Advanced Bio-Analysis Test Kits for the Food, Feed, Fermentation, Wine, Brewing & Dairy Industries Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, Ireland. T + 353 1 286 1220 F + 353 1 286 1264 E [email protected]www.megazyme.com Food, Feed, Fermentation, Wine, Brewing & Dairy Analysis Setting New Standards in Test Technology
View Megazyme's current catalogue for 2013 which lists our advanced bio-analysis test kits for the food, feed, fermentation, wine, brewing & dairy industries. Megazyme is committed to the development of improved test kits for these industries and is established as a world leader in this field. For more information on products we supply and countries we distribute to visit http://www.megazyme.com
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Advanced Bio-Analysis Test Kits for the Food, Feed, Fermentation, Wine, Brewing & Dairy Industries
Megazyme International Ireland,Bray Business Park, Bray, Co. Wicklow, Ireland.
Barry V. McCleary, PhD, DScAgrInnovative test methods with exceptional technical support and customer service.The Megazyme Promise.
Megazyme was founded in 1988 with the specific aim of developing and supplying innovative test kits and reagents for the cereals, food, feed and fermentation industries. There is a clear need for good, validated methods for the measurement of the polysaccharides and enzymes that affect the quality of plant products from the farm gate to the final food. The commitment of Megazyme to “Setting New Standards in Test Technology” has been continually recognised over the years, with Megazyme and founder, Dr McCleary receiving a number of business and scientific awards. Today, Megazyme is a recognised world leader in the development of high quality, innovative test technology; eight of its tests are USA recommended and/or Official Methods and many of its methods are industry standards worldwide. Success in achieving these goals has been due to innovative research together with an understanding of the needs of our customers. Megazyme has its research and manufacturing facilities in Bray, Ireland and has sales and distribution agreements in over 30 countries. The company exports over 98% of its products to over 60 countries including North America, Europe, Australasia, Middle East and the Far East. The success of Megazyme is not just related to our impressive product portfolio (~ 500 products), but to the standard of excellence achieved in developing, manufacturing and marketing quality products that meet market demands. In our creed, the customer truly does come first. We demonstrate this through the services we offer, above and beyond the products we supply. We offer worldwide express delivery on all our shipments. In general, technical queries are answered within 48 hours. To make information immediately available to our customers, we established a website in 1994, and this is continually updated. Today, it acts as the source of a wealth of information on Megazyme products, but also is the hub of our commercial activities. It offers the possibility to purchase and pay on-line, to view order history, to track shipments, and many other features to support customer needs. From a technical point of view, it holds a host of product information such as Data Booklets, MSDS, Certificates of Analysis (COA), Frequently Asked Questions (FAQ), Product Performance information and Mega-Calc™ EXCEL™ based calculation tools. The value and functionality of this site has been recognised through several business awards.Since 2003, Megazyme has expanded through the introduction of a Molecular Biology Division, allowing the production of enzymes for incorporation into new test kits. This new platform technology has resulted in the launch of a range of new research/analytical enzymes and many new and improved test kits which are finding rapid adoption in the wine, dairy and food industries worldwide. More recent research has been directed towards the needs of the Biofuels industry, with the production and supply of a wider range of enzyme substrates, oligosaccharides and research enzymes.
Megazyme Test Kits and ReagentsPurity. Quality. Innovation.
Analyte Cat. No. Analyte Significance Advantages of Megazyme Test Kits
Acetic Acid K-ACETRMK-ACETAK Common food component
1. K-ACETRM is a rapid, manual assay kit employing AK and phosphotransacetylase2. K-ACETAK (auto-analyser) new, stable R1 and R2, and very rapid AK based kit
Ammonia K-AMIAR Common food component K-AMIAR has a very rapid reaction rate (~ 3 min at room temperature). Manual and auto-analyser applications
Amylose / Amylopectin K-AMYL Ratio of these components affects the rate of digestion
and utilisation of starch Novel kit, stable reagents
L-Asparagine / L-Glutamine / Ammonia
K-ASNAM Acrylamide precursors in the production of fried, roasted, toasted potato or other food products
Novel product, enabling all three analytes to be determined in less than 20 min. Manual and microplate format procedures given
L-Ascorbic Acid K-ASCO Naturally found in fruits and vegetables, or supplemented in processed foods Rapid reaction, stable reagents
Available Carbohydrates / Dietary Fibre
K-ACHDF Sugars rapidly digested and absorbed, and dietary fibre Novel procedure, stable reagents
Beta-Glucan (Mixed linkage) K-BGLU Major cell-wall polysaccharide of barley and oats
Rapid reaction, stable reagents, only enzymatic kit available. AOAC Method 995.16; AACC Method 32-23.01; ICC Standard No. 166; RACI Standard Method
Citric Acid K-CITR Common food component / additive Ideal for manual and auto-analyser applications
Ethanol K-ETOH Found in small amounts in many foods Rapid reaction, stable reagents (AlDH supplied as a stable suspension)
Fructan K-FRUCK-FRUCHK
Common component in many foods such as onions and seeds
Sucrose K-SUFRGK-SUCGL Common food component Choice of simple formats available, based either on
glucose oxidase / peroxidase, or hexokinase / G-6-PDH
SweetenersK-ASPTMK-MANOLK-SORB
Aspartame, D-mannitol, D-sorbitol and xylitol are common sweeteners found in a variety of foods
1. K-ASPTM - novel method, only test kit available2. K-MANOL - new method, only test kit available3. K-SORB - diaphorase supplied as a stabilised suspension rather than a lyophilised powder, thus less wasted enzyme
Total Dietary Fibre
K-TDFRK-INTDF Carbohydrate not digested in small intestine
1. K-TDFR: AOAC Methods 985.29, 991.42, 991.43 & 993.19; AACC Methods 32-07.01, 32-21.01, 32-05.01 2. K-INTDF is consistent with the CODEX Alimentarius definition of dietary fibre. AOAC Method 2009.01, 2011.25; AACC Methods 32-45.01 & 32-50.11
Total Starch K-TSTAK-TSTAHK Major food component
Rapid assay formats with options of measuring D-glucose with GOPOD reagent or with hexokinase / G-6-PDH. Stable reagents. AOAC Method 996.11; AACC Method 76-13.01; ICC Method No. 168; RACI Standard Method
Purchase Online at www.megazyme.com 5
Feed Industry Test Kits
Analyte Cat. No. Analyte Significance Advantages of Megazyme Test Kits
Acetic Acid K-ACETRM Commonly found in feed or fermented feed K-ACETRM is a rapid, manual assay kit employing AK and phosphotransacetylase. Stable reagents
Ammonia K-AMIAR Commonly found in feed or fermented feed K-AMIAR has a very rapid reaction rate (~ 3 min at room temperature). Ideal for manual and auto-analyser applications. Stable reagents
Alpha-Amylase K-CERA A key enzyme in most feeds and plant products
Novel assay employing a defined oligosaccharide substrate. High sensitivity and specificity. AOAC Method 2002.01; AACC Method 22-02.01; ICC Standard Method no. 303; RACI Standard Method; CCFRA Flour Testing Working Group Method 0018
Available Carbohydrates / Dietary Fibre
K-ACHDF Rapidly and slowly available sugars for digestion or fermentation Novel procedure. Stable reagents
Beta-Glucan (Barley and oats)
K-BGLU Major cell-wall polysaccharide of barley and oats
Rapid reaction, stable reagents, only enzymatic kit available. AOAC Method 995.16; AACC Method 32-23.01; ICC Standard No.166; RACI Standard Method
Beta-Glucanase K-MBGL Cellulase and b-glucanase levels in feeds Only kit available. Stable reagents. RACI Standard Method
Fructan K-FRUC Fructo-oligosaccharides in grasses and grains Only kit available. Stable reagents; AOAC Method 999.03; AACC Method 32-32.01
D-Fructose / D-Glucose
K-FRUGLK-FRGLMQK-FRGLQR
Major digestible carbohydrates in feedsRapid reaction times, choice of simple formats available, ideal for manual and auto-analyser applications. Stable reagents
