Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. One important group is the fructooligosaccharides (FOS), a special kind of prebiotic associated to the selective stimulation of the activity of certain groups of colonic bacteria. They have a positive and beneficial effect on intestinal microbiota, reducing the incidence of gastrointestinal infections and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of FOS, such as solid-state fermentations utilizing various agro-industrial by-products. By optimizing the culture parameters, FOS yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of FOS. This article is an overview of the results considering recent studies on FOS biosynthesis, physicochemical properties, sources, biotechnological production and applications.     

Colonization of Aspergillus japonicus on synthetic materials and application to the production of fructooligosaccharides

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale.     

Microbiological production of enzymes and their industrial applications

Bacteria and other fungi are industrially cultivated in a variety of ways for the commercial production of some 25 enzymes utilized in many industries ranging from the conversion of starch to fermentable sugars, through chill-proofing of beer to bating of hides.     

Recent progress towards the application of hyperthermophiles and their enzymes

The discovery of extremophiles has drastically changed our understanding towards the diversity of life itself and the conditions under which it can be sustained. Extremophiles have evolved to withstand and multiply under the extremes of temperature, pressure, pH and salinity. Hyperthermophiles are the group that have adapted to high temperature; many have been found to grow at temperatures above the boiling point of water. This review focuses on recent advances in application-based research on hyperthermophiles and their thermostable enzymes.     

Dietary-fiber-degrading enzymes from a human intestinal Clostridium and their application to oligosaccharide production from nonstarchy polysaccharides using immobilized cells

The secretion of nonstarchy polysaccharide-degrading enzymes from an anaerobic human intestinal bacterium, Clostridium butyricum- beijerinckii (isolated from human feces), was investigated. Growth of the bacterium was found when laminarin, konjac glucomannan, and pectic acid were added separately to the culture media as sole carbon source. The corresponding degrading enzymes for these dietary fibers, laminarinase (endo-1,3- beta-glucanase), endo-1,4-beta-mannanase, endo- and exo-pectate lyases, and pectin methylesterase, were then purified and characterized. These extracelluar enzymes, which were secreted by the bacterium in the human large intestine, were considered to contribute to digestion of the ingested dietary fibers to their oligosaccharides, following by short-chain fatty acid fermentation by the bacterium. We have developed cell immobilization techniques of the bacterium on cellulose-foam carriers that are effective for continuous production of the oligosaccharides from the dietary fibers in a fed-batch reactor system. From 9 g of pectic acid, a total of 3.96 g of 4,5-unsaturated digalacturonic acid was produced over 40 h in four 500-ml batchcultures. In the same manner, the corresponding oligosaccharides were obtained from konjac glucomannan and laminarin with average conversion rates of around 30-40%.     

Feasibility study on the use of calcium alginate-entrapped hybridoma cells for IgM production

In order to test the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production, HO-22-1 hybridoma cells entrapped into calcium alginate beads with varying alginate concentrations were cultivated in spinner flasks. It was observed that the IgM produced by the entrapped cells could diffuse out of the calcium alginate beads regardless of alginate concentrations tested (0.8–2.5%). Since the increase in alginate concentrations showed an adverse effect on cell growth and maximum cell concentration, the use of lower alginate concentration was desirable for higher volumetric monoclonal antibody (MAb) productivity. When the entrapped cells in 0.8% alginate beads were cultivated in repeated-fed batch mode, the reduction of serum concentration in the medium from 10% to 1% did not decrease the volumetric IgM production. Taken together, the data obtained here showed the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production.Alginate was generously provided by the Kelco company. This work was supported by the Ministry of Science and Technology, Korea.     

Microbial production of levansucrase for synthesis of fructooligosaccharides and levan

A newly isolated thermophilic bacterial strain from Tunisian thermal source was identified as Bacillus sp. and was selected for its ability to produce extracellular levansucrase. Following the optimization of carbon source, nitrogen source, temperature and initial pH of the growth medium in submerged liquid cultures. In fact, sucrose was found to be a good inducer of levansucrase enzymes. The optimal temperature and pH of the levansucrase were 50°C and 6.5, respectively and its activity increased four folds in the presence of 50mM Fe(2+). This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for more than 1h at pH 6.5. The half-life of the enzyme was 1h at 90°C. Crude enzyme of Bacillus sp. rich in levansucrase was established for the synthesis of fructooligosaccharides and levan. Bacillus sp. could therefore be considered as a satisfactory and promising producer of thermostable levansucrases. Contrary to other levansucrases, the one presented in the current study was able to produce high levels of levan with high molecular weight at 50°C and having an important effect as a hypoglycemic agent which was demonstrated in our previous publications (Dahech et al., 2011 [25]) and as a hypo-cholesterolemic agent which will be investigated in further research.     

