Determination Of Lactose In Milk Research Articles

A simple and rapid ReMALDI MS assay for the determination of lactose in milk

Lactose is an important nutrient but its content in milk products poses concerns for people with lactose intolerance. Thus, a simple, rapid, and high-throughput assay for lactose determination is needed to maintain specified lactose levels during production and in final milk products. Here, we report a reactive matrix-assisted laser desorption/ionization mass spectrometry (ReMALDI MS) method for the determination of lactose in milk products. The selective reactivity and the excellent laser absorption properties of 4-hydrazinoquinazoline (4-HQ) matrix increase the sensitivity of traditional MALDI MS analysis of lactose by 100 folds. After optimization of the method, lactose was detected distinctly in milk without pretreatment. Using xylobiose as an internal standard, the assay accurately quantified lactose in the concentration range of 1.00−700μg/mL (R2 = 0.994). Due to the high sensitivity and specificity, the lactose content in different types of milk samples was determined successfully, as well as the residual lactose in low-lactose and lactose-free milk.

Optical bi-enzyme-titania biosensor system: A new way to detect lactose

Lactose concentration is a key parameter for assessing the quality of raw and processed milk and identifying abnormal milk. Current methods for lactose determination are laborious, time-consuming and oftentimes require skilled lab workers, expensive equipment, and special reagents. In this study, an optical biosensor system based on nanosized titanium dioxide (TiO2) is presented for routine measurement of lactose in milk without sample preparation. TiO2 was obtained via sol-gel synthesis and characterized using dynamic light scattering, microelectrophoresis, X-ray diffraction, transmission electron microscopy, and nitrogen adsorption-desorption analysis. The system, whose colorimetric response was based on the chromogen-free TiO2-based detection of hydrogen peroxide (H2O2), used β-galactosidase and/or glucose oxidase as the biorecognition elements. The selectivity and sensitivity of the biosensors for lactose and glucose was investigated by diffuse reflectance spectroscopy using standard aqueous lactose and glucose solutions and whole milk samples. The results showed that the proposed biosensors had high optical signal reproducibility (RSD = 5–6%), high sensitivity (LOD = 0.005 wt%), and high selectivity. The linear dynamic ranges of lactose and glucose detection were from 0.005 wt% to 0.100 wt%, and the optimal response time of the biosensor was 25 min. The listed features and advantages of label-free detection make the biosensor attractive for simple, inexpensive, sensitive, and selective determination of lactose in milk.

A novel approach for lactose determination in cow's milk exploiting smartphone-based digital-image photometry.

Lactose, the main carbohydrate in cow's milk, may cause health problems for consumers with intolerance. Lactose determination in milk is hindered by the matrix complexity and lack of chromophore groups. Chromatography, volumetric, and spectrophotometric approaches involving chemical derivatization are time-consuming and require laborious sample preparation, which is incompatible with the high analytical demand. In this context, a novel approach is presented for lactose determination in milk exploiting smartphone-based digital-image photometry. It was based on a modification of the Benedict's method, involving formation of the violet Cu(I)/2,2'-biquinoline-4,4'-dicarboxylate (BCA) complex instead of the copper(I) oxide precipitate, aiming at improvement of sensitivity and precision. Sample pretreatment and analyte derivatization were performed in Eppendorf tubes with minimal reagent amounts and a smartphone camera was used for image acquisition under controlled conditions. Measurements were based on the RGB color system, taking channel G as the analytical response because of the complementarity with the color of the complex. Under the optimized conditions, the proposed procedure yielded a linear response up to 20 mg L-1 (r = 0.999), with a limit of detection of 1.5 mg L-1, which is compatible with determination of lactose in milk and dairy products categorized with low content of the sugar. The procedure takes less than 10 min, with a coefficient of variation of 3.0% (n = 12) and consumes as low as 160 μg Cu and 430 μg BCA per determination, thus being a more practical, fast, cost-effective, and environmental friendly analytical method.

