Lactose Residues Research Articles

Разработка технологии получения безлактозного молока методом диафильтрации

Abstract. The purpose. The research is aimed at solving the problem related to the development of technology for producing an environmentally friendly product of animal origin – lactose-free milk. This will solve the problem of consumption of milk and dairy products by people who do not tolerate milk sugar for physiological reasons. Analyzing, on the basis of a literary review, the current state of the issue of lactose-free milk production, it can be concluded that existing technologies have certain disadvantages due to changes in the composition of the initial product, or the significant complexity of the technological chain. The proposed development is based on a method of milk diafiltration consisting of several repetitive cycles. The cycle consists in the separation of milk by the ultrafiltration process into permeate (aqueous lactose solution) and concentrate (proteins, fat, lactose residues). Then pure water is added to the concentrate in a volume equal to the volume of the discharged permeate. For a scientifically based approach to the development of the proposed technology, it is necessary to conduct a study of ultrafiltration separation of milk, obtain optimal operating parameters of the process, determine the change in permeability and selectivity of membranes from lactose concentration, consider the possibility of obtaining lactose-free milk with different mass fraction of fat, determine the number of cycles of the membrane diafiltration process. Research methods. The study of the process of ultrafiltration separation of milk was carried out in laboratory conditions on a membrane installation using organic and inorganic membranes. The experiments determined the main characteristics of ultrafiltration membranes – permeability and selectivity, with varying parameters (parameter range: P = 0.15…0.5 MPa, t = 35…65 °C). Results. Based on the experimental data obtained, by processing them and optimizing the parameters, optimal modes of the milk separation process by ultrafiltration were obtained. The following parameters are defined: the velocity of the product in the supramembrane space, the operating pressure of the separation process, its temperature. The preferred ultrafiltration membranes for the membrane-milk system have been selected. Scientific novelty. To develop a technology for producing lactose-free milk, for the first time a number of studies and experiments were carried out, such as changing the permeability and selectivity of membranes from the concentration of lactose, obtaining lactose-free milk with mass fraction of fat the number of cycles of the membrane diafiltration process was determined.

Sustainable 3D-printed β-galactosidase immobilization coupled with continuous-flow reactor for efficient lactose-free milk production

The production of low-lactose and lactose-free milk remains a critical challenge. Novel enzyme immobilization integrated with the flow reactor that could be applied to hydrolyze milk with cost-effectiveness, facilitation, and safety is highly expected. Here, we present a novel continuous-flow bioreactor loaded with β-galactosidase (β-Gal) covalently immobilized 3D-printed scaffolds for the production of lactose-free milk. 3D-printed polylactic acid (PLA) scaffolds modified by self-assembly of polyethyleneimine and sodium hyaluronate layers are rapidly branched with functional groups, followed by covalent immobilization of β-Gal using genipin as the cross-linker. The use of genipin allows efficient and stable immobilization of the enzyme (92.87 % immobilization efficiency), and the immobilized enzyme retains 90.53 % activity after 6th repeated use. Moreover, the 3D-printed scaffolds integrated into the flow reactor perturbed the fluids in the different reactors through directional channels, which improved the internal mass transfer to achieve high catalytic activity (94.20 % lactose hydrolysis efficiency). The lactose residues of milk reacted in the β-Gal flow reactor for 6 and 12 h were 1.87 g dL-1 and 0.38 g dL-1, which fully met the requirements of the national food safety standards for low-lactose and lactose-free milk. Due to the high efficiency of enzyme immobilization and the stable catalytic activity of the reactor, it has a broad application prospect for the production of low-lactose and lactose-free milk and other biocatalytic fields.

Influence of Pasteurization on Maillard Reaction in Lactose-Free Milk.

