Flavocytochrome b₂‐Based Enzymatic Method of L‐Lactate Assay in Food Products

Abstract

L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b 2 (EC 1.1.2.3; FC b 2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005–0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 μM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b 2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b 2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

Sodium alginate hydrogelation mediated paper-based POCT sensor for visual distance reading and smartphone-assisted colorimetric dual-signal determination of l-lactate

“…2 Moreover, l -lactate is proven to enhance the digestive function of the human gastrointestinal tract 3 and is widely used as a natural or artificial food additive. 4 For example, l -lactate is often added to yogurt and other fermented foods to adjust their acidity. At optimum concentrations, l -lactate can increase the flavor of yogurt, while too high or too low concentration of l -lactate would result in an undesired taste.…”

Microbial L‐ and D‐Lactate Selective Oxidoreductases as a Very Prospective but Still Uncommon Tool in Commercial Biosensors

“…FC b 2 and DLDH, due to their unique catalytic properties (absolute specificity to the L-or D-enantiomer, respectively, absence of need of the exogenous cofactor, and non-selectivity in respect to the nature of electron acceptors) [17] are very important bioanalytical tools and can replace NAD + -dependent LDH or bacterial LO in quantitative analysis of L-and D-lactate in biological fluids and foods, using spectrophotometric [18,19] and biosensor approaches. [20] However, despite the high prospects in the design of commercial biosensor systems, these enzymes have not been widely used yet.…”