Initial Lactose Concentration Research Articles

Optimization of ricotta cheese whey (RCW) fermentation by response surface methodology

A central composite design (CCD) was performed to evaluate the effects of four factors, i.e. temperature (T), pH, agitation rate (K) and initial lactose concentration (L), on ricotta cheese whey batch fermentation and to optimize the process leading to the formation of bio-ethanol. Anaerobic batch fermentation experiments were carried out by using the yeast Kluyveromyces marxianus. After a preliminary experimental analysis, the values of the chosen factors were 32 and 40 degrees C for T, 4 and 6 for pH, 100 and 300 rpm for K, 40 and 80 g L(-1) for L. Response surface methodology (RSM) was used to optimize the fermentation process and an empirical polynomial model was used to fit the experimental data. The best operating conditions resulted to be T=32.35 degrees C, pH 5.41, K=195.56 rpm and L=40 g L(-1) and the model ensured a good fitting of the observed data.

Optimization of process parameters by Taguchi method in the recovery of lactose from whey using sonocrystallization

AbstractAnti‐solvent crystallization of lactose in the presence of ultrasound will reduce crystal size and the level of agglomeration as compared to the commercial cooling crystallization. It offers a potential route to enhance the physical properties as well as the rapid recovery of lactose. Since lactose recovery itself can reduce biological oxygen demand of whey by more then 80%, recovery of lactose from dairy waste stream (whey) solves the problems of dairy industries by improving economics of whey utilization and pollution reduction. In the present study, recovery of lactose from partially deproteinated whey using an anti‐solvent (acetone) by sonocrystallization was optimized for finding the most influencing operating parameters; such as sonication time, anti‐solvent concentration, initial lactose concentration in the whey and initial pH of sample mixture at three levels using L9‐orthogonal method. The responses were analyzed for recovery of lactose from whey. The anti‐solvent concentration and the sonication time were found to be most influencing parameters for the recovery of lactose and the recovery of lactose was found to be 89.03% at the identified optimized level. The crystal size distribution of recovered lactose was found to be narrower (2.5 – 6.5 μm) as compared to the commercial lactose crystals (3.5 – 9.5 μm). (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Role of lactose on the production of d-arabitol by Kluyveromyces lactis grown on lactose

There are remarkably few reports on D-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular D-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on D-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of D-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that D-arabitol production was triggered when an initial lactose concentration was above 278 mmol L(-1). D-Arabitol yield increased with an increase in initial lactose concentration. The highest D-arabitol concentration of 79.5 mmol L(-1) was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L(-1) lactose and 40 g L(-1) yeast extract. Lactose was found to play two important roles in D-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and D-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated the flow from D-glucose-6-phosphate to D-arabitol. The arrest of cell growth triggered D-arabitol production.

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β- d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β- d-galactopyranoside ( oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5–8.0. The K m, k cat and k cat /K m values for oNPG and lactose are 0.9 mM, 92 s −1, 130 mM −1 s −1 and 29 mM, 98 s −1, 3.3 mM −1 s −1, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β- d-Gal p-(1→6)- d-Lac, accounting for 34% of total GOS, and β- d-Gal p-(1→6)- d-Glc, making up 29% of total GOS.

Continuous production of lactulose by immobilized thermostable β-glycosidase from Pyrococcus furiosus

A continuous enzymatic process for the production of the prebiotic disaccharide lactulose through transgalactosylation was developed using free and immobilized β-glycosidase from Pyrococcus furiosus. The hyperthermostable β-glycosidase (CelB) was immobilized onto an anion-exchange resin (Amberlite IRA-93) or onto Eupergit ® C with immobilization yields of 72% and 83%, respectively. The immobilized biocatalysts demonstrated specific activities of 920 nkat g −1 dry carrier and 1500 nkat g −1 dry carrier at 75 °C with p-nitrophenyl-β- d-galactopyranoside as substrate. Continuous biotransformations in packed-bed reactors using carrier bound CelB and in an enzyme membrane reactor using free CelB were carried out at 75 °C. Maximum lactulose yields of 43% related to the initial lactose concentration were reached with the carrier bound CelB preparations. The corresponding productivities were 52 g lactulose l −1 h −1 (Amberlite IRA-93) and 15 g lactulose l −1 h −1 (Eupergit ® C), respectively. The free enzyme tested in an enzyme membrane reactor showed a product yield of 41% and a productivity of 12 g lactulose l −1 h −1 in the first day. While both carrier bound CelB preparations were 100% stable for at least 14 days, the half-life of the free CelB in the enzyme membrane reactor was only about 1.5 days.