Galactomannan K-GALM Reserve carbohydrate in many legume seeds Only kit available. Stable reagents
L-Lactic Acid K-LATE Commonly found in fermented feed Rapid reaction, stable reagents. Ideal for manualand auto-analyser applications
Phytic Acid K-PHYT Found in most plant materials. Major form of bound phosphate in plant materials Novel procedure. Rapid reaction, stable reagents
Raffinose / D-Galactose K-RAFGA Found in high levels in legume seeds. Causes
discomfort and flatulence in pigs Rapid reaction, stable reagents
Resistant Starch K-RSTAR Starch that is not digested in the small intestine of
monogastric animals
Only kit available. Stable reagentsAOAC Method 2002.02; AACC Method 32-40.01,
Total Dietary Fibre
K-TDFRK-INTDF Carbohydrate not digested in small intestine
1. K-TDFR: AOAC Methods 985.29, 991.42, 991.43 and 993.19; AACC Methods 32-07.01, 32-21.01, 32-05.01, and 32-45.01. 2. K-INTDF is consistent with the CODEX Alimentarius definition of dietary fibre. AOAC Method 2009.01
Total Starch K-TSTAK-TSTAHK Starch content of grain and feed
Rapid assay formats with options of measuring D-glucose with GOPOD reagent or with hexokinase / G-6-PDH. Stable reagents. AOAC Method 996.11; AACC Method 76-13.01; ICC Method No. 168; RACI Standard Method
endo-b-Xylanase K-XYLS b-Xylanase in feed High sensitivity. Stable reagents
Beta-Glucanase S-ABG100 b-Glucanase in feed Rapid reaction. Sensitive. Stable reagent
endo-b-Xylanase S-AXBP b-Xylanase in feed Sensitive. Easy to use. Stable reagent
Protease S-AZCAS endo-Protease added to feed Easy to use. Stable reagent
Setting New Standards in Test Technology6
Fermentation Industry Test Kits
Analyte Cat. No. Analyte Significance Advantages of Megazyme Test Kits
Acetic Acid K-ACETRM A common fermentation product K-ACETRM is a rapid, manual assay kit employing AK and phosphotransacetylase. Stable reagents
Ammonia K-AMIAR Commonly measured in fermentation brothsK-AMIAR has a very rapid reaction rate (~ 3 min at room temperature). Ideal for manual and auto-analyser applications. Stable reagents
Alpha-Amylase K-CERA A major fermentation product
Novel assay employing a defined oligosaccharide substrate. High sensitivity and specificity. AOAC Method 2002.01; AACC Method 22-02.01; ICC Standard Method no. 303; RACI Standard Method; CCFRA Flour Testing Working Group Method 0018
L-Asparagine / L-Glutamine / Ammonia
K-ASNAM Common components of animal cell culture mediaNovel product, enabling all three analytes to be determined in less than 20 min. Manual and microplate format procedures given
Beta-Glucanase K-MBGL A major fermentation product Rapid reaction, stable reagents; RACI Standard Method
Citric Acid K-CITR A product of fermentationIdeal for both manual and auto-analyser applications. Reconstituted citrate lyase stable for > 6 months at -20°C. Stable reagents
Ethanol K-ETOH Produced during alcoholic fermentation Rapid reaction, stable reagents (AlDH supplied as a stable suspension)
D-Glucose K-GLUCK-GLUHKR/L Common component of fermentation broths Rapid reaction, stable reagents
Glucose Oxidase K-GLOX A major fermentation product Rapid reaction. Simple format. Stable reagents
Glycerol K-GCROLK-GCROLGK A product of fermentation Rapid reactions, stable reagents
L-Glutamine / Ammonia K-GLNAM Common components of animal cell culture media Simple and rapid test kit gives values for ammonia and
L-Lactic Acid K-LATE Produced predominantly from L-malic acid during malolactic fermentation
Rapid reaction, stable reagents. Ideal for manual and auto-analyser applications
Succinic Acid K-SUCC Wine acid produced during fermentation Rapid reaction (~ 6 min at room temperature), stable reagents
Sucrose K-SUFRGK-SUCGL
Added to increase the amount of alcohol. Use only permitted in certain situations
Choice of simple formats available, based either on glucose oxidase / peroxidase, or hexokinase / G-6-PDH
Urea K-URAMR
Source of Yeast Available Nitrogen (YAN) and precursor of the carcinogen ethyl carbamate. Over-supplementation with diammonium phosphate (DAP) can result in elevated levels
Simple, very rapid (both urea and ammonia measured in < 10 min at room temperature) and sequential / efficient (only one cuvette required per sample)
Alpha-Amylase T-AMZ200 A product of fermentation Rapid reaction, stable reagentAACC Method 22.05; RACI Standard Method
endo-Arabinanase T-ARZ200 A product of fermentation Rapid reaction, stable reagent
Beta-Glucanase S-ABG100 A product of fermentation Rapid reaction, stable reagent
Cellulase S-ACMC A product of fermentation Rapid reaction, stable reagent
Cellulase T-CTZ200 A product of fermentation Rapid reaction, stable reagent
Pullulanase S-RPUL A product of fermentation Rapid reaction, stable reagent
endo-b-Xylanase S-AXBP A product of fermentation Rapid reaction, stable reagent
Purchase Online at www.megazyme.com 7
Analyte Cat. No. Analyte Significance Advantages of Megazyme Test Kits
Acetaldehyde K-ACHYD A sensory compound that adds flavour and complexity, but spoils wine at high concentrations
AcDH supplied as a stabilised solution rather than a lyophilised powder, thus less wasted enzyme
Acetic Acid
K-ACETK-ACETAFK-ACETAKK-ACETRM
A sensory compound that adds flavour and complexity in small amounts, but spoils wine at high concentrations. Produced naturally by yeast in small amounts and by spoilage organisms such as Acetobacter aceti in large quantities. This is the predominant of the acids comprising ~ 85 % volatile acidity (VA)
All kits contain PVP to prevent tannin inhibition.1. K-ACET (manual, efficient) contains stable ACS suspension2. K-ACETAF (auto) very stable R1 and R23. K-ACETAK (auto) is a stable, and very rapid acetate kinase (AK) based kit with excellent linearity4. K-ACETRM is a rapid, manual assay kit employing AK and phosphotransacetylase Stable reagent
Ammonia K-AMIARK-LARGE
Most important inorganic source of Yeast Available Nitrogen (YAN)
Novel enzyme employed is not inhibited by tannins, endpoint reaction time ~ 3 min. Ideal for manual and auto-analyser applications
L-Arginine K-LARGE Most important amino acid in grape juice with respect to YAN
Simple and rapid test kit gives sequential values for ammonia, urea and L-arginine. No tannin inhibition
L-Ascorbic Acid K-ASCO Present naturally in grapes and can be added as an anti-oxidant Rapid reaction, stable reagents
Citric Acid K-CITRNaturally present in small amounts; large amounts indicate addition for acidification (EU limit is 1 g/L)
Ideal for both manual and auto-analyser applications. Reconstituted citrate lyase stable for > 6 months at -20°C. Stable reagents
Ethanol K-ETOH Produced during alcoholic fermentation. Amounts > 17.5 % (v/v) indicate supplementation
Rapid reaction, stable reagents (AlDH supplied as a stable suspension)
D-Fructose / D-Glucose
K-FRUGLK-FRGLMQK-FRGLQR
Grape quality indicator. One of the two principle fermentable sugars of grape juice
Contains PVP to prevent tannin inhibition. Ideal for manual and auto-analyser use. Stable reagents
D-Gluconic Acid K-GATE Grape quality indicator for the production of
certain wines Rapid reaction, stable reagents
Glycerol K-GCROLK-GCROLGK
Quality indicator of finished wine, important for “mouth feel” Novel tablet format offers superior stability, rapid reaction
D-Lactic Acid K-DATEK-DLATE
Produced predominantly by lactic acid spoilage bacteria Rapid reaction, stable reagents
L-Lactic Acid K-LATE K-DLATE
Produced predominantly from L-malic acid during malolactic fermentation
Rapid reaction, stable reagents. Ideal for manual and auto-analyser applications
D-Malic Acid K-DMAL Only present in significant quantities in adulterated wine
D-MDH supplied as a stabilised suspension rather than a lyophilised powder, thus less wasted enzyme
L-Malic Acid
K-LMALR/LK-LMALAFK-LMALMQK-LMALQR
Grape quality indicator. Very important grape acid, converted to less acidic L-lactic acid during malolactic fermentation
K-PANOPA Primary amino nitrogen (PAN) is the most important organic source of YAN Novel kit, rapid reaction, stable reagents, simple format
D-Sorbitol K-SORB High levels indicate addition of fruit Diaphorase supplied as a stabilised suspension rather than a lyophilised powder, thus less wasted enzyme
Succinic Acid K-SUCC Wine acid produced during fermentation Rapid reaction (~ 6 min even at room temperature), stable reagents
Sucrose K-SUFRGK-SUCGL
Added to increase the amount of alcohol. Use only permitted in certain situations
Choice of simple formats available, based either on glucose oxidase / peroxidase, or hexokinase / G-6-PDH
