Whey Lactose Research Articles

Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts

To evaluate the feasibility of producing kefiran industrially, whey lactose, a by-product from dairy industry, was used as a low cost carbon source. Because the accumulation of lactic acid as a by-product of Lactobacillus kefiranofaciens inhibited cell growth and kefiran production, the kefir grain derived and non-derived yeasts were screened for their abilities to reduce lactic acid and promote kefiran production in a mixed culture. Six species of yeasts were examined: Torulaspora delbrueckii IFO 1626; Saccharomyces cerevisiae IFO 0216; Debaryomyces hansenii TISTR 5155; Saccharomyces exiguus TISTR 5081; Zygosaccharomyces rouxii TISTR 5044; and Saccharomyces carlsbergensis TISTR 5018. The mixed culture of L. kefiranofaciens with S. cerevisiae IFO 0216 enhanced the kefiran production best from 568 mg/L in the pure culture up to 807 and 938 mg/L in the mixed cultures under anaerobic and microaerobic conditions, respectively. The optimal conditions for kefiran production by the mixed culture were: whey lactose 4%; yeast extract 4%; initial pH of 5.5; and initial amounts of L. kefiranofaciens and S. cerevisiae IFO 0216 of 2.1×10(7) and 4.0×10(6)CFU/mL, respectively. Scaling up the mixed culture in a 2L bioreactor with dissolved oxygen control at 5% and pH control at 5.5 gave the maximum kefiran production of 2,580 mg/L in batch culture and 3,250 mg/L in fed-batch culture.

SOLVENT EXTRACTION OF β-GALACTOSIDASE FROM KLUYVEROMYCES LACTIS YIELDS A STABLE AND HIGHLY ACTIVE ENZYME PREPARATION

ABSTRACT The yeast Kluyveromyces lactis was cultured in cheese whey and β-galactosidase was extracted from cells by an organic solvent procedure. Under optimum conditions (pH 8.5, 25C), approximately 90% of the intracellular β-galactosidase activity was released into 0.1 M potassium phosphate solution with 2% chloroform after 9 h. The cell-free extract containing β-galactosidase activity was concentrated and the enzyme partially characterized. This enzyme has exhibited an optimum activity at a pH range of 6.2–6.6 and temperature of 40–45C on O-nitrophenyl-β-D-galctopyranoside. The maximum enzyme activity in milk was found at 45C and the concentrated enzymatic extract was found to have 19 U/mg protein. A feasible preparation for commercial application containing 20% glycerol maintained 89% of activity after 2 months at 4C, demonstrating stability. This preparation analyzed on 5% nondenaturing polyacrylamide gel electrophoresis exhibited two bands suggesting two aggregation forms with β-galactosidase activity. PRACTICAL APPLICATIONS Kluyveromyces lactis yeast cells are a safe source of β−galactosidase for food and pharmaceutical industries. Their application to prior hydrolysis of lactose in milk products has become commonplace in alleviating the symptoms of lactose intolerance. It is also of great interest to prevent lactose crystallization in concentrated or frozen dairy products such as condensedmilk, ice cream and “dulce de leche,” thereby increasing consumers' acceptability. In addition, treatment of cheese whey with β-galactosidase increases its fermentability to convert cheese whey lactose into ethanol helping to reduce the pollution caused by large volumes of this by-product released in the environment. Solvent extraction of β-galactosidase from K. lactis is a low-cost process and yields a stable and highly active enzyme preparation.

Formulation of Culture Medium with Agroindustrial Waste for β-Galactosidase Production from Kluyveromyces marxianus ATCC 16045

