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Lactose Assay Kit - Sequential/High Sensitivity

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00:04  Introduction
01:00  Principle
03:19   Reagent Preparation
04:23  Sample Preparation
05:37  Glucose Oxidase / Catalase Pre-Treatment
07:45  Procedure
11:08   Calculations

Lactose Assay Kit K-LOLAC Scheme
Product code: K-LOLAC

65 assays per kit

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Available for shipping

Content: 65 assays per kit
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Lactose
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 1 to 50 µg of lactose (or 0.50 to 25 µg of D-glucose)
Limit of Detection: 1.62 mg/L
Reaction Time (min): ~ 10 min

The K-LOLAC test kit offers a rapid, novel, sequential measurement of free-glucose and lactose in conventional, low-lactose and lactose-free dairy products. This sequential assay format reduces the manual input required by an analyst when compared to traditional lactose assay formats and therefore improves both accuracy and efficiency. When used in combination with the Megazyme Creep Calculator provided, the β-galactosidase employed in this kit allows for the selective measurement of lactose in the presence of galacto-oligosaccharides (GOS) which are commonly found in lactose-free dairy products. This constitutes a significant improvement over existing commercially available lactose assay kits which typically overestimate lactose content in lactose-free samples due to the unselective hydrolysis of GOS by β-galactosidase. Lastly, the sensitivity of the K-LOLAC assay kit has been doubled through the use of a cascade biochemical pathway, helping to significantly reduce the LOD and LOQ for the assay. British Patent Application No. 1710170.0.

We offer more monosaccharide and oligosaccharide assay kit products.

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Scheme-K-LOLAC LOLAC Megazyme

  • World’s first sequential assay for lactose, i.e. improves accuracy and efficiency 
  • Contains a specific β-galactosidase for the selective measurement of lactose in dairy products 
  • Efficient pre-treatment step allows for accurate measurement of lactose in “low-lactose” and “lactose-free” dairy products 
  • Lower limit of detection (LOD) than any other commercially available enzymatic lactose detection method. LOD at 1.62 mg/L 
  • Very competitive price (cost per test)
Certificate of Analysis
Safety Data Sheet
Assay Protocol Data Calculator Validation Report
Megazyme publication

Determination of Lactose Concentration in Low-Lactose and Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients: Single Laboratory Validation of an Enzymatic Method, First Action Method 2020.08.

Ivory, R., Delaney, E., Mangan, D. & McCleary, B. V. (2021).  Journal of AOAC International, qsab032.

Background: The AOAC Stakeholder Panel on Strategic Food Analytical Methods issued a call for methods for the measurement of lactose in low-lactose and lactose-free products under SMPR 2018.009 (1). Megazyme’s Lactose Assay Kit (K-LOLAC) was developed specifically to address the need for accurate enzymatic testing in lactose-free samples. Objective: The Lactose Assay (K-LOLAC) was validated for measurement of lactose in low-lactose and lactose-free milk, milk products, and products containing dairy ingredients. A single-laboratory validation of the method is reported. Method: The Lactose Assay (K-LOLAC) is an accurate and sensitive enzymatic method for the rapid measurement of lactose in low-lactose or lactose-free products. Validation analysis was performed on a sample set of 36 commercial food and beverage products and a set of 10 certified reference materials. Parameters examined during the validation included working range and linear range, selectivity, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness and stability. Results: For all samples tested within the lower range (10-100 mg/100 g or mL), recoveries varied from 93.21-14.10%. Recoveries obtained for samples in the higher range (>100 mg/100 g or mL) varied from 94.44-108.28%. All materials had repeatability relative standard deviations (RSDr and RSDir) of < 9%. Conclusions:  The commercial Lactose Assay Kit (K-LOLAC) as developed by Megazyme meets the requirements set out under SMPR 2018.009. Highlights: The Lactose Assay (K-LOLAC) is a robust, quick and easy method for analysis of lactose in foodstuffs and beverages.

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Megazyme publication

A novel enzymatic method for the measurement of lactose in lactose‐free products.

