Continuous Hydrolysis of Fructans in Jerusalem Artichoke Extracts using Immobilized Nonviable Cells of Kluyveromyces marxianus

Non-viable immobilized cells of Kluyveromyces marxianus in alginate beads having inulase (ß-2,1-fructan fructano-hydrolyase E.C.3.2.1.7) acitivity were used as biocatalyst in a packed bed reactor. Extracts of Jerusalem artichoke tubers were contacted with the biocatalyst for continuous conversion of the fructan component to fructose. In a bed reactor packed with 100 mL of beads, a volumetric productivity of 136 g/L/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a 55% loss in the original activity was observed, giving a half life for the biocatalyst of 28 days.     

固定化菊粉酶的研究

报道了克鲁维酵母突变株（ｋｌｕｙｖｅｒｏｍｙｃｅｓ－ＵＶ－Ｇ－４０－３）所产菊粉酶的固定化及固定化酶的性质，并用固定化菊粉酶酶解洋姜提取液生产果糖，在分批式反应器中，当底物和固定化酶体积比为３．５∶１时，２．５ｈ水解率达到９２．４％，产率为２８．７ｇ／Ｌｈ，在连续填充床反应器中，在稀释率为０．５ｈ－１，转化率达９３．６％，产率为１５．７ｇ／Ｌｈ，半衰期达１１０ｄ以上。     

Production of soya milk containing low flatulence‐causing oligosaccharides in a packed bed reactor using immobilised α‐galactosidase

Peanut α‐galactosidase was immobilised in calcium alginate beads and used to hydrolyse the flatulence‐causing oligosaccharides, raffinose and stachyose, in soya milk in batch and in packed bed reactor with recycle. The immobilised enzyme exhibited a slightly lower activity than the free enzyme. The activity yield of immobilised α‐galactosidase was 75.1% and the immobilisation yield was 82.6%. Batch hydrolysis using immobilised enzyme at 55 °C resulted in 96% reduction in the oligosaccharides after 12 h. For the continuous process, a packed bed reactor with recycle was used. More than 98% of the oligosaccharides were hydrolysed after 6 h of reaction at 55 °C. The immobilised enzyme also proved to be stable up to three repeated hydrolysis reactions.     

Interesterification of milkfat with soybean oil catalysed by Rhizopus oryzae lipase immobilised on SiO2‐PVA on packed bed reactor

The potential of the lipase from Rhizopus oryzae immobilised on SiO₂‐PVA to catalyse the interesterification of the milkfat with soybean oil in a packed bed reactor running on continuous mode was evaluated. The reactor operated continuously for 35 days at 45 °C, and during 12 days, no significant decrease in the initial lipase activity was verified. Interesterification yields were in the range from 35 to 38%wt, which gave an interesterified product having 59% lower consistency in relation to non‐interesterified blend. Results showed the potential of the lipase from Rhizopus oryzae to mediate the interesterification of milkfat with soybean oil in packed bed reactor, attaining a more spreadable product under a cool temperature. The biocatalyst operational stability was assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 34 days and 0.034 day^?1, for half‐life and a deactivation coefficient, respectively.     

Configuration of a bioreactor for milk lactose hydrolysis

Permeabilized microbial cells can be used as a crude enzyme preparation for industrial applications. Immobilization and process recycling can compensate for the low specific activity of this preparation. For biomass immobilization, the common support is alginate beads; however, its low surface area and the low biomass concentration limit the activity. We here describe a biocatalyst consisting of a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen. A ratio of wet permeabilized biomass to alginate of 50:4 (wt/wt) resulted in a paste with maximum immobilized beta-galactosidase activity and maximum gel biomass retention. The biocatalysts retained activity better when stored in milk at 4 degrees C than in 50% glycerol. The unused biocatalysts stored in milk did not lose activity after 50 d. However, repeated use of the same biocatalyst 40 times resulted in almost 50% loss of activity. A bioreactor design with two different conditions of operation were tested for milk lactose hydrolysis using this biocatalyst. The bioreactor was operated at 40 degrees C as packed bed or with recirculation, similar to a continuous stirred tank reactor. The continuous system with recirculation resulted in 82.9% lactose hydrolysis at a residence time of 285.5 min (flow of 2.0 ml/min), indicating the potential of this system for processing low lactose milk, or even in processing other substrates, using an appropriate biocatalyst.     

