Immobilization ofLactobacillus bulgaricus in a hollowfiber bioreactor for production of lactic acid from acid whey permeate

Lactobacillus bulgaricus was immobilized in the shell side of an industrial hollow-fiber ultrafiltration module. Acid whey permeate, containing 46 g/L lactose supplemented with 10 g/L yeast extract, was pumped through the tube side at dilution rates of 0.2–2.5/h. At a cell concentration of 100 g/L, productivity was 1.5–5 g lactic acid/L/h.     

Lactic acid recovery from cheese whey fermentation broth using combined ultrafiltration and nanofiltration membranes

The separation of lactic acid from lactose in the ultrafiltration permeate of cheese whey broth was studied using a cross-flow nanofiltration membrane unit. Experiments to test lactic acid recovery were conducted at three levels of pressure (1.4, 2.1, and 2.8 MPa), two levels of initial lactic acid concentration (18.6 and 27 g/L), and two types of nanofiltration membranes (DS-5DK and DS-5HL). Higher pressure caused significantly higher permeate flux and higher lactose and lactic acid retention (p<0.0001). Higher initial lactic acid concentrations also caused significantly higher permeate flux, but significantly lower lactose and lactic acid retention (p<0.0001). The two tested membranes demonstrated significant differences on the permeate flux and lactose and lactic acid retention. Membrane DS-5DK was found to retain 100% of lactose at an initial lactic acid concentration of 18.6 g/L for all the tested pressures, and had a retention level of 99.5% of lactose at initial lactic acid concentration of 27 g/L when the pressure reached 2.8 MPa. For all the test when lactose retention reached 99–100%, as much as 64% of the lactic acid could be recovered in the permeate.     

Effect of the variation of the level of lactose conversion in an immobilized lactase reactor upon operating costs for the production of baker’s yeast from hydrolyzed cottage cheese whey permeate

Operating costs for the production of Baker’s yeast from hydrolyzed permeate from the ultrafiltration of cottage cheese whey were calculated as a function of the level of lactose conversion in the immobilized lactase reactor. These costs were calculated for the case of 90% conversion of lactose in the reactor and compared to those that result when running the reactor at lower conversions with recycle of unreacted lactose. Total operating costs were estimated by combining individual operating costs for the immobilized enzyme reactor, costs associated with processing a lactose recycle stream, and energy costs associated with cooling the reactor feed stream and sterilizing the hydrolysate stream. It was determined that operating costs are minimized at about 9.9 ￠/lb. of lactose when the reactor is run at approx. 72% conversion. This represents a savings of 2.4 ￠/lb. of lactose over the case of a once-through 90% conversion of lactose in the reactor.     

Integrated Downstream Processing of Lactoperoxidase from Milk Whey Involving Aqueous Two-Phase Extraction and Ultrasound-Assisted Ultrafiltration

The present work involves the adoption of an integrated approach for the purification of lactoperoxidase from milk whey by coupling aqueous two-phase extraction (ATPE) with ultrasound-assisted ultrafiltration. The effect of system parameters of ATPE such as type of phase system, polyethylene glycol (PEG) molecular mass, system pH, tie line length and phase volume ratio was evaluated so as to obtain differential partitioning of contaminant proteins and lactoperoxidase in top and bottom phases, respectively. PEG 6000-potassium phosphate system was found to be suitable for the maximum activity recovery of lactoperoxidase 150.70% leading to 2.31-fold purity. Further, concentration and purification of enzyme was attempted using ultrafiltration. The activity recovery and purification factor achieved after ultrafiltration were 149.85% and 3.53-fold, respectively. To optimise productivity and cost-effectiveness of integrated process, influence of ultrasound for the enhancement of permeate flux during ultrafiltration was also investigated. Intermittent use of ultrasound along with stirring (2 min acoustic and 2 min stirring) resulted in increased permeate flux from 0.94 to 2.18 l/m² h in comparison to the ultrafiltration without ultrasound. The use of ultrasound during ultrafiltration resulted in increase in flux, but there was no significant change in activity recovery and purification factor. The integrated approach involving ATPE and ultrafiltration may prove to be a feasible method for the downstream processing of lactoperoxidase from milk whey.     