Mangan, D., McCleary, B. V., Culleton, H., Cornaggia, C., Ivory, R., McKie, V. A., Delaney, E. & Kargelis, T. (2018). Journal of the Science of Food and Agriculture, 99, 947-956.

Background: In recent years there has been a surge in the number of commercially available lactose‐free variants of a wide variety of products. This presents an analytical challenge for the measurement of the residual lactose content in the presence of high levels of mono‐, di‐, and oligosaccharides. Results: In the current work, we describe the development of a novel enzymatic low‐lactose determination method termed LOLAC (low lactose), which is based on an optimized glucose removal pre‐treatment step followed by a sequential enzymatic assay that measures residual glucose and lactose in a single cuvette. Sensitivity was improved over existing enzymatic lactose assays through the extension of the typical glucose detection biochemical pathway to amplify the signal response. Selectivity for lactose in the presence of structurally similar oligosaccharides was provided by using a β-galactosidase with much improved selectivity over the analytical industry standards from Aspergillus oryzae and Escherichia coli (EcLacZ), coupled with a ‘creep’ calculation adjustment to account for any overestimation. The resulting enzymatic method was fully characterized in terms of its linear range (2.3-113 mg per 100 g), limit of detection (LOD) (0.13 mg per 100 g), limit of quantification (LOQ) (0.44 mg per 100 g) and reproducibility (≤ 3.2% coefficient of variation (CV)). A range of commercially available lactose‐free samples were analyzed with spiking experiments and excellent recoveries were obtained. Lactose quantitation in lactose‐free infant formula, a particularly challenging matrix, was carried out using the LOLAC method and the results compared favorably with those obtained from a United Kingdom Accreditation Service (UKAS) accredited laboratory employing quantitative high performance anion exchange chromatography - pulsed amperometric detection (HPAEC‐PAD) analysis. Conclusion: The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose‐free samples.

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Fermentation Ability of Bovine Colostrum by Different Probiotic Strains.

Hyrslova, I., Krausova, G., Michlova, T., Kana, A. & Curda, L. (2020). Fermentation, 6(3), 93.

Over the past decade, the use of bovine colostrum and its bioactive components as the basis of functional food and dietary supplements for humans has substantially increased. However, for developing new products enriched with probiotics and bovine colostrum, the influence of colostrum composition on the growth promotion of bacteria still needs to be tested. Therefore, we decided to study the influence of bovine colostrum chemical and mineral composition as well as the content of bioactive compounds (immunoglobulins, lactoferrin, lactoperoxidase) on the growth of ten selected strains from genera Lactobacillus, Lacticaseibacillus, Bifidobacterium, and Enterococcus. After 24 h of fermentation, the growth was assessed based on lactic and acetic acids production evaluated using isotachophoresis, bacterial counts determined by the agar plate method, and change of pH. The production of acids and bacterial counts were significantly (P<0.05) different between selected genera. The change of bacterial counts was correlated with pH, but the correlation between growth and bovine colostrum composition was not proven. The highest growth and production of lactic acid was observed after the fermentation of bovine colostrum by the strains Enterococcus faecium CCDM 922A and CCDM 945.

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Exploring the potential of microalgae in the recycling of dairy wastes.

Gramegna, G., Scortica, A., Scafati, V., Ferella, F., Gurrieri, L., Giovannoni, M., Bassi, R., Sparla, F., Mattei, B. & Benedetti, M. (2020). Bioresource Technology Reports, 12, 100604.

Culturing microalgae using dairy-wastes offers the opportunity of producing valuable biomass for different industrial applications. The capability of four Chlorella species and a recombinant Chlamydomonas reinhardtii strain to mixotrophically grow in wastewaters from an Italian dairy factory was investigated. A robust algal growth could be efficiently sustained in these wastes, despite the abundance of D-Lactose (~4% w/v), that could not be metabolized by any microalgal species. Non axenic cocultivation of microalgae together with microbial communities from the dairy wastes resulted in a marked decrease of their pollution load, thus reducing the necessity of expensive treatments before their discharge in the municipal sewage system. Microalgae cultivated using dairy-wastes were characterized by a lipid content ranging from 12% to 21% (w/w), with Auxenochlorella protothecoides reaching the highest lipid productivity (~0.16 g/L/d) whereas the transplastomic C. reinhardtii strain expressing a thermostable β-glucosidase reached a recombinant enzyme productivity of 0.18 mg/L/d.