Determination of lactose in milk by high resolution ultrasonic spectroscopy

The application of high-resolution ultrasonic spectroscopy for determination of the amount of lactose in milk and milk-based drinks is described. The methodology is based on high precision real time ultrasonic monitoring of hydrolysis of lactose in milk by the enzyme β-galactosidase, allowing for quantitation of lactose present at reaction completion. The method can also be used to control in real-time the hydrolysis of lactose for the precise preparation of milks with preferred lactose concentrations. The elimination of sample preparation steps and the minimal need for user interaction represent significant simplification and time savings over existing methods. Comparison of the ultrasonic data with results from HPLC-RI showed excellent agreement in the determination of lactose in normal, skimmed and chocolate milks.

Fe-N-C nanozyme mediated bioactive paper-3D printing integration technology enables portable detection of lactose in milk

Portable 3D printing biosensing devices have great potential as rapid detection and analytical platforms due to their unique characteristics such as high precision and low cost. In this study, a method for rapid determination of lactose in milk was developed based on bioactive paper-3D printing integration technology. The bioactive paper was prepared by combining three enzymes cascade system composed of β -galactosidase, glucose oxidase and Fe-N-C nanozyme (with excellent peroxidase-like activity and superior affinity). In the presence of lactose, the test paper changed from colorless to blue under the condition of TMB, which can be identified by naked eye. With the help of portable chromometer and smart phone, the RGB color index was analyzed and quantified. The entire detection process can be integrated into the 3D printing device that we have designed and completed in 25 min, with the detection limits of qualitative and quantitative were 0.04 g 100 g−1 and 5.61 μg 100 g−1, which could simultaneously satisfy the detection requirements of rapid, sensitive and portable operation. The bioactive paper and 3D printing device are long shelf life (six months) and cost-effective, which means this integration technology has a good application prospect in areas lacking experimental conditions.

Technological and methodological aspects of the production of low- and lactose-free dairy products

The features of technologies for low- and lactose-free dairy products, which provide for special operations to hydrolyze lactose or remove it using ultra- or nanofiltration followed by hydrolysis of the residual amount, are considered. Dairy products manufactured using these technologies in different countries as well as enterprises leading in this field of production are presented. The analysis of the methods used to determine the quantitative content of residual lactose in low- and lactose-free dairy products is carried out: enzymatic, HPLC, HPAEC-PAD, amperometric biosensors, Raman spectroscopy. Due to the dairy industry’s need for analytical methods for the determination of lactose in milk and dairy products with low- or lactose-free content, the AOAC Stakeholder Group on Strategic Food Analysis Methods approved Standard Performance Requirements for Biosensor Methods (SMPR®) 2018.009. These requirements were introduced for the quantitative determination of lactose in milk as well as in dairy and milk-containing products with a low or no lactose content. The biosensor method is recommended for use as the official first step of AOAC method. Additionally, it is advisable to use high performance liquid chromatography (HPLC) with mass spectrometric detection, as well as high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as an international standard method of analysis for the determination of lactose in milk with low- or lactose-free content.

Development of a new two‐enzyme biosensor based on poly(pyrrole‐co‐3,4‐ethylenedioxythiophene) for lactose determination in milk

A new lactose biosensor was developed by preparing a suitable copolymer of polypyrrole and poly(3,4‐ethylenedioxythiophene) synthesized using the electropolymerization method. Pyrrole and 3,4‐ethylenedioxythiophene monomers were deposited in the presence of sodium dodecylbenzene sulphonic acid on a platinum disc electrode, which was used as the working electrode. The sensor is based on the serial reactions of β‐galactosidase and galactose oxidase immobilized on a copolymer‐modified platinum disc electrode. Successful synthesis of the enzyme‐immobilized copolymer was confirmed by FT‐IR spectrometry, SEM, and electrochemical analysis. The response of the enzyme electrode to lactose was determined by cyclic voltammetry at + 0.40 V. The response time of the biosensor was found to be from 8 to 10 s, and the upper limit of the linear working portion was found to be at a lactose concentration of 2.30 mM with a detection limit of 1.4 × 10−5 M. The apparent Michaelis–Menten constant was found to be 0.65 mM of lactose. The effects of interferents were also investigated. Lactose concentrations determined by the biosensor were in good agreement with those measured by the reference methods. Our results show that the developed biosensor has a significant potential to the determination of lactose concentration in milk. POLYM. ENG. SCI., 58:839–848, 2018. © 2017 Society of Plastics Engineers