A novel ceramic membrane reactor system for the continuous enzymatic synthesis of oligosaccharides

The continuous enzymatic production of galactosyl-oligosaccharides (GOS) from lactose as a substrate using a new type of ceramic membrane reactor system was investigated. GOS are non-digestible oligosaccharides and have recently attracted interest as prebiotics. However, the composition of oligosaccharides fraction and the variability in β-glycosidic linkages depend on the enzyme source. In the study presented below, native, physically immobilized, β-galactosidase from Kluyveromyces lactis (EC 3.2.1.23) was used as enzyme to catalyse transgalactosylation reaction to produce GOS, competed against the hydrolysis of lactose into its two component monosaccharides, glucose and galactose. To optimize GOS yielded, process conditions were varied: the average residence time of the enzyme was varied in the range of 13 to 24 min, the trans-membrane pressure (TMP) was in the range of 1 to 2 bar and the initial concentration of substrate was varied from 10 to 30% (w/w). Regarding the conditions investigated here, the maximum oligosaccharide concentration exceeded 38% (w/w) when the average residence time was 24 min, the TMP was 2 bar and an initial lactose concentration of 30% (w/w) was adjusted.

Lactose uptake rate by kefir yeast using 14C-labelled lactose to explain kinetic aspects in its fermentation

Τhe present work shows the relation between kefir fermentation ability and carbohydrate uptake rate. This was examined in a model system containing kefir co-culture and lactose in order to study fermentation induced by yeasts and bacteria at the same time. Lactose uptake rate was recorded by using 14C-labelled lactose. The effect of lactose, cell concentration and pH on lactose fermentation was examined. Results have shown increase of lactose uptake rate at lower cell concentrations and specifically the maximum values of lactose uptake rate were obtained at 30 °C, 5.5 pH value and initial lactose and cell concentration 10% w/v and 16 g/L, respectively. Likewise, lighten that the increase of the fermentation rate by immobilized cells can be attributed also, in addition to other factors, to lower cell concentration on the surface of the support or of the promoter. Besides, it is shown that the effect of pH value on the biochemical reactions, carried out by intracellular enzymes can be attributed, except to the effect of pH on enzyme ability, in addition to the effect of pH on carbohydrate uptake rate.

Galactooligosaccharides production by β-galactosidase immobilized onto magnetic polysiloxane–polyaniline particles

Magnetized polysiloxane coated with polyaniline (mPOS–PANI) was used as a support for β-galactosidase immobilization via glutaraldehyde. The galactooligosaccharides (GOS) production by this derivative was investigated under different initial lactose concentrations (5–50%) and temperatures (30–60 °C). The initial lactose concentration in the reaction media affected the total amounts of produced GOS and their time course production was described as a “bell-shaped” curve as a result of the balance between transgalactosylation and hydrolysis. No significative difference was observed for the free and immobilized enzymes. The reaction rates for lactose hydrolysis and GOS formation increased with increasing temperature from 30 °C to 60 °C, but GOS production at all lactose conversion levels was almost unchanged with changing temperature. The mPOS–PANI matrix was also characterized by scanning electronic microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometry (VSM), thermomagnetization, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).

Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.

Isothermal batch crystallization of alpha-lactose: A kinetic model combining mutarotation, nucleation and growth steps

A kinetic model combining first-order differential equations of the three consecutive steps of lactose crystallization, i.e., mutarotation, nucleation and crystal growth rate, was developed. Numerical solutions successfully fitted the variations of crystal mass growth rate as a function of lactose concentration during unseeded isothermal batch crystallization, at different initial lactose concentrations and temperatures. The model allowed the induction phase and the influence of seeding to be simulated by taking into account the dependency of crystal growth rate on total crystal surface area and, consequently, on nucleation rate. The proposed model highlights the large influence of the mutarotation step, even in unseeded crystallization kinetics. The effects of high lactose supersaturations that prevail at industrial scale during whey powder manufacturing and the effects of alkaline pH (9.5) on lactose crystallization kinetic were successfully predicted.