Continuous Synthesis of Glucoamylase by Immobilized Fungal Mycelium of Aspergillus niger

The extracellular glucoamylase enzyme (EC 3.2.1.3) was synthesized continuously by the immobilized mycelial fragments of A. niger. Of the several polymeric matrices attempted for immobilization k-carrageenan and alginate were found to be the most effective. However, the enzyme activity exhibited by the immobilized mycelia (I.M.) was 15-20% lower than that of free cells under batch conditions. The immobilized cells have retained nearly the same enzymatic activity (120IU/g of I.M.) for 6 repeated batches and thereafter decline in activity was noticed. An aerated packed bed reactor with I.M. was operated continuously for 360 h. The volumetric productivity of the reactor was 1600IU/L/h for 192 h and reduced to 25% in 360 h.     

Temperature Dependence of Stability and Activity of an Immobilized Glucose Isomerase in a Packed Bed

A mathematical model describing the kinetics of the conversion of glucose to fructose by an immobilized glucose isomerase has been developed. The model is a straight-forward one and based on known kinetic behaviour of glucose isomerase and on limitations posed by diffusional resistance when a spherical particle containing the enzyme is considered. The model has been used to derive a relationship between initial enzyme activity and temperature. It can be demonstrated that the relationship between activity and temperature shows deviations from the familiar Arrhenius plot. The same holds for the activity half life time in a packed bed column. Experimental material confirming the theoretical considerations is presented. The experimental results have been applied to estimate some important parameters of the material used. An Arrhenius-type relationship is shown to hold for the estimated enzyme activity as well as for the kinetics of the deactivation process. The parameters obtained can be used in temperature optimization studies for the packed bed conversion process. The influences of initial activity, particle size and temperature on the production of fructose are discussed.     

STABILIZATION OF THE ACTIVITY OF β-GALACTOSIDASE IN PERMEABILIZED IMMOBILIZED CELLS FOR HYDROLYSIS OF LACTOSE IN MILK

A potentially low cost β‐galactosidase was prepared as a crude permeabil‐ized cell mass of the yeast Kluyveromyces lactis that had been grown in ultrafiltered cheese whey. The enzyme preparation was immobilized in alginate beads. Milk lactose hydrolysis rates were 25% higher in manganese alginate beads than in calcium alginate beads. The immobilized biocatalyst lost activity when stored in calcium chloride solution, however, storage in 5 mM DTT or 50% glycerol allowed the biocatalyst to be recycled at least 5 times without any loss of activity.     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

CHARACTERIZATION OF A CHEMICALLY CONJUGATED β-GALACTOSIDASE BIOREACTOR1

A chemically prepared conjugate of avidin and E. coli β-galactosidase was adsorbed to biotinylated controlled-pore glass beads and used in a fluidized-bed bioreactor to assess the feasibility of bioselective adsorption immobilization technology. Biotinylated 200 nm pore diameter porous glass beads were prepared by reaction of 3-aminopropyl-glass beads with sulfosuccinimidyl-6-(biotinamido) hexanoate. Avidin and biotinylated β-galactosidase were sequentially adsorbed to the matrix. The fluidized-bed bioreactor was characterized with respect to β-galactosidase activity using both a lactose solution and o-nitrophenyl β-D-galactopyranoside (ONPG) as substrates. A lactose solution (4.5%, pH 7) was assayed for lactose hydrolysis at various flow rates. The bioreactor was operated for three months at 65–75% lactose hydrolysis with no loss in enzyme activity. The biocatalyst was characterized by amino acid analysis to determine the amount of each of the two proteins adsorbed. Results indicated 162 μ protein/mg beads of which 36% was avidin and 64% was β-galactosidase corresponding to 1 mole of avidin per mole of β-galactosidase monomer. Biocatalyst activity using ONPG as the substrate was 430 μmoles/min/mg protein, yielding a specific activity of 672 μmoles/min/mg β-galactosidase. These results lead to the conclusion that biospecific adsorption of the β-galactosidase conjugate onto biotinylated glass beads via avidin results in a biocatalyst that is stable and retains a high specific activity.     

Immobilization of Aspergillus oryzae with κ-carrageenan for soybean oligosaccharide hydrolysis

The immobilized Aspergillus oryzae spores in κ-carrageenan were used for production of α-galactosidase. The immobilized cells could be used up to 5 repeated cycles for enzyme production. They were employed for raffinose family oligosaccharides (RFO) hydrolysis in batch, repeated batch, and continuous operations after 5 days of fermentation. In batch mode, 65, 73, and 80% of RFOs were hydrolyzed after 3, 6, and 12 h, respectively; in repeated batch; 70, 63, 52, and 45% were hydrolyzed in 1, 2, 3, and 4 repeated cycles. In the fluidized bed reactor, 65, 58, 53, 48, and 44% RFOs were hydrolyzed at flow rates of 25, 50, 75, 100, and 125 mL/h respectively. The κ-carrageenan beads maintain good mechanical strength up to 4 repeated uses for RFO hydrolysis in soymilk, and their use in the hydrolysis of RFOs of soybean is a promising solution to overcome flatulence and to increase consumption of soy products.     