Determination of lactose in milk products: a comparison of three-enzyme amperometric biosensor and gas chromatography/tandem mass spectrometry

Two completely different analytical methods for lactose determination are presented. Three-enzyme biosensor involving β-galactosidase, glucose oxidase, and horseradish peroxidase was constructed with successive steps of optimization. Primary experiments were carried out with horseradish peroxidase biosensor in solution of hydrogen peroxide. Addition of poly(ethylene glycol) diglycidyl ether was examined and the best constant potential for chronoamperometric experiments was selected at −0.05 V using cyclic voltammetry. Ferrocene was chosen as the best from three tested mediators and optimum concentration of ferrocene was found out at 2 g dm−3. Two electrode materials were compared. Spectroscopic graphite electrode proved better electrochemical response for its twice larger electro-active surface than screen-printed carbon electrode. Phosphate buffer solution of pH 7.5, at which the three-enzyme system provided the highest current signal, was used for lactose determination in milk, milk powder, curd cheese, and yogurt. Gas chromatography with tandem mass spectrometric detection was used for determination of lactose, glucose, and galactose in the real samples with internal standard salicine using single ion monitoring mode. Determined content of lactose in the milk products was compared with declared values. Gas chromatography with tandem mass spectrometric detection offers higher sensitivity and orthogonal selectivity in comparison to enzyme biosensor.

Determination of Lactose in Milk by Raman Spectroscopy

A Raman spectroscopy-based approach was developed for the rapid determination of lactose in milk using crystal violet as an internal standard. The crystal violet peak at 1173 reciprocal centimeters did not interfere with the lactose peak at 1085 reciprocal centimeters; therefore, crystal violet can be used to quantify lactose in milk. This developed method allowed the determination of lactose from 0.028–0.1 mole per liter, with a limit of detection of 0.019 mole per liter. The results revealed that Raman spectroscopy is a simple and rapid method for the determination of lactose in milk.

Co-immobilized glucose oxidase and β-galactosidase on bovine serum albumin coated allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer as a biosensor for lactose determination in milk

Bovine serum albumin (BSA) was adsorbed on allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) at pH 8.0 (16% w/w). The amino, thiol and carboxylic acid functional groups available on protein coated surface were utilized for covalent immobilization of glucose oxidase and β-galactosidase, both independently, and in a step-wise manner on the same matrix, with no more than 10% loss of enzyme activity during immobilization. Glutaraldehyde cross-linking after immobilization provided stable enzyme preparations. The pH-optima of the immobilized enzymes were similar to those for free enzyme but their thermal stability was vastly improved. The co-immobilized enzyme support was used as a biosensor for determination of lactose in milk with excellent reproducibility and reusability.

Artificial Enzyme Mimics for Catalysis and Double Natural Enzyme Co-immobilization

This work presents a new chemiluminescent (CL) probe array assay. The new type CL probe array is based on enzyme mimics of Co3O4-SiO2 mesoporous nanocomposite material, which not only have an excellent catalytic effect on the luminol-H2O2 CL reaction in an alkaline medium but also can be used for the immobilization of enzymes. The linear range of the lactose concentration is 3.0 × 10(-7) to 1.0 × 10(-5) g mL(-1) and the detection limit is 6.9 × 10(-8) g mL(-1). β-Galactosidase and glucose oxidase were selected as a model for enzyme assays to demonstrate the applicability of Co3O4-SiO2 mesoporous nanocomposite material in multienzyme immobilization. The novel bifunctional CL probe array has been successfully applied to the determination of lactose in milk.