Seven strains of the genus Kluyveromyces were screened for β-galactosidase activity and Kluyveromyces marxianus ATCC 16045 was selected as the best enzyme producer for culture medium optimization. The production of β-galactosidase by submerged cultivation was evaluated using a factorial design and response surface methodology. The culture medium containing whey and parboiled rice effluent was formulated to maximize the production of β-galactosidase. The effects of the initial pH and the concentrations of whey lactose, peptone, (NH4)2SO4, yeast extract, and parboiled rice effluent on enzyme production were studied using a 2 IV 6-2 fractional design. A CCRD (24 trials plus axial and central points) was used for the four variables selected from the fractional design (lactose, peptone, (NH4)2SO4 and yeast extract), with β-galactosidase activity as the response. The optimum conditions established for production were a whey (lactose) concentration of 120 g/L, a yeast extract concentration of 5 g/L, a peptone concentration of 15 g/L, a (NH4)2SO4 concentration of 15 g/L, a parboiled rice effluent concentration of 30 g/L, and a pH value of 4.0. Under these conditions, the highest enzymatic activity of 10.4 U/mL was measured, being 9.5–9.7 as the values predicted by the proposed model, showing an enzymatic activity increase of 30% using alternative sources of lactose and nitrogen for β-galactosidase production.

Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris

Abstract A β-galactosidase gene (designated PaGalA ) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX 1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml −1 having an activity of 9500 U ml −1 from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg −1 had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.

Immobilization of Aspergillus oryzae β galactosidase on concanavalin A-layered calcium alginate-cellulose beads and its application in lactose hydrolysis in continuous spiral bed reactors

Immobilization of Aspergillus oryzae β galactosidase on concanavalin A-layered calcium alginate-cellulose beads and its application in lactose hydrolysis in continuous spiral bed reactors In this study, Aspergillus oryzae β galactosidase was immobilized on concanavalin A layered calcium alginate-cellulose beads as a bioaffinity support. Immobilized enzyme showed a remarkable broadening in temperature-activity profiles as compared to the native enzyme and exhibited 65% activity in the presence of 5% galactose. Michaelis constant (Km) was 2.57 mM and 5.38 mM for the free and the immobilized β galactosidase, respectively. Crosslinked β galactosidase showed greater catalytic activity in the presence of Mg2+ and was more stable during storage at 4°C for 6 weeks. Immobilized enzyme hydrolyzed 67% lactose in milk in 8 h and 85% lactose in whey in 9 h in the stirred batch process at 50°C. The continuous hydrolysis of lactose by crosslinked β galactosidase in spiral bed reactor exhibited 93% and 88% hydrolysis of lactose at flow rate of 20 ml/h and 30 ml/h, after 1 month operation, respectively.

Potential Applications of Immobilized β-Galactosidase in Food Processing Industries.

The enzyme β-galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β-galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the different types of techniques used for the immobilization of β-galactosidase and its potential applications in food industry.

Research perspectives and role of lactose uptake rate revealed by its study using 14C-labelled lactose in whey fermentation

The present investigation examines the effect of pH, temperature and cell concentration on lactose uptake rate, in relation with kinetics of whey fermentation using kefir and determines the optimum conditions of these parameters. Lactose uptake rate was measured by adding 14C-labelled lactose in whey. The results reveal the role of lactose uptake rate, being the main factor that affects the rate of fermentation, in contrast to the activity of the enzymes involved in lactose bioconversion process. Lactose uptake rate results discussion showed that mainly Ca2+ is responsible for the reduced whey fermentation rate in comparison with fermentations using synthetic media containing lactose. Likewise, the results draw up perspectives on whey fermentation research to improve whey fermentation rate. Those perspectives are research to remove Ca2+ from whey, the use of nano and microtubular biopolymers and promoters such as γ-alumina pellets and volcan foaming rock kissiris in order to accelerate whey fermentation.

The Effect of pH, Temperature and Alkali Metal Ions on the Hydrolsis of Whey Lactose Catalysed by β-Galactosidase from Kluyveromyces marxianus*

The Effect of pH, Temperature and Alkali Metal Ions on the Hydrolsis of Whey Lactose Catalysed by β-Galactosidase from Kluyveromyces marxianus*