In order to improve the safety and quality of lactose-free milk (LFM) Maillard reaction products (MRPs), this study used raw cow's milk as raw material and lactase hydrolysis to prepare LFM, which was heat-treated using pasteurization and then placed in storage temperatures of 4 °C, 25 °C and 37 °C to investigate the changes in the Maillard reaction (MR). The results of the orthogonal test showed that the optimal conditions for the hydrolysis of LFM are as follows: the hydrolysis temperature was 38 °C, the addition of lactase was 0.03%, and the hydrolysis time was 2.5 h. Under these conditions, the lactose hydrolysis rate reached 97.08%, and the lactose residue was only 0.15 g/100 g as determined by high-performance liquid chromatography (HPLC), complying with the standard of LFM in GB 28050-2011. The contents of furoamic acid and 5-hydroxymethylfurfural were determined by high-performance liquid chromatography, the color difference was determined by CR-400 color difference meter, and the internal fluorescence spectrum was determined by F-320 fluorescence spectrophotometer. The test results showed that the variation range of furosine in lactose-free milk after pasteurization was 44.56~136.45 mg/100g protein, the range of 5-hydroxymethylfurfural (HMF) was 12.51~16.83 mg/kg, the color difference ranges from 88.11 to 102.53 in L*, from -0.83 to -0.10 in a*, and from 1.88 to 5.47 in b*. The furosine content of LFM during storage at 4, 25, and 37 °C ranged from 44.56 to 167.85, 44.56 to 287.13, and 44.56 to 283.72 mg/100 g protein, respectively. The average daily increase in protein content was 1.18-3.93, 6.46-18.73, and 15.7-37.66 mg/100 g, respectively. The variation range of HMF was 12.51~17.61, 12.51~23.38, and 12.51~21.1 mg/kg, and the average daily increase content was 0.03~0.07, 0.47~0.68, and 0.51~0.97 mg/kg, respectively. During storage at 4 °C, the color difference of LFM ranged from 86.82 to 103.82, a* ranged from -1.17 to -0.04, and b* ranged from 1.47 to 5.70. At 25 °C, color difference L* ranges from 72.09 to 102.35, a* ranges from -1.60 to -0.03, b* ranges from 1.27 to 6.13, and at 37 °C, color difference L* ranges from 58.84 to 102.35, a* ranges from -2.65 to 1.66, and b* ranges from 0.54 to 5.99. The maximum fluorescence intensity (FI) of LFM varies from 131.13 to 173.97, 59.46 to 173.97, and 29.83 to 173.97 at 4, 25, and 37 °C. In order to reduce the effect of the Maillard reaction on LFM, it is recommended to pasteurize it at 70 °C-15 s and drink it as soon as possible during the shelf life within 4 °C.

Chitosan-based Maillard products for enzyme immobilization in multilayers structure: Its application in electrochemical sensing

In this work we demonstrate for the first time the advantages of using water-soluble chitosan polycation obtained by Maillard reaction with glucose (G-Chit) or lactose residues for the immobilization of glucose oxidase (GOx) and lactate oxidase by the layer-by-layer adsorption method onto carbon paste electrodes. Besides its solubility in enzyme-compatible media, these polymers have a suitable charge density for electrostatic adsorption and allow adequate charge transfer at the electrode-solution interface. Based on the analysis of the effect of the type of chitosan derivative on the enzymatic response; we built an efficient biosensor based on G-Chit and GOx. The multilayered structure made of three bilayers of G-Chit and GOx showed the best performance; the biosensor exhibits a sensitivity of (12.4 ± 0.3) µA mM−1 cm−2 in a glucose concentration range from 180 µM to 1.75 mM. In addition, the analytical response of this biosensor was tested in milk samples with negligible effects of the matrix. The results confirm the suitability of these chitosan derivatives for the adsorption of enzymes and the construction of supramolecular structures. The favorable environment for the enzymatic activity results in bioelectrodes with fast responses for quantification of glucose even in a complex matrix like milk.

Sequence-Defined Heteromultivalent Precision Glycomacromolecules Bearing Sulfonated/Sulfated Nonglycosidic Moieties Preferentially Bind Galectin-3 and Delay Wound Healing of a Galectin-3 Positive Tumor Cell Line in an In Vitro Wound Scratch Assay.

Within this work, a new class of sequence-defined heteromultivalent glycomacromolecules bearing lactose residues and nonglycosidic motifs for probing glycoconjugate recognition in carbohydrate recognition domain (CRD) of galectin-3 is presented. Galectins, a family of β-galactoside-binding proteins, are known to play crucial roles in different signaling pathways involved in tumor biology. Thus, research has focused on the design and synthesis of galectin-targeting ligands for use as diagnostic markers or potential therapeutics. Heteromultivalent precision glycomacromolecules have the potential to serve as ligands for galectins. In this work, multivalency and the introduction of nonglycosidic motifs bearing either neutral, amine, or sulfonated/sulfated groups are used to better understand binding in the galectin-3 CRD. Enzyme-linked immunosorbent assays and surface plasmon resonance studies are performed, revealing a positive impact of the sulfonated/sulfated nonglycosidic motifs on galectin-3 binding but not on galectin-1 binding. Selected compounds are then tested with galectin-3 positive MCF 7 breast cancer cells using an in vitro would scratch assay. Preliminary results demonstrate a differential biological effect on MCF 7 cells with high galectin-3 expression in comparison to an HEK 293 control with low galectin-3 expression, indicating the potential for sulfonated/sulfated heteromultivalent glycomacromolecules to serve as preferential ligands for galectin-3 targeting.