Determination of lactose by a novel third generation biosensor based on a cellobiose dehydrogenase and aryl diazonium modified single wall carbon nanotubes electrode

In this paper a new third-generation amperometric biosensor for lactose determination is described. The biosensor is based on the highly efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and single walled carbon nanotubes (SWCNT). The SWNCTs were surface modified with aryl diazonium salts of p-phenylenediamine (NH2-PD) and deposited on top of a glassy carbon (GC) electrode. The PsCDH NH2-PD/SWCNT-GC biosensor showed very efficient DET and exhibited an extraordinary high current density of 500 mu M cm(-2) in a 5 mM lactose solution at pH 3.5. The biosensor has a detection limit for lactose of 0.5 mu M, a large linear range from 1 to 150 mu M lactose and a high sensitivity (476.8 nA mu M-1 cm(-2)). It shows also a fast response time (4s), good reproducibility (RSD =1.75%) and good stability (half-life 12 days). In addition, it is easy, simple to manufacture, and cheap because a low amount of enzyme is required and highly selective, as no significant interference was observed. For these reasons, it can represent a valid alternative to HPLC measurements for lactose determination in milk and dairy products. (c) 2012 Elsevier B.V. All rights reserved. (Less)

A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase

A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05mM and 30mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also “lactose free” milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.

An Integrated Amperometric Biosensor for the Determination of Lactose in Milk and Dairy Products

An integrated amperometric biosensor for the determination of lactose is reported. The bioelectrode design is based on the use of a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode on which the enzymes beta-galactosidase (beta-Gal), glucose oxidase (GOD), peroxidase (HRP) and the mediator tetrathiafulvalene (TTF) are coimmobilized by a dialysis membrane. beta-Gal catalyzes the hydrolysis of lactose, and the produced glucose is catalytically oxidized to gluconic acid and H(2)O(2), which is reduced in the presence of HRP. This enzyme reaction is mediated by TTF, and the reduction of TTF(+) at 0.00 V (vs Ag/AgCl) gives rise to an amperometric signal proportional to the lactose concentration. The biosensor exhibits a good repeatability of the measurement carried out with the same biosensor, a good reproducibility of the responses obtained with different biosensors and a useful lifetime of 28 days. A linear calibration plot was obtained for lactose over the 1.5 x 10(-6) to 1.2 x 10(-4) M concentration range, with a limit of detection of 4.6 x 10(-7) M. The effect of potential interferents (sucrose, lactulose, fructose, arabinose, maltose, galactose, glucose and uric and ascorbic acids) on the biosensor response was evaluated. Furthermore, the bioelectrode exhibits a suitable performance in flow-injection systems in connection with amperometric detection. The developed biosensor was applied to the determination of lactose in milk and other foodstuffs (chocolate, butter, margarine, yogurt, cheese and mayonnaise), and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

Two-enzyme lactose biosensor based on β-galactosidase and glucose oxidase deposited by AC-electrophoresis: Characteristics and performance for lactose determination in milk

In this paper, we have investigated the deposition of two enzymes β-galactosidase (β-Gal) and glucose oxidase (GOx) using alternating current electrophoretic deposition (AC-EPD) to manufacture a lactose sensor. Using the optimal deposition parameters and optimal testing conditions, the sensor has a sensitivity up to 111 nA/mM mm 2. This sensitivity is high considering the low activity of the enzymes employed in this study (9 units/mg for β-Gal and 5.6 units/mg for GOx). The sensor has a large linear range up to 14 mM lactose, fast response time (∼8 s) and reasonable stability without employing any stabilizers or outer polymer membrane. In addition, it is easy and simple to manufacture, highly reproducible and cheap because low activity enzymes are used. The sensor was tested to determine lactose in milk samples and is demonstrated to be accurate.

