Production Of Lactobionic Acid Research Articles

Selective production of lactobionic acid by aerobic oxidation of lactose over gold crystallites supported on mesoporous silica

Partial oxidation of lactose over Au-based catalyst system using nanostructured silica materials with improved activity, selectivity and stability was investigated as a novel chemo-catalytic approach for selective synthesis of lactobionic acid (LBA) for therapeutic, pharmaceutical and food grad applications. Highly active gold crystallites dispersed on mesoporous silica (SiO 2-meso) using bis-[3-(triethoxysilyl) propyl] tetrasulfide (BTSPT), a silane coupling agent to immobilize gold, were successfully formulated, and their catalytic activity was evaluated in an agitated semi-batch reactor. The catalysts were characterized by N 2 physisorption, XRD, XPS and TEM. The influence of the reaction conditions, i.e., temperature, pH value, metal loading and catalyst/lactose ratio on lactose conversion were investigated. After 100 min of reaction, the catalyst containing 0.7% Au showed the highest catalytic activity (100% lactose conversion) and a 100% selectivity towards LBA, when it was used at a catalyst/lactose ratio of 0.2 under alkaline (pH 9.0) and mild reaction temperature (65 °C).

Production of lactobionic acid and sorbitol from lactose/fructose substrate using GFOR/ GL enzymes from Zymomonas mobilis cells: A kinetic study

In this work, we have investigated the kinetics of the biotechnological production of lactobionic acid ( LBA) and sorbitol by the catalytic action of glucose–fructose oxidoreductase ( GFOR) and glucono-δ-lactonase ( GL) enzymes. The cells of bacterium Zymomonas mobilis ATCC 29191 containing this enzymatic complex were submitted to permeabilization and reticulation procedures. The effect of the concentration of substrates on the rate of product formation using a mobilized cell system was investigated. The application of higher fructose concentration seems to not affect the initial rate of formation of the bionic acid. Under conditions of low initial concentration of lactose, the experimental kinetic data of the bi-substrate reaction were modelled by assuming a rate equation of the classical ping-pong mechanism. The found kinetic parameters displayed a low affinity of the GFOR enzyme for both substrates. The enzymatic system did not exhibit normal Michaelis–Menten kinetics in response to a change of concentration of lactose, when fructose was held constant, presenting a sigmoid relationship between initial velocity and substrate concentration. A rate equation based on Hill kinetics was used to describe the kinetic behaviour of this enzyme-substituted reaction at higher lactose concentrations. The results from batch experiments using immobilized cells within Ca-alginate beads revealed that there is no pronounced occurrence of mass transfer limitations on LBA production for beads with 1.2 mm in average diameter. This discussion aids for defining the best operating conditions to maximize the productivity for LBA and sorbitol in this bioconversion and also for reducing the complexity of downstream separation processes.

Analysis of experimental errors in bioprocesses. 1. Production of lactobionic acid and sorbitol using the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of Zymomonas mobilis

The proper determination of experimental errors in bioprocesses can be very important because experimental errors can exert a major impact on the analysis of experimental results. Despite this, the effect of experimental errors on the analysis of bioprocess data has been largely overlooked in the literature. For this reason, we performed detailed statistical analyses of experimental errors obtained during the production of lactobionic acid and sorbitol in a system utilizing as catalyst the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of the bacteria Zymomonas mobilis. The magnitude of the experimental errors thus obtained were then correlated with the process operation conditions and with the composition of the culture media used for bacterial growth. It is shown that experimental errors can depend very significantly on the operation conditions and affect the interpretation of available experimental data. More specifically, in this study, experimental errors depended on the nutritional supplements added to the cultivation medium, the inoculation process, and the reaction time, which may be of fundamental importance for actual process development. The results obtained also indicate, for the first time, that GFOR activity can be affected by the composition of the medium in which cells are cultivated.

Simulated moving bed technology to improve the yield of the biotechnological production of lactobionic acid and sorbitol

This work presents an analysis on the suitability of the Simulated Moving Bed (and pseudo-SMB) technology on separating the mixture obtained from the lactose oxidation catalyzed by glucose-fructose oxidoreductase and glucono-α-lactonase enzymes. These enzymes from the Zymomonas mobilis bacteria are able to oxidize lactose in presence of fructose to its respective organic acid—lactobionic acid—and sorbitol. Some alternative arrays of chromatographic systems, as fixed bed column, SMB unit, 4 section pseudo-SMB, have been explored to separate the multi-component mixture, in such way to make possible the product recovery and the recycle of substrates to the enzymatic reactor. This study involved the definition of appropriate operating conditions and the prediction of the performance of the separation units, or arrangement of units, through modeling and simulations tools. To define the proper operating conditions, inequalities from equilibrium theory and the concept of the separation volume analysis have been considered. In this analysis, equilibrium and kinetic parameters for the compounds adsorbing on DOWEX 50W-X4 resin, in K+ and Ca+2 ion-loadings, have been obtained from chromatographic methods (pulse and adsorption-desorption techniques). The enzymatic kinetic of production of lactobionic acid and sorbitol using permeabilized cells of Z. mobilis is shown. The strategy of keeping the highest value of reaction rate by the integration of a chromatographic system proved to be viable when it was found the feasibility to apply the SMB system in cascade.

