Initial Lactose Concentration Research Articles

Kinetic modelling of lactic acid production from whey by Lactobacillus casei (NRRL B‐441)

AbstractThe biomass growth, lactic acid production and lactose utilisation kinetics of lactic acid production from whey by Lactobacillus casei was studied. Batch fermentation experiments were performed at controlled pH and temperature with six different initial whey lactose concentrations (9‐77 g dm−3) in a 3 dm3 working volume bioreactor. Biomass growth was well described by the logistic equation with a product inhibition term. In addition, biomass and product inhibition effects were defined with corresponding power terms, which enabled adjustment of the model for low‐ and high‐substrate conditions. The Luedeking‐Piret equation defined the product formation kinetics. Substrate consumption was explained by production rate and maintenance requirements. A maximum productivity of 2.5 g dm−3 h−1 was attained with an initial lactose concentration of 35.5 g dm−3. Copyright © 2006 Society of Chemical Industry

Kinetics of lactose fermentation using a recombinant Saccharomyces cerevisiae strain

This work presents a multi-route, non-structural kinetic model for interpretation of ethanol fermentation of lactose using a recombinant flocculent Saccharomyces cerevisiae strain expressing both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces lactis. In this model, the values of different metabolic pathways are calculated applying a modified Monod equation rate in which the growth rate is proportional to the concentration of a key enzyme controlling the single metabolic pathway. In this study, three main metabolic routes for S. cerevisiae are considered: oxidation of lactose, reduction of lactose (producing ethanol), and oxidation of ethanol. The main bioprocess variables determined experimentally were lactose, ethanol, biomass, and dissolved oxygen concentrations. Parameters of the proposed kinetic model were established by fitting the experimental data obtained in a small lab-scale fermentor with the initial lactose concentrations ranging from 5 g/dm3 to 50 g/dm3. A very good agreement between experimental data and simulated profiles of the main variables (lactose, ethanol, biomass, and dissolved oxygen concentrations) was achieved.

Reactor Performance in the Presence of Catalyst Deactivation

AbstractThe main variable of enzymatic processes is often found to be the operating temperature. An increase in temperature leads to higher rates for the catalytic transformation. However, beyond a certain temperature catalyst deactivation is winning the game. Therefore, processes should be optimized in order to determine the temperature which leads to a minimal demand of enzyme preparation. For the prediction of such optimal reactor operation, modeling of the temperature dependence of the process has to be performed. Examples of such modeling are given for the hydrolysis of lactose in UHT milk by means of three different β‐galactosidases – those from Aspergillus oryzae, Kluyveromyces lactis, and Escherichia coli. The reaction kinetics for a constant initial lactose concentration can be described by a model of two parameters, of which only one depends on temperature. For the lactase of E. coli the reaction can be described as a simple reaction with first order kinetics. The deactivation mechanism includes a reversible as well as an irreversible path of denaturation. The temperature dependent parameters follow Arrhenius' and van't Hoff's law, respectively. On the basis of their particular reaction models all three enzymes can be compared with respect to their optimum use. The models have been verified under laboratory conditions and have shown their usefulness for the prediction of optimum operating variables. Quite remarkable features have been found for the lactase of E. coli.

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10-60 g L(-1)) at 30 degrees C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L(-1) and 60 g L(-1) initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g(-1) to 0.41 g g(-1), indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L(-1)) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h(-1)), biomass yield (0.38 g g(-1)) and overall productivity (2.9 g L(-1) h(-1)) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.

Batch kinetics and modelling of ethanolic fermentation of whey

SummaryThe fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L−1, the production of ethanol was 0.618 g L−1 whereas at 50 g L−1 it was 3.98 g L−1. However, an increase in lactose concentration to 100 g L−1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L−1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S0 = 10 and 50 g L−1 but failed in case of S0 = 100 g L−1 because of strong substrate inhibition.