CHARACTERIZATION OF IMMOBILIZED LIPASE IN ALUMINOSILICATE FOR LACTOSYL PALMITATE SYNTHESIS

Whey lactose can be esterified enzymatically by using immobilized lipase. The lipase can be isolated from Rhizopus oryzae, purified and immobilized in mesoporous aluminosilica. The use of immobilized lipase has advantages, there are longer shelf life and repeatable use. It is necessary to characterize the immobilized lipase dan ester product. The aim of the research was to characterize immobilized lipase, including determination lipase adsorption type in mesoporous aluminosilicate, immobilized lipase stability during storage time, efficiency of repetitive use of immobilized lipase. The result showed that lipase adsorption in mesoporous aluminosilicate was physical adsorption type through hydrogen bound and electrostatic interaction. Immobilized lipase stability was relatively constant at storage temperature 5 °C for 25 days resulting in 98.16% of initial activity. The repetitive use of immobilized lipase showed efficient until 5 uses within activity of 50.22%. The IR spectra of lactosyl palmitate from both whey and pure lactose result showed bands at wavelength number of 3462 cm-1(OH bond), 1739 cm-1 and 1747 (C=O ester bond) 1295 cm-1 dan 1242 cm-1 (C-O ester bond). In addition, the HLB value for lactosyl palmitate (whey) 4.708 and lactosyl palmitate (pure lactose) 4.715, therefore both lactosyl palmitate is appropriate as emulgator in W/O. Keywords: immobilized lipase, aluminosilica, lactose, whey, lactosyl palmitate

Fermentation of deproteinized cheese whey powder solutions to ethanol by engineered Saccharomyces cerevisiae: effect of supplementation with corn steep liquor and repeated-batch operation with biomass recycling by flocculation

The lactose in cheese whey is an interesting substrate for the production of bulk commodities such as bio-ethanol, due to the large amounts of whey surplus generated globally. In this work, we studied the performance of a recombinant Saccharomyces cerevisiae strain expressing the lactose permease and intracellular beta-galactosidase from Kluyveromyces lactis in fermentations of deproteinized concentrated cheese whey powder solutions. Supplementation with 10 g/l of corn steep liquor significantly enhanced whey fermentation, resulting in the production of 7.4% (v/v) ethanol from 150 g/l initial lactose in shake-flask fermentations, with a corresponding productivity of 1.2 g/l/h. The flocculation capacity of the yeast strain enabled stable operation of a repeated-batch process in a 5.5-l air-lift bioreactor, with simple biomass recycling by sedimentation of the yeast flocs. During five consecutive batches, the average ethanol productivity was 0.65 g/l/h and ethanol accumulated up to 8% (v/v) with lactose-to-ethanol conversion yields over 80% of theoretical. Yeast viability (>97%) and plasmid retention (>84%) remained high throughout the operation, demonstrating the stability and robustness of the strain. In addition, the easy and inexpensive recycle of the yeast biomass for repeated utilization makes this process economically attractive for industrial implementation.

Efficient bioconversion of lactose in milk and whey: immobilization and biochemical characterization of a β-galactosidase from the dairy Streptococcus thermophilus LMD9 strain

The gene encoding β-galactosidase from dairy Streptococcus thermophilus strain LMD9 was cloned, sequenced and expressed in Escherichia coli. The recombinant enzyme was purified and showed high specific activity of 464 U/mg. This protein displays a homotetrameric arrangement composed of four 118 kDa monomers. Monitoring of the activity showed that this enzyme was optimally active at a wide range of temperatures (25–40 °C) and at pH from 6.5 to 7.5. Immobilization of the recombinant E. coli in alginate beads clearly enhanced the enzyme activity at various temperatures, including 4 and 50 °C, and at pH values from 4.0 to 8.5. Stability studies indicated that this biocatalyst has high stability within a broad range of temperatures and pH. This stability was improved not only by addition of 1 mM of Mn 2+ and 1.2 mM Mg 2+, but essentially through immobilization. The remarkable bioconversion rates of lactose in milk and whey at different temperatures revealed the attractive catalytic efficiency of this enzyme, thus promoting its use for lactose hydrolysis in milk and other dairy products.

A modelling study on hydrolysis of whey lactose and stability of β-galactosidase

In the present study, the effect of process conditions on whey lactose hydrolysis and enzyme inactivation were investigated. The experiments were carried out in 250 mL of 25 mM phosphate buffer solution by using β-galac- tosidase produced from Kluyveromyces marxianus lactis in a batch reactor system. The degree of lactose hydrolysis (%) and residual enzyme activity (%) against time were investigated versus lactose concentration, enzyme concentra- tion, temperature and pH. The mathematical models were derived from the experimental data to show the effect of process conditions on lactose hydrolysis and residual enzyme activity (in the presence and absence of lactose). At the optimum process conditions obtained (50 g/L of lactose concentration, 1 mL/L of enzyme concentration, 37 o C of tem- perature and pH 6.5), 81% of lactose was hydrolyzed and enzyme lost its activity by 32%. The activation energy for hydrolysis reaction (EA) and the enzymatic inactivation energy (ED) were calculated as 52.7 kJ/mol and 96.7 kJ/mol. Mathematical models at various process conditions have been confirmed with the experimental results.

