Production of high fructo-oligosaccharide syrup with two enzyme system of fructosyltransferase and glucose oxidase

Summary A novel two enzyme system of fructosyltransferase and glucose oxidase to enhance the content of the net fructo—oligosaccharide (FOS) fractions in the industrial production of FOS syrup from sucrose was devised. The net FOS content in the commercial FOS syrup has been limited only to 55–60 % due to the accumulation of glucose which acts as a feedback inhibitor of the fructosyltransferase. By supplementing glucose oxidase to the conventional FOS reaction system, we could convert the glucose to gluconic acid readily separable from neutral sugars by simple ion exchange operation in the next step. The simultaneous removal of glucose was proved effective in proceeding the reaction by fructosyltransferase further by relieving the product inhibition caused by glucose. By this way, we could raise the net FOS content as high as 90 %.     

Fructooligosaccharide production by <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Fructooligosaccharides (FOS) production by Penicillium expansum was evaluated. In a first stage, the best conditions for P. expansum growth and sporulation were established with potato/dextrose/agar being the most suitable medium at between 22 and 25°C, giving good growth and good sporulation. The inocula from this medium were used for FOS production using shake-flask cultures, and yielded 0.58 g FOS/g sucrose (3.25 g FOS/l.h), demonstrating the potential of this strain for sucrose conversion to FOS.     

Expression of an Aspergillus niger glucose oxidase in saccharomyces cerevisiae and its use to optimize fructo-oligosaccharides synthesis

Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.     

Kinetics of sucrose conversion to fructo-oligosaccharides using enzyme (invertase) under free condition

The study reports the synthesis of fructo-oligosaccharide (FOS) from sucrose using invertase derived from Saccharomyces cerevisiae. The reaction was conducted in a batch mode under free enzyme condition. Fructo-oligosaccharide formation was detected at a high sucrose concentration of over 200 g/L. The investigation was extended to study the effect of different parameters such as initial sucrose concentration (ISC), pH, and enzyme concentration. A maximum FOS yield of 10 % (dry basis) was observed using 525 g/L of ISC, with 6 U/mL of the enzyme, and pH 5.5 at 40 °C. 1-Kestose was the major product of among different forms of FOS. The FOS yield increased with an increase in sucrose concentration up to 525 g/L, beyond which it started to decrease. However, the maximum FOS yield was not affected by the increasing concentration of the enzyme beyond a certain level (2 U/mL). Furthermore, the activity of enzyme slightly increased with an increase in the pH up to 6, and thereafter it declined. Addition of glucose decreased the FOS yield because of enzyme inhibition. A five-step, ten-parameter model was developed, for which the simulation was performed in COPASI. The results predicted by the model were consistent with the experimental data.     

Sugar Beet Juice Fermentation by Zymomonas mobilis Attached to Stainless Steel Wire Spheres

The fermentation of sugar beet juice as well as juice syrup medium by Zymomonas mobilis inoculum attached to stainless steel wire spheres was investigated. A semi‐synthetic sucrose medium enriched with mineral salts and yeast extract was used as the control. It was established that raw sugar beet juice ensured good Zymomonas mobilis culture growth and slightly decreased ethanol synthesis applying both flame‐burned and TiCl₄‐treated wire spheres as carriers (Q_x = 0.05—0.06 g/l × h; Q_eth = 1.02—1.22 g/l × h). High ethanol yield was also observed in juice medium (Y = 0.45‐0.46 g/g), however, levan synthesis with this medium decreased. The application of juice syrup brought about less growth effect and ethanol synthesis as compared to juice medium. The use of semi‐synthetic sucrose medium resulted in high levan production (Q_lev = 0.6—0.7 g/l × h), however, reduced ethanol production by 40%. In conclusion, sugar beet juice or syrup is recommendable for the preparation of Zymomonas mobilis inoculum. The levan production stage has to be realized using an optimized semi‐synthetic sucrose medium. The installed wire spheres filled with inocula provided the possibility for a repeated batch fermentation process, which could be recommended for both juice and semi‐synthetic sucrose medium fermentation.     

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.     

Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii

High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L^?1 yeast extract and 300 g L^?1 sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h^?1, FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L^?1 was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days.     