Synthesis of prebiotic galactooligosaccharides using whole cells of a novel strain, Bifidobacterium bifidum NCIMB 41171

A novel strain of Bifidobacterium bifidum NCIMB 41171, isolated from a faecal sample from a healthy human volunteer and able to express beta-galactosidase activity, was used in synthesis reactions for the production of galactooligosaccharide from lactose. The beta-galactosidase activity of whole bifidobacterial cells showed an optimum activity at pH 6.8-7.0 and 40 degrees C. The transgalactosylation activity of the B. bifidum cells from 50% (w/w) lactose resulted in a galactooligosaccharide mixture (20% w/w) comprising (w/w): 25% disaccharides, 35% trisaccharides, 25% tetrasaccharides and 15% pentasaccharides. Using different initial lactose concentrations, the conversion rate to galactooligosaccharides was maximum (35%) when 55% (w/w) lactose was used. In fermentation experiments, B. bifidum showed an increased preference towards the produced galactooligosaccharide mixture, displaying higher growth rate and short-chain fatty acid production when compared with commercially available oligosaccharides.

Synthesis of galacto‐oligosaccharide from lactose using β‐galactosidase from Kluyveromyces lactis: Studies on batch and continuous UF membrane‐fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40 degrees C and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40 degrees C, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAE-PAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal GOS).

Estudo da produção de beta-galactosidase por fermentação de soro de queijo com Kluyveromyces marxianus

The enzymatic hydrolysis of lactose by b-galactosidase plays an important role in the processing of milky products such as the production of lactose-hydrolyzed milk for consumption by intolerant person to lactose and the prevention of the crystallization in dairy products. In this work, the influences of nutrient concentrations in the culture medium based on cheese whey were studied with the objective of producing b-galactosidase from Kluyveromyces marxianus. The fermentations were carried out in a shaker at 30°C and initial pH 5.5 under agitation, starting with an initial cellular concentration of 107 cells/mL, varying the initial concentrations of lactose and yeast extract. For extraction of the enzyme of the cells it was used autolysis with chloroform in potassium phosphate buffer. In the medium with a initial lactose concentration of 50g/L, supplemented with salts, yeast extract 12g/L, the enzymatic activity and cellular concentration were 28 UGl/mL and 5.3g/L respectively.

Enzymatic Production and Complete Nuclear Magnetic Resonance Assignment of the Sugar Lactulose

The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.

Production of β‐galactosidase from recombinant Saccharomyces cerevisiae grown on lactose

AbstractImproved productivity and costs reduction in fermentation processes may be attained by using flocculating cell cultures. The production of extracellular heterologous β‐galactosidase by recombinant flocculating Saccharomyces cerevisiae cells, expressing the lacA gene (coding for β‐galactosidase) of Aspergillus niger under the ADHI promotor and terminator in a bioreactor was studied. The effects of lactose concentration and yeast extract concentration on β‐galactosidase production in a semi‐synthetic medium were analysed. The extracellular β‐galactosidase activity increased linearly with increasing initial lactose concentrations (5–150 g dm−3). β‐Galactosidase production also increased with increased yeast extract concentration. During the entire fermentation, no accumulation of the hydrolysed sugars, glucose and galactose, was observed. The catabolic repression of the recombinant strain when cultured in a medium containing equal amounts of glucose and galactose was confirmed. In complete anaerobiosis, the fermentation of lactose resulted in a very slow fermentation pattern with lower levels of β‐galactosidase activity. The bioreactor operation together with optimisation of culture conditions (lactose and yeast extract concentration) led to a 21‐fold increase in the extracellular β‐galactosidase activity produced when compared with preliminary Erlenmeyer fermentations. Copyright © 2004 Society of Chemical Industry

Aspergillus niger β-galactosidase production by yeast in a continuous high cell density reactor

The continuous production of extracellular heterologous β-galactosidase by a recombinant flocculating Saccharomyces cerevisiae, expressing the lacA gene (coding for β-galactosidase) of Aspergillus niger was investigated. A continuous operation was run in a 6.5 l airlift bioreactor with a concentric draft tube using lactose as substrate. Data on the operation with semi-synthetic medium with 50 and 100 g/l initial lactose concentrations are presented. The best result for β-galactosidase productivity—6.2×10 5 U/1 h—was obtained for a system operating at 0.24 h −1 dilution rate and for a lactose concentration in the feed of 50 g/l. This value represents a 11-fold increase in β-galactosidase productivity when compared to batch culture. Together with extracellular β-galactosidase production an ethanol productivity of 9 g/1 h was obtained for the bioreactor fed with 50 g/l initial lactose concentration at 0.45 h −1 dilution rate. In addition to β-galactosidase and ethanol production, this system allowed for complete lactose metabolism. The feasibility and advantages of using continuous high-cell-density systems operating with flocculent yeast cells for extracellular protein production is clearly shown.