Biodegradation kinetics and modeling of whey lactose by bacterial hemoglobin VHb-expressing Escherichia coli strain

Abstract The batch fermentation of cheese whey lactose was achieved using Escherichia coli:pUC8:16 recombinant strain that was transformed with Vitreoscilla hemoglobin gene(vgb). In this process, 70% of the initial whey lactose was biodegraded during 24 h of incubation time. Biodegradation was accompanied with a turnover of glucose intermediate and a production of lactic acid. Total lactic acid produced by this recombinant strain was 57.8 mmol/L compared with a reference lactic acid producing strain, Lactobacillus acidophilus, that yielded only 55.3 mmol/L of lactic acid from the same initial whey lactose concentration. The engineering of vgb gene transformation in E. coli strain has led to increase in bacterial biomass and boosted lactic acid production, relative to other strains that lack the vgb gene like E. coli:pUC9 or E. coli wild type or Enterobacter aerogenes. Contrary to Monod's, Haldane's model gave a good fit to the growth kinetics data. Kinetic constants of the Haldane equation were μm = 0.5573 h−1, Ks = 4.8812 g/L, KI = 53.897 g/L. Biomass growth was well described by the logistic equation while Luedeking–Piret equation defined the product formation kinetics. Substrate consumption was explained by production rate and maintenance requirements. In simulation studies including the Haldane model, an evident agreement was observed between measured and calculated biomass, product, and substrate concentrations.

Diacetyl and acetoin production from whey permeate using engineered Lactobacillus casei

The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products.

OPTIMIZATION OF PERMEABILIZATION PROCESS FOR LACTOSE HYDROLYSIS IN WHEY USING RESPONSE SURFACE METHODOLOGY

ABSTRACT To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 in relation toβ‐galactosidase activity was optimized using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. Permeabilized whole cells can be advantageous over pure enzyme preparations in terms of cost‐effectiveness and increased stability maintained by the intracellular environment. Response surface methodology (RSM) was applied to optimize concentration of CTAB, temperature and the treatment time for maximum permeabilization of yeast cells. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity obtained by RSM were 0.06% (w/v) CTAB concentration, 28C temperature and process duration of 14 min. At these conditions of process variables, the maximum value of enzyme activity was found to be 1,334 IU/g. The permeabilized yeast cells were highly effective and resulted in 90.5% lactose hydrolysis in whey.PRACTICAL APPLICATION β‐Galactosidase is one of the most promising enzymes, which has several applications in the food, fermentation and dairy industry. However, the industrial applications of β‐galactosidase have been hampered by the costs involved in downstream processing. The present investigation was focused on developing the low‐cost technology for lactose hydrolysis based on permeabilization process. Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. Keeping this in view, lactose hydrolysis in whey has been successfully performed using permeabilized Kluyveromyces marxianus cells. Hydrolysis of lactose using β‐galactosidase converts whey into a potentially very useful food ingredient, which has immense applications in food industries. Its use has increased significantly in recent years, mainly in the dairy products and in digestive preparations. Lactose hydrolysis causes several potential changes in the manufacture and marketing of dairy products, including increased solubility, sweetness and broader fermentation possibilities.

Optimal immobilization of β-galactosidase from Pea ( PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications

Response surface methodology (RSM) and centre composite design (CCD) were used to optimize immobilization of β-galactosidase (BGAL) from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads. The immobilization efficiency of 75.66% and 75.19% were achieved with Sephadex G-75 and chitosan, respectively. There was broad divergence in physico-chemical properties of Sephadex- PsBGAL and chitosan- PsBGAL. Chitosan- PsBGAL was better suited for industrial application based on its broad pH and temperature optima, higher temperature stability, reusability etc. Sephadex- PsBGAL and chitosan- PsBGAL showed much variation in their catalytic properties with respect to soluble enzyme. About 50% loss in activity of Sephadex- PsBGAL and chitosan- PsBGAL were observed after 12 and 46 days at 4 °C, respectively. Chitosan- PsBGAL showed higher rate of lactose hydrolysis present in milk and whey at room temperature and 4 °C than Sephadex- PsBGAL. In both cases, lactose of milk whey was hydrolyzed at higher rate than that of milk.

