Initial Lactose Concentration Research Articles

Effect of Nutrient Supplementation on Biobutanol Production from Cheese Whey by Abe (acetone–butanol–ethanol) Fermentation

Cheese whey is a liquid effluent obtained from the cheese manufacturing process, presenting a high volumetric production and high organic loads related to its lactose content. The aim of this research was to increase the productivity of ABE fermentation with the bacterial strain Clostridium beijerinckii CECT 508, when using nanofiltered sheep cheese whey as a substrate. Thermal sterilization of the whey was essential to avoid the proliferation of lactic acid bacteria. An experimental design including Plackett-Burman and Response Surface Methodology (RSM) was applied to figure out which additional nutrients were necessary for the fermentation process and their optimum concentrations. For this specific whey, it was established that 1 g/L yeast extract, 5 g/L CaCO3, 0.019 g/L FeSO4·7H2O, 0.2 g/L MgSO4·7H2O and 2.1 g/L NH4Cl needed to be added. Under optimal nutrient conditions, the highest butanol production (9.11 g/L) was obtained with an initial lactose concentration of 57 g/L; achieving 49% lactose consumption and 0.311 g/g YB/L yield. However, the best lactose consumption rate (87%) was recorded for lactose initial concentrations of 30 g/L, which also allowed satisfactory fermentation values (8.51 g/L butanol and 0.328 g/g YB/L yield). On the other hand, the best YB/L yields (0.428 g/g; which is close to the theoretical value) were obtained for initial lactose concentrations of 40 g/L, attaining a value of 52% lactose consumption and 8.91 g/L butanol. Therefore, the ABE fermentation could be a feasible solution to treat cheese whey and remove lactose, obtaining acceptable amounts of butanol.

A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor owensensis with potential applications in production of biofuels and biochemicals.

BackgroundThermophilic enzymes have attracted much attention for their advantages of high reaction velocity, exceptional thermostability, and decreased risk of contamination. Exploring efficient thermophilic glycoside hydrolases will accelerate the industrialization of biofuels and biochemicals.ResultsA multifunctional glycoside hydrolase (GH) CoGH1A, belonging to GH1 family with high activities of β-d-glucosidase, exoglucanase, β-d-xylosidase, β-d-galactosidase, and transgalactosylation, was cloned and expressed from the extremely thermophilic bacterium Caldicellulosiruptor owensensis. The enzyme exerts excellent thermostability by retaining 100 % activity after 12-h incubation at 75 °C. The catalytic coefficients (kcat/Km) of the enzyme against pNP-β-D-galactopyranoside, pNP-β-D-glucopyranoside, pNP-β-D-cellobioside, pNP-β-D-xylopyranoside, and cellobiose were, respectively, 7450.0, 2467.5, 1085.4, 90.9, and 137.3 mM−1 s−1. When CoGH1A was supplemented at the dosage of 20 Ucellobiose g−1 biomass for hydrolysis of the pretreated corn stover, comparing with the control, the glucose and xylose yields were, respectively, increased 37.9 and 42.1 %, indicating that the enzyme contributed not only for glucose but also for xylose release. The efficiencies of lactose decomposition and synthesis of galactooligosaccharides (GalOS) by CoGH1A were investigated at low (40 g L−1) and high (500 g L−1) initial lactose concentrations. At low lactose concentration, the time for decomposition of 83 % lactose was 10 min, which is much shorter than the reported 2–10 h for reaching such a decomposition rate. At high lactose concentration, after 50-min catalysis, the GalOS concentration reached 221 g L−1 with a productivity of 265.2 g L−1 h−1. This productivity is at least 12-fold higher than those reported in literature.ConclusionsThe multifunctional glycoside hydrolase CoGH1A has high capabilities in saccharification of lignocellulosic biomass, decomposition of lactose, and synthesis of galactooligosaccharides. It is a promising enzyme to be used for bioconversion of carbohydrates in industrial scale. In addition, the results of this study indicate that the extremely thermophilic bacteria are potential resources for screening highly efficient glycoside hydrolases for the production of biofuels and biochemicals.

Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides in batch operation

The production of prebiotic galacto-oligosaccharides (GOS) considers the enzymatic synthesis by lactose transgalactosylation and the purification from the reacted mixture (raw GOS). Simultaneous synthesis and purification of GOS (SSP) was conducted in a one-pot operation using β-galactosidase from Aspergillus oryzae and Saccharomyces cerevisiae or Kluyveromyces marxianus cells, allowing the selective removal of unwanted carbohydrates (monosaccharides and lactose). The reaction was carried out in a stirred reactor at 40 °C, pH 4.5 and 200 rpm, using lactose without additional supplements, evaluating the effect of yeast genus (S. cerevisiae or K. marxianus), enzyme-substrate ratio (20–400 IUH/g lactose), initial lactose concentration (20–50% w/w) and substrate mass-biomass ratio (0.05–0.4). The best GOS yield was obtained with S. cerevisiae at 50 IUH/g lactose and 0.25 g cell biomass/g carbohydrates. These conditions were used for scaling-up into a bioreactor (1000 g of 40% w/w lactose) operated under temperature and pH control at an aeration rate of 5 vvm, obtaining a GOS yield of 40% at 24 h of reaction, which is significantly higher than obtained in the conventional system of GOS synthesis. SSP may represent a technological advantage in terms of productivity and yield of GOS production.

A study on the effect of parameters on lactic acid production from whey

Abstract In batch fermentation of whey, selection of suitable species at desired conditions such as substrate, product concentrations, temperature and inoculum size were investigated. Four Lactobacillus species and one Lactococcus species were screened for lactic acid production. Among them L. bulgaricus ATCC 11842 were selected for further studies. The optimal growth of the selected organism for variable size of inocula was examined. The results indicated that inoculum size had insignificant effect on the cell and lactic acid concentration. The effect of temperature was also studied at 32, 37, 42 and 47°C. Results showed that the concentration of cell dry weight increased with increment of temperature from 32 to 42°C. The maximum cell and lactic acid concentration was obtained at 42°C. The effect of initial substrate concentration on lactic acid production was also examined. The optimum initial lactose concentration was found to be 90 g/l.

Experimental studies and two-dimensional modelling of a packed bed bioreactor used for production of galacto-oligosaccharides (GOS) from milk whey.

In the present study, extensive experimental investigations and detailed theoretical analysis of a two-dimensional packed bed bioreactor, employed for the production of galacto-oligosaccharides (GOS) from milk whey were performed. Model equations, in one- and two-dimensions, capable of predicting the substrate concentration distribution in the bioreactor were developed by coupling mass balance equation with appropriate velocity distribution equation and solved numerically. Validation of the proposed model equations was done by a set of experimental data obtained from the bioreactor. The effects of reactor to catalyst particle diameter ratio (d t/d p), feed flowrate (10(-6)-10(-9)m(3)s(-1)), and initial lactose concentration (50-200kgm(-3)) on substrate concentration distribution were investigated in detail. While, the distribution of substrate concentration in axial direction was independent of d t/d p, it was observed that for d t/d p <40, significant radial concentration distribution existed. It was further observed that the substrate conversion and product yield obtained experimentally showed an excellent agreement (97±2%) with the results predicted by the two-dimensional model equation, whereas, the results predicted by the one-dimensional model equation did not lie within the desired confidence level (<90%). The results were confirmed by both curve fitting and statistical analysis. The prediction of substrate concentration distribution in axial and radial directions using the developed two-dimensional model equation is necessary for computing the bioreactor volume to achieve the desired GOS yield.

