Removal Of Lactose Research Articles

Lactose Intolerance and Other Disaccharidase Deficiency

Intolerance to foods which contain lactose can cause a range of intestinal and systemic symptoms. These symptoms are caused by Lactase deficiency which is encoded by a single gene (LCT) of ≈ 50kb located on chromosome 2q21. In some food items, lactose has been missed because of "hidden" lactose due to inadequately labeled, confusing diagnosis of lactose intolerance based on dietary restriction of dairy foods. Two polymorphisms, C/T13910 and G/A22018, linked to hypolactasia, correlate with breath hydrogen and symptoms after lactose. The key in the management of lactose intolerance is the dietary removal of lactose. Patients diagnosed as lactose intolerant must be advised of "risk" foods, inadequately labeled, including processed meats, bread, cake mixes, soft drinks, and lagers. This review highlights the types, symptoms and management of lactose intolerance and also highlights differences from milk allergy which closely mimics the symptoms of lactose intolerance.

EXPLORING THE OPTIMUM CONDITIONS FOR MAXIMIZING THE MICROBIAL GROWTH OF Candida intermedia BY RESPONSE SURFACE METHODOLOGY

Exploring optimum and cost-efficient medium composition for microbial growth of Candida intermedia Y–1981 yeast culture growing on whey was studied by applying a multistep response surface methodology. In the first step, Plackett–Burman (PB) design was utilized to determine the most significant fermentation medium factors on microbial growth. The medium temperature, sodium chloride and lactose concentrations were determined as the most important factors. Subsequently, the optimum combinations of the selected factors were explored by steepest ascent (SA) and central composite design (CCD). The optimum values for lactose and sodium chloride concentrations and medium temperature were found to be 18.4 g/L, 0.161 g/L, and 32.4°C, respectively. Experiments carried out at the optimum conditions revealed a maximum specific growth rate of 0.090 1/hr; 42% of total lactose removal was achieved in 24 h of fermentation time. The obtained results were finally verified with batch reactor experiments carried out under the optimum conditions evaluated.

Chronic Diarrhea in Children

Chronic Diarrhea in Children

Comparison between homologous human milk supplements and a commercial supplement for very low birth weight infants

To describe the methodology for the preparation of two additives derived from human milk, liquid and powdered, and to compare this composition with the commercial additive FM85®. For the preparation of the liquid and powdered supplements, 40 samples of human milk were used. Both supplements have been through three preparation phases: skimming, evaporation and lactose removal. After these phases, the liquid supplement is ready, and the powdered requires a fourth phase - lyophilization. To each sample of the liquid and powdered supplements were added, respectively, 80 mL (group I) and 100 mL (group II) of pooled banked human milk. For comparison, 20 samples of 100 mL of the pool were added to 5 g of the FM85® supplement (Nestlé) (group III). Analyses of carbohydrates, protein, lipids, calcium, phosphorus, sodium, osmolality and caloric content were performed, considering a significant difference p < 0.05. Groups I, II, and III showed, respectively, the following results: protein = 1.81, 2.38 and 1.96 g/dL (p < 0.001); carbohydrates = 6.70, 7.25 and 10.06 g/dL (p = 0.006); fat = 3.75, 3.75 and 3.73 g/dL (p = 0.96); calcium = 36.92, 44.75 and 79.37 mg/dL (p = 0.001); phosphorus = 20.02, 23.28 and 56.30 mg/dL (p = 0.02); sodium = 14.32, 14.40 and 20.33 mEq/L (p = 0.143); osmolality = 391.45, 412.47 and 431.00 mOsmol/kgH(2)O (p = 0.074); and caloric content = 67.78, 72.27 and 81.65 kcal (p = 0.001). The studied additives differ significantly from the commercial additive FM85® in some of its components, and its composition may or may not meet the quantity of nutrients suggested by the most recent recommendations.

Structural prediction and comparative docking studies of psychrophilic β- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes.

