Rapid and accurate determination of d- and l-lactate, lactose and galactose by enzymatic reactions coupled to formation of a fluorochromophore: Applications in food quality control

Abstract

A fluorometric-coupled reaction for the accurate and rapid determination d- and l-lactate and lactose, galactose in foods is presented. The method was found useful for an accurate determination of these metabolites in heterogeneous, opaque and colourful foods without pretreatments. Example for the determination of lactose, galactose, d- and l-lactate in milk, and yogurts and d- and l-lactate in milk, wine and beer is provided. Unexpectedly, we found that the composition of some commercial bio-yogurts produced in Israel is not consistent with the classical definition of yogurts. Thus, this method offers rapid and accurate methodology, which should be particularly valuable in food quality control.

Introduction

Lactic acid (lactate) is generated from glucose in eukaryote and prokaryotic organisms (Berg, Tymoczko, & Stryer, 2002). In mammals, l (+)-lactate (l-lactate) is the major lactate stereoisomer formed in intermediary metabolism and is present in blood. d (−)-Lactate (d-lactate) is also present but only at about 1–5% of the total lactate concentration. d-Lactate found in human physiologic fluids originates from bacterial production in the intestinal tract (Bongaerts et al., 1995, Ewaschuk et al., 2005).

Bacteria can produce both d- and l-lactate and a group of bacteria commonly known as lactic acid bacteria (LAB) produce lactate as the major metabolic end product of carbohydrate fermentation (Salminen, von Wright, & Ouwehand, 2004). Yogurt is a dairy product produced by LAB fermentation of milk lactose to produce lactic acid. Classically, to be named yogurt, the product should be made with the bacterial species’ Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus (Tamime & Deeth, 1980). These bacteria typically form a mixture of l-lactate and d-lactate, with significant contribution of d-lactate to total lactate production. Modern dairy products including yogurts frequently contain added live cultures of LAB such as L. acidophilus, L. casei and Bifid bacterium species, which are commonly known as probiotics (i.e. their consumption confers benefit to the host). The above-mentioned bacteria produce mostly l-lactate as the principal end product (Sarkar, 2008).

Lactose is a disaccharide, mainly found in milk and milk products. d-Galactose is a monosaccharide that can be found as a degradation product of carbohydrates such as lactose and β-galactosidic oligosaccharides in foods; thus both lactose and galactose are found in biological fluids (Berg et al., 2002).

Virtually, all varieties of the yogurts available nowadays in stores in Israel and other countries do not include in their labeling information on lactose, galactose and lactate contents as well as the distribution of lactate among the d- and l-subtypes. Thus, consumers cannot know to which extend yogurts bought in modern markets comply with its original definition. Fermentative products such as wine, pickled vegetables and cured meats and fish produce a mixture of d- and l-lactate. As d-lactate is a specific indicator of bacteria fermentation, it serves as a common indicator for the freshness quality of milk, meat and fruit juice (Soga & Ross, 1997). In the wine industry, the content of d-lactate can indicate wine spoilage by LAB.

Analysis of lactose galactose and lactate involves classical NADH-linked reactions (Berg et al., 2002, Shapiro et al., 2002). Thus, the oxidation of these metabolites may be estimated from the stoichiometrically linked increased absorption of NADH. A common alternative consists of measuring NADH disappearance by coupling its oxidation to NAD with enzyme such as diaphorase.

However, applying monochromatic absorbance photometry for analysis of foods is frequently difficult and problematic (Nielsen, 2003) because many of them are heterogeneous and compound substances: (i) that contain fat droplets of varying size that scatters light in an unpredictable way (e.g., milk and yogurt), (ii) that is opaque and colloidal solution of proteins (e.g., milk and yogurt) that scatter and absorb light, (iii) that contain intense colorant (e.g., red wine) that interfere with the monochromatic absorbance. Overcoming these problems necessitate the use of various pretreatments procedures of samples to minimize the above described problems. However, in many cases such pretreatments only partially resolve the problem, in addition to the fact that many of these procedures are cumbersome and time consuming.

In the present report, we present a fluorometric-coupled reaction for the accurate and rapid determination of lactose, galactose, d- and l-lactate in foods. Thus, this method is useful for their accurate determination in heterogeneous, opaque and colourful foods without pretreatments. Example for the determination of lactose, galactose, d- and l- lactate in milk, and yogurts and d- and l-lactate in wine and beer is provided.