Technical note: Application of immobilized cells of Kluyveromyces marxianus for continuous hydrolysis to fructose of fructans in Jerusalem artichoke extracts

Dead cells of Kluyveromyces marxianus having inulase (β-D-fructo fructanohydrolase E.C.3.2.1.7) activity were immobilized in alginate beads and used as a biocatalyst in a packed bed reactor and a stirred batch reactor. Fructosans of Jerusalem artichoke tubers, after extraction, were utilized for continuous or semi-continuous production of fructose. In a bed reactor packed with 100 ml of beads, a volumetric productivity of 36 g/l/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a half life of 28 days was observed for the biocatalyst. Using artichoke extract containing 20% fructan solution, 98% conversion could be achieved in a batch reactor in 20 hr. Repeated cycling of beads resulted in considerable loss of catalyst from the reactor and subsequent loss in catalytic activity, thus giving a half life of only 14 days.     

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.     

Composition of Fermented Grape Juice Continuously Produced by Immobilized Lactobacillus casei

Continuous fermentation of grape juice was carried out in a packed bed column system using Lactobacillus casei adsorbed onto ceramic beads. Chemical components in the reaction vessel of this system were compared with those of a free-cell fermentation. No major differences were noted in the principal components. Diacetyl and methionine levels decreased, however, whereas aspartic acid and cysteine increased. Silica levels increased immediately after initiation of the immobilized cell culture due to elution from the ceramic carrier beads. In two of the five trials the reactor was contaminated with Bacillus megaterium.     

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.     

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。     

Strategies to remove water formed as by-product on the monoolein synthesis by enzymatic esterification performed on packed bed reactor

Different strategies to remove water generated on the monoolein synthesis by enzymatic esterification of oleic acid with glycerol were evaluated. Esterification reactions were catalyzed by Penicillium camembertii lipase immobilized on SiO₂-PVA and runs carried out on a packed bed reactor (PBR) under continuous mode. The use of molecular sieves was the selected technique to remove the water formed, and for this purpose, two experimental setups were performed: (1) reactor packed with alternate layers of molecular sieves and immobilized lipase and (2) three reactors in series having a water column extractor between two immobilized packed beds, making it possible to replace the saturated molecular sieves by a regenerated one at a given time. Continuous run without applying dehydration techniques to remove the formed water was used as a control. The best performance was obtained using a water column extractor as verified by an increase in the monoolein formation from 22 to 35%. This strategy also provided a good performance of the bed reactor as a result of the high operational stability estimated for the immobilized derivative (half-life = 19 days).     

Continuous Production of Lactic Acid from Whey Perméate by Free and Calcium Alginate Entrapped Lactobacillus helveticus

Lactobacillus helveticus strain milano was used for the continuous fermentation of lactic acid in cheese whey-yeast extract permeate medium. The best productivity of lactic acid was with the free cell system, which was 9.7 g/L per h at a dilution rate of .352 h⁻¹. Under such conditions, lactose conversion was 87.5%, based on the lactose concentration of 37.4 g/L in feed. Under high dilution rates, the cells were elongated to several times their normal size, resulting in wall growth. The cell growth on the fermentor wall caused system instability; however, it prevented cell wash-out under high dilution rates. The packed bed column system using Ca alginate entrapped cells is not suitable for practical application. Nonuniform pH control, plugging of the column by leaked cells, and decalcification of Ca alginate beads were major drawbacks of the packed bed system.     

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.     

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。      

Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V_max values were 1.37 and 1.06 mmol min⁻¹ mg⁻¹, respectively. The K_m and K_i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.     