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GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway.

Liu, Y., Wang, X., Zhen, Z., Yu, Y., Qiu, Y. & Xiang, W. (2019). Cellular & molecular biology letters, 24(1), 1-12.

Methods: The expressions of GRP78 in BMECs stimulated with methionine, leucine, estrogen and prolactin were determined using western blotting and immunofluorescence assays. To explore the function of GRP78 in BMECs, the protein was overexpressed or knocked down, respectively using an overexpression vector or an siRNA mixture transfected into cells cultured in vitro. Flow cytometry was used to analyze cell proliferation and cell activity. The contents of lactose and triglyceride (TG) secreted from the treated BMECs were measured using lactose and TG assay kits, respectively. Western blotting analysis was used to measure the β-casein content and the protein levels of the signaling molecules known to be involved in milk biosynthesis and cell proliferation. Results: GRP78overexpression significantly stimulated milk protein and milk fat synthesis, enhanced cell proliferation, positively regulated the phosphorylation of mammalian target of rapamycin (mTOR), and increased the amount of protein of cyclinD1andsterol regulatory element-binding protein 1c (SREBP-1c). GRP78 knockdown after siRNA transfection had the opposite effects. We further found that GRP78 was located in the cytoplasm of BMECs, and that stimulating methionine, leucine, estrogen and prolactin expression led to a significant increase in the protein expression of GRP78 in BMECs. Conclusions: These data reveal that GRP78 is an important regulator of milk biosynthesis and the proliferation of BMECs through the mTOR signaling pathway.

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Comparative study of two lactases by K-Lolac enzymatic method in skimmed milk. 

Benbouziane, B., Bentahar, M. C., Takarly, H. & Benakriche, B. M. (2019). South Asian Journal of Experimental Biology, 9(1), 1-6.

Lactose absorption at the level of the small intestine depends on its hydrolysis by β-galactosidase. The activity of this enzyme, which gets to the peak at the beginning and halflife, decreases progressively after weaning. This activity loss (hypolactasie) is a physiological phenomenon observed in 70 to 75% of the world's population. Hypolactasy is transmitted according to an autosomal recessive mode to an incomplete penetrance and is linked to polymorphosis located in the promoter region of the gene coding the lactase. A solution is proposed regarding ingestion of dairy dislactosed products or products with unduly low lactose rates. In this study, two different enzymes were used, a β-galactosidase of Bifidobacterium [β-gal Bb] source and another β-galactosidase of Kluyveromyces lactis [β-gal Kl] source with different concentrations on lactose degradation in a preparation based on skimmed milk at 4°C during 18h with a 39 g/l lactose rate. Determining hydrolysis rate in lactose was achieved with an enzymatic method using a Megazyme K-lolac kit. The results demonstrated that β-gal Kl (Maxilat) in a 100 µl/L dose gives an optimal performance as compared to β-gal Bb (Nola fit) in residual lactose concentrations 1.85 g/L and 2.78 g/L respectively. However, in a dose that was superior to 1500 and 2000, the β-gal Bb was significantly more performing than β-gal Kl. To sum up, the enzymatic method used to define the residual lactose rate, the kit KLolac, gives very reliable results with a low threshold (LOD 1.62 mg/L).

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Lactose-free Yogurts do not Show any Benefits for Lactose-Intolerant Subjects, Compared with Lactose-Containing Yogurts. 

Ghio, B., Márquez, D., Peche, B., Peña, F. & Saavedra, F. (2019). Journal of Food & Nutritional Disorders, 8(3), 2.