A thermo-responsive supramolecular hydrogel that senses cholera toxin via color-changing response.

A Pyrene-based amphiphile with C4-alkanoyl spacer and lactose (PyLac) self-assembles in the aqueous media to form an injectable hydrogel. It shows preferential binding with Cholera Toxin (CT) via its terminal galactose residue, and hence can be employed for the selective detection of CT via color-changing response.

One-pot synthesis of sugar-decorated gold nanoparticles with reduced cytotoxicity and enhanced cellular uptake

The unique properties of gold nanoparticles (AuNPs) enable their use for instance in medical diagnostics and therapeutic applications. The straightforward applicability of AuNPs requires accordingly the development of facile synthesis approaches towards the manufacturing of ready-to-use biomedical devices. Additionally, the behavior of nano-sized gold colloids in contact with living systems needs further and deeper investigations since nanoparticle-cell interactions, cellular uptake and cytotoxicity, particularly with regard to surface chemistry effects, are still poorly understood. Taken it into account, we herein demonstrate a facile one-pot synthesis of polymer-stabilized gold nanoparticles (AuNPs) using branched polyethyleneimine (BPEI) with primary amine groups substituted by lactose residues. The polymer chains are simultaneously responsible for the Au3+ reduction and stabilization of the produced metallic colloids. The approach enables the manufacturing of highly stable ∼ 20 nm AuNPs with the surface decorated by sugar moieties. The use of sugar substituted polymer chains leads to gold nanocolloids with reduced cytotoxicity and enhanced cellular uptake compared to those stabilized by unmodified polymer chains. These findings can lead to significant advances towards the application of AuNPs-based therapies, diagnostics and imaging.

Sweet Vector for Gene Delivery: the Sugar Decoration of Polyplexes Reduces Cytotoxicity with a Balanced Effect on Gene Expression.

The use of sugar-functionalized polyplexes as a nonviral gene delivery vector with lower cytotoxicity than the well-known polymeric carrier branched polyethyleneimine (BPEI) is investigated. The substitution of primary amine groups in the BPEI chains with lactose residues leads to larger polyplexes, presumably due to the higher amount of polymer required to complete DNA condensation. Nevertheless, the sugar functionalization substantially reduces the cytotoxicity of the assemblies. The nanocomplexes are taken up by the cells to a greater extent, whereas the levels of gene expression are maintained compared to those obtained using BPEI, which is known for its excellent transfection efficiency. Accordingly, the preparation of lower-cytotoxicity polyplexes while maintaining gene expression, which is highly relevant to the field, is demonstrated.

Synthesis of a series of new glycoclusters and the evaluation of their anti-adhesion activities

According to our early researches, some glycoclusters having glucose, mannose, cellose and lactose residues showed good anti-adhesion activity of leukocytes to endothelial cells and exerted anti-inflammatory effects. Based on these results and combination principles of drugs, a series of new glycoclusters modifying with potentially anti-oxidant activity pharmacophores have been synthesized, and their anti-adhesion activities were assessed by static state cell-based adhesion assay. The results showed that some modified glycoclusters displayed better activities than their leading compound.

Technological aspects of lactose-hydrolyzed milk powder

Few reports describe the effect of lactose hydrolysis on the properties of milk powder during production and storage. Hence, the aim of this study was to evaluate the effects of five different levels of enzymatic lactose hydrolysis during the production and storage of milk powder. As the lactose hydrolysis rate increased, adhesion to the drying chamber also increased, due to higher levels of particle agglomeration. Additionally, more brown powder was obtained when the lactose hydrolysis rate was increased, which in turn negatively affected rehydration ability. Using Raman spectroscopy, crystallization of the lactose residues in various samples was assessed over 6weeks of accelerated aging at a room temperature environment with 75.5% of air moisture. Products with 25% or greater lactose hydrolysis showed no signs of crystallization, in contrast to the non-hydrolyzed sample.