Enzyme immobilization/bioconjugation in producing galactio-oligosaccharidies from lactose: developments of kinetic models and bio-reactors

Most studies use analytical grade enzymes (β-galactosidase, etc) under free-state for enzymatic conversion of lactose into galacto-oligosaccharide (GOS). This paper is a summary of earlier published works [1-4] which had four-fold objectives; i) use of an industrial grade β-galactosidase and, ii) use of a membrane as a support for enzyme immobilization, iii) use of smart polymers for enzyme bioconjugation, and finally, iv) kinetic studies with model developments to develop efficient bio-reactor(s). Enzyme immobilization/bioconjugation was carried out for its better stability, reusability, and ease of separation after the reaction.Firstly, the immobilization of enzymes (β-galactosidase) was carried out on a PVDF disc membrane by cross-linking with glutaraldehyde under two different feed conditions; stagnant and flowing. Total GOS formation was high with highest initial lactose concentration under stagnant feed; whereas; with flowing feed, the selectivity of GOS formation increased by increasing both the initial lactose concentration and the feed flow rate. A five-step, nine-parameter kinetic model was developed (based on Michaelis-Menten kinetics) and simulated results matched exceeding well with the experimental results. Secondly, smart/responsive polymers were bioconjugated with β-galactosidase and its property estimations were made. Comparison were made for bioconjugation with thermo-responsive poly-N-isopropylacrylamide (PNIPAAm) to a non-responsive polymer, poly-acrylamide. The process of bioconjugation decreases the lower critical solution temperature (LCST). The PNIPAAm-β-galactosidase bioconjugate showed much-improved operational, thermal and storage stabilities. Finally, the application of thermo-responsive PNIPAAm-β-galactosidase bioconjugate was carried out for the synthesis of GOS from lactose. A higher GOS yield was observed as compared to enzyme immobilized membrane surface. High temperatures led to enzyme deactivation. The apparent kinetic parameters were estimated from the same five-step, nine-parameter kinetic model, and the simulated results (using COPASI®) excellently matched with the experimental data.

Studies on production of ethanol from cheese whey using Kluyveromyces marxianus

Whey, the residue remaining following cheese/casein production, represents a major disposal problem for the dairy industry as this has been considered as a highly polluting stream. Typically, whey contains lactose, proteins and fat. Due to lactose content, liquid whey can be considered as a cheap resource for production of bio-ethanol. Bio-ethanol can help to meet global fuel demand in the present scenario of depleting fossil fuel reserve. Direct fermentation of cheese whey or whey permeate yields low ethanol concentration due to poor lactose content, thus making the process uneconomical. Cheese whey powder (CWP), a dried and concentrated form of cheese whey, containing high concentration of lactose, can be an attractive source for ethanol production. In the present study, ethanol was produced from CWP with initial lactose concentration of 150 g/L at a temperature of 35°C and pH 4.5 using lactose positive microorganism, Kluyveromyces marxianus NCIM 3217. Maximum ethanol production was found to be 43.71 g/L and almost all the lactose was consumed within 72 h. Maximum biomass concentration at the end of fermentation was found to be 6.02 g/L. From the experimental data, it was found that the microbial growth followed Monod kinetic model. Ethanol production using K. marxianus NCIM 3217 was found to follow Luedeking – Piret equation and it was mixed-growth-associated. Thus, cheese whey powder could be an attractive substrate for bio-ethanol production and and hence can solve the environmental pollution problem created by whey surplus.