Separate and Concentrate Lactic Acid Using Combination of Nanofiltration and Reverse Osmosis Membranes

The processes of lactic acid production include two key stages, which are (a) fermentation and (b) product recovery. In this study, free cell of Bifidobacterium longum was used to produce lactic acid from cheese whey. The produced lactic acid was then separated and purified from the fermentation broth using combination of nanofiltration and reverse osmosis membranes. Nanofiltration membrane with a molecular weight cutoff of 100–400 Da was used to separate lactic acid from lactose and cells in the cheese whey fermentation broth in the first step. The obtained permeate from the above nanofiltration is mainly composed of lactic acid and water, which was then concentrated with a reverse osmosis membrane in the second step. Among the tested nanofiltration membranes, HL membrane from GE Osmonics has the highest lactose retention (97 ± 1%). In the reverse osmosis process, the ADF membrane could retain 100% of lactic acid to obtain permeate with water only. The effect of membrane and pressure on permeate flux and retention of lactose/lactic acid was also reported in this paper.     

Succinic Acid Production from Cheese Whey using Actinobacillus succinogenes 130 Z

Actinobacillus succinogenes 130 Z was used to produce succinic acid from cheese whey in this study. At the presence of external CO₂ supply, the effects of initial cheese whey concentration, pH, and inoculum size on the succinic acid production were studied. The by-product formation during the fermentation process was also analyzed. The highest succinic acid yield of 0.57 was obtained at initial cheese whey concentration of 50 g/L, while the highest succinic acid productivity of 0.58 g h⁻¹ L⁻¹ was obtained at initial cheese whey concentration of 100 g/L. Increase in pH and inoculum size caused higher succinic acid yield and productivity. At the preferred fermentation condition of pH 6.8, inoculum size of 5% and initial cheese whey concentration of 50 g/L, succinic acid yield of 0.57, and productivity of 0.44 g h⁻¹ L⁻¹ were obtained. Acetic acid and formic acid were the main by-products throughout the fermentation run of 48 h. It is feasible to produce succinic acid using lactose from cheese whey as carbon resource by A. succinogenes 130 Z.     

Bacterial production of PHA from lactose and cheese whey permeate

Due to the large availability of agro-industry wastes containing potentially exploitable substrates, such as whey from dairy industry, a study on the bacterial conversion of lactose and whey permeate to poly(β-hydroxyalkanoate) (PHA) was undertaken. A first approach was carried out on culture collection strains. Among a number of strains tested, Hydrogenophaga pseudoflava DSM 1034 and Sinorhizobium meliloti 41 were found to grow on lactose and produce PHA. These findings suggested to investigate among a wider range of microorganisms by directly isolating new strains from soil. A number of soil bacteria were first isolated on a minimal medium containing lactose as unique carbon source and PHA-accumulating traits were then investigated. Three isolates, identified by 16S rDNA sequence analysis as Sinorhizobium sp., Bacillus megaterium and Bacillus sp., were selected for their efficient growth and PHA production using lactose as carbon source. The same strains were also tested for their ability to accumulate PHA by direct fermentation of whey and whey permeate. Our results suggest that production of the polymer from cheese whey or whey permeate may be possible, although further research is needed to determine whether these microorganisms have the potential for commercial production of such biodegradable polymers.     

Cheese whey tangential filtration using tubular mineral membranes

Membrane separation techniques are extensively used in dairy industry both for milk and cheese whey processing. However, cheese whey might still be considered as a problematic waste despite its high content of many valuable substances, such as proteins, lactose or minerals, which can be further used, e.g. in human nutrition, pharmacy or biotechnologies. Another problem, which food technologists have to face, is variable quality, composition and properties of food materials bringing high demands on manufacturing industry. In this paper, filtration kinetics and separation efficiency during purification and fractionation of cheese whey (sweet and salty) from Czech dairies by pilot-plant filtration (Bollene, France) was studied using tubular membranes (Membralox, USA). Various mineral membranes’ cut-offs were tested and all experiments ran in the retentate recycling mode. The obtained mass concentration factors were between 1.9 and 16.5. Steady state fluxes were calculated from the experimental data using a mathematical model. Fine ultrafiltration on a 5 kDa membrane gave steady state fluxes of 14–19 L m^?2 h^?1. The coarse pre-filtration on 100 nm, 200 nm or 500 nm membranes showed various permeate fluxes between 22 L m^?2 h^?1 and 153 L m^?2 h^?1. Despite the high pore sizes of the used membranes, lactose was partially rejected by all membranes tested.     