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.

Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. K m values of BgalC for p-nitrophenyl-β-galactopyranoside ( pNPGal), o-nitrophenyl-β-galactopyranoside ( oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.

Practical Preparation of Epilactose Produced with Cellobiose 2-Epimerase fromRuminococcus albusNE1

A practical purification method for a non-digestible disaccharide, epilactose (4-O-beta-galactosyl-D-mannose), was established. Epilactose was synthesized from lactose with cellobiose 2-epimerase and purified by the following procedure: (i) removal of lactose by crystallization, (ii) hydrolysis of lactose by beta-galactosidase, (iii) digestion of monosaccharides by yeast, and (iv) column chromatography with Na-form cation exchange resin. Epilactose of 91.1% purity was recovered at 42.5% yield.

Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus

The putative beta-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli beta-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two beta-galactosidase genes. No functional characteristic of the putative beta-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had beta-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus beta-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37 degrees C and could hydrolyze 73% of lactose in milk in 30 h at 10 degrees C. The L. acidophilus beta-galactosidase (LacZ) was identified as cold-adapted beta-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.

A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene cloning, overexpression, purification and properties

BackgroundThe development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production.ResultsIn this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this β-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of β-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50°C, 60% of the maximum activity of the enzyme was determined at 25°C and 15% of the maximum activity was detected at 0°C.ConclusionThe properties of Arthrobacter sp. 32cβ-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.

Amendment of the Activity of Microbial Preparation Laktazym to Protein Biodegradation for Dairy Wastewater

ABSTRACTThere exist various biological products in the markets for augmentation of wastewater treatment process, but their specificity toward definite substrates is low. In the present study the amendment and the enrichment of biodegradation activity of commercial preparation Laktazym toward a target contaminant in dairy wastewater were investigated. Two microbial dominants—AS-05 and LB-16 with high biodegradation activity to the milk proteins were isolated by the applied selection pressure with casein. Both cultures were detailed characterized by biomass accumulation, effectiveness of organics, protein and lactose removal, as well as by key enzyme activities—total dehydrogenase, phosphatase and nitrate reductase.Anaerobic biodegradation process for dairy wastewater in a critical situation of destabilized biofilm has been simulated in lab with different biological systems. The experimental data showed greater positive effect on the biodegradation of the organic matter, particularly of the milk proteins for the two modifications of investigated preparation with the selected cultures, than for the variant Laktazym. Moreover at the 48th and the 72nd hour the effectiveness of protein removal for the combination Laktazym + AS-05 (Peptostreptococcus sp.) was higher with 67%, than for the variant only with Laktazym.

Fractionation of dairy wastewater into a protein‐rich and a lactose‐rich product

AbstractFractionation of dairy wastewater into lactose‐enriched and protein‐enriched streams using an ultrafiltration membrane technique was examined. Three different sizes (3, 5 and 10 kDa) of regenerated cellulose membranes were used to determine the most efficient membrane for the process. Performance was determined under various operating conditions that included membrane size, temperature and transmembrane pressure (TMP) on the lactose and protein separation. The experiments were initially conducted with a made‐up solution of (1) pure lactose, and (2) lactose solution with protein at different concentrations. After the behaviour of the membrane towards lactose and protein were assessed the experiments were performed with dairy wastewater supplied by a local dairy processing plant.From the results it was found that the 3, 5 and 10 kDa membranes provided 70–80, 95–100 and 100% recovery of lactose, respectively, from the made‐up solution of pure lactose. The 10 kDa membrane results also showed a 100% recovery of lactose from the wastewater sample. At high temperatures, the lactose removal was higher by a minimum of 5% and a maximum of 18%. In general, increase in TMP increased the permeate flux which increased the percentage of lactose being removed. Other experiments conducted with wastewater showed that the permeate flux decreased initially for 25 min until a steady state was reached. The addition of protein to the made‐up lactose solution decreased the percentage of lactose being removed. The 5 kDa membrane shows 90‐95% rejection of protein, whereas the 10 kDa membrane yields low protein rejection at lower TMPs, but high rejection at higher TMPs.For the membranes considered (i.e. cellulose‐based material and the cassette‐type module), the most effective membrane for lactose recovery from made‐up solution, as well as from dairy wastewater, is 10 kDa. Protein rejection from a made‐up solution of lactose and protein was low while using a 10 kDa membrane. Fouling was found to be very significant from the experiments with wastewater. Copyright © 2007 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Molecular Cloning and Characterization of the Gene Encoding Cold-Active β-Galactosidase from a Psychrotrophic and Halotolerant Planococcus sp. L4