Membrane chromatography reactor system for the continuous synthesis of galactosyl-oligosaccharides

Galactosyl-oligosaccharides (GOS) are non-digestible oligosaccharides which are qualified as functional foods. GOS are enzymatically synthesized from lactose by transgalactosylation catalyzed by the enzyme β-galactosidase. In this study, a method for immobilization of β-galactosidase from Kluyveromyces lactis on a chromatography membrane was developed. The immobilization was performed at 4, 15 and 40°C. A strong basic anion exchange membrane was investigated. In static experiments the highest enzyme activity was measured at an immobilization procedure temperature of 15°C. The immobilization on the chromatography membranes was found to be very rapid likely due to ionic adsorption. The synthesis of GOS was carried out in a Continuous Membrane Chromatography Reactor System (CMCRS) at 40°C and pH 7.0 using 20 wt% initial lactose concentration. The chromatography membranes proved to be a good support for the continuous process at high convective flow rates in the enzyme reactor system. Up to 82 % lactose conversion with 24 % GOS yield was achieved at different fluxes. The corresponding reactor productivity for the production of GOS from lactose in the CMCRS was 98.7 g GOS per hour and cubic centimeter membrane volume, which significantly exceeds previously reported results.

Whey fermentation by thermophilic lactic acid bacteria: Evolution of carbohydrates and protein content

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 °C for 24 h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS–PAGE. The studied strains grew well (2–3 log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24 h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 μg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, α-lactalbumin being degraded in a greater extent (2.2–3.4-fold) than β-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 μg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 μg/ml). Regarding α-lactalbumin and β-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.

Immobilization of β-Galactosidase in Adsorptive Membranes for the Continuous Production of Galacto-Oligosaccharides from Lactose

A method for immobilization of β-galactosidase from Kluyveromyces lactis on an adsorptive membrane for the continuous synthesis of galacto-oligosaccharides from lactose was carried out. The immobilization was performed at 4, 10, 15 and 40°C. Two strongly basic anion exchange membranes with tradename Mustang TM Q and Sartobind TM Q were investigated. In static experiments, the highest enzyme activity was measured on Mustang TM Q membranes at the immobi- lization temperature of 10°C. The synthesis of GOS was performed in a Continuous Membrane-Chromatography- Reactor-System (CMCRS) at 40°C and pH 7.0 using 20 wt% initial lactose concentration. The investigated membranes proved to be a good support for the continuous process at high convective flow rates in the enzyme reactor system. Up to 82% lactose conversion with 24% GOS yield was achieved at different fluxes. The corresponding reactor productivity for the production of GOS from lactose in the CMCRS was 98.7 grams GOS per hour and cubic centimeter membrane vol- ume, which significantly exceeds previously reported results.

Simultaneous biosynthesis of (+)-geodin by a lovastatin-producing fungus Aspergillus terreus

The simultaneous biosynthesis of lovastatin (mevinolinic acid) and (+)-geodin by Aspergillus terreus ATCC 20542 with regard to the medium composition, i.e. the concentrations of carbon and nitrogen source, was described in this paper. A. terreus is a lovastatin producer but the formation of lovastatin was accompanied by the significant amounts of (+)-geodin, when the elevated concentration of carbon source (lactose) was still present in the medium in the idiophase and nitrogen source (yeast extract) was deficient. It was observed for runs, in which the higher (above 20 g l −1) initial lactose concentration was applied or when the nitrogen deficiency led to the decrease of biomass content in the system. In contrast to lovastatin, there was not optimum initial concentration of yeast extract, as its lowest tested initial concentration (2 g l −1) led to the highest (+)-geodin volumetric formation rates and final yield. What is more, even higher final (+)-geodin concentrations were achieved at elevated initial lactose concentration (40 g l −1) and in the lactose-fed fed-batch run. In the fed-batch run lovastatin concentration increased significantly too, as this metabolite formation is also carbon source dependent. Finally, (+)-geodin occurred to be a metabolite, whose formation, in contrast to lovastatin, is non-growth associated.

Enzymatic Production of Galactooligosaccharides by β-Galactosidase from Bifidobacterium longum BCRC 15708

The production of galactooligosaccharides (GOSs) by transgalactosylation using beta-galactosidase from Bifidobacterium longum BCRC 15708 was studied. Other than lactose, galactose, and glucose, two types of GOSs, tri- and tetrasaccharides, were formed after beta-galactosidase action on 40% lactose. Trisaccharides were the major type of GOS formed. Generally, an increase of the initial lactose concentration in the reaction mixture resulted in a higher GOS production. A maximum yield of 32.5% (w/w) GOSs could be achieved from 40% lactose solution at 45 degrees C, pH 6.8, when the lactose conversion was 59.4%. The corresponding productivity of GOSs was 13.0 g/(L.h). Transgalactosylation activity of beta-galactosidase from a test organism showed a relatively lower sensitivity toward glucose and galactose than that from other organisms. The addition of 5% or 10% glucose or galactose to the reaction mixture did not significantly (p>0.05) reduce the transgalactosylation reaction of beta-galactosidase.

