose and inulin as co-substrates for oligosaccharide production through inulinase hydrolysis and synthesis is viable in comparative to a method using a single substrate. Maximum yields (674.82?mg/g substrate) were obtained with 5.95% of inulin, 59.87% of sucrose, and 5.68 U/g of inulinase, with an incubation period of 9?hr. The use of sucrose and inulin as co-substrates in the reaction simultaneously produced FOS and IOS from sucrose and inulin. Total conversion yield was approximately 67%. Our results support the high value-added production of oligosaccharides using Jerusalem artichoke, which is generally used as a substrate in prebiotics and/or bioethanol production.     

菊芋粕菊粉的酶法提取、组成及抗氧化活性研究

本研究拟应用酶法提取技术解决菊芋菊粉工业化生产中的废弃物——菊芋粕再利用程度低的问题,并评价菊芋粕菊粉的抗氧化活性功效。对菊芋粕菊粉的果胶酶酶法提取的最佳条件通过响应面法进行了优化,并对菊芋初次水提菊粉(primary water-extracted inulin,PWI)和二次酶提菊粉(secondary enzymatic-extracted inulin,SEI)的组成成分和抗氧化活性进行了比较分析。响应面法优化确定的菊芋粕菊粉最佳酶法提取条件为:pH4.5、提取温度50 ℃、酶底比7.5 U/g、提取时间2 h,该提取方法所得菊芋粕菊粉的得率为35.30%±0.85%,与传统热水浸提法相比菊粉得率提高38.16%。组成分析结果显示,SEI的总糖和菊粉含量均显著高于PWI( P <0.05)。在菊粉聚合度方面,PWI中蔗果三糖和蔗果四糖含量较高,而SEI中蔗果五糖、蔗果六糖及其以上聚合度菊粉的含量较高。此外,SEI的抗氧化活性优于PWI。因此,果胶酶辅助提取方法有望为菊芋粕菊粉的再利用难题提供新的解决思路。     

Effect of acid or enzymatic hydrolysis on ethanol production by Zymomonas mobilis growing on Jerusalem artichoke juice

Summary Ethanol production from the inulin of Jerusalem artichoke byZ. mobilis was studied in batch and continuous fermentations. Both acid or enzymatic hydrolysis were used. In continuous cultures enzymatic hydrolysis showed better results. Ethanol productivities of 17.7 and 29.0 g/l.h were obtained at output concentrationsca 35 g/l (% of conversion 99 and 83; ethanol yield 0.45 g/g). The hydrolysed juice could be used without any nutrient addition.     

Single cell oil production from hydrolysates of inulin and extract of tubers of Jerusalem artichoke by Rhodotorula mucilaginosa TJY15a

In this study, we found that Rhodotorula mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.     

Production of high concentrations of ethanol from inulin by simultaneous saccharification and fermentation using Aspergillus niger and Saccharomyces cerevisiae.

Pure nonhydrolyzed inulin was directly converted to ethanol in a simultaneous saccharification and fermentation process. An inulinase-hyperproducing mutant, Aspergillus niger 817, was grown in a submerged culture at 30 degrees C for 5 days. The inulin-digestive liquid culture (150 ml) was supplemented with 45 g of inulin, 0.45 g of (NH4)2SO4, and 0.15 g of KH2PO4. The medium (pH 5.0) was inoculated with an ethanol-tolerant strain, Saccharomyces cerevisiae 1200, and fermentation was conducted at 30 degrees C. An additional 20 g of inulin was added to the culture after 15 h of fermentation. S. cerevisiae 1200 utilized 99% of the 65 g of inulin during the fermentation, and produced 20.4 and 21.0% (vol/vol) ethanol from chicory and dahlia inulins, respectively, within 3 days of fermentation. The maximum volumetric productivities of ethanol were 6.2 and 6.0 g/liter/h for chicory and dahlia inulins, respectively. The conversion efficiency of inulin to ethanol was 83 to 84% of the theoretical ethanol yield.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Synthesis of new oligosaccharides from raffinose by Aspergillus niger fructosyltransferase

Purified fructosyltransferase from Aspergillus niger exhibited transfructosylation activities, producing fructose, DP2, DP4, and DP5 from raffinose. The structures of two products synthesized from raffinose were identified as O--d-galactopyranosyl (16)--d-glucopyranose and O--d-galactopyranosyl (16)--d-glucopyranosyl-[O--d-fructofuranosyl (21)]--D-fructofuranoside, which means that C-2 hydroxyl group of fructose released from one raffinose molecule were linked to the C-1 hydroxyl group of fructose of another raffinose.     