填充床反应器固定化菊糖果糖转移酶催化合成双果糖酐Ⅲ的研究

本文建立了填充床反应器固定化菊糖果糖转移酶水解菊糖的工艺。以菊糖为底物,探究该工艺水解菊糖的条件,以单因素实验为基础,依据正交实验优化,考察菊糖浓度、反应时间、反应温度及底物流速等因素对双果糖酐Ⅲ含量的影响,获得最佳的工艺条件。该工艺采用填充床反应器的容积为20m L,固定化酶的装载量为15m L。结果表明:菊糖浓度为100g/L,反应时间为1h,温度为60℃,底物流速为20m L/h,在该工艺条件下制取双果糖酐Ⅲ的浓度为67.38g/L。该工艺能持续操作48d以上,半衰期为48d,为该工艺大规模生产提供理论与操作的基础。      

Continuous hydrolysis of raffinose family oligosaccharides in soymilk by fluidized bed reactor

Soybean is an important pulse crop for human and animal nutrition. However, raffinose family oligosaccharides (RFOs) found in soybean seeds specifically hinder its consumption as it is not digested by normal human carbohydrases and is fermented by intestinal microflora, leading to flatulence. Immobilized spores of Aspergillus oryzae were grown for 5 days in calcium alginate gel beads, then harvested and employed in RFOs hydrolysis in batch mode, repeated batch and continuous mode in a fluidized bed reactor. Effective hydrolysis of 244.4 mg/60 ml was obtained after 6 h incubation. And in continuous mode 465.6 mg/100 ml was obtained at a flow rate of 25 ml/h. The use of immobilized A. oryzae cells for hydrolysis of RFOs is a promising solution to overcome flatulence and to increase the consumption of soy products.     

Stability evaluation of an immobilized enzyme system for inulin hydrolysis

The stability of free and Amberlite-immobilized inulinase, aiming at inulin hydrolysis was evaluated. The apparent activation energy of the biotransformation decreased when the immobilized biocatalyst was used, suggesting diffusional limitations, despite a decrease in the optimal temperature for catalytic activity for the immobilized biocatalyst. Thermal deactivation, of both forms of the biocatalyst, was evaluated by the linear inverted model. Inulinase immobilization consistently enhanced half-life of the enzyme, which increased up to 6-fold, as compared to the free form. Mean enzymatic activity was computed for both forms of the biocatalyst, and evidenced a decrease of optimal temperature with increased incubation periods. The deactivation energies estimated by an Arrhenius plot, evidenced a decrease of roughly 20% when free inulinase was used. The immobilized biocatalyst was effectively reused in successive batch runs for the hydrolysis of a 5% inulin solution.     

Continuous Saccharification of Corn Starch in a Membrane Reactor. Part I: Conversion,Capacity and Productivity

Effects of enzyme concentration, substrate concentration and residence time on substrate conversion, reactor capacity and productivity were evaluated. As enzyme concentration increased from 1 to 6 g/L, substrate conversion also increased. Substrate conversion was essentially unchanged at enzyme concentrations greater than 6 g/L. Reactor capacity was significantly affected by both substrate and enzyme concentration. As residence time increased, substrate conversion increased 15–22%. Productivities of the continuous reactor were 10 to 20 times greater than those obtained in a batch reactor. The continuous membrane reactor was capable of producing a highly pure glucose syrup at low residence times. Due to the high levels of substrate conversion, minimal operational problems were experienced.     

Immobilization of amyloglucosidase on polystyrene anion exchange resin I. preparation and properties

Amyloglucosidase (exo‐1,4‐ α‐D‐glucosidase, E C 3.2. 1.3) was coupled to glutaraldehyde activated Indion 48‐R (a cross‐linked macroporous anion exchanger) by Schiff base reaction. The bound enzyme exhibited 60–70% activity of the free enzyme. Substrate concentrations as high as 32% (w/w) liquefied tapioca starch could be quantitatively converted into 96–98% (w/w) dextrose in 24 h at 50°C and pH 4.5. Though immobilization lowered the temperature optimum to 50–60°C from 65°C for the free enzyme, it increased the temperature stability. However, there was no change either in the pH optimum or pH stability after immobilization. In batch operations, the immobilized preparation showed a half life of 32 and 12 days at 50°C and 60°C respectively.     