Supply Chain of Bioethanol Production from Whey: A Review

Whey, a major byproduct of the cheese and dairy industry, has valuable nutritional constituents but poses a major environmental risk, if disposed off without prior treatment. Whey has a high organic content, its major constituent being lactose (4.5-5% w/v). With a very high BOD and COD load (30,000 – 50,000 ppm and 60.000 – 80,000 ppm respectively) and annual production of over 160 million tonnes, with an estimated growth rate of 1–2% yearly, fermentation of whey to bioethanol has proved to be a lucrative option, both for bioremediation of whey and production of ethanol from waste sources; reducing the costs incurred in otherwise advanced effluent treatment processes required for its disposal. Moreover, with increasing global demand for bioethanol as an alternative to scarce fossil fuels and advocacy by governmental institutions, due to its non-polluting nature aiding in reduced environmental pollution and global warming; conversion of whey to ethanol is progressively more desirable. The aim of this review is to discuss the supply chain involved in the production of ethanol from whey. The study will involve literature survey, experimentation and developing a model.This study looks at the supply chain involved in the production and subsequent distribution of whey, for the purpose of valorization and extraction of valuable constituents, as well as its utilization in production of bioethanol. Based on existing studies, it has been seen that an initial lactose concentration of 200 gL-1 in the whey permeate, provides the maximum yield for ethanol. Under such conditions, maximum ethanol obtained from fermentation of whey was found to be 80.95 kg m-3, proving the economic viability of the process. However, the supply chain of whey for bioethanol production is not well defined, making effectiveness and implementation of ethanol production from whey constricted. Thus, although the environmental risk posed by the vast production of whey, is mitigated by its conversion to ethanol, providing adequate bioremediation of its polluting components, this process is yet to be industrialized widely.

Dendrimer-like nanoparticles based β-galactosidase assembly for enhancing its selectivity toward transgalactosylation

Functional nanomaterials have been pursued to assemble nanobiocatalysts since they can provide unique hierarchical nanostructures and localized nanoenvironments for enhancing enzyme specificity, stability and selectivity. Functionalized dendrimer-like hierarchically porous silica nanoparticles (HPSNs) was fabricated for assembling β-galactosidase nanobiocatalysts for bioconversion of lactose to galacto-oligosaccharides (GOS). The nanocarrier was functionalized with amino (NH2) and carboxyl (COOH) groups to facilitate enzyme binding, benchmarking with non-functionalized HPSNs. Successful conjugation of the functional groups was confirmed by FTIR, TGA and zeta potential analysis. HPSNs-NH2 showed 1.8-fold and 1.1-fold higher β-galactosidase adsorption than HPSNs-COOH and HPSNs carriers, respectively, with the highest enzyme adsorption capacity of 328mg/g nanocarrier at an initial enzyme concentration of 8mg/ml. The HPSNs-NH2 and β-galactosidase assembly (HPSNs-NH2-Gal) demonstrated to maintain the highest activity at all tested enzyme concentrations and exhibited activity up to 10 continuous cycles. Importantly, HPSNs-NH2-Gal was simply recycled through centrifugation, overcoming the challenging problems of separating the nanocarrier from the reaction medium. HPSNs-NH2-Gal had distinguished catalytic reaction profiles by favoring transgalactosylation, enhancing GOS production of up to 122g/l in comparison with 56g/l by free β-galactosidase. Furthermore, it generated up to 46g/l GOS at a lower initial lactose concentration while the free counterpart had negligible GOS production as hydrolysis was overwhelmingly dominant in the reaction system. Our research findings show the amino-functionalized HPSNs can selectively promote the enzyme activity of β-galactosidase for transgalactosylation, which is beneficial for GOS production.

Chitosan crosslinked with genipin as support matrix for application in food process: Support characterization and β-d-galactosidase immobilization

In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested for the immobilization of β-d-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of the enzyme and crosslinking with genipin. The particles were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The immobilization of the enzyme by crosslinking with genipin provided biocatalysts with satisfactory activity retention and thermal stability, comparable with the ones obtained with the traditional methodology of immobilization using glutaraldehyde. β-d-Galactosidase–chitosan–genipin particles were applied to galactooligosaccharides synthesis, evaluating the initial lactose concentration, pH and temperature, and yields of 30% were achieved. Moreover, excellent operational stability was obtained, since the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. Thus, the food grade chitosan–genipin particles seem to be a good alternative for application in food process.