Novel method for the production of a mixture containing fructooligosaccharides and isomaltooligosaccharides

A sugar mixture containing fructooligosaccharides and isomaltooligo-saccharides was produced. Sucrose was converted to fructooligosaccharides by a commercial enzyme preparation. The sugar mixture contained kestose (33.5%), nystose (13.3%), fructofuranosyl nystose (5.7%), glucose (20.9%), and unreacted sucrose (26.6%). The unreacted sucrose was converted to isomaltooligosaccharides by reacting the sugar mixture with Leuconostoc mesenteroides B-512FM dextransucrase. The final product comprised fructooligosaccharides (kestose, nystose, fructofuranosyl nystose), isomaltooligosaccharides (isomaltose through isomaltodecaose), glucose, and fructose.     

Theoretical calculation of the sugar concentrations during enzymatic production of fructooligosaccharides

Summary In a batch production of fructooligosaccharides from sucrose, the concentrations of residual sucrose, glucose and fructooligosaccharides at a given reaction time(t) and initial sucrose concentration(S₀) were theoretically calculated by the following correlation equations: Glucose(t) = 0.0653 S₀ × ln(t); Fructooligosaccharides(t) = 0.1636 S₀ × ln(t); Sucrose(t)=S₀ - Glucose(t) + FOS(t).     

The isolation of lytic enzymes from Cytophaga and their application to the rupture of yeast cells

Yeast–lytic enzymes have been isolated on a pilot scale from Cytophaga species by precipitation and the light, enzyme-rich solid phase recovered by liquid-liquid separation. The enzyme complex was immobilized to soluble polymeric carbohydrates and the effectiveness of the free and immobilized enzyme for protein release and cell debris dissolution has been assessed.     

Optimal production of cellulolytic enzymes and their location in trichoderma pseudokonigii

The maximum production of cellulose enzymes (FPactivity, CMcellulase and β-glucosidase) in the culture filtrate was observed at 27°C and pH 5.0, except for β-glucosidase, being at 6.0. The levels of extracellular enzymes in shake and stationary culture conditions was almost equal although they reached their maxima earlier in the former. The distribution of the three enzymes in extracellular, cytosol and cell debris fractions revealed most of the FPactivity and CMcellulase occurring extracellulary. More than 50% of the β-glucosidase was present in the cell debris fraction.     

Continuous production of fructooligosaccharides from sucrose by immobilized fructosyltransferase

The productivity of the continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized onto a high-porous ion exchange resin was optimal with 600 g sucrose/l at a flow rate of 2.7 h^–1 expressed as a superficial space velocity. When the column was operated at 50 °C, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days continuous operation, achieving high productivity of 1174 g/l · h.     

Continuous production of high-content fructooligosaccharides by a complex cell system

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high-content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with beta-fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module. The complex cell system was run at 30 degrees C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7-day continuous production process with a dilution rate of 0.04 h(-1), and the volumetric productivity for total FOS was more than 160 g L(-1) h(-1).     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

Lipid-metabolizing enzymes of myelin and their relation to the axon

The old concept of myelin as a metabolically inert membrane has been considerably revised as a result of the discovery of numerous enzyme activities in the isolated membrane. The high degree of purification and low levels of contamination markers leave little doubt that the measured activities are intrinsic to myelin itself. Slightly more than half of the discovered activities involve lipid metabolism. One such enzyme, neutral cholesterol esterase, is myelin-specific, while the rest occur in other subcellular fractions as well as myelin. These include activities involved in synthesis of cerebrosides, phospholipids, and cholesteryl esters; only a few degradative enzymes are presently known. In vivo studies have shown that various substrates utilized by lipid-synthesizing enzymes of myelin can originate in the axon. Six such substrates have been characterized. The possibility exists that these enzymes may be wholly or partially dependent on the axon as the primary source of substrate, thereby suggesting a possible form of metabolic dependency of myelin on the axon.