Long-chain vitamin K2 production in LAB is of interest for food industry, but there has not been much effort made to optimize conditions for vitamin K2 production and to elucidate the influencing environmental factors. In this study we examined the levels and forms of MKs (MK-5 to MK-10) from different LAB strains, and the influence of cultivation conditions. In our study, the levels of total vitamin K2 production are expressed as specific concentrations (in nmol vitamin K2 per gram cell dry weight). The values of specific concentrations show the natural vitamin K2 producing capacity of bacteria and may provide insights into the bacterial physiology related to the synthesis of these membrane-embedded menaquinones. Titres were derived from the amount of vitamin K2 recovered from the biomass harvested from the culture medium, expressed as nmol vitamin K2/L medium, and therefore showed a combined effect of vitamin K2 accumulation in the cells and biomass accumulation in the media. The values of titres provide clear information on the product output for a certain amount of medium/material input, and therefore is of strong industrial interest.
We screened L. lactis ssp. cremoris, L. lactis ssp. lactis and Lc. mesenteroides strains, as these LAB (sub)species are known to possess the complete set of genetic elements for menaquinone synthesis  and are frequently used in food fermentation processes. From each (sub)species, the prototype (MG1363) or type strain (DSM20481, DSM20343), together with two other strains isolated from fermented foods, were selected to show a representative picture of the diversity among vitamin K2-producing LAB species and strains. Comparative analysis of vitamin K2 production showed that the specific concentrations and titres of the six selected L. lactis strains varied largely, demonstrating a wide strain diversity in the natural vitamin K2 producing capacity (Fig. 1a, b). The specific concentration values were in the same order of magnitude as reported previously for L. lactis . It is also clear that specific concentrations and titres may not show the same trend. As vitamin K2 is cell membrane-associated, the titres are not only determined by specific concentrations but also biomass yields. For example, strain FM03 showed one of the highest specific concentrations but its titre value was among the lowest due to its low biomass accumulation in the culture media (Additional file 1: Figure S1). We detected no significant amounts of vitamin K2 in Lc. mesenteroides FM06 and DSM20343 and only very minor amounts in FM08 in the tested conditions. Although a complete set of menaquinone-synthesis genes have been identified in Lc. mesenteroides, Brooijmans et al.  also observed the discrepancy that they could not induce respiratory growth in the tested Lc. mesenteroides strain.
Analysis of the relative abundance of long-chain MKs in L. lactis strains showed very similar distributions, and MK-8 and MK-9 were found to be the most abundant (Fig. 1c). The minor amount of vitamin K2 produced by Lc. mesenteroides strain FM08 was found to contain MK-10 as the major form. Given the large strain diversity, strain selection can be an efficient first step towards improved vitamin K2 fortification in fermented foods or food supplements. Both high vitamin K2 producing capacity and high biomass accumulation under selected cultivation conditions should be criteria for strain selection to achieve vitamin K2 fortification. For this reason, we selected prototype strain L. lactis MG1363, which showed both high specific concentration and high titre, to further analyse the impact of selected cultivation parameters on vitamin K2 production in L. lactis.
Using L. lactis MG1363 as the model strain, we demonstrated that vitamin K2 production is influenced by temperature, carbon source, aeration and mode of energy metabolism. Temperature influenced both the total amount and relative abundance of different long-chain MK forms in MG1363. At 30 °C to 33.5 °C the specific concentrations and titres were the highest (Fig. 2a, b), temperatures above or below this range resulted in lower vitamin K2 production. As the temperature increased from 20 to 37 °C, MK-8 became more abundant and MK-9 less (Fig. 2c). This could be caused by the influence of temperature on the activity of the enzyme that controls the isoprenoid side chain elongation during menaquinone synthesis. As MKs are membrane embedded, their relative abundance could also be influenced by the dynamics in biomass accumulation, including the growth rate (shown in Additional file 1: Table S2), composition of lipids and fatty acid side chains, and membrane fluidity, which are all affected by temperature.
The impact of different carbon sources on vitamin K2 production was also significant. Fructose, trehalose, maltose and mannitol all resulted in about twofold increase of vitamin K2 production compared to glucose (Fig. 3a, b). Trehalose stood out for the highest titre level, as it also lead to the highest biomass accumulation (Additional file 1: Figure S3). This is one of the first studies to examine the effect of using trehalose for metabolite production in LAB. Our study demonstrates a positive effect of various carbon sources compared to glucose, on vitamin K2 production in L. lactis.