Nowadays, there is constant increase of commercial lactose-free yogurts for lactose-intolerant individuals. However, the real interest of these yogurts is unclear considering that several clinical trials have shown that the living bacteria present in the yogurt improved lactose tolerance in hypolactasic subjects, due to their β-galactosidase activity that remains functional in the small intestine of these individuals. The aim of this study was to determine whether the intake of lactose-free yogurt (LFY) is beneficial for hypolactasic lactose-intolerant subjects compared with that of traditional, lactose-containing yogurt (LCY). Twenty-two subjects with auto-reported digestive symptoms after milk consumption carried out a hydrogen breath test (HBT) with 25g lactose to confirm their hypolactasic status. Fourteen subjects (63.6%) who exhibit a positive HBT accompanied by digestive symptoms were finally incorporated to the study. In two independent days, they have to ingest, in a double-blind and randomized form, 250g of LFY or LCY. These products brought 0.5g and 19.8g of lactose, respectively and both exhibited total counts of lactic acid bacteria higher than 107 CFU/g. Changes in breath H2 excretion and digestive symptoms were registered during 180 min. When the volunteers carried out the HBT with LFY and LCY, no differences were detected in H2 excretion or the intensity of digestive symptoms (individual or total). Accordingly, our results suggest than the intake of LFY that are more expensive than LCY, does not bring any supplementary detectable benefits for the lactose intolerant subjects.

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Influence of particle size on the physicochemical properties and stickiness of dairy powders.

O'Donoghue, L. T., Haque, M. K., Kennedy, D., Laffir, F. R., Hogan, S. A., O'Mahony, J. A. & Murphy, E. G. (2019). International Dairy Journal, 98, 54-63.

The compositional and physicochemical properties of different whey permeate (WPP), demineralised whey (DWP) and skim milk powder (SMP) size fractions were investigated. Bulk composition of WPP and DWP was significantly (P < 0.05) influenced by powder particle size; smaller particles had higher protein and lower lactose contents. Microscopic observations showed that WPP and DWP contained both larger lactose crystals and smaller amorphous particles. Bulk composition of SMP did not vary with particle size. Surface composition of the smallest SMP fraction (<75 µm) showed significantly lower protein (−9%) and higher fat (+5%) coverage compared with non-fractionated powders. For all powders, smaller particles were more susceptible to sticking. Hygroscopicity of SMP was not affected by particle size; hygroscopicity of semi-crystalline powders was inversely related to particle size. This study provides insights into differences between size fractions of dairy powders, which can potentially impact the sticking/caking behaviour of fine particles during processing.

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Accurate analysis of residual lactose in low-lactose milk: Comparing a variety of analytical techniques.

Churakova, E., Peri, K., Vis, J. S., Smith, D. W., Beam, J. M., Vijverberg, M. P., Stor, M. C. & Winter, R. T. (2019). International Dairy Journal, 96, 126-131.

To receive the designation “lactose-free”, milk should contain <0.01% (w/w) lactose. As the analysis of such low levels of lactose is often hampered by other saccharides present or formed during milk processing, methods are required that are highly sensitive, accurate and precise. Currently, there is no international standard analysis method for the determination of lactose in low- or lactose-free milk, despite such a need from the dairy industry. We validated the analysis of residual lactose in lactase-treated UHT milk using HPAEC-PAD on a CarboPac PA100 column and compared it with a variety of commonly used analytical techniques for measuring lactose, including HPLC-RI, NMR, enzymatic kits, cryoscopy, and lactose biosensors. The results show that only one analytical technique, namely the Biomilk300, an amperometric biosensor, has performance comparable with analysis by HPAEC-PAD, which remains one of the most accurate, precise and sensitive methods to assess low levels of lactose in milk.

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Safety Information
Symbol : GHS05, GHS07, GHS08
Signal Word : Danger
Hazard Statements : H302, H302+H332, H314, H334, H360
Precautionary Statements : P201, P202, P260, P261, P264, P270, P271, P280, P284, P301+P312, P301+P330+P331, P304+P340, P342+P311, P501
Safety Data Sheet
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