SulphiteK-SULPHK-TSULPHK-ETSULPH
Sulphites are used as an essential additive in the control of microbial contamination during aging and to also protect the wine against detrimental “oxidative and enzymatic browning”
Choice of simple formats available, based either on liquid ready reagent chemical reactions (K-SULPH & K-TSULPH) or an enzymatic reaction (K-ETSULPH). Stable reagents
Tartaric Acid K-TARTOccurs naturally in grapes and is one of the most prevalent organic acids. Key indicator of total (titratable) acidity (TA)
Stable liquid ready reagents. Simple, rapid chemical reaction for manual, auto-analyser and microplate formats
Urea K-URAMRSource of YAN and precursor of the carcinogen ethyl carbamate. Over-supplementation with DAP can result in elevated levels
Simple, very rapid (both urea and ammonia measured in < 10 min at room temperature) and sequential / efficient (only one cuvette required per sample)
Wine Industry Test Kits
Setting New Standards in Test Technology8
Brewing Industry Test Kits
Analyte Cat. No. Analyte Significance Advantages of Megazyme Test Kits
Alpha-Amylase K-CERA A key indicator of malt quality
Novel assay employing a defined oligosaccharide substrate. High sensitivity and specificity. AOAC Method 2002.01; AACC Method 22-02.01; ICC Standard Method no. 303; RACI Standard Method; CCFRA Flour Testing Working Group Method 0018
Beta-Amylase K-BETA3 A key indicator of malt quality Only kit available. Stable reagents; RACI Standard Method
Beta-Glucan (Barley and oats)
K-BGLU Major cell-wall polysaccharide of barley and oatsRapid reaction, stable reagents, only enzymatic kit available. AOAC Method 995.16; AACC Method 32-23.01; ICC Standard Method No. 166; RACI Standard Method
Beta-Glucanase K-MBGL b-Glucanase level in maltRapid reaction, stable reagents, only enzymatic kit available; RACI Standard Method
D-Glucose K-GLUCK-GLUHKR/L Major component of fermentation mixture Rapid reaction, stable reagents
Malt Amylase K-MALTA Measurement of a-/b-amylase. Key indicators of malt quality
Combination of both K-CERA and K-BETA3
Total Starch K-TSTAK-TSTAHK Starch content of grain and feed
Rapid assay formats with options of measuring D-glucose with GOPOD reagent or with hexokinase / G-6-PDH. Stable reagents. AOAC Method 996.11; AACC Method 76-13.01; ICC Method No. 168; RACI Standard Method
Alpha-Amylase T-AMZBG200 Allows measurement of a-amylase in pre-harvest sprouted barley
Novel procedure. Rapid reaction, stable reagent
Beta-Glucanase T-BGZ200 Key enzyme in hydrolysis of malt b-glucansNovel substrate. Rapid reaction, stable reagent; RACI Standard Method
Limit-Dextrinase T-LDZ200 Key enzyme in hydrolysis of 1,6-linkages in starch
and branched malto-dextrinsNovel substrate. Rapid reaction, stable reagent; RACI Standard Method
endo-b-Xylanase T-XAX200 Key enzyme in hydrolysis of malt xylans Novel substrate. Rapid reaction, stable reagent
Purchase Online at www.megazyme.com 9
Dairy Industry Test Kits
Analyte Cat. No. Analyte Significance Advantages of Megazyme Test Kits
Acetaldehyde K-ACHYDOne of the most important aroma compounds in yogurt, responsible for the characteristic taste. Also formed in milk during storage
AlDH supplied as a stabilised solution rather than a lyophilised powder, thus less wasted enzyme.Stable reagents
Acetic Acid
K-ACETK-ACETAFK-ACETAKK-ACETRM
Fermentation product of yogurt and cheese
All kits contain PVP to prevent tannin inhibition.1. K-ACET (manual, efficient) contains stable ACS suspension2. K-ACETAF (auto) very stable R1 and R23. K-ACETAK (auto) is a stable, and very rapid acetate kinase (AK) based kit with excellent linearity4. K-ACETRM is a rapid, manual assay kit employing AK and phosphotransacetylase Stable reagents
Ammonia K-AMIAK-AMIAR
Important indicator of the hygienic quality (microbial load) of milk
K-AMIAR has a very rapid reaction rate (~ 3 min at room temperature). Ideal for manual and auto-analyser applications. Stable reagents
Aspartame K-ASPTM Common milkshake and yogurt sweetener Rapid reaction, stable reagents, only enzymatic kit available
Citric Acid K-CITRImportant quality indicator of milk, especially for butter and cheese production.Permitted additive
Ideal for both manual and auto-analyser applications. Reconstituted citrate lyase stable for > 6 months at -20°C. Stable reagents
Ethanol K-ETOH Produced during the fermentation of kefir Rapid reaction, stable reagents (AlDH supplied as a stable suspension)
Formic Acid K-FORM Minor acid in dairy productsFDH supplied as a stabilised suspension rather than a lyophilised powder, thus less wasted enzyme.Stable reagents
D-Fructose / D-Glucose
K-FRUGLK-FRGLMQ Common milkshake and yogurt sweetener
Rapid reaction times, choice of simple formats available, ideal for manual and auto-analyser applications. Stable reagents
D-Gluconic Acid K-GATE Weak organic acid found in dairy products. High
levels found in certain cheeses Rapid reaction, stable reagents
D-Glucose K-GLUCK-GLUHKR/L
Low levels expected in unprocessed / unadulterated milk and in cheese. Useful marker when producing lactose depleted dairy products
Choice of simple formats available, based either on glucose oxidase / peroxidase, or hexokinase / G-6-PDH. Stable reagents
L-Glutamic Acid K-GLUT Found in high concentrations, especially in cheese
No wasted diaphorase solution (stable suspension supplied). Stable reagents
D-Lactic Acid K-DATE Quality indicator of milk, yogurt and cheese Rapid reaction, stable reagents
L-Lactic Acid K-LATE Quality indicator of fresh milk. High levels in yogurt and cheese
Rapid reaction, stable reagents. Ideal for manualand auto-analyser applications
D-/L-Lactic Acid K-DLATE Quality indicator of fresh milk, yogurt and cheese Rapid reaction, flexible concurrent format.
Stable reagents
Lactose /D-Galactose K-LACGAR Key quality (value) indicator of milk Very rapid reaction (~ 5 min even at room temperature).
Succinic Acid K-SUCC Minor dairy acid Rapid reaction (~ 6 min even at room temperature). Stable reagents
Sucrose K-SUFRGK-SUCGL Not present naturally in dairy products
Choice of simple formats available, based either on glucose oxidase / peroxidase, or hexokinase / G-6-PDH. Stable reagents
Urea K-URAMRQuality indicator of milk, especially that used for cheese production. Used as a metabolic marker of bovine blood urea levels
Simple, very rapid (both urea and ammonia measured in < 10 min at room temperature) and sequential / efficient (only one cuvette required per sample)
Setting New Standards in Test Technology10
Enzymatic Procedures Based on NAD+ / NADH / NADP+ / NADPH In general terms, enzymes are catalytic proteins that convert one compound into another, and such reactions frequently occur without any visible sign that they have taken place. However, certain enzymatic reactions result in either a “colour” being produced or consumed, and the intensity of the colour change can be measured using a common spectrophotometer. Enzymes commonly selected for analytical applications are those that either directly, or indirectly (via other enzymes), produce or consume a compound called NADH (or NADPH), that although invisible to the human eye, absorbs light strongly at a wavelength of 340 nm (extinction coefficient [ε] = 6300 M-1 cm-1). Figure 1 depicts the various types of enzymatic reactions, either consuming or producing NADH (or NADPH), that are employed in many enzymatic test kits (for example see the L-malic acid assay kit biochemical pathway). As can be seen from figure 1, it is sometimes necessary to include an additional reaction in order to obtain quantitative results. This reaction is catalysed by the enzyme
diaphorase, in the presence of a compound called INT, which converts the NADH (or NADPH) produced in the first reaction into a red coloured compound called INT-formazan. Invented in the 1950s, the spectrophotometer in its various guises is today one of the most commonly used analytical
instruments. The spectrophotometer is a powerful analytical instrument because it can measure changes in absorbance very accurately and quickly. The enzymatic analysis reaction itself is performed in a plastic or glass cuvette that sits between the
source of light and the detector inside the spectrophotometer (as depicted simplistically in figure 2). A known amount of light travels through the cuvette and the amount that emerges is quantified by the detector. The change in intensity as the light passes through the reaction solution in the cuvette is recorded as an absorbance reading.
