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Enzymatic Food AnalysisBy: Jvo Siegrist, Product Manager Microbiology ivo.siegrist@sial.com, AnalytiX Volume 9 Article 2

Enzymatic methods for food analysis are highly specific and offer considerable time and cost savings over othermethods, especially from the sample preparation standpoint. Sigma-Aldrich offers a wide variety of convenient kits andreagents for rapid and reliable enzymatic food analysis.

Because of the importance of enzymatic methods in food production and assurance of quality and safety, there aremany textbooks, national and international industry associations and government agencies devoted to the subject. Likemany other analytical methods, enzymatic analysis was used first in clinical diagnosis, then adapted for thedetermination of food ingredients. The high specificity of enzymes enables the analysis in complex sample matrixeswithout complicated sample preparation techniques. This makes enzymatic food analysis a highly valuable toolbecause it saves time, reduces costs and gives reliable results independent of the sample matrix. Additionally,enzymatic methods use non-hazardous reagents, are environmentally friendly and can be automated for in-line processmonitoring.

Applications: Carbohydrate and dietary fibre levels

Qualitative and quantitative analysis in the food and beverage industry is extremely important from quality, storage,nutrition and safety standpoints. The levels of certain carbohydrates, like glucose, lactose, fructose, sucrose and starch,affect intolerance conditions, diabetes and obesity. The presence of unwanted carbohydrates or their hydrolysisproducts can alter the manufacturing process or reduce product shelf life. They can also indicate microbialcontamination (e.g. yeasts) or improper processing (e.g. overheating). For fruit juice and wine, raw materials that havevariable sugar content influence the quality of the finished product and should therefore be monitored.

Figure 1 Enzyme assay

Brand Cat. No. Kit Pack SizeDetection method &wavelength

Sigma SCA20 Sucrose Assay KitSufficient for ~20assays NADH; 340 nm

Sigma GAGO20 Glucose (GO) Assay KitSufficient for ~20assays

H2O2; 540 nm

Sigma GAHK20 Glucose (HK) Assay KitSufficient for ~20assays

NADH; 340 nm

Sigma FA20 Fructose Assay KitSufficient for ~20assays

NADH; 340 nm

Sigma STA20 Starch (GO/P) Assay KitSufficient for ~20assays

H2O2; 540 nm

Sigma SA20 Starch (HK) Assay KitSufficient for ~20

assaysNADH; 340 nm

assays

Sigma TDF100A Total Dietary fibre Assay KitSufficient for ~100assays

gravimetric

Fluka TDFC10Total Dietary fibre AssayControl Kit

Sufficient for >=10assays

gravimetric

Table 1 Enzymatic assay kits

Brand Cat. No. Product Required for Kit Number

Sigma-Aldrich 258105 Sulphuric acid, ACS reagent GAGO20, STA20

Sigma-Aldrich 154938 Dimethyl sulphoxide, ACS reagent STA20

Sigma-Aldrich 459844 Ethyl alcohol, ACS reagent STA20, TDF100A

Sigma-Aldrich 184519 Petroleum ether, ACS reagent TDF100A

Sigma-Aldrich 320110 Acetone, ACS reagent TDF100A

Sigma-Aldrich S0876 Sodium phosphate dibasic, anhydrous TDF100A

Sigma-Aldrich S0751 Sodium phosphate monobasic, anhydrous TDF100A

Sigma-Aldrich S2567 Sodium hydroxide, 1.0 M TDF100A

Sigma H3162 Hydrochloric acid, 1.0 M TDF100A

Table 2 Additional required reagents

Determination of dietary fibre is another important food analysis. Consuming high-fibre foods, like fruits, vegetables,nuts and grains, is recommended to treat or prevent such maladies as constipation, haemorrhoids and diverticulitis.Water-soluble fibre also helps decrease blood cholesterol levels. From a chemical perspective, dietary fibre is a mixtureof complex organic substances, including hydrophilic compounds, like soluble and insoluble polysaccharides and non-digestable oligosaccharides, and a range of non-swellable, relatively hydrophobic compounds, like cutins, suberins andlignins. Verifying a high content of dietary fibre in food permits a higher quality grading and access to higher-endproduct markets.

Figure 2 Natural fruit juice contains dietary fibre and many different sugars

Table 3 Standard Official Methods

Detection principle of enzymatic reactions

Enzymatic methods to determine analyte concentration typically employ photometry to measure the concentrationchanges of specific products or substrates during the enzyme-catalysed reaction. Concentration of compound of interestis measured using the reaction stoichiometry.

Some commonly employed enzyme systems include:

Nicotinamide adenine dinucleotide (NADH/NAD+) coenzyme systemActivity of dehydrogenases using the NAD+/NADH system is measured by monitoring the changes inabsorbance at 340 nm. NADH has an absorption maximum at 340 nm, while the oxidised form NAD+ doesnot absorb at this wavelength.

H2O2 production (Oxidase/peroxidase system)Oxidases cleave H2O2 from molecules (e.g. glucose) and o-dianisidine is oxidised with the enzymeperoxidase and H2O2. Oxidized o-dianisidine reacts with sulphuric acid to form a more stable pink-colouredproduct that can be measured at 540 nm.