Production of microbial levan from sucrose,sugarcane juice and beet molasses

Summary Bacillus polymyxa (NRRL-18475) produced a levan-type fructan (B, 26 fructofuranoside) when grown on sucrose, sugarcane juice, and sugarbeet molasses. The organism converted about 46% of the fructose moiety of sucrose to levan when grown on sucrose medium, however, the yields of levan from sugarcane juice and beet molasses were much less than sucrose solution. Such sugarcane juice and beet molasses can be made a good substrate for levan production by various modifications. Adding peptone to sugarcane juice or passing beet molasses through a column of gel filtration media improved levan yield to a level almost comparable to that obtained from sucrose.     

Fructan Biosynthesis by Intra‐ and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra‐ and extracellular levansucrase produced by the Z. mobilis strain 113 “S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that the cell free culture liquid, containing ethanol, levan, gluconic acid and sucrose (15%) at 25 °C, did not show any additional levan synthesising activity. At optimum FOS synthesis conditions (45 °C and 70% sucrose), the cell‐free culture liquid exhibited a high FOS synthesising activity (31% from total carbohydrates), with slightly reduced biomass activity. It was concluded that as a result of the simultaneous ethanol and levan production, the remaining biomass as well as the cell‐free culture liquid could be used for FOS production.     

Mature‐stem expression of a silencing‐resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally‐controlled expression of a silencing‐resistant gene encoding a vacuole‐targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole‐cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high‐isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value‐added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing‐resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field‐grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.     

Structural analysis and optimised production of fructo- oligosaccharides by levansucrase from Acetobacter diazotrophicus SRT4

Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C-NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40°C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.     

Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40 degrees C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35 degrees C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38 degrees C.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Enzymatic synthesis of biodiesel from palm oil assisted by microwave irradiation

Optimal conditions for enzymatic synthesis of biodiesel from palm oil and ethanol were determined with lipase from Pseudomonas fluorescens immobilized on epoxy polysiloxane–polyvinyl alcohol hybrid composite under a microwave heating system. The main goal was to reduce the reaction time preliminarily established by a process of conventional heating. A full factorial design assessed the influence of ethanol-to-palm oil (8:1–16:1) molar ratio and temperature (43–57 °C) on the transesterification yield. Microwave irradiations varying from 8 to 15 W were set up according to reaction temperature. Under optimal conditions (8:1 ethanol-to-oil molar ratio at 43 °C), 97.56 % of the fatty acids present in the palm oil were converted into ethyl esters in a 12-h reaction, corresponding to a productivity of 64.2 mg ethyl esters g^?1 h^?1. This represents a sixfold increase from the process carried out under conventional heating, thus proving to be a potential tool for enhancing biochemical modification of oils and fats. In general, advantages of the new process include: (1) microwaves speed up the enzyme-catalyzed reactions; (2) there are no destructive effects on the enzyme properties, such as stability and substrate specificity, and (3) the microwave assistance allows the entire reaction volume to be heated uniformly. These bring benefits of a low energy demand and a faster conversion of palm oil into biodiesel.     

AtRBOH I confers submergence tolerance and is involved in auxin-mediated signaling pathways under hypoxic stress

Satsuma mandarin fruit (Citrus unshiu Mark.) photosynthesizes as comparable to leaf at about 100 days after full bloom (DAFB). In this study, translocation and accumulation of fruit-fixed photosynthate were investigated by using ¹⁴CO₂. When fruit at 108 DAFB was exposed to ¹⁴CO₂ for 48 h under 135 photosynthetic photon flux density (PPFD), ¹⁴C-sucrose, ¹⁴C-glucose and ¹⁴C-fructose were detected not only in flavedo but juice sac; more than 50?% of fruit assimilated ¹⁴C-sugars were present in juice sac. Thus, majority of rind-fixed photosynthate are infiltrated into juice sac and accumulated there within 48 h after assimilation. Although ¹⁴C-sucrose was predominant at flavedo where high SS (sucrose synthase) activity toward synthesis was present, the amount decreased gradually from the outside (flavedo) to the inside (juice sac) of fruit. In vascular bundle, strong SS toward cleavage and soluble acid invertase activities were involved, and ¹⁴C-fructose was predominant in juice sac. Accordingly, rind-fixed photosynthate is once converted to sucrose, the translocated sugar in Citrus, at flavedo by SS toward synthesis, and loaded on vascular bundle through symplastic and/or apoplastic movement in the albedo tissue. In the vascular bundle, sucrose may be degraded by SS toward cleavage and invertase, and resulting hexoses transported symplastically to the juice sac through juice stalk.     