The gene bgaP encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the BgaP gene revealed an open reading frame of 2031 bp encoding for a protein of 677 amino acid residues. The BgaP was heterologously expressed in E. coli and purified followed by Ni2+ affinity chromatography. The molecular mass of the native enzyme was approximately 156 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the BgaP indicated molecular masses of 78 and 77.311 kDa, respectively, suggesting that the BgaP is a dimer. The purified BgaP had an isoelectric point of 4.8 and exhibited maximal activity at 20 degrees C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 degrees C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG); the Km values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 degrees C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 degrees C had 14 and 47 times more than that of E. coli beta-galactosidase at 20 degrees C, respectively. Therefore, cold-active beta-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4 could conceivably be developed to fulfill the practical requirements to enable its use for lactose removal in milk and dairy products at low temperature or a reporter enzyme for psychrophilic genetic systems.

Natural sweetening of food products by engineering Lactococcus lactis for glucose production

We show that sweetening of food products by natural fermentation can be achieved by a combined metabolic engineering and transcriptome analysis approach. A Lactococcus lactis ssp. cremoris strain was constructed in which glucose metabolism was completely disrupted by deletion of the genes coding for glucokinase ( glk), EII man/glc ( ptnABCD), and the newly discovered glucose-PTS EII cel ( ptcBAC). After introducing the lactose metabolic genes, the deletion strain could solely ferment the galactose moiety of lactose, while the glucose moiety accumulated extracellularly. Additionally, less lactose remained in the medium after fermentation. The resulting strain can be used for in situ production of glucose, circumventing the need to add sweeteners as additional ingredients to dairy products. Moreover, the enhanced removal of lactose achieved by this strain could be very useful in the manufacture of products for lactose intolerant individuals.

Optimization of lactose utilization in deproteinated whey by Kluyveromyces marxianus using response surface methodology (RSM)

Kluyveromyces marxianus Y-8281 yeast culture was utilized for the biological treatment of deproteinated whey wastewater in a batch system. Removal of lactose was optimized by the utilization of response surface methodology, RSM. The empirical model developed through RSM in terms of effective operational factors of medium pH, temperature, lactose and ammonia concentrations was found adequate to describe the treatment of deproteinated whey. Through the analysis, medium pH and temperature were found to be the most significant factors and an increment in both had a positive effect on lactose utilization, while lactose and ammonia concentrations had the least weight within the ranges investigated. Based on contour plots and variance analysis, optimum operational conditions for maximizing lactose removal were found to be 31 °C, 45 g/L whey powder concentration, 4 g/L total ammonium salt concentration and medium pH 6. Under the optimum operating conditions determined, 95% lactose removal was achieved after an 18-h fermentation.

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.

Lactose surface modification by decantation: are drug-fine lactose ratios the key to better dispersion of salmeterol xinafoate from lactose-interactive mixtures?

