Abstract
Diacetyl and acetoin are key aroma components of fermented milk but are produced in low concentrations by starter cultures. In this study, we expressed NADH oxidase, acetolactate synthase, and inactivated acetolactate decarboxylase in Lacticaseibacillus casei TCS to generate recombinant L. casei strains, and investigated the effects of the genes encoding these enzymes on diacetyl and acetoin production during milk fermentation. In the single-gene recombinant strains tested, diacetyl concentrations were highest in milk fermented by L. casei TCSI-nox (nox gene overexpressed, 3.68 mg/kg), whereas acetoin concentrations were highest in milk fermented by L. casei TCS-ΔalsD (alsD gene deleted, 32.94 mg/kg). Moreover, diacetyl and acetoin concentrations were higher in the inducible strains than in the corresponding constitutive strains (e.g., TCSI-nox vs. TCSC-nox, and TCSI-ΔalsD-nox vs. TCSC-ΔalsD-nox). This phenomenon was also reflected in the protein expression levels and enzyme activities. In the double-gene recombinant strains tested, the highest concentrations of diacetyl and acetoin were produced by L. casei TCSI-ΔalsD-nox (nox overexpressed and alsD deleted, 4.66 mg/kg, 69.62 mg/kg, respectively). The triple-gene recombinant L. casei TCS-ΔalsD-nox-alsS produced the highest concentrations of diacetyl and acetoin, which were 2.38 and 11.19 times, respectively, the concentrations produced by the original strain. These results show that the nox, alsS, and alsD genes make key contributions to the biosynthesis of diacetyl and acetoin by L. casei. The modification of multiple genes had a synergistic effect, leading to greatly increased synthesis of diacetyl and acetoin by L. casei during its fermentation of milk.
Highlights
Fermented milk is a dairy product made by fermenting raw bovine, caprine, or ovine milk, or the corresponding powdered milk, with lactic acid bacteria (LAB) as the starter cultures (Tamime, 2002)
When L. casei TCSI-nox was induced for 59 h and L. casei TCSI-∆alsD-nox was induced for 42 h, an intense protein band was seen at 45 to 60 kDa (Figure 2a, b), which is close to the expected size of recombinant NOX
Recombinant strains with a single-gene modification produced more diacetyl and acetoin than the original strain during milk fermentation, and this was accompanied by increased expression levels and activities of key enzymes involved in the synthesis of diacetyl and acetoin
Summary
Fermented milk is a dairy product made by fermenting raw bovine, caprine, or ovine milk, or the corresponding powdered milk, with lactic acid bacteria (LAB) as the starter cultures (Tamime, 2002). Fermented milk is one of the most popular fermented dairy products globally, due to its texture, nutrition, and flavor (Shiby and Mishra, 2013; Chen et al, 2017). Diacetyl and acetoin can be synthesized by microorganisms during fermentation (Sieuwerts et al, 2008; Chen et al, 2017; Figure 1). The biosynthetic precursors for diacetyl and acetoin are usually citric acid and glucose. Diacetyl is generated from α-acetolactate through nonenzymatic oxidative decarboxylation under acidic conditions. Acetoin is generated either from α-acetolactate by α-acetolactate decarboxylase or from diacetyl by diacetyl reductase. Diacetyl and acetoin are either excreted as end products or partially reduced to butanediol catalyzed by acetoin reductase (Papagianni, 2012; Yang et al, 2017). The application and development of genomic and metabolomic technologies have enabled
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