Modern enzymatic analysis test kits generally contain all reagents necessary to perform the assay, e.g. buffer, cofactor(s), trigger enzyme and standard solution, in an easy to use form that is stable for > 2 years (even while in use). During a typical enzymatic analysis, purified water is mixed with buffer, cofactor and sample. Then an absorbance reading (A
1) is taken just
before addition of the “trigger” enzyme (specific for the analyte in question), after which the reaction takes place (see figure 3). When the reaction has finished (i.e. the “endpoint” has been achieved), a second absorbance reading is taken (A
2). The
difference between these two absorbance readings (i.e. A1 - A
2)
is called the change in absorbance (or ΔA), due to the analyte in question. It is this value, after correction with a blank reading (reaction containing no sample), that is used to calculate the concentration of the analyte in the sample (typically as g/L), by way of a simple factor (e.g. 0.2535 in the case of the the acetic acid AK / PTA format). For example (a typical acetic acid calculation),
A1 (blank) = 1.400, A
2 (blank) = 1.398,
A1 (sample) = 1.420, A
2 (sample) = 0.650.
ΔAacetic acid = (1.420 - 0.650) - (1.400 - 1.398) = 0.768Thus the concentration of acetic acid = 0.768 x 0.2535 = 0.1947 g/LWhen using Megazyme test kits, calculations can either be performed manually as illustrated above or by using a free EXCEL™-based calculator, called “Mega-Calc™”, downloadable from where each assay kit appears on the Megazyme website (www.megazyme.com).
Principles of Test Procedures
Figure 2. Simplistic representation of enzymatic analysis being conducted using a spectrophotometer.
Figure 3. Decrease in absorbance at 340 nm on incubation of 0-35 µg of acetic acid with acetate kinase in the acetic acid AK / PTA format.
Figure 1. Enzymatic reaction pathways employed in analytical test kits that either consume or produce NADH (NADPH).
Purchase Online at www.megazyme.com 11
Chromogenic SubstratesChromogenic, or dye-labelled polysaccharides are useful in the specific measurement of polysaccharide endo-hydrolases in crude plant extracts or industrial enzyme preparations. Traditionally, such enzymes have been measured using the native polysaccharide, followed by quantification of the increase in reducing sugar or decrease in viscosity on hydrolysis. Since a range of enzymes, including endo- and exo-polysaccharidases and glycosidases, act on any given polysaccharide, reducing-sugar methods are not specific. Viscosity reduction methods are specific for endo-hydrolase activity, but are tedious to perform, and require specialist equipment. Chromogenic polysaccharide substrates offer the advantages of being specific and sensitive, and can form the basis of accurate, reliable and robust assay procedures.
With soluble chromogenic substrates (e.g. Azo-Barley Glucan or Red Pullulan), enzyme is incubated with the soluble substrate, the reaction is terminated and high molecular weight, partially hydrolysed fragments are precipitated from solution with an organic solvent such as ethanol or methoxyethanol. The suspension is mixed thoroughly, centrifuged, and the colour in the supernatant solution measured. With the aid of a standard curve, the concentration of enzyme can be determined.
With insoluble chromogenic substrates, the insoluble substrate (gelatinous particles) is depolymerised and solubilised by the action of the endo-hydrolase. The reaction is terminated by adding an alkaline solution to stop enzyme activity and the reaction slurry is filtered or centrifuged. Colour in the filtrate or supernatant is measured in a spectrophotometer and the colour intensity is directly related to enzyme activity. For convenience in dispensing, insoluble chromogenic substrates are supplied in tablet form (e.g. Xylazyme AX and Amylazyme tablets).
Colourimetric SubstratesThe colourimetric substrates supplied by Megazyme (e.g. Ceralpha reagent, Betamyl-3® reagent and Amyloglucosidase assay reagent) are based on a defined oligosaccharide which is covalently linked to p-nitrophenol through the reducing D-glucosyl residue of the oligosaccharide. In the measurement of a-amylase using Ceralpha reagent, the substrate is composed of end-blocked p-nitrophenyl maltoheptaoside in the presence of an excess quantity of thermostable a-glucosidase. When the substrate is cleaved by a-amylase, the a-glucosidase removes the remaining D-glucosyl residues releasing free p-nitrophenol, which in the presence of an alkaline solution is converted to the yellow phenolate ion. The assay for b-amylase (“Betamyl-3® reagent”) employs p-nitrophenyl maltotrioside in the presence of b-glucosidase. Amyloglucosidase assay reagent contains p-nitrophenyl b-maltoside in the presence of b-glucosidase. When amyloglucosidase removes the terminal a-linked D-glucosyl residue, the second D-glucosyl unit is released by the excess level of b-glucosidase present in the reagent mixture. Free p-nitrophenol is also released and this is converted to the yellow phenolate ion by addition of an alkaline solution.
The Megazyme Advantage – Assay Format ChoiceManufacturers of enzymatic test kits generally only offer a single “manual” assay format for each analyte. It thus becomes necessary for users of auto-analyser equipment to adapt such products. In contrast, Megazyme offers either specific analyser formats or instructions for the use of manual kits in “analyser format”. Such a choice is available for measurement of acetic acid, ammonia, L-ascorbic acid, citric acid, D-fructose and D-glucose, glycerol, L-lactic acid, L-malic acid and primary amino nitrogen. Use of manual or auto-analyser formats requires a spectrophotometer or automated equipment. This may preclude analysts in small wineries from performing these analyses. To counter this, Megazyme developed the MegaQuant™ formats for measurement of L-malic acid and D-fructose plus D-glucose. In these assays, the final reaction product is a coloured formazan compound (INT-formazan), thus allowing measurement with an inexpensive colourimeter.
While Megazyme offers the traditional biochemistry in some of its bio-analysis test kits, many contain advanced enzymes, offering reduced reaction times and/or increased stability. Where necessary, cofactor stability is enhanced by offering these in a stable tablet form. Also, alternative biochemical reactions are evaluated and have been implemented. Some of these modifications and advances are highlighted in test kits for acetic acid ammonia, D-fructose / D-glucose and L-malic acid.
1. Acetic acidFour kits for acetic acid determination are offered by Megazyme and the differences between these are:
a. K-ACET is an advanced format of the traditional ACS based kits offered by other manufacturers for manual analysis. The advantages of this kit include (i) ACS is supplied as a very stable (> 2 years) ready-to-use ammonium sulphate suspension and thus wastage that occurs with other kits is avoided, (ii) PVP has been incorporated to prevent sample interference (e.g. tannins in wine analysis) and (iii) an EXCEL™ based calculator (Mega-Calc™) is available free on-line for hassle-free raw data processing (see page 14).
b. K-ACETAF is an advanced format of the traditional ACS based kits offered by other manufacturers for analyser use. The advantages of this kit include (i) and (ii) above, plus (iii) R1 is stable (no rising absorbance value) at 4˚C for > 3 days due to unique stabilisation system (see page 13).
c. K-ACETRM is a novel assay format that we strongly recommend for all manual acetic acid analysis applications. This kit offers many advantages over traditional kits, including (i) stoichiometric change in absorbance and thus no complex calculations required, (ii) very rapid reaction (~ 4 min), (iii) only two absorbance readings are required, (iv) PVP is incorporated to prevent sample interference (e.g.
Setting New Standards in Test Technology12
from tannins in wine), (v) simple format, (vi) all reagents are stable for > 2 years during use, (vii) extended linearity, (viii) reduced creep from ester hydrolysis and (ix) an EXCEL™ based calculator (Mega-Calc™) is available on- line for hassle-free raw data processing (see page 15).
d. K-ACETAK is a novel assay format that we strongly recommend for all auto-analyser applications. This kit offers many advantages over traditional kits, including (i) very rapid reaction (~ 10 min), (ii) PVP is incorporated to prevent sample interference (e.g. from tannins in wine), (iii) simple format, (iv) all reagents are stable for > 2 years during use, (v) linear calibration (R2 ~ 0.9995) up to 30 μg/mL in final reaction solution and (vi) R1 is stable at 4˚C for > 7 days (see page 14). Note: this kit is not recommended for manual analysis applications (see K-ACETRM).
2. AmmoniaMegazyme offers an advanced format (K-AMIAR) for the measurement of ammonia employing microbial glutamate dehydrogenase. This enzyme is not inhibited by any food or beverage sample component, such as tannin, and thus this kit is strongly recommended for all manual and auto-analyser applications (see page 15).