Carbohydrate assay kits

The kits for the enzymatic assay of sucrose, fructose and starch are based on the enzymatic determination of glucose.The first step is the conversion or degradation of the carbohydrate to glucose or glucose derivate. The glucoseconcentration is then determined according to the two different detection systems: glucose oxidase (GO) or hexokinase(HK) in combination with the glucose-6-phosphate dehydrogenase reaction.

Figure 3 Glucose (GO) assay kit Detection of glucose via glucose oxidase (GO) and peroxidase, monitored at 540 nm.

Figure 4 Glucose (HK) assay kit Detection of glucose via hexokinase (HK) and glucose-6-phosphate dehydrogenase byformation of NADH, monitored at 340 nm.

Figure 5 Sucrose assay kit Sucrose is hydrolysed to glucose and fructose by invertase. Glucose and fructose arephosphorylated with ATP in the hexokinase reaction. Glucose-6- phosphate and NAD are then converted to 6-phosphogluconateand NADH via glucose-6-phosphate dehydrogenase, monitored at 340 nm.

Figure 6 Fructose assay kit Fructose is phosphorylated by ATP in a reaction catalysed by hexokinase. The resulting fructose-6-phosphate is then converted to glucose-6- phosphate by phosphoglucose isomerase. Glucose-6-phosphate and NAD is convertedto 6-phosphogluconate and NADH, monitored at 340 nm.

Figure 7 Starch (GO/P) assay kit The hydrolysis of starch to glucose is catalysed by �-amylase and amyloglucosidase. Glucoseis then converted to gluconic acid and H2O2 by glucose oxidase; detection of H2O2 via peroxidase reaction at 540 nm(proportional to the original starch concentration).

Figure 8 Starch (HK) assay kit The hydrolysis of starch to glucose is catalysed by amyloglucosidase. Glucose is phosphorylatedby ATP in a reaction catalysed by hexokinase. Glucose-6-phosphate and NAD is converted to 6-phosphogluconate and NADH;detection of NADH at 340 nm (proportional to the original starch concentration).

Total dietary fibre assay kit

The total dietary fibre content is determined by a combination of enzymatic and gravimetric methods. Samples of dried,fat-free foods are gelatinised with heat-stable amylase and then enzymatically digested with protease andamyloglucosidase to remove protein and starch in the sample. Ethanol is added to precipitate the soluble dietary fibre.The residue is then filtered and washed with ethanol and acetone. After drying, the residue is weighed. Half of thesample is analysed for protein and the other half is ashed. Total dietary fibre equals weight of residue minus weight ofprotein and ash.

Figure 9 Flow chart of Total dietary fibre assay kit

Additional information and instruction bulletins to all our enzymatic assay kits are available on our website sigma-aldrich.com/enzym-food-kits

Materials

Product # Image Description Molecular Formula Add to Cart

320110 Acetone ACS reagent,≥99.5%

C3H6O pricing

TDFC10 Dietary Fiber, Total, AssayControl Kit sufficient for≥10 assays

pricing

154938 Dimethyl sulfoxide ACSspectrophotometric grade,≥99.9%

C2H6OS pricing

459844 Ethanol ACS reagent,≥99.5% (200 proof),absolute

C2H6O pricing

FA20 Fructose Assay Kit sufficientfor 20 assays

pricing

GAGO20 Glucose (GO) Assay Kitsufficient for 20 assays

pricing

GAHK20 Glucose (HK) Assay Kitsufficient for 20 assays

pricing

184519 Petroleum ether ACSreagent

pricing

S2567 Sodium hydroxide solutionvolumetric, 1.0 M NaOH(1.0N)

HNaO pricing

S0876 Sodium phosphate dibasicReagentPlus®, ≥99.0%

HNa2O4P pricing

S0751 Sodium phosphatemonobasic ReagentPlus®,≥99.0%

H2NaO4P pricing

STA20 Starch (GO/P) Assay Kitsufficient for 20 assays

pricing

SA20 Starch (HK) Assay Kitsufficient for 20 assays

pricing

SCA20 Sucrose Assay Kit sufficientfor 20 assays

pricing

258105 Sulfuric acid ACS reagent,95.0-98.0%

H2O4S pricing

TDF100A Total Dietary Fiber Assay Kitsufficient for ~100 assays

pricing

References

1. Trowell, H. Definitions of Fibre. The Lancet 1974, 303 (7856), 503.

1. Trowell, H. Definitions of Fibre. The Lancet 1974, 303 (7856), 503.2. Trowell, H., Southgate, D. A.; Wolever, T. M. S.; Leeds, A. R., Gassull, M. A.; Jenkins, D. A. Dietary Fibre

Redefined. The Lancet, 1976, 307 (7966), 967.3. Van Soest, P.J. and McQueen, R.W. The chemistry and estimation of fiber. Proc. Nutr. Soc.1973, 32, 123–130.4. Official Methods of Analysis of AOAC International, 16th Edition, Volume II, Section 45.4.07, Method 985.29

(1997).5. Matissek, R.; Schnepel, F.; Steiner, G. Lebensmittelanalytik. 2nd Edition (1992), p.397.