Galacto-oligosaccharides Synthesis from Lactose and Whey by β-Galactosidase Immobilized in PVA

The synthesis of galacto-oligosaccharides (GOS) by ??-galactosidase immobilized in both polyvinyl alcohol (PVA) lenses and sol?Cgel carriers was studied and compared with the performance of the free enzyme. PVA-immobilized ??-galactosidase retained 95?% of the initial activity after seven repeated uses and retained 51?% of the initial activity after 3?months of storage, while sol?Cgel-immobilized ??-galactosidase only retained 39?% of the initial activity under storage. Lactose conversion takes place at a higher rate in the PVA-immobilized ??-galactosidase, while the lowest rate of lactose conversion was noticed with immobilized ??-galactosidase in sol?Cgel. Continuous production of GOS from either lactose or whey, with PVA-immobilized ??-galactosidase, was performed in a packed-bed reactor. A maximum GOS production of 30?% of total sugars was attained for a 40-% lactose feed solution with a feed rate of 10.8?ml/h, at pH 4.5 and 40?°C, corresponding to a productivity of 117?g/l?h. The maximum GOS productivity of 344?g/l?h was obtained at a flow rate of 28.7?ml/h. 3-OS and 4-OS were the major types of GOS formed. Conversion of whey in continuous mode resulted in GOS production of 15?% of total sugars and formation of 45?% 3-OS, 40?% 4-OS, and 15?% 5-OS.     

β-Galactosidase from <Emphasis Type="Italic">Penicillium canescens</Emphasis>. Properties and immobilization

β-galactosidase from Penicillium canescens was immobilized on chitosan, sepharose-4B, foamable polyurethane and some other carriers. The highest yield of immobilization (up to 98%) was obtained by using chitosan as a carrier. The optimum pH and temperature were not significantly altered by immobilization. High stability of immobilized β-galactosidase during storage was demonstrated. Efficient lactose saccharification (over 90%) in whey was achieved by using immobilized β-galactosidase.     

Inexpensive isolation of beta-D-glucopyranosidase from alpha-L-arabinofuranosidase,alpha-L-rhamnopyranosidase,and o-acetylesterase

β-d-Glucopyranosidase (βG, EC 3.2.1.21) has been isolated from some collateral activities, α-l-arabinofuranosidase (Ara, EC3.2.1.55), α-l-rhamnopyranosidase (Rha, EC 3.2.1.40), and o-acetylesterase (Est, EC 3.1.1.53), using a commercial enzyme preparation and a simple method economically sustainable for the food industry. The procedure comprises precipitation of extraneous substances by adding ethanol and CaCl₂, ultrafiltration, and adsorption, first on bentonite and then on chitosan. The results obtained were the complete isolation of βG from the above-mentioned activities, a drastic reduction in extraneous compounds, such as brown substances and polysaccharides, and a slight increase in purification.     

Downstream processing of lactic acid-whey permeate fermentation broths by hollow fiber ultrafiltration

Membrane filtration is a suitable method for cell harvesting and clarification of fermentation broths. Hollow fiber ultrafilters gave essentially 100% rejection ofL. bulgaricus cells from a whey permeate fermentation broth. A combination of low pressures and high velocity generally gave the best permeate flux. Fermentation media components (in this case, from the whey permeate) contributed significantly to fouling. Considering the pressure limitations of the current generation of asymmetric hollow fiber modules and the changes in physical properties of the fermentation broths, a cell concentration of 100–150 g/L could be obtained with the flux still relatively high (above 20LMH), although the chemical compatibility of the membrane module itself under long-term exposure to high acid conditions should be considered.     