A Kinetic Modeling of the Liquid-Phase Oxidation of Lactose Over Pt- and Au-Supported Catalysts

Pt and Au catalysts, 2 wt% metal loading, supported on SiO2 and Al2O3 were used to study the effect of metal and support on the liquid-phase oxidation of lactose. Pt-based catalysts were prepared by incipient wetness impregnation while Au-based catalysts were obtained by the precipitation-deposition method. Catalytic tests were carried out in aqueous phase at 65 °C, using O2 as oxidizing agent and keeping pH constant at 9 by controlled addition of NaOH aqueous solution. In all of the cases, the only product of reaction detected and quantified was lactobionic acid. It was found that Pt supported on Al2O3 was more active than Pt supported on SiO2. This was explained on the basis that metal Pt dispersion on Al2O3 was three times higher than on SiO2. At the same time, Au/Al2O3 catalyst was more active than Pt/Al2O3 catalysts. The higher activity of Au/Al2O3 was attributed to Au nanoparticles interacting with the support, as determined by transmission electron microscopy. It was also verified that Au/Al2O3 activity was almost the same after two consecutive runs, indicating a good stability of the Au active phase. Kinetic studies were carried out by varying the initial concentration of lactose in the reaction mixture. A negative order respect to the reactant, determined applying a pseudo-homogeneous model, was estimated, which indicates that lactose molecules are strongly adsorbed on the surface of metal Au nanoparticles. A LHHW model, assuming that oxygen chemisorption was the controlling step, allowed to explain the negative order respect to lactose.

Galacto-oligosaccharides formation during manufacture of different varieties of yogurt. Stability through storage

Galacto-oligosaccharides (GOS) have interest in the food industry due to their recognized functional properties. In this work, we studied the effect of a commercial β-galactosidase enzyme from Kluyveromyces lactis (YNL-2, GODO) and Lactobacillus acidophilus La-5, on GOS formation during the manufacture and storage of drinkable and stirred yogurts. In a preliminary step, GOS synthesis and lactose hydrolysis by β-galactosidase was evaluated at different initial lactose concentrations and doses of enzyme. The GOS formation was favored with increasing of lactose concentration and enzyme doses, while the hydrolysis dominated at lower level of lactose. In turn, the presence of GOS was already evident at 45 min of fermentation in yogurts with addition of β-galactosidase. Mean concentrations were 0.36 and 0.62 g/100 g for fresh drinkable and stirred yogurts, respectively. No changes in the GOS levels were observed through storage, indicating that they were stable in the products. The probiotic bacteria added were not able to produce GOS. The diminution of lactose was significant in yogurts with β-galactosidase; contents of residual lactose were around 1.3 g/100 g. We obtained different varieties of reduced-lactose yogurts enriched in galacto-oligosaccharides. The presence of probiotic and prebiotic would increase the functional properties of yogurts.

Continuous ultrafiltration membrane reactor coupled with nanofiltration for the enzymatic synthesis and purification of galactosyl-oligosaccharides

Continuous enzymatic production of galactosyl-oligosaccharides (GOS) from a lactose substrate in an ultrafiltration membrane reactor (UMR) coupled with a nanofiltration separation system (CPNSS) was investigated. The overall rate of production over 4h of continuous production was 80–104mg GOS formed/U with an average residence time of 66min, an initial lactose concentration of 300g/L, an inlet pressure of 2.0bar inlet pressure, and an outlet pressure of 1.5bar. In the continuous diafiltration (CD) process, the concentration of various sugars and the relationship between the yield and purity of oligosaccharides was well predicted by mathematical models, the increased rate of sugar rejections was less than 10%, and the decreased rate of concentrations in the tank was less than 15%. In the CPNSS, 33.4wt.% GOS was obtained in the UMR, and 1.24kg of high-purity GOS (specifically, GOS purity of 57.2% and lactose content less than 20%) was achieved. This final yield was 80.1% GOS, which meets industry standards.