3. D-Fructose and D-GlucoseThree formats are offered for the measurement of D-fructose and D-glucose:
a. K-FRUGL is an advanced format containing PVP to prevent sample interference and is strongly recommended for all manual and auto-analyser applications (see page 24).
b. K-FRGLQR is a “ready to use” format supplied as a liquid stable formulation recommended for high throughput use with auto-analysers and microplate readers. The advantages of this kit include (i) no reagent preparation required (ii) very simple format, (iii) very cost effective (iv) PVP is incorporated to prevent sample interference (e.g. from tannins in wine) (iv) all reagents are stable for > 1 year (see page 25).
c. K-FRGLMQ is a novel assay format strongly recommended for users who do not possess a laboratory and / or analytical expertise. The advantages of this kit include (i) samples do not need to be sent out for contract analysis, (ii) no spectrophotometer is required, (iii) very simple format, (iv) very cost effective, and (v) accurate and reliable with all samples (including red wine) (see page 25).
4. D-GlucoseTwo assay formats are offered:
a. K-GLUHKR/L is a traditional assay format based on glucose-6-phosphate dehydrogenase and hexokinase (see page 31).
b. K-GLUC is a traditional assay format based on glucose oxidase and peroxidase that is more cost effective than methods based on glucose-6-phosphate dehydrogenase and hexokinase (see page 30).
5. L-Malic acidL-Malic acid is the most commonly measured analyte in winery applications, thus four assay formats are offered by Megazyme:
a. K-LMALR/L is an advanced format of the traditional kits offered by other manufacturers for manual analysis. The advantages of this kit include (i) PVP is incorporated to prevent sample interference (e.g. from tannins in wine), (ii) all reagents are stable for > 2 years during use (both enzymes are supplied as ammonium sulphate suspensions), (iii) very rapid reaction (~ 3 min) and (iv) an EXCEL™ based calculator (Mega-Calc™) is available on-line for hassle-free raw data processing. This kit is thus strongly recommended for all manual analysis applications (see page 40).
b. K-LMALAF is designed for auto-analyser applications. It is an advanced format of traditional kits offered by other manufacturers. The advantages of this kit include (i) PVP is incorporated to prevent sample interference (e.g. from tannins in wine), (ii) very stable R1 and R2 reagents, (iii) all reagents are stable for > 2 years during use (both enzymes are supplied as ammonium sulphate suspensions), and (iv) linear calibration (R2 ~ 0.9994) up to 80 μg/mL in final reaction solution. This kit is thus strongly recommended for all auto-analyser applications (see page 40).
c. K-LMALQR is a “ready to use” format supplied as a liquid stable formulation recommended for high throughput use with auto-analysers and microplate readers. The advantages of this kit include (i) no reagent preparation required (ii) very simple format, (iii) very cost effective (iv) PVP is incorporated to prevent sample interference (e.g. from tannins in wine) (iv) all reagents are stable for > 1 year (see page 41).
d. K-LMALMQ is a novel assay format that is strongly recommended for users who do not possess a laboratory and / or analytical expertise. The advantages of this kit include (i) samples do not need to be sent out for contract analysis, (ii) no spectrophotometer is required, (iii) very simple format, (iv) very cost effective, and (v) accurate and reliable with all samples (including red wine) (see page 41).
Purchase Online at www.megazyme.com 13
Advanced Bio-Analysis Test Kits
Setting New Standards in Test Technology14
Advantages
• No wasted aldehyde dehydrogenase solution (stable suspension supplied)
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
UV-method for the determination of Acetaldehyde in foodstuffs, beverages, and other materials
(e.g. yogurt), bread, fruit juices, soft drinks, cocoa, vegetable and fruit products, coffee, and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by MEBAK, and in CH
• Very stable reagent when prepared for auto-analyser applications (> 7 days at 4°C)
• PVP incorporated to prevent tannin inhibition
• Linear calibration (R2 ~ 0.9995) up to 30 μg/mL of acetic acid in final reaction solution
• Validated by the University of Wine, Suze la Rousse, France
• Very rapid reaction
• Very competitive price (cost per mL of reagent)
• All reagents stable for > 2 years
Setting New Standards in Test Technology16
Acetic Acid (Rapid manual format) Cat. No. K-ACETRM Manual format UV-method for the determination of Acetic Acid in foodstuffs, beverages, and other materials
Principle: (acetate kinase)(1) Acetic acid + ATP acetyl-phosphate + ADP
(phosphotransacetylase)(2) Acetyl-phosphate + CoA acetyl-CoA + Pi
dairy products (e.g. cheese), meat, processed meat, vegetable and fruit products, bakery products, paper, pharmaceuticals, cosmetics and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by MEBAK, OIV, EU, ISO2963, AOAC and IFU22
(Note: If the enzyme oxaloacetate decarboxylase is present in the sample, some of the oxaloacetate productis converted to pyruvate. Therefore, to ensure citric acid is measured quantitatively, D-lactate dehydrogenase(D-LDH) is employed to efficiently convert any pyruvate produced into D-lactate and NAD+).
Advantages
• Reconstituted citrate lyase stable for 4 weeks at 4°C / 6 months at -20°C
• Buffer / cofactor / enzyme tablets for efficient use of kit components
• PVP incorporated to prevent tannin inhibition
• Ideal for both manual and auto-analyser applications
• Very competitive price (cost per test)
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Ethanol Cat. No. K-ETOH
UV-method for the determination of Ethanol in foodstuffs, beverages, and other materials
low-alcoholic / non-alcoholic beverages, pickles, fruit and fruit juice, chocolate products, vinegar, jam, bread and bakery products, honey, soy sauce, dairy products, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by IFU, EBC, MEBAK, ASBC, and in D, CH, A, and F
Advantages
• Simple format – aldehyde dehydrogenase supplied as stable suspension
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 23
Total Dietary Fibre Cat. No. K-TDFR
For the determination of Total Dietary Fibre in cereal products, foodstuffs, feeds and other materials
Principle: (a-amylase + amyloglucosidase)(1) Starch + H
2O D-glucose
(protease)(2) Protein + H
2O peptides
(3) Dietary fibre determined gravimetrically following alcohol precipitation (4) Ash and residual protein determined on DF residues and subtracted
Kit size: 200 assaysMethod: Hydrolysis / removal of non-dietary fibre componentsTotal assay time: ~ 100 minDetection limit: 0.5-100 % of sample weightApplication examples: Food ingredients, food products and other materialsMethod recognition: AOAC (Methods 985.29, 991.42, 991.43 and 993.19) and
AACC (Methods 32-05.01, 32-07.01 and 32-21.01)
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years
• High purity / standardised enzymes employed
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Simple format
Total Dietary Fibre (Integrated) Cat. No. K-INTDFFor the determination of Total Dietary Fibre in cereal products, foodstuffs, feeds and other materials. CODEX Type I method (2011)
Principle: (Pancreatic a-amylase + amyloglucosidase)(1) Non-resistant starch + H
dietetic foods, bread, bakery products, candies, desserts, confectionery, ice-cream, fruit and vegetables, condiments, tobacco, cosmetics, pharmaceuticals, paper and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by AOAC, EN, NEN, NF, DIN, GOST, OIV, IFU, AIJN, MEBAK, IOCCC, and in D, CH, A, and I
Advantages
• PVP incorporated to prevent tannin inhibition
• Ideal for both manual and auto-analyser applications (excellent on-machine stability)
• Validated by the University of Wine, Suze la Rousse, France
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation (manual analysis applications)
• Rapid reaction at either 25 or 37°C
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
UV-method suitable for auto-analyser and microplate formats for the determination of D-Fructose and D-Glucose in foodstuffs, beverages, and other materials
dietetic foods, bread, bakery products, candies, desserts, confectionery, ice-cream, fruit and vegetables, condiments, tobacco, cosmetics, pharmaceuticals, paper and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by AOAC, EN, NEN, NF, DIN, GOST, OIV, IFU, AIJN, MEBAK, IOCCC, and in D, CH, A, and I
Kit size: 100 assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 10 minDetection limit: 15.4 mg/LApplication examples: L-Fucose is present as the main component in fucoidan (a
marine polysaccharide), foods, pharmaceuticals and other materials (e.g. biological samples etc.)