Fractionation of whey-derived peptides using a combination of ultrafiltration and nanofiltration

This paper describes the fractionation and further isolation and characterisation of peptides and proteins present in sweet whey by means of ultrafiltration using a regenerated cellulose membrane with a nominal molar mass cut-off value of 10 kg/mol and nanofiltration through sulphonated polyether sulphone membrane with a cut-off of 1 kg/mol. The concentration of whey proteins was done below the critical flux. The sieving coefficients for the whey components (proteins, lactose and salts) were estimated. Whey proteins were completely rejected by the ultrafiltration membrane. Size exclusion chromatography (SEC) and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to evaluate the molar masses of the peptide fractions that were present in the whey permeates. Nanofiltration of whey permeates obtained after ultrafiltration was conducted at two pH values (9.5 and 3.0) that corresponded to the different charged states of the membrane and of the peptides. The transmission of peptides, amino acids and lactose was found to be mainly affected by the permeability of the fouling layer. The selectivity of the nanofiltration membranes toward peptides compared to lactose was calculated as 0.82 and 6.81 at pH 9.5 and 3.0, respectively.     

Separation ofN-carbamoyl aspartate andl-dihydroorotate from serum by high-performance liquid chromatography with fluorimetric detection

Summary A high-performance liquid chromatographic method, with 9-anthryldiazomethane as derivatizing agent, has been developed for the simultaneous determination ofN-carbamoyl aspartate andl-dihydroorotate in serum. Sample preparation for 1 mL serum was by simple liquid-liquid extraction and then derivatization. The compounds were separated on a Luna C18(2) column by use of a gradient prepared from acetonitrile and 10 mM sodium acetate buffer, pH 6.0, and fluorimetric detection was performed at excitation and emission wavelengths of 365 nm and 412 nm, respectively. The response was found to be linearly dependent on concentration between 0.8 and 60 μg mL⁻¹ forl-dihydrooratate and between 0.9 and 90 μg mL⁻¹ forN-carbamoyl aspartate; the mean recovery rates were 50 and 51%, respectively. The limits of detection and quantification were 0.33 μg mL⁻¹ and 0.6 μg mL⁻¹, respectively, forl-dihydroorotate and 0.4 μg mL⁻¹ and 0.7 μg mL⁻¹ forN-carbamoyl aspartate. This method can be used to assess accumulation ofN-carbamoyl aspartate andl-dihydroorotate in body fluids in situations where cellular pyrimidine de novo synthesis is impaired.     

Analysis of free carbohydrates in milk using micropacked columns

Summary A gas chromatographic method using a micropacked column is described for the analysis of lactose, galactose, lactulose and epilactose in processed milks. The method is evaluated for precision and accuracy using phenyl-β-glucoside as an internal standard. Recoveries near 100% were found for lactulose concentrations higher than 0.1 mgml⁻¹, showing coefficients of variation from 5.9 to 9.4%.     

Removal of cadmium ions using micellar-enhanced ultrafiltration with mixed anionic-nonionic surfactants

Micellar-enhanced ultrafiltration (MEUF) was used to remove cadmium ions from wastewater efficiently. In this study the nonionic surfactants polyoxyethyleneglycol dodecyl ether (Brij35) and polyoxyethylene octyl phenyl ether (TritonX-100) were for micellar-enhanced ultrafiltration to lower the dosage of the anionic surfactant sodium dodecyl sulfate (SDS). The surfactant critical micelle concentration (CMC) and the degree of micelle counterion binding were investigated. The effects of nonionic surfactant addition on the efficiency of cadmium removal, the residual quantities of surfactant, the permeate flux and the secondary membrane resistance were investigated. A comparison between MEUF with SDS and MEUF with mixed anionic–nonionic surfactants was undertaken. The results show that the addition of Brij35 or TritonX-100 reduced the CMC of SDS and the degree of counterion binding for the micelles. Due to these variations the Cd²⁺ rejection efficiency was at a maximum when the Brij35:SDS and the TritonX-100:SDS molar ratio was 0.5. The Cd²⁺ rejection efficiency in MEUF with SDS is higher than for MEUF with mixed surfactants when the total dose of surfactant is constant. The permeate flux of MEUF with SDS is higher than that for MEUF with mixed surfactants while the secondary resistance of MEUF with SDS is less than that of MEUF with mixed surfactants.     