Synthesis of galactooligosaccharides using sweet and acid whey as a substrate

Two commercial available lactases from Aspergillus oryzae and Kluyveromyces lactis were used to study the synthesis of galactooligosaccharides (GOS) in sweet and acid whey. At 38 g L−1 initial lactose concentration, the A. oryzae enzyme gave a GOS yield of 10.91 ± 0.01% in lactose solution, 10.93 ± 0.18% in sweet whey and 11.32 ± 0.59% in acid whey. Thus, the components in whey did not influence the enzymes transgalactolytic activity. On the other hand, the K. lactis enzyme showed a strong dependence on whey type and whey concentration. At 38 g L−1 initial lactose concentration, GOS yields were 10.93 ± 0.26% in lactose solution, 4.30 ± 0.17% in sweet whey and 10.56 ± 0.41% in acid whey. However, with increasing initial lactose concentration, the inhibitory effect of sweet whey was decreasing, which resulted in even higher yields than in lactose solution.

Kinetic studies and model development for the formation of galacto-oligosaccharides from lactose using synthesized thermo-responsive bioconjugate

In an earlier study by us [47], thermo-responsive bioconjugate (poly-N-isopropylacrylamide-β-galactosidase) was synthesized and characterized. This study utilizes the prowess of such smart bioconjugate for the enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose at various initial lactose concentrations (ILC), enzyme concentrations, and temperatures, while maintaining a constant pH of 6. A maximum GOS yield of 35% (on dry basis) was observed at 100g/L ILC and 0.275mg/mL (0.055U/mL) conjugated protein. The GOS yield remained approximately the same for 50 and 100g/L ILC, beyond which, it decreased. As the enzyme concentration increased, the equilibrium formation of GOS increased and eventually attained a plateau when the concentration of conjugated protein exceeded 0.275mg/mL (0.055U/mL). GOS yield increased on raising the temperature from 30 to 40°C, and declined thereafter. The apparent kinetic parameters were estimated from a five-step, nine-parameter kinetic model, which was then simulated using the COPASI package. The simulated results demonstrated an excellent match with the experimental data.

Oleaginous yeast Cryptococcus curvatus exhibits interplay between biosynthesis of intracellular sugars and lipids

AbstractPatterns of the biosynthesis of major metabolites of the oleaginous yeast Cryptococcus curvatus NRRL Y‐1511 were investigated during cultivation on sugar‐based media. When lactose or sucrose was employed as substrate under nitrogen‐limited conditions, the yeast strain accumulated high quantities of intra‐cellular total sugars (ITS) at the beginning of fermentation (up to 68% w/w), with ITS values progressively decreasing to 20%, w/w, at the end of the fermentation. Decrease in ITS content and consumption of extra‐cellular lactose led to a subsequent rise in lipid accumulation, reaching 29.8% in dry cell weight at 80 g/L of initial lactose concentration. Lactose was a more favorable substrate for lipid production than sucrose. In nitrogen‐excess conditions, ITS were produced in significant quantities despite the continuous presence of nitrogen in the medium. Growth on lactose was not followed by secretion of extra‐cellular β‐galactosidase. High quantities of extra‐cellular invertase were observed during growth on sucrose. The composition of ITS was highly influenced by the sugar used as substrate. Cellular lipids contained mainly palmitic and to lesser extent linoleic and stearic acids. This is the first report in the literature that demonstrates the interplay between the biosynthesis of intra‐cellular total sugars and lipid synthesis for oleaginous yeast strains.Practical applications: Cryptococcus curvatus represents a promising candidate for the successful production of microbial lipids and polysaccharides during growth on several types of low‐cost sugars utilized as substrates. Thus, C. curvatus could be used to produce a variety of metabolites useful for industrial biotechnology.Interplay between the biosynthesis of intra‐cellular total sugars produced per unit of dry weight (YITS/X %, w/w) and intra‐cellular lipids produced per unit of dry weight (YL/X %, w/w) of Cryptococcus curvatus, during growth on lactose, under nitrogen‐limited conditions.