Hydrolysis of whey lactose by immobilized β-Galactosidase

Hydrolysis of whey lactose to glucose and galactose by immobilized galactosidase comes as an alternative to enlarge the possibilities of commercial use of this feedstock. To be applied at industrial scale, the process should be performed continuously .This work aimed to study the hydrolysis of whey lactose by an immobilized enzyme reactor. b-Galactosidase from Aspergillus oryzae was immobilized on silica and activity and stability were evaluated. The best immobilization results were attained by using glutaraldehyde as support's activator and enzyme stabilizer. The optimized enzyme proportion for immobilization was 15-20 mg g-1 of support. Treatments of whey were performed (microfiltration, thermal treatment and ultrafiltration), seeking the elimination of sludge, and the effects on operating the fixed bed reactor were evaluated. Ultrafiltration was the best treatment towards a proper substrate solution for feeding the reactor.

Polyhydroxyalkanoate (PHA) Biosynthesis from Whey Lactose

AbstractSummary: The potential of three different microbial wild type strains as polyhydroxyalkanoate (PHA) producers from whey lactose is compared. Homopolyester and co‐polyester biosynthesis was investigated by the archaeon Haloferax mediterranei and the eubacterial strains Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava. H. mediterranei accumulated 50 wt.‐% of poly‐3‐(hydroxybutyrate‐co‐6%‐hydroxyvalerate) in cell dry mass from hydrolyzed whey without addition of 3‐hydroxyvalerate (3HV) precursors (specific productivity qp: 2.9 mg/g h). Using P. hydrogenovora, the final percentage of poly‐3‐hydroxybutyrate (PHB) amounted to 12 wt.‐% (qp: 0.03 g/g h); co‐feeding of valeric acid resulted in the production of 12 wt.‐%. P‐3(HB‐co‐21%‐HV) (qp: 0.02 g/g h). With H. pseudoflava, it was possible to reach 40 wt.‐% P‐3 (HB‐co‐5%‐HV) on not‐hydrolyzed whey lactose plus valeric acid as 3HV precursor (qp: 9.1 mg/g h); on hydrolyzed whey lactose without addition of valeric acid, the strain produced 30 wt.‐% of PHB (qp: 0.16 g/g h). The characterization of the isolated biopolyesters completes the study.

Immobilization of β-galactosidase by bioaffinity adsorption on concanavalin A layered calcium alginate–starch hybrid beads for the hydrolysis of lactose from whey/milk

Aspergillus oryzae β-galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate–starch beads. The maximum activity of the immobilized β-galactosidase was obtained at 60 °C, approximately 10 degrees higher than that of the free enzyme. The immobilized β-galactosidase exhibited significantly higher stability to heat, urea, MgCl 2, and CaCl 2 than the free enzyme. An enhancement of the activity of immobilized β-galactosidase by up to 5.0% MgCl 2 was seen, whereas the activity of the free enzyme decreased above 3.0% MgCl 2. Immobilized β-galactosidase retained 61%, 50% and 43% activity in the presence of 5% CaCl 2, 5% galactose and 4 m urea, respectively, when incubated for 1 h at 37 °C. The immobilized β-galactosidase had a much higher K iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose. The immobilized β-galactosidase preparation was superior to the free enzyme in hydrolysing lactose in whey or milk in a batch process: it hydrolyzed 89% of the lactose in whey in 3 h and 79% of the lactose in milk in 4 h. The immobilized β-galactosidase retained 61% of its original activity after 2 months storage at 4 °C, while the soluble enzyme showed only 37% of the initial activity under identical conditions.

Hydrolysis of whey lactose using CTAB-permeabilized yeast cells

Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by beta-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to beta-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 degrees C.