Method recognition: Novel method
Advantages
• Very cost effective
• All reagents stable for > 2 years after preparation
• Only enzymatic kit available
• Simple format
• Rapid reaction time (~ 10 min)
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Galactomannan Cat. No. K-GALM
UV-method for the determination of Galactomannan in legume seeds, foodstuffs and plant products
Principle: (b-mannanase)(1) Galactomannan + H
2O galactomanno-oligomers
(b-mannanase + a-galactosidase)(2) Galactomanno-oligomers + H
Kit size: 40 assaysMethod: Spectrophotometric at 510 nmReaction time: ~ 100 minDetection limit: 1-100 % of sample weightApplication examples: Yeast preparations and other materialsMethod recognition: Novel method
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 12 months after preparation
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Beta-Glucanase (Malt and microbial) Cat. No. K-MBGLColourimetric method for the determination of Beta-Glucanase in malt, foodstuffs and fermentation products
Kit size: 50 assaysMethod: Spectrophotometric at 340 nmTotal assay time: 120 minDetection limit: 1-100 % of sample weightApplication examples: Jelly sweets, cosmetics, food gums and other materialsMethod recognition: Novel method
Advantages
• Very cost effective
• Only enzymatic kit available
• Simple format
• All reagents stable for > 2 years after preparation
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
D-Gluconic Acid / D-Glucono-δ-lactone Cat. No. K-GATEUV-method for the determination of D-Gluconic Acid and D-Glucono-δ-lactone in foodstuffs, beverages, and other materials
Principle: (gluconate kinase)(1) D-Gluconate + ATP gluconate-6-phosphate + ADP
(gluconate-6-phosphate dehydrogenase)(2) Gluconate-6-phosphate + NADP+ ribulose-5-phosphate + NADPH + CO
juice, dairy products, pharmaceuticals, paper and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by ISO, DIN, GOST, and in D and CH
Advantages
• All reagents stable for > 2 years after preparation
• Very competitive price (cost per test)
• Very rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 31
D-Glucosamine (plus Glucosamine-Sulphate and -Acetate) Cat. No. K-GAMINE
UV-method for the determination of D-Glucosamine, D-Glucosamine sulphate and N-Acetyl Glucosamine in food supplements, foodstuffs, beverages and other materials
foods, bakery products, candies, fruit and vegetables, tobacco, cosmetics, pharmaceuticals, feed, paper and other materials (e.g. biological cultures, samples etc)
Method recognition: Widely used and accepted in clinical chemistry and food analysis
Advantages
• All reagents stable for > 12 months after preparation
• Very competitive price (cost per test)
• Simple format
• Standard included
Setting New Standards in Test Technology32
D-Glucose (Hexokinase format) Cat. No. K-GLUHKR/L
UV-method for the determination of D-Glucose in foodstuffs, beverages, and other materials
Principle: (hexokinase)(1) D-Glucose + ATP G-6-P + ADP
Kit size: 110 (K-GLUHKR) or 220 (K-GLUHKL) assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 5 minDetection limit: 0.66 mg/LApplication examples: Wine, beer, fruit juices, soft drinks, milk, jam, dietetic
foods, bakery products, candies, fruit and vegetables, tobacco, cosmetics, pharmaceuticals (e.g. infusions), feed, paper (and cardboard) and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by AOAC, EN, NEN, NF, DIN, GOST, OIV, IFU, AIJN, MEBAK, and in D, CH, A, and I
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Glucose Oxidase Cat. No. K-GLOXColourimetric method for the determination of Glucose Oxidase in foodstuffs and fermentation products
• All reagents stable for > 12 months after preparation
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 33
D-Glucuronic Acid / D-Galacturonic Acid Cat. No. K-URONICUV-method for the determination of D-Glucuronic Acid or D-Galacturonic Acid in hydrolysates of plant material and polysaccharides and other materials
Principle: (Uronate dehydrogenase; UDH)(1) D-Glucuronic acid + NAD+ + H
2O D-glucarate + NADH + H+
(Uronate dehydrogenase; UDH)(2) D-Galacturonic acid + NAD+ + H
2O D-galactarate + NADH + H+
Kit size: 100 assaysMethod: Spectrophotometric at 340 nmTotal assay time: ~ 10 min at 25˚C or ~ 5 min at 37˚CDetection limit: ~ 17 mg/LApplication examples: Hydrolysates of plant material and polysaccharides and
other materialsMethod recognition: Novel method
Advantages
• Very cost effective
• All reagents stable for > 2 years during use
• Only test kit available
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Alpha-Glucuronidase Cat. No. K-AGLUAUV-method or the measurment of Alpha-D-Glucuronidase in various enzyme preparations
Principle: (a-D-glucuronidase)(1) Aldouronic acid (tri:tetra:penta) + H
(uronate dehydrogenase; UDH)(2) D-Glucuronic acid + NAD+ + H
2O D-glucarate + NADH + H+
Kit size: 50 assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 25 minDetection limit: 17 mU/mLApplication examples: Enzyme preparations and other materials Method recognition: Novel method
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years as supplied
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Setting New Standards in Test Technology34
L-Glutamic Acid Cat. No. K-GLUTColourimetric method for the determination of L-Glutamic Acid (Monosodium Glutamate; MSG) in foodstuffs and other materials
(diaphorase)(2) INT + NADH + H+ NAD+ + INT-formazan
Kit size: 60 assaysMethod: Spectrophotometric at 492 nmReaction time: ~ 9 minDetection limit: 0.21 mg/LApplication examples: Fruit and vegetables (e.g. tomato), processed fruit and
vegetables (e.g. tomato puree / juice, ketchup, soy sauce), condiments, processed meat products (e.g. extracts, bouillon and sausages), soup, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by ISO, GOST, NMKL, and in D, CH, and B
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Glutamate dehydrogenase solution stable at -20ºC
• No wasted diaphorase solution (stable suspension supplied)
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
L-Glutamine / Ammonia (Rapid) Cat. No. K-GLNAM
UV-method for the determination of L-Glutamine and Ammonia in cell culture media, foodstuffs and other materials
Principle: (glutaminase)(1) L-Glutamine + H
2O L-glutamate + NH
4+
(microbial glutamate dehydrogenase)(2) NH
4+ + 2-Oxoglutarate + NADPH L-glutamate + NADP+ + H
2O
Kit size: 50 assays of eachMethod: Spectrophotometric at 340 nmReaction time: ~ 15 minDetection limit: 0.54 mg/L (L-glutamine) 0.06 mg/L (ammonia)Application examples: Cell culture media and cultures, dietary supplements,
vegetables and other materials (e.g. biological samples etc)
Method recognition: Novel method
Advantages
• Very rapid reaction due to use of high activity glutaminase and uninhibited glutamate dehydrogenase
• All enzymes supplied as stabilised suspensions
• Only enzymatic kit available
• Very cost effective
• All reagents stable for > 2 years after preparation
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 35
UV-method for the determination of Glycerol in foodstuffs, beverages, and other materials
Principle: (glycerokinase)(1) Glycerol + ATP L-glycerol-3-phosphate + ADP
(diaphorase)(2) INT + NADH + H+ NAD+ + INT-formazan
Kit size: 50 assaysMethod: Spectrophotometric at 492 nmReaction time: ~ 10 minDetection limit: 0.8 mg/LApplication examples: Animal feeds, food, baby milk formulation and other
materialsMethod recognition: Novel method
Advantages
• Very cost effective
• Reagents stable for > 2 years after preparation
• Only enzymatic kit available
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 37
D-Isocitric Acid Cat. No. K-ISOC
UV-method for the determination of D-Isocitric Acid in foodstuffs
Principle: (isocitrate dehydrogenase)(1) D-Isocitric acid + NADP+ 2-oxoglutarate + CO
2 + NADPH + H+
(pH 9-10)(2) D-Isocitric acid ester + H
2O D-isocitric acid + alcohol
(pH 9-10)(3) D-Isocitric acid lactone + H
2O D-isocitric acid
Kit size: 100 assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 3 minDetection limit: 0.35 mg/LApplication examples: Fruit juices, fruit products, soft drinks and other
materials (e.g. biological cultures, samples etc)Method recognition: Methods based on this principle have been accepted by
EN, NEN, NF, DIN, GOST, IFU, AIJN, and in D, E, and CH
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• No wasted isocitrate dehydrogenase solution (stable suspension supplied)
• Very rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
D-Lactic Acid Cat. No. K-DATE
UV-method for the determination of D-Lactic Acid in foodstuffs, beverages, and other materials
Kit size: 50 assays of eachMethod: Spectrophotometric at 340 nmReaction time: ~ 10 min (L-lactic acid) and ~ 5 min (D-lactic acid)Detection limit: 0.21 mg/LApplication examples: Wine, soft drinks, milk, dairy products, foods containing milk