Use of electrodialysis technique for the separation of lactobionic acid produced by Zymomonas mobilis

The separation of lactobionic acid (LBA), a product of a biotechnological process catalyzed by the enzymes glucose-fructose oxidoreductase and glucono-δ-lactonase of the bacteria Zymomonas mobilis, was studied using ion selective membranes in electrodialysis (ED). The recovery of LBA, present in a solution with a concentration of 20 g L –1, was 38.7% in 250 min of testing for an electrical potential difference of 15 V. When compared with a solution containing all the components present in the bioconversion (lactose, fructose, and sorbitol), the recovery was inhibited by the presence of non-ionic substances and the LBA recovery efficiency decreased to 16.2%. The same behavior was observed when a Z. mobilis bioconversion medium was used, with a recovery of 14%. Despite the low recovery efficiency the high specificity in relation to the other bioconversion components makes this process suited for LBA production.

Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt (“Caspian Sea yogurt”) in Japan

Lactobionic acid was first found in a Caucasian fermented milk product popularly known as “Caspian Sea yogurt” in Japan. The presence of lactobionic acid in the fermented milk was indicated by the results of both high-performance anion-exchange chromatographic analysis with pulsed amperometric detection and mass spectrometric analysis. Thereafter, the acid was purified from the yogurt and analyzed by nuclear magnetic resonance. A substantial amount of lactobionic acid was found to be accumulated in the upper layer of the yogurt, especially within 10mm from the surface. A total of 45mg of lactobionic acid per 100g of the upper yogurt layer was collected after 4 d of fermentation. The annual intake of lactobionic acid in individuals consuming 100g of the yogurt every day would be 0.5 to 1.0g. A lactose-oxidizing bacterium was isolated from the fermented milk and was identified as Acetobacter orientalis. Washed A. orientalis cells oxidized monosaccharides such as d-glucose at considerable rates, although their activities for substrates such as lactose, maltose, and cellobiose were much lower. When A. orientalis cells were cultivated in cow's milk, they exhibited lactose-oxidizing activity, suggesting that this bacterium was the main organism involved in the production of lactobionic acid in the yogurt.

Gluconobacter UTBC-427株休止菌体を用いたラクトビオン酸の生産

Gluconobacter UTBC-427株休止菌体を用いたラクトビオン酸の生産

Scale‐up of enzymatic production of lactobionic acid using the rotary jet head system

Enzymatic oxidation of lactose to lactobionic acid (LBA) by a carbohydrate oxidase from Microdochium nivale was studied in a pilot-scale batch reactor of 600 L working volume using a rotary jet head (RJH) for mixing and mass transfer (Nordkvist et al., 2003, Chem Eng Sci 58:3877-3890). Both lactose and whey permeate were used as substrate, air was used as oxygen source, and catalase was added to eliminate the byproduct hydrogen peroxide. More than 98% conversion to LBA was achieved. Neither enzyme deactivation nor enzyme inhibition was observed under the experimental conditions. The dissolved oxygen tension (DOT) was constant throughout the tank for a given set of operating conditions, indicating that liquid mixing was sufficiently good to avoid oxygen gradients in the tank. However, at a given oxygen tension measured in the tank, the specific rate of reaction found in the RJH system was somewhat higher than previously obtained in a 1 L mechanically stirred tank reactor (Nordkvist et al., 2007, in this issue, pp. 694-707). This can be ascribed to a higher pressure in the recirculation loop which is part of the RJH system. Compared to mechanically stirred systems, high values of the volumetric mass transfer coefficient, k(L)a, were obtained when lactose was used as substrate, especially at low values of the specific power input and the superficial gas velocity. k(L)a was lower for experiments with whey permeate than with lactose due to addition of antifoam. The importance of mass transfer and of the saturation concentration of oxygen on the volumetric rate of reaction was demonstrated by simulations.

Production of lactobionic acid with process integrated electrochemical enzyme regeneration and optimisation of process variables using response surface methods (RSM)

This study focussed on the continuous electrochemical regeneration of cellobiose dehydrogenase, which has excellent biocatalytic properties. Conversion of lactose to latobionic acid was used to measure catalytic activity. The results obtained were compared with the results for the enzymatic regeneration method. Moreover, medium components were optimised using response surface methods for the turnover frequency of the biocatalyst. No loss of activity for the biocatalyst was observed due to contact with electrodes. Overall, this electrochemical process promises to be easier to control, more cost-effective and environmentally friendly than enzymatic processes. The product lactobionic acid can be widely applied because of its non-toxic and biodegradable character.