Doxorubicin and paclitaxel co-bound lactosylated albumin nanoparticles having targetability to hepatocellular carcinoma

Anticancer drug targeting to liver asialoglycoprotein receptors (ASGPR) is viewed as a good approach for hepatocellular carcinoma (HCC) treatment. Lactose residue is a promising ASGPR ligand due to its high receptor affinity. Herein, we introduce doxorubicin and paclitaxel co-bound lactosylated albumin (Lac-BSA) nanoparticles (Dox/Pac Lac-BSA NPs) with good liver targetability. Lac-BSA was synthesized by conjugating lactobionic acid to naïve BSA then characterized by mass spectrometry. Dox/Pac Lac-BSA NPs were fabricated utilizing high-pressure homogenization and evaporation with Nab® (nanoparticle albumin bound) technology. Dox/Pac Lac-BSA NPs were spherical and well-dispersed, with a 148.7±13.8nm particle size and −54.1±0.7mV zeta potential at a 100% Lac-BSA feed ratio. Combined Dox and Pac synergistic cytotoxicity was confirmed in Hep G2 cells. Specifically, the inhibitory concentration (IC50; 0.21±0.02μg/ml) for Dox/Pac Lac-BSA NPs was 3.2 time lower than plain Dox/Pac BSA NPs (IC50; 0.68±0.04μg/ml). Also, Dox/Pac Lac-BSA NPs exhibited better internalizing in Hep G2 cells (61.8% vs. 14.4% for Dox) and spheroids compared to Dox/Pac BSA NPs. Finally, Dox/Pac Lac-BSA NPs displayed much greater localization into ICR mice livers compared to Dox/Pac BSA NPs. This was indicated by the presence of NP lactose residues revealed by a galactose inhibition study. Based on these results, we suggest that lactose-modified albumin-based nanoparticles fabricated with the Nab® technique can be a potential therapeutic vector for treating HCC via hepatocyte targeting.

Multivalent glycoconjugate as the vector of target delivery of bioactive compounds

New amphiphilic glycoconjugate with seven terminal lactose residues was synthesized. The properties of delivery systems from bioactive substance containing this conjugate were investigated.

Neoglycoenzyme-Gated Mesoporous Silica Nanoparticles: Toward the Design of Nanodevices for Pulsatile Programmed Sequential Delivery.

We report herein the design of a stimulus-programmed pulsatile delivery system for sequential cargo release based on the use of a lactose-modified esterase as a capping agent in phenylboronic acid functionalized mesoporous silica nanoparticles. The dual-release mechanism was based on the distinct stability of the cyclic boronic acid esters formed with lactose residues and the long naturally occurring glycosylation chains in the modified neoglycoenzyme. Cargo delivery in succession was achieved using glucose and ethyl butyrate as triggers.

Coiled coil type neoglycoproteins presenting three lactose residues

Scaffold design, synthesis and application are relevant for biomedical research. For example, multivalent interactions, such as those between cell surface glycoproteins and lectins can influence the potency and duration of signalling. The spacing between carbohydrates on their native protein scaffold could be important. Herein, the coiled coil design principle is used to generate synthetic coiled coil type glycoproteins, where three lactose residues are grafted to the coil via N-linkages to asparagine. Molecular modelling indicates that the distance between the galactose anomeric carbon atoms on the neoglycoproteins is ∼30Å. The inclusion of lactose was accommodated in both the final heptad towards the N-terminus, or more centrally in the penultimate heptad. In either case, neither the helicity nor the assembly to the trimeric form was unduly altered by the presence of the disaccharide.

Design and synthesis of an octavalent bolaamphiphile with terminal lactose residues

The synthesis of an octavalent bolaamphiphile with D-glucose moiety as branching unit has been designed and accomplished via 1,3-dipolar cycloaddition of bis(tetra-O-propargylglucoside) and 8 equiv of 2-azidoethyl β-D-lactoside.

"Click chemistry" in the synthesis of the first glycoconjugates of bacteriochlorin series

A regioselective synthesis of glycoconjugates based on bacteriochlorophyll a and lactose derivatives has been carried out. The conjugation was achieved via 1,3-dipolar cycloaddition of bacteriochlorins containing a terminal triple bond and a lactose azide derivative. The conjugates obtained in this way had one or two disaccharide fragments attached to pyrrol A, the exocyclic imide ring of the tetrapyrrolyc macrocycle, or to both positions. Exhaustive NMR analysis by 1D and 2D NMR experiments ( 1H-1H COSY, TOCSY, ROESY, 1H-13C HSQC, HMBC, and 1H-15N HMBC) allowed us to determine the structures and configurations of the glycoconjugates obtained. A bioassay of the glycoconjugates using the Hep2 cell line showed that the highest efficiency was observed for the glycosylated bacteriopurpurinimide containing a lactose residue at pyrrole ring A.