(e.g. dietetic foods, bakery products, baby food, chocolate, sweets and ice-cream), vinegar, fruit and vegetables, processed fruit and vegetables, meat products, food additives, paper (and cardboard), cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by DIN, GOST, IDF, EEC, EN, ISO, OIV, IFU, AIJN, MEBAK, and in D, NL, CH, and I
Advantages
• Rapid total analysis time (concurrent / flexible D- and L-lactic acid reaction format)
• D-lactate dehydrogenase reaction very rapid with most samples (~ 5 min)
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standards included
L-Lactic Acid Cat. No. K-LATEUV-method for the determination of L-Lactic Acid in foodstuffs, beverages and other materials
Kit size: 58 (K-LMALR) or 116 (K-LMALL) assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 3 minDetection limit: 0.25 mg/LApplication examples: Wine, beer, fruit juices, soft drinks, candies, fruit and
vegetables, bread, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by AOAC, EEC, EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN, MEBAK, and in D, CH, and I
Advantages
• PVP incorporated to prevent tannin inhibition
• Both enzymes supplied as stable suspensions
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Very rapid reaction (~ 3 min)
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
L-Malic Acid (Analyser format) Cat. No. K-LMALAF
Analyser format UV-method for the determination of L-Malic Acid in foodstuffs, beverages and other materials
Kit size: 250.8 mL of prepared reagent (R1 + R2)Method: Spectrophotometric at 340 nmReaction time: ~ 3 minDetection limit: 20 mg/L (recommended assay)Application examples: Wine, beer, fruit juices, soft drinks, candies, fruit and
vegetables, bread, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by AOAC, EEC, EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN, MEBAK, and in D, CH, and I
Advantages
• PVP incorporated to prevent tannin inhibition
• Very stable reagent when prepared for auto-analyser applications
• Linear calibration (R2 ~ 0.9994) up to 80 μg/mL of L-malic acid in final reaction solution
• Validated by the University of Wine, Suze la Rousse, France
• Very competitive price (cost per mL of reagent)
• Both enzymes supplied as stable suspensions
• Very rapid reaction (~ 3 min)
Setting New Standards in Test Technology42
L-Malic Acid Liquid Ready Reagents Cat. No. K-LMALQRUV-method suitable for auto-analyser and microplate formats for the determination of L-Malic Acid in foodstuffs, beverages, and other materials
Kit size: 1100 assays (auto-analyser format)Method: Spectrophotometric at 340 nmReaction time: ~ 3 minDetection limit: 166 mg/L (recommended format)Application examples: Wine, beer, fruit juices, soft drinks, candies, fruit and
vegetables, bread, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by AOAC, EEC, EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN, MEBAK, and in D, CH, and I
Advantages
• PVP incorporated to prevent tannin inhibition
• “Ready to use” liquid stable formulation
• Very competitive price (cost per test)
• All reagents stable for > 18 months
• Very rapid reaction (~ 3 min)
• Standard included
L-Malic Acid (MegaQuant™ format) Cat. No. K-LMALMQSimple colourimetric method for the determination of L-Malic Acid in foodstuffs, beverages and other materials
(diaphorase)(3) INT + NADH + H+ NAD+ + INT-formazan
Kit size: 60 assaysMethod: Spectrophotometric at 505 nmReaction time: ~ 6 minDetection limit: 0.25 mg/LApplication examples: Wine, beer, fruit juices, soft drinks, candies, fruit and
vegetables, bread, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Novel method
Advantages
• Novel product, patented technology
• Highly stable reagents (at least three seasons use)
• Very competitive price (cost per test)
• Spectrophotometer /laboratory / expertise not required
• Very simple procedure
• Rapid reaction time (~ 6 min)
• Standard included
Purchase Online at www.megazyme.com 43
Malt Amylase Cat. No. K-MALTAColourimetric method for the determination of Alpha-Amylase and Beta-Amylase in cereal grains, malt, food, beverages and fermentation products
Principle:
(1) Alpha-Amylase is measured using the “Ceralpha” Method as used in K-CERA
(2) Beta-Amylase is measured using the “Betamyl-3” Method as used in K-BETA3
Kit size: 50 assays of eachMethod: Spectrophotometric at 400 nmTotal assay time: ~ 20 min (Ceralpha Method) ~ 10 min (Betamyl-3 Method)Detection limit: 0.05 U/mLApplication examples: Cereal flours, malts, fermentation broths, and other
pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Novel method
Advantages
• Novel product (only enzymatic kit available)
• Very cost effective
• All reagents stable for > 2 years after preparation
• Simple format
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
D-Mannose / D-Fructose / D-Glucose Cat. No. K-MANGLUV-method for the determination of D-Mannose, D-Fructose and D-Glucose in foodstuffs, yeast cell preparations and other materials
Kit size: 500 assaysMethod: Spectrophotometric at 235 nmReaction time: ~ 30 minDetection limit: N/AApplication examples: Food ingredients (e.g. citrus fruit and apple) and other
materialsMethod recognition: JECFA
Setting New Standards in Test Technology46
Primary Amino Nitrogen (NOPA) Cat. No. K-PANOPA
UV-method for the determination of Primary Amino Nitrogen in grape juice, must, wine and other materials
manufacture and other materialsMethod recognition: Used and accepted in food analysis
Advantages
• Very rapid reaction due to inclusion of galactose mutarotase (patented technology)
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Raffinose / Sucrose / D-Glucose Cat. No. K-RAFGLColourimetric method for the determination of Raffinose (also stachyose and verbascose), Sucrose and D-Glucose in legume seeds, plant materials, foodstuffs and feed
Principle: (a-galactosidase)(1) Raffinose + stachyose + verbascose + H
Kit size: 50 assaysMethod: Spectrophotometric at 340 nmTotal assay time: ~ 20 minDetection limit: ~ 1.3 mg/LApplication examples: Hydrolysates of plant material and polysaccharides and
other materialsMethod recognition: Novel method
Advantages
• Very cost effective
• All reagents stable for > 2 years during use
• Only test kit available
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
D-Sorbitol / Xylitol Cat. No. K-SORBColourimetric method for the determination of D-Sorbitol and Xylitol in foodstuffs and wine
(diaphorase)(3) INT + NADH + H+ NAD+ + INT-formazan
Kit size: 58 assaysMethod: Spectrophotometric at 492 nmReaction time: ~ 15 minDetection limit: 0.20 mg/LApplication examples: Diabetic foods (e.g. honey, jam and chocolate),
dietetic foods, chewing gum, candies, fruit juice (e.g. apple juice), ice-cream, sweets, bakery products (e.g. desserts), marzipan, paper (and cardboard), cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by IFU, AIJN, and in D
Advantages
• Each vial of sorbitol dehydrogenase is stable for > 2 months at 4ºC after dissolution
• No wasted diaphorase solution (stable suspension supplied)
• Very competitive price (cost per test)
• Reagents stable for > 2 years as supplied
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 49
Starch Damage Cat. No. K-SDAMColourimetric method for the determination of Starch Damage in cereal flours
Principle: (fungal a-amylase)(1) Damaged (or gelatinised) starch + H
Kit size: 100 assaysMethod: Spectrophotometric at 510 nmTotal assay time: ~ 120 min (plus overnight incubation) Detection limit: 2-100 % of sample weightApplication examples: Plant materials, starch samples and other materialsMethod recognition: AOAC (Method 2002.02 and AACC (Method 32-40.01)
Advantages
• Very cost effective
• All reagents stable for > 2 years as supplied
• Only enzymatic kit available
• Measures enzyme resistant starch
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Setting New Standards in Test Technology50
Total Starch (GOPOD format) Cat. No. K-TSTAColourimetric method for the determination of Total Starch in cereal products, feeds, foodstuffs and other materials
Principle: (a-amylase, 100oC + DMSO)(1) Starch granules + H
Kit size: 100 assaysMethod: Spectrophotometric at 510 nmTotal assay time: ~ 90 minDetection limit: 1-100 % of sample weightApplication examples: Cereal flours, food products and other materialsMethod recognition: AOAC (Method 996.11), AACC (Method 76-13.01),
ICC (Standard Method No. 168), and RACI (Standard Method)
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 12 months after preparation
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Total Starch (Hexokinase format) Cat. No. K-TSTAHKUV-method for the determination of Total Starch in grains, animal feeds, foodstuffs and other materials
Principle: (a-amylase, 100oC + DMSO)(1) Starch granules + H