Optimising chitosan–pectin hydrogel beads containing combined garlic and holy basil essential oils and their application as antimicrobial inhibitor

Chitosan–pectin hydrogel beads that trap and release the maximal amount of combined garlic and holy basil essential oils to inhibit food microorganisms were developed based on the central composite design, with chitosan (0.2–0.7% w/v), pectin (3.5–5.5% w/v) and calcium chloride (CaCl₂) (5.0–20.0% w/v) contents. The optimal bead consisted of 0.3–0.6% w/v chitosan, 3.9–5.1% w/v pectin and 8.0–17.0% w/v CaCl₂, which had a high encapsulation efficiency (62.16–79.06%) and high cumulative release efficiency (31.55–37.81%) after storage at 5 °C for 15 days. Optimal hydrogel beads were packed into a cellulose bag to evaluate antimicrobial activity by the disc volatilisation method. The beads inhibited Bacillus cereus, Clostridium perfringens, Escherichia coli, Pseudomonas fluorescens, Listeria monocytogenes and Staphylococcus aureus but did not affect Lactobacillus plantarum and Salmonella Typhimurium. The oil-containing beads could potentially be applied in food packaging to inhibit the mentioned microorganisms.     

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.     

Comparative Production of Sugarcane Vinegar by Different Immobilization Techniques

Sugarcane juice was converted to ethanol by Saccharomyces cerevisiae producing 8% (v/v) ethanol. This ethanol was used for vinegar production using adsorbed (bagasse, corn cobs and wood shavings) and entrapped (calcium alginate) cells of Acetobacter aceti NRRL 746. All three adsorbed carrier materials were statistically similar for acetic acid production and produced acidity from 5.9 to 6.7% after 28 days of submerged fermentation. By recycling bagasse adsorbed cells, the time of acetic acid fermentation was reduced to 13 days. Semi‐continuous fermentation of bagasse adsorbed cells using a packed bed column further reduced the fermentation time to 80 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.     

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.     

影响填充床酶反应器酸解合成结构脂质因素研究进展

结构脂质因具有独特营养和生理功能而引起人们关注;填充床酶反应器酸解合成结构脂质具有工艺简捷、酶利用率高、副反应较少,适于连续化生产等优点而成为研究热点。该文概述影响填充床酶反应器酸解合成结构脂质主要因素,为其实现工业化生产提供参考。     

渗透性K.lactis细胞内乳糖酶水解牛乳中乳糖的研究

应用填充床细胞反应器,以渗透性K．lactis细胞内乳糖酶连续催化牛乳中乳糖的水解。渗透性K．lactis细胞内乳糖酶的热失活遵循一级反应动力学,40℃时酶的半衰期为9．5h,表观米氏常数为0．39mmol/ml,最大反应速率Vmax为0．188mmol/min。在2．6cm×50cm填充床生物反应器上,渗透性K．lactis细胞内乳糖酶催化牛乳中乳糖水解的适宜体积流速为1．0ml/min,酶反应的适宜温度40℃,反应速率达到动态平衡后生成的葡萄糖的质量浓度为45．2mg/ml,牛乳中乳糖的水解率为91．3%。     

Immobilised yeast grape must deacidification in a recycle fixed bed reactor

Maloalcoholic fermentation (MAF) of grape must by Schizosaccharomyces pombe immobilised in calcium‐alginate double‐layer beads (ProMalic^®) was studied in Erlenmeyer flasks and in a total recycle fixed‐bed reactor operating in batch mode. The reaction is pseudo‐first order with respect to l ‐malic acid and under similar conditions deacidification is faster in the recycle reactor. This was attributed to mass transfer limitations which were confirmed in the recycle reactor by studying the influence of yeast load on the rate of MAF. Mass transfer limitations are also responsible for the lower activation energy of fermentation with the immobilised yeast (67 ± 9 kJ mol^?1) in comparison with the free cells (126 ± 19 kJ mol^?1). Alcoholic fermentation and MAF were performed simultaneously, both in the recycle reactor and in the industrial trials, confirming the efficacy of immobilised S. pombe to reduce grape must acidity without interfering with the main fermentation. Altogether, the present results are useful for the scale‐up of a recycle reactor to process large volumes of grape must.     

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.