Vitamin K2 is known for its function as electron carrier in the bacterial ETC, and L. lactis can switch to respiratory metabolism when both oxygen and heme are supplied . Moreover, the presence of oxygen alone has impact on metabolism by altering the balance of redox cofactors, as well as expression and activity of key enzymes . Therefore, it was highly relevant to examine whether vitamin K2 production was influenced by aeration only (fermentation under aerobic conditions) and respiratory metabolism (aerobic respiration). We demonstrated production of MK-5 to MK-10 under static fermentation, aerobic fermentation and aerobic respiration conditions, and a shift towards longer chained MKs under the two aerobic conditions (Fig. 4). This is in agreement with the study from Brooijmans et al.  regarding the quantity of MK-5 to MK-10. We observed increased specific concentrations of MK-5 to MK-10 under both aerobic conditions compared to static fermentation, which could suggest specific roles of long-chain MKs in aerobic conditions. Moreover, as the increase was observed for both aerobic fermentation and aerobic respiration, it is likely that MK production responded to oxygen, and not necessarily a functional respiratory ETC. A transcriptomic analysis from Cretenet et al.  also revealed that the first gene in menaquinone-synthesis pathway, menF, is upregulated by 2.7-fold during the early stage of cultivation under oxygen condition compared to anaerobic condition in MG1363. Pedersen et al.  also detected up-regulation of another menaquinone-synthesis enzyme, menB, under aerobic condition compared to static cultivation in a L. lactis strain. Increased activity of the menaquinone-synthesis pathway could explain the increased vitamin K2 content in aerobically cultivated L. lactis MG1363 cells. The increase of MK production was even more obvious in terms of titres, since besides higher specific concentrations, the biomass accumulations in aerobic fermentation and aerobic respiration were also higher than in static fermentation (Additional file 1: Figure S4). A higher biomass yield under aerobic conditions was also reported by Nordkvist et al. and they propose that this was because of less energy limitation for biomass synthesis . Respiratory metabolism is known to enhance the growth efficiency in L. lactis , and indeed we obtained the highest biomass accumulation with aerobic respiration, reaching a more than twofold increase compared to static fermentation.
When we further examined the aerobic fermentation conditions by applying different degrees of aeration, we observed that all aerobic fermentation conditions clearly resulted in enhanced vitamin K2 production compared to static fermentation (Fig. 5a, b), and the enhancement did not seem to be a function of the degree of aeration.
When the effects of aeration and respiratory metabolism were examined with carbon sources other than glucose, differences in response were observed (Fig. 6a, b). With fructose as the carbon source, aerobic fermentation resulted in the highest vitamin K2 production, while aerobic respiration was similar to static fermentation. When trehalose was used, aerobic fermentation resulted in similar level as static fermentation, while aerobic respiration resulted in much lower vitamin K2 production. Therefore, the effect of aerobic fermentation and aerobic respiration on vitamin K2 production was found to be carbon source-dependent. It was found to be consistent though, for all 3 carbon sources tested, the biomass accumulation was highest under aerobic respiration, followed by aerobic fermentation, and lowest under static fermentation (Additional file 1: Figure S6). The MK profile also reflected that for all 3 carbon sources, aerated conditions lead to a shift towards longer chained MKs compared to static fermentation (Fig. 6c).
The reason why each carbon source or cultivation condition influenced vitamin K2 production in this way remains to be elaborated. As a secondary metabolite, the metabolic fluxes towards vitamin K2 are complicated with a variety of enzymatic reactions involved. The flux towards chorismate, which is the first compound in menaquinone biosynthesis pathway, could be influenced by the central carbon metabolism. As different carbon sources are taken up, converted and directed to the central carbon metabolism, the conversion rate, energy transduction, redox factor regeneration and regulation of the sugar catabolism all vary [34, 35]. This could globally explain why the amount of vitamin K2 production in L. lactis responded to aeration as well as respiration in a carbon source-dependent manner. A series of genes are required for menaquinone synthesis, some dedicated to the naphthoquinone synthesis, some to the elongation of the isoprenoid tail. Together, they influence the quantity and composition of the menaquinone pool. However, to date, regulations of these genes under different conditions have been hardly investigated , and little is known about feedback regulation in this pathway, especially in LAB. A mechanistic explanation for the response towards each specific carbon source or cultivation condition requires further studies and the construction of a comprehensive metabolic model.
We further demonstrated that the obtained knowledge can be transferred to food fermentation processes by fortifying quark with vitamin K2. Consistently, cultivation temperature of 30 °C, altered carbon source (fructose) and aerobic cultivation of the pre-culture, resulted in higher vitamin K2 content in the quark product.
The insights obtained from this study show a proof of principle that strain selection and combination of favourable cultivation parameters, namely temperature, aeration, carbon source and mode of energy metabolism, can contribute to improved production of long-chain vitamin K2 in LAB strains. As hydrophobic, membrane-embedded compounds, menaquinones are not produced extracellularly and continuously, but remain cell-associated in all producing bacteria. Nevertheless, simple and efficient procedures are being continuously developed and optimized for extracting vitamin K2 and other valuable cell-associated molecules from biomass [37,38,39], that, together with the knowledge obtained from this study, will facilitate the biotechnological production of long-chain vitamin K2.
The effect of different carbon sources also demonstrate the potential of long-chain vitamin K2 fortification during fermentation of different raw food materials, enabling utilization of low-value substrates in industrial processes or development of vitamin K2-enriched fermentation-based food products as exemplified in the current study with the production of vitamin K2-fortified quark. Since vegetables like cabbage , beetroots and carrots  contain high levels of fructose, they could serve as excellent sources for in situ vitamin K2 fortification through fermentation with selected LAB.