Abstract
BackgroundDiacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl.ResultsBy expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate.ConclusionsAn efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.
Highlights
Diacetyl provides the buttery aroma in products such as butter and margarine
The excess pyruvate is subsequently consumed via the acetoin pathway, comprised of α-acetolactate synthase (ALS) and α-acetolactate decarboxylase (ALD) (Fig. 1). α-Acetolactate, the intermediate in this pathway, is inherently unstable and can either be enzymatically converted into acetoin or undergo spontaneous decarboxylation to diacetyl under aerobic condition [4, 8,9,10,11]
We envisioned that using a production strategy based on whole cell based biocatalysis might be a superior approach, e.g. by using pyruvate as a substrate
Summary
Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Lactis biovar diacetylactis citrate is transported into the cell via the citrate permease (CitP) and cleaved by citrate lyase (CL) into acetate and oxaloacetate. The latter is subsequently decarboxylated by oxaloacetate decarboxylase (OD) into pyruvate. Thereby a long-lasting taste is obtained, and high initial concentrations of diacetyl are avoided [21]
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