Continuous Enzymatic Regeneration of Electron Acceptors Used by Flavoenzymes: Cellobiose Dehydrogenase-Catalyzed Production of Lactobionic Acid as an Example

An efficient enzymatic bioprocess is described in which lactose, an abundant renewable resource produced by the dairy industry, is completely and efficiently converted with a specific productivity of up to 32 g (kU h)−1 into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the fungal enzyme cellobiose dehydrogenase which oxidizes several β-1,4-linked disaccharides including lactose specifically at position C-1 of the reducing sugar moiety to the corresponding lactones. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H2O-producing, copper-containing oxidase, and therefore has to be added in low, catalytic amounts only. Redox mediators that were successfully employed in this novel process and hence are compatible with the laccase regeneration system include benzoquinone, ABTS, ferricyanide, or ferrocene, amongst others. Factors affecting operational stability of the biocatalysts employed in this process include the redox mediator used, the temperature, and importantly the volumetric gas flow necessary for maintaining the dissolved oxygen tension. Lactobionic acid is a mild and sweet tasting acid with excellent chelating properties. These useful characteristics have lead to a growing number of patents for diverse applications in the food, pharmaceutical and detergent industries.

Screening of Lactobionic Acid Producing Microorganisms.

Lactobionic acid (LA) is derived from lactose and expected to be a versatile material for grow-ing bifidobacterium and forming mineral salts with high solubility in water for supplements. We aimed to develop microbial or enzymatic production systems of LA. To this aim, we screened lactose-oxidizing microorganisms, and obtained a strain of Burkholderia cepacia. The lactose-oxidizing activity existed in the membrane fraction of disrupted cell preparation of the strain. Only oxygen was necessary for lactose-oxidizing activity as a proton acceptor. A crude cell-free enzyme preparation was prepared, and its oxidizing ability and other properties on several saccharides were examined. The cell-free preparation oxidized D-glucose, D-mannose, D-galactose, D-xylose, L-arabinose and D-ribose. It also reacted with lactose, cellobiose, maltose, maltotriose, maltotetaose and maltopentaose. The strain accumulated LA in the culture supernatant with no loss of lactose. The strain is advantageous to production of LA by both fermentation and enzymatic reaction.

Production of lactobionic acid from whey by Pseudomonas sp. LS13-1

Pseudomonas sp. LS13-1 was isolated as a producer of lactobionic acid from whey and when grown with 207 g whey l−1 (150 g lactose l−1 equivalent) and three intermittent additions of 69 g whey l−1 (50 g lactose l−1 equivalent) in a fed-batch culture at pH 5.5 in a 2-l jar fermenter, it produced 175 g lactobionic acid l−1 after 180 h. In a lactose medium it produced 240 lactobionic acid l−1 from a total of 300 g lactose l−1 after 155 h. With the addition of 20 CaCO3 l−1 instead of pH control, 290 g lactobionic acid l−1 was produced in the lactose medium after 155 h with a yield of higher than 90% (mon mol−1).

On some mechanistic aspects of the electrochemical oxidation of lactose at platinum and gold electrodes in alkaline medium

Abstract The electrocatalytic oxidation of lactose was performed in alkaline medium at both platinum and gold electrodes. Electrolysis carried out on Au electrodes showed that the conversion yield decreases when the initial concentration of lactose increases. This does not affect the selectivity in the production of lactobionic acid which is evaluated to be nearly 100%. The kinetic study on lead modified platinum electrodes showed that the oxidation of lactose into lactobionic acid follows an overall first order law with k0 = 8.2 × 10−4 min−1 cm−2. An infrared reflectance spectroscopic investigation reveals the presence of a lactone intermediate at low potentials. On a gold electrode, the chromatographic analyses confirm the IR spectroscopic studies, leading us to assume that the reaction mechanism depends on the applied electrode potential.

Continuous enzymatic production of lactobionic acid using glucose-fructose oxidoreductase in an ultrafiltration membrane reactor

Glucose-fructose oxidoreductase from Zymomonas mobilis catalyzed the oxidation of various aldose sugars to the corresponding aldonic acids. The enzyme was used for the selective and high-yield conversion of lactose to lactobionic acid in batch, fed-batch and continous reaction mode. A productivity of 110 g L d was obtained in an ultrafiltration membrane reactor, operated for 70 h.

Use of lactose in the pharmaceutical and chemical industry

Lactose is used in the pharmaceutical industry for tabletting. For this purpose it must be purified to pharmaceutical grade. Many of the properties of crystallized lactose make it suitable for tabletting, but further research is needed into the mechanisms involved in the prom. Lactose can also be used as a basis for the production of lactulose lactitol and lactobionic acid. These processes are described, and possible commercial applications for the products are discussed.