Sugar-Functionalized Water-Soluble Cyclotriveratrylene Derivatives: Preparation and Interaction with Fullerene

Cyclotriveratrylene (CTV) has attracted much attention because of its good chemical stability, small cavity, stable conformation, and facile modification. In this article, two water-soluble CTV derivatives (CTV-G and CTV-L) functionalized by glucose and lactose residues were synthesized, respectively. Unexpectedly, sugar-bearing CTVs exhibit a distinct photoluminescence, which might be ascribed to the enhanced planar conformation of cyclotriveratrylene ring derived from the spatial effect of bulky branch groups. The interaction between the water-soluble CTV derivatives and C(60) was investigated in organic solvent and aqueous solution, which was further characterized by fluorescence spectra, ultraviolet-visible spectra, and Raman spectra. CTV-G can associate with C(60) to form supramolecular complex with 1:1 molar ratio (K(a) = 1.38 × 10(5) M(-1), 298 K). As for CTV-L, a similar complex with a lower association constant (K(a) = 5.09 × 10(4) M(-1), 298 K) can also be formed.

Mannose-Functionalized “Pathogen-like” Polyanhydride Nanoparticles Target C-Type Lectin Receptors on Dendritic Cells

Targeting pathogen recognition receptors on dendritic cells (DCs) offers the advantage of triggering specific signaling pathways to induce a tailored and robust immune response. In this work, we describe a novel approach to targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide "pathogen-like" properties that ensure nanoparticles engage C-type lectin receptors on DCs. The surface of polyanhydride nanoparticles was functionalized by covalent linkage of dimannose and lactose residues using an amine-carboxylic acid coupling reaction. Coculture of functionalized nanoparticles with bone marrow-derived DCs significantly increased cell surface expression of MHC II, the T cell costimulatory molecules CD86 and CD40, the C-type lectin receptor CIRE and the mannose receptor CD206 over the nonfunctionalized nanoparticles. Both nonfunctionalized and functionalized nanoparticles were efficiently internalized by DCs, indicating that internalization of functionalized nanoparticles was necessary but not sufficient to activate DCs. Blocking the mannose and CIRE receptors prior to the addition of functionalized nanoparticles to the culture inhibited the increased surface expression of MHC II, CD40 and CD86. Together, these data indicate that engagement of CIRE and the mannose receptor is a key mechanism by which functionalized nanoparticles activate DCs. These studies provide valuable insights into the rational design of targeted nanovaccine platforms to induce robust immune responses and improve vaccine efficacy.

Surface hydrophilic modification with well-defined glycopolymer for protein imprinting matrix

Well-defined lactose-containing glycopolymer has been synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with (4-cyanopentanoic acid)-4- dithiobenozoate (CAD) as chain transfer agent. The glycopolymer was introduced onto the exterior surfaces of the bovine serum albumin (BSA) imprinted polymer beads by grafting copolymerization with methyl methacrylate and ethylene glycol dimethacrylate. After alcoholysis, the hydrophilic lactose residues of glycopolymer will stretched on the surface of the MIP beads and then the hydrophilicity of the surface will be enhanced. Rebinding test shows that the glycopolymer hydrophilic modified BSA imprinted polymer presents higher performance selectivity than that of unmodified one, which means that the hydrophobic–hydrophilic balance of the imprinted polymer surface is in favor of the improvement of specific recognition property of the material.

Preparation of glycopolymer hollow particles by sacrificial dissolution of colloidal templates

In this paper, a simple method for fabrication of glycopolymer hollow particles was demonstrated. The P(St-CPEM)- g-PVBSAELA core–shell particles were prepared by emulsifier-free emulsion polymerization of styrene (St) with 2-chloropropionyloxyethyl methacrylate (CPEM) using potassium persulfate as an initiator, followed by surface-initiated activator generated electron transfer–atom transfer radical polymerization (AGET–ATRP) of a styrene derivative bearing a lactose residue, i.e., N-2-4-(vinylbenzenesulfonamido)ethyl lactobionamide (VBSAELA). Dynamic light scattering measurement showed that the P(St-CPEM)- g-PVBSAELA core–shell particles possess graft layers of ca. 160 nm in thickness on the P(St-CPEM) core of 455 nm in diameter. By taking advantages of large difference in solubility between the PSt-based core and the PVBSAELA shell, the submicron-sized PVBSAELA hollow particles were obtained through a selective extraction of the core part from the P(St-CPEM)- g-PVBSAELA particles in tetrahydrofuran. The hydrodynamic diameter of the resulting hollow particles decreased by 15% compared to that of the corresponding core–shell particles. Finally, the micron-sized, raspberry-shaped, PVBSAELA hollow particles were successfully fabricated by a sacrificial dissolution of the PSt-based components from the PVBSAELA-grafted, core/shell heterocoagulates.