Bone-Bound Glucoamylase as a Biocatalyst in Bench-Scale Production of Glucose Syrups from Liquefied Cassava Starch

This research assesses the bench-scale application of a non-conventional support, bone particles, for glucoamylase (GA) immobilization and its subsequent use in cassava starch hydrolysis. Upon determining the appropriate conditions to immobilize GA onto chicken bone particles, such as pH, ionic strength, particle size, and enzyme load, bench-scale immobilization of commercial GA without further purification was performed. Under the selected conditions, 270 GA units per gram of support were adsorbed. Optimal temperature and thermal stability of immobilized GA were only slightly different from those of the free enzyme, while optimal pH became more acidic by about one unit. The feasibility of the use of this immobilized biocatalyst for high glucose syrup production from liquefied cassava starch, at bench scale in batch process using a stirred-tank reactor, was demonstrated. Repeated use of the GA-bone derivative showed that similar conversions to those achieved with soluble enzyme (dextrose equivalent = 98) were reached until the third batch and over 90% until the 25th batch.     

Lipase-catalyzed synthesis of fatty acid fructose esters

Fatty acid sugar esters are non-ionic surfactants which are widely used in food, cosmetic and pharmaceutical industry. Lipase-catalyzed synthesis of fructose with fatty acid in organic medium was performed in a batch reactor at atmospheric pressure. Influence of different reaction parameters, such as different kinds of lipase preparations and organic solvents, biocatalyst concentration, molecular sieve concentration, temperature, stirring rate and the use of different fatty acids as acyl donors was studied. Optimum conditions were found using 10% (w/w of substrates) lipase from Candida antarctica B (Novozym 435) and 12.1% (w/w) of molecular sieves at 60 °C and stirring rate of 600 rpm. The highest conversion was obtained in ethyl methylketon (82%), which is permitted for general use in the manufacture of food additives.     

Cell-recycle membrane bioreactor for conducting continuous malolactic fermentation

A cell-recycle membrane bioreactor system has been evaluated for conducting rapid, continuous malolactic conversion in wine. A reactor (300 mL) containing 10¹⁰ cfu/mL of Leuconostoc oenos and operating at a flow rate of 6 mL/min for periods up to 56 h gave greater than 95% degradation of L-malic acid in a range of red and white wines. Stability of malic acid degrading activity and long term performance of the reactor varied with strain of Leuc. oenos , wine being processed and temperature. Increasing operating temperature from 20°C to 30°C caused rapid loss of activity. When grape juice was processed, reactor performance (>95% degradation of malic acid) was stable for at least 125 h. Addition of 10% ethanol to the juice caused a loss in reactor activity. Some off-flavours were acquired by the wine during processing in the reactors, but this problem was minimised by using reactors containing a mixture of cells of Leuc. oenos and Saccharomyces cerevisiae .     

The chilled storage life and retail display performance of vacuum and carbon dioxide packed hot deboned beef striploins

Two cooling regimes that complied with the New Zealand meat hygiene requirement that hot deboned meat be chilled to +7 °C or less within 24 hr of leaving the slaughter floor were evaluated for the production of chilled table meats. Electrically stimulated hot deboned bull beef half striploins were either vacuum or carbon dioxide packed before being cooled in accordance with either Regime 1 (cool at +5 °C for 24 hr, transfer to chiller operating at −1.0 ± 0.5 °C) or Regime 2 (cool at +5 °C for 24 hr, hold at 5 °C for 6 days, transfer to chiller operating at −1.0 ± 0.5 °C). Striploins were removed from −1.0 °C storage 8, 28, 42, 56, 70, 84 and 98 days after slaughter and subjected to microbiological, tenderness, sensory and retail display performance evaluations.

Both Regimes 1 and 2 produced meat of acceptable mean tenderness, 8 kgF (MIRINZ Tenderometer) in either vacuum or carbon dioxide packs within 28 and 8 days of slaughter, respectively. However, 70 days after slaughter the first signs of over-ageing became apparent. Steaks from Regimes 1 and 2 maintained acceptable visual appearance during retail display at 5 °C for 48 hr and 24 hr, respectively. After these times, the product was judged by the panel to be unacceptable because of its dull dark lean tissue and grey to green discoloration of the fat. Poor colour stability during retail display was mirrored by deterioration of sensory attributes, particularly aroma which is indicative of incipient spoilage. While carbon dioxide packaging in combination with Regime 1 offered an initial microbiological advantage over vacuum packaging, this advantage was not, however, carried over into retail display.

Poor colour and sensory stability during retail display suggest that chilled table cuts derived from hot deboned bull beef are more suited to the Hotel-Restaurant-Institutional (HRI) trade than supermarket retailing. To serve the HRI, vacuum packed hot deboned bull beef primal cuts processed by Regime 1 appear to be the combination of choice. This combination would enable commercial processors to produce quality table beef with a chilled storage life of up to 70 days.