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l−1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l−1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l−1 although Lactobacillus helveticus appeared to tolerate up to 100 g l−1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l−1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The β-galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.

Batch and continuous culture of Lactococcus lactis NZ133: experimental data and model development

A model has been developed for the batch and continuous fermentation of Lactococcus lactis strain NZ133. It was observed that the growth kinetics of L. lactis was predominantly influenced by lactose limitation and lactate product inhibition with the growth of this particular strain showing a relatively high sensitivity to lactate inhibition. The Luedeking–Piret equations for growth and for lactate production were successfully incorporated into the model. Parameters for the model were determined based on experimental data using a program developed in Microsoft EXCEL. The values of the key kinetic constants were: maximum specific growth rate ( μ max), 1.10 h −1; growth associated constant for lactate production ( α), 0.932 g g −1; non-growth associated constant for lactate production ( β), 3.02 g g −1 h −1; average lactate yield based on lactose ( Y p/ s ), 0.93 g g −1. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles on media with initial lactose concentrations ranging from 20 to 100 g l −1. The model also provided good prediction of continuous culture data on medium with an initial lactose concentration of 40 g l −1.

Optimization of the enzymic process for manufacturing low-lactose milk containing oligosaccharides

Methods and enzymic processing conditions for manufacturing low-lactose milk containing oligosaccharides were evaluated. Comparisons were made between the method of using β-galactosidase to transform the lactose in milk into oligosaccharides directly and the method of applying ultrafiltration techniques to separate lactose from milk proteins and then transform the lactose in the permeate into oligosaccharides. Since the β-galactosidase exhibited higher transgalactosylative activity in the permeate than in the milk and the higher the initial lactose concentration the higher the conversion to oligosaccharides, a useful procedure for manufacturing low-lactose high-oligosaccharides milk would be to separate milk proteins from lactose by ultrafiltration first, concentrate the UF permeate by evaporation, apply enzymic reaction in the concentrated permeate, and add the hydrolysate to the retentate to obtain the final milk product. The optimal conditions for the enzymic reaction in the permeate was 25.3% of initial lactose concentration, an E/ S ratio of 6.7%, and carried out at 50 °C for 3.5 h.

A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus

A 5 l packed bed bioreactor was used to study the effect of initial lactose concentration and hydraulic retention time (HRT) on cell growth, lactose utilization and lactic acid production. Up to 95% of the initial lactose concentration was utilized at longer HRTs (30-36 h). The study showed that lactic acid production increased with increases in HRT (12-36 h) and initial lactose concentrations. The highest lactic acid production rate (3.90 g l(-1) h(-1)) was obtained with an initial lactose concentration of 100 g/l and an HRT of 18 h, whereas the lowest lactic acid production rate (1.35 g l(-1) h(-1)) was obtained with an initial lactose concentration of 50 g/l and an HRT of 36 h. This suggested that optimal lactic acid production can be achieved at an HRT of 18 h and initial lactose concentration of 100 g/l.

Fed-batch fermentation of Ganoderma lucidum for hyperproduction of polysaccharide and ganoderic acid