Kit size: 20 assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 6 minDetection limit: 0.26 mg/LApplication examples: Wine, fruit and vegetables, soy sauce, cheese, egg, egg
products and other materials (e.g. biological cultures, samples etc)
Method recognition: Methods based on this principle have been accepted by EEC, and in D and CH
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years as supplied
• Very rapid reaction (even at room temperature)
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Sucrose / D-Fructose / D-Glucose Cat. No. K-SUFRGUV-method for the determination of Sucrose, D-Fructose and D-Glucose in foodstuffs, beverages and other materials
Kit size: 50 assays of eachMethod: Spectrophotometric at 340 nmReaction time: ~ 30 minDetection limit: 1.38 mg/LApplication examples: Beer, fruit juices, soft drinks, milk, jam, honey, dietetic foods,
bread, bakery products, dairy products, candies, desserts, confectionery, sweets, ice-cream, fruit and vegetables (e.g. potato), meat products (e.g. sausage), condiments (e.g. ketchup and mustard), feed, tobacco, cosmetics, pharmaceuticals, paper and other materials
Method recognition: Methods based on this principle have been accepted by NF, EN, NEN, DIN, GOST, IFU, AIJN, MEBAK, IOCCC, and in D and A
Advantages
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation
• Rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Stabilised D-glucose / D-fructose standard solution included
Setting New Standards in Test Technology52
Sucrose / D-Glucose (GOPOD format) Cat. No. K-SUCGLColourimetric method for the determination of Sucrose and D-Glucose in foodstuffs, beverages and other materials
materialsMethod recognition: Validated for red and white wines at the Bundesamt für
Weinbau, Austria. Used widely in the wine industry
Advantages
• “Ready to use” liquid stable formulation
• Very competitive price (cost per test)
• All reagents stable for > 18 months
• Very rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 53
Total Sulphite (Enzymatic) Cat. No. K-ETSULPH
UV-method for the determination of Total Sulphite (SO2
-) in beverages, foodstuffs and other materials
Principle: (sulphite oxidase)(1) SO
3- + O
2 + H
2O SO
4- + H
2O
2
(NADH-POD)(2) H
2O
2 + NADH + H+ 2 H
2O + NAD+
Kit size: 30 assaysMethod: Spectrophotometric at 340 nmTotal assay time: ~ 30 minDetection limit: 0.3 mg/LApplication examples: Wine, beer, fruit juices, soft drinks, jam, fruit and
vegetables, pharmaceuticals and other materialsMethod recognition: Methods based on this principle have been accepted by
DIN, EN, MEBAK and NMKL
Advantages
• Very cost effective
• All reagents stable for > 2 years during use
• Simple format
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Total and Free Sulphite Cat. No. K-SULPH
Colourimetric methods for the determination of Total and Free Sulphite in wine, fruit juice, foodstuffs and other materials
Principle:
The Total Sulphite assay is based on the reaction principle between thiol groups and Ellman’s reagent
The Free Sulphite assay is based on the reaction principle of SO2, fuchsin and
aldehyde binding
Kit size: 40 assays of each (manual) 400 assays of each (auto-analyser)Method: Total sulphite: Spectrophotometric at 405 nm Free sulphite: Spectrophotometric at 575 nmTotal assay time: Total sulphite: ~ 6 min Free sulphite: ~ 9 minDetection limit: Total sulphite: ~ 5 mg/L Free sulphite: ~ 2 mg/LApplication examples: Wine, fruit juice, seafood, food stuffs and other materialsMethod recognition: Validated for red and white wines at the Bundesamt für
Weinbau, Austria. Used widely in the wine industry
Advantages
• “Ready to use” liquid stable formulation
• Very competitive price (cost per test)
• All reagents stable for > 18 months
• Very rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Setting New Standards in Test Technology54
Tartaric Acid Cat. No. K-TART
Colourimetric method for the determination of Tartaric Acid in wine, fruit juice and other materials
Principle:
The Tartaric acid assay is based on the reaction principles between tartaric acid and vanadate
Kit size: 200 assays (manual) 2000 assays (auto-analyser)Method: Spectrophotometric at 505 nmTotal assay time: ~ 4 minDetection limit: ~ 108 mg/LApplication examples: Wine, fruit juice and other materialsMethod recognition: Used widely in the wine industry
Advantages
• “Ready to use” liquid stable formulation
• Very competitive price (cost per test)
• All reagents stable for > 1 year
• Very rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Trehalose / D-Glucose Cat. No. K-TREH
UV-method for the determination of Trehalose and D-Glucose in foodstuffs, beverages, and other materials
and shrimp), fruit juices, purees and fillings, nutrition bars, surimi, dehydrated fruits and vegetables, fruit products, white chocolate, sports drinks, dairy products, egg products, soups and sauces, confectionery, chewing gum, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples etc)
Method recognition: Novel method
Advantages
• Only enzymatic kit available
• Very cost effective
• All reagents stable for > 2 years after preparation
• Very rapid reaction
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 55
Urea / Ammonia (Rapid) Cat. No. K-URAMR
UV-method for the determination of Urea and Ammonia in foodstuffs, beverages and other materials
Kit size: 100 assaysMethod: Spectrophotometric at 340 nmReaction time: ~ 6 minDetection limit: 0.7 mg/LApplication examples: Analysis of D-xylose in fermentation broths and
hydrolysates of plant material and polysaccharidesMethod recognition: Novel method
Advantages
• Very cost effective
• Reagents stable for > 2 years after preparation
• Only enzymatic kit available
• Rapid reaction (~ 6 min)
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
Purchase Online at www.megazyme.com 57
Key to Method Recognition
Official Bodies:
AIJN Association of the Industry of Juices and Nectars from Fruits and VegetablesAACC American Association of Cereal ChemistsAOAC AOAC INTERNATIONALASBC American Society of Brewing ChemistsCCFRA Campden & Chorleywood Food Research Association GroupDIN Deutsche Industrie Norm (German Standard)EBC European Brewery ConventionEEC Council of European CommunitiesEN European NormsGOST GOSSTANDART (State Committee of the Russian Federation for Standardisation
and Metrology)ICC International Association for Cereal Science and TechnologyICUMSA International Commission for Uniform Methods of Sugar Analysis
IDF International Dairy FederationIFU International Federation of Fruit Juice ProducersIOCCC Office International du Caccao, du Chocolat et de la ConfiserieISO International Standard OrganisationIUPAK International Union of Pure and Applied ChemistryJECFA Joint FAO / WHO Expert Committee on Food AdditivesMEBAK Central European Brewing Committee for AnalysisNBN Norme Belge (Belgian Standard)NEN Nederlandske Norm (Dutch Standard)NF Normes Françaises (French Standard)NMKL Nordisk Metodikkomité for Næringsmidler (Nordic Committee of Food Analysis)OIV Office International de la Vigne et du Vin (International Wine Office)RACI Royal Australian Chemical InstituteUKMBI United Kingdom Milling and Baking Industries
Countries:
A AustriaB BelgiumCH SwitzerlandD GermanyE SpainF FranceI ItalyNL Netherlands
PROTEASE TABLET TESTST-PRAK200 Protazyme AK (200 tablets)T-PRAK1000 Protazyme AK (1000 tablets)T-PROL200 Protazyme OL (200 tablets)T-PROL1000 Protazyme OL (1000 tablets)
GENERAL CHEMICALSG-AMBOH Amberlite FPA OH- Ion Exchange ResinG-AMBH Ambersep 200 H+ Ion Exchange ResinG-CEL100 Celite (100 g)G-CEL500 Celite (500 g)G-LCYST200 L-Cysteine Hydrochloride Monohydrate
BUFFERSB-BISTRIS250 BIS-TRIS Buffer SaltB-CAPS200 CAPS Buffer SaltB-CAPSO250 CAPSO Buffer SaltB-GLYGLY250 Glycylglycine Buffer SaltB-HEPES250 HEPES Buffer SaltB-MES250 MES Monohydrate Buffer SaltB-MOPS250 MOPS Buffer SaltB-PIPES250 PIPES Buffer SaltB-TRIS500 TRIS Buffer Salt
DVDs, EQUIPMENT & BOOKSTraining DVDsD-AMYL Amylose/Amylopectin DVDD-WINE Assay Format for the Wine Testing Kits DVDD-BETA3 b-Amylase (BETAMYL-3 Method) DVDD-BGLU b-Glucan (Mixed Linkage) DVDD-CERA Ceralpha : a-Amylase DVDD-INTDF Integrated Total Dietary Fibre DVDD-MBGL Malt b-Glucanase DVDD-PRET Sample Pre-treatment Format DVDD-SDAM Starch Damage DVDD-TSTA Total Starch DVD
EquipmentD-FRGLMQ MegaQuant™ Meter plus D-Fructose & D-Glucose ReagentsD-LMALMQ MegaQuant™ Meter plus L-Malic Acid ReagentsD-MQTUB Tubes for MegaQuant™ Meter (24 tubes)D-IBMKIII Megazyme Incubation Bath MK IIID-INTDFB Water Bath for Integrated Total Dietary Fibre Procedure
BookD-ADFTB Advanced Dietary Fibre Technology Book
Cat. No. Product Cat. No. Product
Setting New Standards in Test Technology64
Megazyme International IrelandBray Business Park, Bray, Co. Wicklow, Ireland