Production of galacto‐oligosaccharides by immobilized recombinant β‐galactosidase from Aspergillus candidus

The preparation of galacto-oligosaccharides (GOSs) was studied using the immobilized recombinant beta-galactosidase from Aspergillus candidus CGMCC3.2919. The optimal pH and temperature for the immobilized enzyme were observed at pH 6.5 and 40 degrees C, respectively. Increasing the initial lactose concentration increased the yield of GOSs. The dilution rate was found to be a key factor during the continuous production of GOSs. The maximum productivity, 87 g/L.h was reached when 400 g/L lactose was fed at dilution rate of 0.8/h. The maximum GOS yield reached 37% at dilution rate of 0.5/h. Continuous operation was maintained for 20 days in a packed-bed reactor without apparent decrease in GOS production. The average yield of GOSs was 32%, corresponding to the average productivity of 64 g/L.h, which implied that the immobilized recombinant beta-galactosidase has potential application for GOS production.

β-Galactosidase from Talaromyces thermophilus immobilized on to Eupergit C for production of galacto-oligosaccharides during lactose hydrolysis in batch and packed-bed reactor

The b-galactosidase from Talaromyces thermophilus CBS 236.58 immobilized onto Eupergit C produced galacto-oligosaccharides (GalOS) in batch- wise and continuous packed-bed mode of operation. A maximum yield of GalOS of 12, 39 and 80 g l -1 was obtained for initial lactose concentrations of 50, 100 and 200 g l -1 , respectively, for batch conversion experiments. The immobilized enzyme could be re-used for several cycles for lactose hydrolysis and transformation. The maximum GalOS concentration of approximately 50 g l -1 was obtained with the dilution rate of 0.375 h -1 in a packed-bed reactor, when using an initial lactose concentration of 200 g l -1 . Continuous conversion of lactose in the packed-bed reactor resulted in the formation of relatively more trisaccha- rides than when employing the immobilized enzyme in discontinuous mode of operation.

Improved immobilization of Lactobacillus rhamnosus ATCC 7469 in polyacrylamide gel, preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.

Production of Prebiotic Galacto-Oligosaccharides from Lactose Using β-Galactosidases from Lactobacillus reuteri

The beta-galactosidases (beta-Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocatalysts for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. Maximum GOS yields were 38% when using an initial lactose concentration of 205 g/L and at approximately 80% lactose conversion. The product mixtures were analyzed by capillary electrophoresis (CE) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Disaccharides other than lactose and trisaccharides made up the vast majority of GOS formed. The main products were identified as beta-d-Galp-(1-->6)-d-Glc (allolactose), beta-d-Galp-(1-->6)-d-Gal, beta-d-Galp-(1-->3)-d-Gal, beta-d-Galp-(1-->6)-Lac, and beta-d-Galp-(1-->3)-Lac. There were no major products with beta1-->4 linkages formed. Both intermolecular and intramolecular transgalactosylation were observed. d-Galactose proved to be a very efficient galactosyl acceptor; thus, a relatively large amount of galactobioses was formed. Monosaccharides could be conveniently separated from the mixture by chromatography using a strong cation-exchange resin.

Formation of galacto‐oligosaccharides during lactose hydrolysis by a novel β‐galactosidase from the moderately thermophilic fungusTalaromyces thermophilus

Discontinuous and continuous processes of lactose hydrolysis and concomitant galacto-oligosaccharide (GalOS) formation were studied. To this end a wide experimental range of the main variables was evaluated, including the initial lactose concentration, the degree of lactose conversion, the pH value and the temperature for discontinuous transformations, while the initial lactose concentration and the feed rate were varied for the continuous process. For both processes a high-initial lactose concentration proved to be advantageous for the formation of GalOS. The maximum amount of GalOS (100 g/L, corresponding to a yield of approximately 50% based on the initially employed lactose) was obtained after 8 h of incubation when using 200 g/L lactose as substrate and 90% lactose hydrolysis was observed. GalOS productivity in the continuous process (g/L.h) was enhanced by an increase of the flow rate. The maximum GalOS productivity of 70 g/L.h was obtained at a flow rate of 24 mL/h when using a reactor with a total working volume of 21 mL. As was evident from these experiments, this beta-galactosidase from a moderately thermophilic fungus showed a strong transgalactosylation activity and can be used for the formation of GalOS, sugars that are of considerable interest for functional food applications because of their presumed healthpromoting effects.