Synthesis and studies of spectroscopic and electrochemical properties of dinuclear ruthenium(<Emphasis Type="SmallCaps">II</Emphasis>) and manganese(<Emphasis Type="SmallCaps">II</Emphasis>) complexes

New dinuclear ruthenium manganese complexes of general composition (bpy)₂Ru(L)MnCl_x(H₂O)₂ (L is 1,10-phenanthroline-5,6-dione, 3,3′-dicarboxy-2,2′-bipyridyl, or bis(pyrazolyl); x = 2 or 4) were synthesized by the reaction of (bpy)₂Ru(L) with MnCl₂ · 4H₂O. These compounds and the starting mononuclear ruthenium complexes were studied by spectrophotometric and electrochemical methods in MeCN. The position of the charge-transfer band Ru^II → L in the spectra depends on the donor-acceptor characteristics of the ligand L. For the dinuclear complex under study, the formal potentials of reversible one-electron oxidation of Ru^II are in the range of 0.9–1.2 V (vs. the standard hydrogen electrode), whereas oxidation of Mn^II occurs at more positive (by 0.1–0.2 V) potentials. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 2281–2285, October, 2005.     

Enhanced β-Galactosidase Production from Whey Powder by a Mutant of the Psychrotolerant Yeast <Emphasis Type="Italic">Guehomyces pullulans</Emphasis> 17<Emphasis Type="Italic">–</Emphasis>1 for Hydrolysis of Lactose

In order to isolate β-galactosidase overproducers of the psychrotolerant yeast Guehomyces pullulans 17–1, its cells were mutated by using nitrosoguanidine (NTG). One mutant (NTG-133) with enhanced β-galactosidase production was obtained. The mutant grown in the production medium with 30.0 g/l lactose and 2.0 g/l glucose could produce more β-galactosidase than the same mutant grown in the production medium with only 30.0 g/l lactose while β-galactosidase production by its wild type was sensitive to the presence of glucose in the medium. It was found that 40.0 g/l of the whey powder was the most suitable for β-galactosidase production by the mutant. After optimization of the medium and cultivation conditions, the mutant could produce 29.2 U/ml of total β-galactosidase activity within 132 h at the flask level while the mutant could produce 48.1 U/ml of total β-galactosidase activity within 144 h in 2-l fermentor. Over 77.1% of lactose in the whey powder (5.0% w/v) was hydrolyzed in the presence of the β-galactosidase activity of 280 U/g of lactose within 9 h while over 77.0% of lactose in the whey was hydrolyzed in the presence of β-galactosidase activity of 280 U/g of lactose within 6 h. This was the first time to show that the β-galactosidase produced by the psychrotolerant yeast could be used for hydrolysis of lactose in the whey powder and whey.     

Immobilization of the β-galactosidase fromAspergillus niger on chitosan

Crude or purifiedAspergillus niger β-galactosidase preparations were immobilized on chitosan (deacetylated chitin, activated with glutaraldehyde). The most active immobilized systems were obtained withcrude enzyme preparations. The immobilized enzyme hydrolyzed lactose in pure lactose solutions, ultrafiltrate whey permeates, or acid wheys at similar rates. The pH activity profiles and K_m values of the chitosan-bound enzyme were not significantly altered on immobilization, and its stability on repetitive use up to 60°C was increased by reduction with NaBH₄. After 8 weeks on discontinuous operation (8 h use per day), β-galactosidase-chitosan columns were found to retain about 90, 50, or 60% of their initial activities with lactose, ultrafiltrate permeate, or acid whey solutions, respectively. The efficiency of the β-galactosidase-chitosan conjugate appears to be comparable or greater than those of other described systems, and its stability should allow its use on an industrial scale. A preliminary report of this work has already been presented at the 4th International Enzyme Engineering Conference (September 25-30, 1977, Bad Neuenahr, FRG).     

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.