Application of a novel co-enzyme reactor in chemiluminescence flow-through biosensor for determination of lactose

A novel enzyme reactor with co-immobilization of β-galactosidase and glucose oxidase in calcium alginate fiber (CAF) and amine modified nanosized mesoporous silica (AMNMS) was prepared which incorporate the adsorption and catalysis of AMNMS with the cage effect of the polymer to increase catalytic activity and stability of immobilized enzyme. The enzyme reactor was applied to prepare a chemiluminescence (CL) flow-through biosensor for determination of lactose combined with a novel luminol-diperiodatonickelate (DPN) CL system we reported. It shows that the CL flow-through biosensor possesses long lifetime, high stability, high catalytic activity and sensitivity. The relative CL intensity was linear with the lactose concentration in the range of 8 × 10 −8–4 × 10 −6 g mL −1 with the detection limit of 2.7 × 10 −8 g mL −1 (3 σ). It has been successfully applied to the determination of lactose in milk.

The Applications of a Graphite Paste Electrode Containing p-Benzoquinone Combined with Oligosaccharide Dehydrogenase to the Determination of Lactose in Milk and to the Measurement of the Activity of Amylase in Blood Serum

The Applications of a Graphite Paste Electrode Containing p-Benzoquinone Combined with Oligosaccharide Dehydrogenase to the Determination of Lactose in Milk and to the Measurement of the Activity of Amylase in Blood Serum

Conductometric biosensor based on glucose oxidase and beta-galactosidase for specific lactose determination in milk

This paper investigates the development of a biosensor associating two distinct enzymatic activities, that of the beta-galactosidase and that of the glucose oxidase, in order to apply it for the quantitative detection of lactose in milk. To eliminate interferences with glucose, a differential mode of measurement was used. Results show a linear calibration curve for lactose concentration between 60 and 800 μM (0.03 to 0.3 g/L). Tests with real commercial milk samples were carried out to validate the conductometric biosensor.

A Novel Correlation for Rapid Lactose Determination in Milk by a Cryoscopic Technique

Residual lactose in special milk was systematically determined for people with lactose intolerance by means of a rapid on-line measurement of the cryoscopic point. A proposed cryoscopic procedure was compared to 2 conventional yet highly laborious methods: the enzymatic procedure with spectrophotometric control and the polarimetric method. Several experiments with different mixtures of both semi-skimmed and low-lactose milk were performed. A lineal relationship was found between lactose concentration and freezing point, the analytical equation for which shows a close relationship regarding the 3 methods used. The advantages of the cryoscopic procedure include speed in obtaining results and operational simplicity at a low cost, better monitoring of enzymatic hydrolysis kinetics, and greater control over the production process for delactosed milk. The equation obtained also enables prediction of the lactose percentage in commercial milk by a simple measurement of freezing point.

Multi-enzyme biosensors with amperometric detection for determination of lactose in milk and dairy products

In home-made sensors coimmobilizing enzymes in thin-layer plexi-cells on natural protein membranes, three enzyme cells: β-galactosidase and galactose oxidase (A), β-galactosidase and glucose oxidase (B) and β-galactosidase, galactose oxidase and glucose oxidase (C) were built into a flow-injection-analyzer system. The lactose was decomposed and oxidized by the immobilized enzymes and the hydrogen peroxide generated during the enzymatic reactions was determined by amperometric detection. The parameters for biochemical and electrochemical reactions (concentration of buffer, temperature, flow rate) were optimized in each enzyme cell. The pH optima of the lactose measurement was determined in the three enzyme cells mentioned above. The pH optimum of the cells A, B and C were 6.4, 4.5 and 4.8, respectively. The measuring ranges were 1–5 mM, 2–10 mM and 1–5 mM, while the detection limits were 0.5, 1.0 and 0.5 mM, respectively. More than 600, 1000 and 800 samples could be measured with these cells, respectively. Commercial milk and instant dessert powder products were analysed also. Our results showed that the cells B and C were more suitable for the determination of the lactose content of milk. For samples of dairy products containing added glucose, starch and other carbohydrates, enzyme cell A could be used for the efficient determination of lactose in one step.