Use of acyl phosphonates for the synthesis of inulin esters and their use as emulsion stabilizing agents

Inulin, the polydisperse polyfructose, extracted from chicory, was modified via esterification with acyl phosphonates. The grafting of an acyl chain onto the inulin backbone under different conditions led to a highly efficient synthesis of a series of inulin esters, with interesting tensioactive properties. The derivatives were evaluated in oil-in-water (O/W) emulsions with isoparaffinic oil, Isopar M. Therefore, a 2% (w/v) aqueous solution of inulin-based surfactant was used in 50/50 O/W emulsions, in nonelectrolyte, and in electrolyte media, using 1 M MgSO4. Longer acyl chains, e.g., dodecanoyl (C12), hexadecanoyl (C16), and octadecanoyl (C18), with degrees of substitution lower than 0.5, gave rise to the highest emulsion stabilities against coalescence.     

Ethanol production from Jerusalem artichoke juice using flocculent cells of Kluyveromyces marxianus

Summary Flocculent cells ofKluyveromyces marxianus SM 16-10 were used for batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 20% initial sugar concentration, a maximum ethanol concentration of 92 g/l was achieved in 7 h, when the flocculent cell concentration was 30 g dry wt./l bioreactor volume. The same flocculent cells were used repeatedly for 7 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at about 94% of the theoretical for all the 7 batch cycles, while the maximum ethanol production rate increased from 17.21 g ethanol/1/h during the first batch run to 21 g ethanol/1/h during the last batch run.     

The effect of fructooligosaccharides with various degrees of polymerization on calcium bioavailability in the growing rat

Maximizing peak bone mass during adolescence may be the key to postponing and perhaps preventing bone fractures due to osteoporosis in later life. One mechanism to maximize peak bone mass is to maximize calcium absorption, and it has been suggested that inulin and oligofructose might be one of the ways of doing so. In this study, fructooligosaccharides with various degrees of polymerization have been compared in terms of impact on calcium absorption, bone density, and excretion of collagen cross-links in the young adult male rat. The various oligosaccharides were oligofructose (DP2-8), inulin (DP>23), and a mixture of 92% inulin and 8% short-chain oligofructose (DP2-8). Measuring ex vivo bone mineral density (BMD) and bone mineral content (BMC) showed that BMD was significantly higher in the group fed inulin (DP>23) in both femurs, whereas BMC was significantly higher in the spine. The excretion of fragments of Type 1 collagen decreased in all groups over the 4 weeks of feeding, but the decrease was most significant in the group fed inulin (DP>23). Several hypotheses have been offered to explain the effect of the fructooligosaccharides on calcium absorption and retention. These include the production of organic acids that would acidify the luminal contents and enhance solubility and hence absorption, or possibly a mechanism via calbindinD9k. This study is unique in that it compares the different fructooligosaccharides in the same model, and it clearly shows that the various fructans do not have the same effect. In our model, inulin (DP>23) had the most significant effect on calcium bioavailability.     

Repeated batch production of ethanol from Jerusalem artichoke tubers using recycled immobilized cells of Kluyveromyces fragilis

Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.     

Synthesis of 2-keto-L-gulonic acid from gluconic acid by co-immobilized Gluconobacter oxydans and Corynebacterium sp.

2-Keto-L-gulonic acid was produced from gluconic acid using co-immobilized cells of Gluconobacter oxydans and Corynebacterium sp. with 2,5-diketo-D-gluconic acid. Gluconobacter oxydans and Corynebacterium sp. were entrapped together with polyvinylalcohol and alginate. 50 g/l glucose, 50 g/l gluconic acid, and the mixture of equal volume of 50 g/l glucose and 50 g/l gluconic acid were used as substrates. When the ratio of two cells was 1 to 1 with 100 mg cells/ml, the conversion of 2-KLG from gluconic acid was 38% (g/g). © Rapid Science Ltd. 1998