Lactose electroisomerization into lactulose: Effect of the electrode material, active membrane surface area-to-electrode surface area ratio, and interelectrode-membrane distance

The aim of the present work was to study and develop an innovative, clean, and environmentally friendly process for lactulose synthesis by electroactivation of lactose. In this work, the electrode material (type 304 stainless steel, titanium, and copper), dimensionless interelectrode-membrane distance at the cathodic compartment (0.36, 0.68, and 1), and the membrane:electrode surface area ratio (0.23, 0.06, and 0.015) were considered to be the factors that could affect the kinetic conversion of lactose into lactulose. The reactions were conducted under an initial lactose concentration of 0.15mol/L at 10°C, Froude number (mixing speed) of 2.05×10−2, and electric current intensity of 300mA for 30min. The highest lactulose formation yield of 32.50% (0.05mol/L) was obtained by using a copper electrode, interelectrode-membrane distance of 0.36, and membrane:electrode surface area ratio of 0.23. The 2-parameter Langmuir, Freundlich, and Temkin isotherm models were used for the prediction of the lactose isomerization kinetics as well as the 3-parameter Langmuir-Freundlich isotherm model. It was shown that the lactose isomerization kinetics into lactulose followed the Temkin and Langmuir-Freundlich models with coefficients of determination of 0.99 and 0.90 and a relative error of 1.42 to 1.56% and 4.27 to 4.37%, respectively.

Enzyme, β-galactosidase immobilized on membrane surface for galacto-oligosaccharides formation from lactose: Kinetic study with feed flow under recirculation loop

The work focuses on producing galacto-oligosaccharides (GOS) through an enzymatic reaction with lactose under a partial recirculation loop by utilizing membrane-immobilized β-galactosidase. Cross-linking through covalent bonding, using gluteraldehyde, was employed to immobilize enzyme on a microporous polyvinylidene fluoride membrane. GOS synthesis was carried out in a laboratory fabricated reaction cell, whereby three immobilized membranes were housed in series. The reaction was conducted at varying initial lactose concentrations (ILCs) and feed flow rates at pH 6 and 40°C. A maximum GOS of 30% (dry basis) was obtained after 60h of reaction time, 50g/L ILC, 241U of enzyme (specific loading of 600U/g-membrane), and 0.5mL/min of feed flow rate at 56% lactose conversion. The GOS yield increased with increased ILC and decreased feed flow rate. The selectivity of GOS formation increased by increasing both the ILC and the feed flow rate, whereas the reverse was true for mono-saccharides. The immobilized enzyme retained ∼50% of its initial activity after 30 days of storage at 20°C, while the native enzyme lost 100% of its activity within 21 days. Furthermore, a five-step, nine-parameter model was developed, and simulated results showed excellent agreement with the experimental data.

Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85°C. A maximum production of 197g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.

OPTIMIZATION OF CULTURAL CONDITIONS FOR LACTIC ACID PRODUCTION BY LACTOBACILLUS BULGARICUS ATTC 11842 GROWN ON WHEY

The effects on the lactic acid (LA) production by thermophile Lactobacillus bulgaricus ATTC 11842 on whey as a basal medium of seven factors namely, temperature °C, pH, Lactose g/l, Yeast extract g/l, corn steep liquor (CSL) g/l, K2HPO4 g/l, and salts g/l (MnSO4, MgSO4 and FeSO4) were investigated, through the statistical analysis of the results by Plackett and Burmann experimental design. pH was found to have the high significant effect on lactic acid production. By response surface methodology (RSM) design the optimal value of pH and concentrations in the medium of yeast extract, K2HPO4 and salts were then investigated, it should be 5.5 of pH, 2.73 g/l of K2HPO4, 1.59 g/l of yeast extract and 0.0326 g/l, 0.1304 g/l, 0.01304 g/l of MgSO4, MnSO4 and FeSO4 respectively. The results obtained with the optimal results were 20.9592 g/l. of lactic acid and the corresponding yields was 0.5665% (ratio between the amount of lactic acid produced and the initial concentration of lactose).