A process for simultaneous efficient production of bioactive ganoderic acid (GA) and Ganoderma polysaccharide by fed-batch fermentation of Ganoderma lucidum was developed. Effects of carbon source and initial sugar concentration on the production of GA and polysaccharide were studied at first. Sucrose as a carbon source was suitable for the extracellular polysaccharide (EPS) production although the cells could not grow well. Lactose was beneficial for the cell growth and production of GA and intracellular polysaccharide (IPS). When the initial lactose concentration exceeded 35 g/l, the GA accumulation was decreased. The GA production was remarkably improved by pulse feeding of lactose, when its residual concentration was between 10 and 5 g/l. The fed-batch fermentation process in shake flasks could be successfully reproduced in stirred bioreactors (STR) with dissolved oxygen (DO) controlled between 20 and 35% of air saturation during fermentation. The maximum cell density reached 21.89±0.35 and 20.95±0.01 g DW/l in STR and flasks, respectively. The production of EPS reached 0.87±0.05 (STR) and 0.75±0.05 g/l (flask), and the total production of IPS reached 2.49±0.07 (STR) and 2.40±0.05 g/l (flask). The total production of GA reached 367.1±17.4 and 297.9±13.7 mg/l for STR and flask, respectively, and their corresponding productivity was 29.6±0.5 and 14.3±0.1 mg/l per day. Not only the cell density but also the content, production and productivity of GA and IPS obtained in this work are the highest reported in agitated fermentation of G. lucidum.

High-temperature alcoholic fermentation of whey using Kluyveromyces marxianus IMB3 yeast immobilized on delignified cellulosic material

A novel system for high-temperature alcoholic fermentation of whey is described. This system consists of Kluyveromyces marxianus yeast immobilized on delignified cellulosic material (DCM). The effect of pH, initial lactose concentration and temperature on the fermentation of a synthetic medium containing lactose was studied. Batch fermentations of whey were also carried out and the formation of volatile by-products was examined. The concentrations of higher alcohols were found to be in very low levels leading to a product of improved quality. The fermented whey had an improved characteristic aroma compared to unfermented whey. The possibility to use fermented whey as raw material for the production of a novel, low alcohol content drink was also investigated.

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae β-galactosidase immobilized on cotton cloth

The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae β-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased ∼25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40°C and 48 days at 50°C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40°C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 8–19, 2002.

Synthesis of galacto-oligosaccharides in AOT/isooctane reverse micelles by β-galactosidase

Synthesis of galacto-oligosaccharides (GOS) from lactose by β-galactosidase was investigated in AOT/isooctane reverse micelles where the enzyme was fairly stable. GOS yield increased with initial lactose concentration in both reverse micelles and aqueous system. Transgalactosylation reaction of the enzyme was strongly dependent on the molar ratio of water to surfactant ( w 0) of reverse micelles. At pH 7.0 and 45°C, a maximum GOS concentration from 45% (w/v) lactose solution was 51.2% (w/w) in the reverse micelles with w 0=15, while a maximum of 31% in aqueous media. Effects of solvents on GOS formation were also examined.

The influence of crystallization conditions on the morphology of lactose intended for use as a carrier for dry powder aerosols.

Lactose has been widely used as a carrier for inhalation aerosols. The carrier morphology is believed to affect the delivery of the drug. The aim of this study was to investigate the effects of crystallization conditions on the morphology of alpha-lactose monohydrate intended for use as the carrier for dry powder aerosols. The crystallization of lactose was carried out from aqueous solutions at different supersaturations, temperatures, different stages of crystallization and in the presence of different water-miscible organic solvents. The majority of lactose crystals were found to be either tomahawk-shaped or pyramidal after crystallization at an initial lactose concentration between 33-43% w/w, but these became prismatic if the lactose concentration was increased to 50% w/w. A further increase in the lactose concentration to 60% w/w led to the preparation of elongated cuboidal crystals. Higher initial lactose concentrations tended to result in the crystallization of more elongated particles. Crystallization at 40 degrees C was shown to prepare lactose crystals with a more regular shape and a smoother surface than those crystallized at 0 degrees C. Lactose particles generated during the later stage of crystallization were found to be more regular in shape with a smoother surface than those prepared in the earlier stage. The addition of 10% (v/v) methanol or ethanol or acetone to the mother liquor increased the growth rate of lactose particles whereas addition of propanol or glycerine inhibited the rate of crystal growth. Lactose crystals prepared in the presence of glycerine were more regularly shaped with a smoother surface than those prepared in the presence of ethanol or acetone. All the resultant crystals were shown to comprise alpha-lactose monohydrate. Lactose crystals could be prepared with a precisely defined morphology by means of carefully controlling the crystallization conditions.