The role of fine lactose in the dispersion of salmeterol xinafoate (SX) from lactose mixtures was studied by modifying the fine lactose concentration on the surface of the lactose carriers using wet decantation. Fine lactose was removed from lactose carriers by wet decantation using ethanol saturated with lactose. Particle sizing was achieved by laser diffraction. Fine particle fractions (FPFs) were determined by Twin Stage Impinger using a 2.5% SX mixture, and SX was analyzed by a validated high-performance liquid chromatography method. Adhesion forces between probes of SX and silica and the lactose surfaces were determined by atomic force microscopy. FPFs of SX were related to fine lactose concentration in the mixture for inhalation grade lactose samples. Reductions in FPF (2-tp 4-fold) of Aeroflo 95 and 65 were observed after removing fine lactose by wet decantation; FPFs reverted to original values after addition of micronized lactose to decanted mixtures. FPFs of SX of sieved and decanted fractions of Aeroflo carriers were significantly different (p < 0.001). The relationship between FPF and fine lactose concentration was linear. Decanted lactose demonstrated surface modification through increased SX-lactose adhesion forces; however, any surface modification other than removal of fine lactose only slightly influenced FPF. Fine lactose played a key and dominating role in controlling FPF. SX to fine lactose ratios influenced dispersion of SX with maximum dispersion occurring as the ratio approached unity.

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO2 of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.

Influence of ambient air temperature on the cooling/heating load of a single cell protein jacketed fermenter operating on cheese whey under continuous conditions.

The heat generated by mixing and lactose metabolism, during the continuous production of single cell protein from cheese whey lactose using a jacketed fermenter with running cooling water, was calculated using a heat balance equation. The technique quantified the heat produced in and lost from the fermentation unit. Most of the heat generated by mixing in the cell-free system (97.47%) was lost with exhaust gas, while a very small amount (2.53%) was lost through the fermenter lid, wall, and bottom. The heat generated by mixing was significant (26.31% of the total heat generated in the fermentation system with an active yeast population present) and, therefore, cannot be ignored in heat balance calculations. About 19.71% of the total heat generated in the reactor was lost through the coolant at an ambient temperature of 22 +/- 0.5 degrees C, showing the need for a cooling system. A yeast population size of 986 million cells/mL and a lactose removal efficiency of 95.6% were observed. About 72.5% and 27.5% of the lactose consumed were used for growth and respiration, respectively. A yield of 0.66 g of cells/g of lactose was achieved. The heat released by unit biomass was 7.05 kJ/g of cells. The results showed the significant impact of ambient air temperature on the cooling load. The heat to be removed from the medium by the cooling system varied from 3.46 to 281.56 kJ/h when the temperature increased from 16 to 30 degrees C. A heating system is needed to maintain the medium temperature at 34 degrees C when the ambient air temperature is below 16 degrees C.

Dental caries-protective agents in milk and milk products: investigations in vitro

Objectives: To characterise and identify the anti-dental caries components that exist in milk and milk products. Methods: Standard enamel or hydroxyapatite demineralisation tests were devised to simulate the action of acid on tooth mineral, and they were used to show which constituents of milk possessed a potential protective action against acid attack. Results: Milk and milk products were fractionated and tested, revealing that minerals, including calcium and phosphorus, played a part in this protective process. The findings also drew attention to the effectiveness of minor milk protein or protein-associated components, the structures of which are mostly known, which were separated and characterised by gel electrophoresis as proteose-peptone fractions 3 and 5. The strength of adsorption of the protein or polypeptides in these fractions to dental enamel was measured and found to be sufficient to reduce the extent of demineralisation of enamel by acid buffer solutions. Conclusions: The removal of lactose, fat, casein and other proteins had little influence on the protective effect of the milk fractions. Besides calcium and phosphorus, milk contains other more powerful protective factors, which were identified as proteose-peptone fractions 3 and 5. Details of their composition and the strength of their adherence to the surface of dental mineral are given.

Effect of various preheat treatments on the heat stability of unconcentrated milk

Abstract The effect of preheating milk and serum-free casein micelle dispersions (SPFCM) was studied over a range of temperatures. Important differences were recorded after preheating at different temperature–time combinations. The effect of pH adjustment after various preheat treatments was also studied. Removal of lactose before preheat treatment had a significant effect.