Production of high-fructose syrup from date by-products in a packed bed bioreactor using a novel thermostable invertase from Aspergillus awamori

Abstract

Dates by-products (discarded dates) from the sucrose-rich variety of ‘Deglet Nour’ were used as starting biomass to produce high-fructose syrup (HFS) based on an immobilized invertase process. A novel extracellular thermostable invertase obtained from Aspergillus awamori cultivated in submerged medium was induced with sucrose at 1% and used for this purpose. A zymogram of the crude extract showed the presence of a unique enzyme form that was optimally produced on the 5^th day. This enzyme preparation was biochemically characterized and immobilized on acetic acid-solubilized chitosan by covalent binding using glutaraldehyde (Yi = 88%, Ya = 54% and 15.53 U/g). When deployed in a packed bed reactor (PBR), HFS was efficiently and continuously produced from sucrose derived from aqueous date extracts. Feeding with an extract initially containing 139.2 g/L total sugar with 78.6 g/L sucrose at a flow rate of 17 ml/h, 50°C and pH 6 resulted in a conversion factor of 0.95 and a final fructose content in the syrup of 69 g/L.     

Continuous hydrolysis of sucrose by invertase adsorbed in a tubular reactor

Studies were made of invertase adsorption on Amberlite ion exchange resins. Up to 4000 units of adsorbed enzymatic activity (aea) were obtainedper g of IRA 93 resin; for an aea of 1600 units, the maximum ratio of aea over units of soluble enzyme used for adsorption was close to 50%. Nodesorption occurred during extensive washing at 30°C with 0.01M sodiumacetate buffer at pH 5. Progressive desorption of aea from the invertase–IRA 93 complex occurred when buffer molarity and temperature were increased. Desorption differed only slightly when the buffer pH was 3 or 5. Theoptimum pH of aea was 3.2 with IRA 93 resin, and varied between 3.2 and 5.1with other resins, depending on their anionic or cationic nature. Batch hydrolysis of sucrose by IRA 93–adsorbed invertase followed 1st order kinetics with respect to the substrate concentration, as in the case of soluble invertase. Continuous sucrose hydrolysis with IRA 93–adsorbed invertase was performed in a tubular reactor, and the percent conversion was experimentally determined as a function of the flow rate. The reaction was experimentally determined 50% (w/v) sucrose solution, at pH4 and 30°C; at the selected flow rate, the ratio of sucrose hydrolysis remained constant and close to 76%. This shows that invertase was not desorbed from the tubular reactor. Some continuous hydrolyses were performed with an industrial sucrose solution: enzymatic activity seemed to be stable for anextended period for time (1 month) at 30°C and pH 3 or 4.     

Use of tropical maize for bioethanol production

Tropical maize is an alternative energy crop being considered as a feedstock for bioethanol production in the North Central and Midwest United States. Tropical maize is advantageous because it produces large amounts of soluble sugars in its stalks, creates a large amount of biomass, and requires lower inputs (e.g. nitrogen) than grain corn. Soluble sugars, including sucrose, glucose and fructose were extracted by pressing the stalks at dough stage (R4). The initial extracted syrup fermented faster than the control culture grown on a yeast extract/phosphate/sucrose medium. The syrup was subsequently concentrated 1.25–2.25 times, supplemented with urea, and fermented using Saccharomyces cerevisiae for up to 96 h. The final ethanol concentrations obtained were 8.1 % (v/v) to 15.6 % (v/v), equivalent to 90.3–92.2 % of the theoretical yields. However, fermentation productivity decreased with sugar concentration, suggesting that the yeast might be osmotically stressed at the increased sugar concentrations. These results provide in-depth information for utilizing tropical maize syrup for bioethanol production that will help in tropical maize breeding and development for use as another feedstock for the biofuel industry.     

Fructosyltransferase activity of commercial enzyme preparations used in fruit juice processing

Summary Of twenty-two commercial fungal enzyme preparations used in fruit juice processing examined, Pectinex Ultra SP-L, was found to possess the highest activity of fructosyltransferase (44.8 units/ml). The enzyme preparation converted sucrose into a high fructooliogosaccharide syrup containing 42.3 % kestose, 17.2% nystose, 10.6% sucrose, 27.8% glucose, and 2.1% fructose. The efficiency was 69% based the amount of sucrose consumed.