A rapid and efficient method for the extraction and identification of menaquinones from Actinomycetes in wet biomass

Background Menaquinones are constituents of prokaryote cell membranes where they play important functions during electron transport. Menaquinone profiles are strongly recommended for species classification when proposing a new Actinomycetes taxon. Presently, the most widely used methods to determine menaquinones are based on freeze-dried cells. Taxonomic research in our lab has revealed that menaquinone concentrations are low for some species of the genus Microbacterium, leading to difficulties in identifying menaquinones. Results Menaquinones extracted using the novel lysozyme-chloroform-methanol (LCM) method were comparable in quality to those obtained using the Collins method, the most widely used method. All tested strains extracted via the LCM method showed higher concentrations of menaquinones than those extracted via the Collins method. For some Microbacterium strains, the LCM method exhibited higher sensitivity than the Collins method, and more trace menaquinones were detected with the LCM method than the Collins method. In addition, LCM method is faster than the Collins method because it uses wet cells. Conclusion The LCM method is a simple, rapid and efficient technique for the extraction and identification of menaquinones from Actinomycetes. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-021-02240-z.

when using 300 mg of freeze-dried cells [11]. Low concentrations of MKs lead to difficulties in identifying MKs. In 2005, a water-soluble MK-7 extraction method for Bacillus subtilis natto was reported that demonstrated an increased MK-7 concentration using lysozyme [12]. In 2019, the effect of pretreatment methods for disrupting Bacillus subtilis natto cells was reported. Higher yields of MK-7 were obtained with chemical pretreatment methods (including the use of methanol, ethanol, n-propanol and 2propanol) than enzyme, freezing and heating or ultrasound treatments [13]. In this study, chemical methods using methanol, ethanol and ethyl acetate were initially used to disrupt wet cells of some Microbacterium strains before MK extraction. However, non-visible MK bands were found. Fortunately, higher yields of MKs were obtained by disrupting wet cells with lysozyme than those obtained using the Collins et al. (1977) methodology. Here, an improved, simple, rapid (1-day) and highresolution MK analysis method is reported for Actinomycetes.

Extraction of menaquinones
The cells used for MK extraction were divided equally into two parts. For the lysozyme-chloroform-methanol (LCM) method proposed in this study, wet cells can be directly used for MK extraction. With this procedure, it takes about 3 h to acquire a crude extract of MKs. However, the Collins method requires hours or even an overnight period to acquire freeze-dried cells and then an overnight extraction is needed to obtain the crude extract of MKs. Therefore, the LCM method is faster than the Collins method.
In addition to the time saving feature, the LCM method obtains higher concentrations of MKs from Actinomycetes. In this study, the MK concentrations of the tested strains ranged from 0.063 to 0.921 mg/g dry cell weight (DCW) for the LCM method and 0.001 to 0.591 mg/g DCW for the Collins method (Table 1). When the LCM method was used, the MK concentration from Saccharopolyspora coralli E2A T was the lowest (0.063 mg/g DCW) among all the tested strains, but this concentration was higher than that of most strains extracted by the Collins method. Microbacterium ureisolvens CFH S00084 T showed the lowest MK concentration (0.001 ± 0.001 mg/g DCW) with an almost invisible MK band on thin layer chromatography (TLC) plates using the Collins method. However, when the LCM method was used, the MK concentration of strain CFH S00084 T was 355fold higher (P < 0.01) than that of the Collins method. In addition, the MK concentration of Brachybacterium squillarum JCM 16464 T extracted via the LCM method was 33.2-fold higher (P < 0.01) than that of the Collins method. The MK concentrations of other strains  extracted by the LCM method were 1.2-17.1-fold higher (P < 0.01) than those extracted by the Collins method.
As an example, Fig. 2 shows MKs extracted from strain Microbacterium yannicii JCM 18959 T using both methods and then detected by ultra-performance liquid chromatography (UPLC). Other strains are shown in Supplementary Figures 1 to 11 (Additional file 1). In general, for the same cell biomass, the MK concentration obtained by the LCM method was higher than that obtained by the Collins method.

Identification of menaquinones
As is well known, the UPLC system is faster and more sensitive than the high-performance liquid chromatography (HPLC) system. Thus, the UPLC system was used first for MK identification. The MK-9 standard and MKs extracted from strains O2 (MK-5 to MK-8), A18JL200 (MK-10 to MK-13) and NY27 (MK-13 to MK-15) were mixed as controls for method establishment. MK-5 to MK-15 were observed using the UPLC system at retention times of 1  3C). In this study, both the HPLC and UPLC systems separated MK-5 to MK-15 well, with no significant difference between the two systems in their ability to separate these MKs. A larger sample volume (10 μL) may be used to acquire a better signal for the HPLC system. The absorption spectrum of each MK exhibited two peaks: 247.5 and 269.5 nm for the UPLC system ( Fig. 3B) and 248.3 and 269.6 nm for the HPLC system (Fig. 3D). The molecular mass of the MKs detected by mass spectrometer (MS), as shown by the mass spectra ( Fig. 4), most accurately matched the ion fragment of [M + Na] + ( Table 2). According to the spectral absorption peak and molecular mass, we could confirm each of the MKs. Unsaturated MKs were observed for most of the strains in this study. Hydrogenated MKs of MK-5 (H2), MK-7 (H2) and MK-8 (H2) were observed in strain O2; MK-9 (H2, H4) was observed in strain E2A T ; and MK-9 (H4, H6, H8) was observed in strain MCCC 1A03308 T . MK-9 (H4), MK-9 (H6) and MK-9 (H8) from Streptomyces indicus MCCC 1A03308 T extracted via the LCM method separated well using the UPLC system, showing retention times of 3.54, 3.97 and 4.52 min, respectively ( Fig. 5). Hydrogenated MKs also separated well using the HPLC system. MKs of the tested strains using the LCM and Collins methods are shown in Table 3. The corresponding UPLC-UV data for these strains are shown in Supplementary Figures1 to 11 (Additional file 1). The main MKs of all the strains detected using both methods were primarily the same as reported ( Table 3). The MK composition of the tested strains extracted with both the LCM and Collins methods were the same except for M. ureisolvens CFH S00084 T and M. ginsengiterrae JCM 15516 T . Specifically, MK-12 and MK-13 were the observed MKs from strains CFH S000084 T and JCM 15516 T using the Collins method; however, along with MK-12 and MK-13, MK-11 and MK-14 were also detected with significant peaks in these two strains when using the LCM method (Additional file 1: Supplementary Figure 6 and 7).

Discussion
To validate the LCM method, the popular MK extraction protocol described by Collins et al. (1977) was used as a control to test the reliability of our MK extraction method from wet cells. During exploratory development of the LCM method, chloroform:methanol (2:1 v/v) was first used to extract MKs directly from wet cells without a lysozyme pretreatment; however, no MKs were obtained from Microbacterium strains. Besides wet cells, freeze-dried cells can also be used for the LCM method, and there is no significant difference in MK concentrations between the wet cells and freeze-dried cells. However, the use of freeze-dried cells will extend the experimental time. For the lysozyme treatment conditions, the optimal concentration of lysozyme and incubation time are also different for different strains. Generally, most of the cells can be lysed with a final lysozyme concentration of 1 mg/mL and an incubation time of 30 min to 1 h [27]. If the concentration of MKs is low for LCM method, increasing the concentration of lysozyme and prolonging the treatment time will provide better results. Water in the lysozyme-treated cells should be removed using methanol or ethanol before the chloroform-methanol extraction step, otherwise the extraction efficiency will be reduced. TLC is an important purification and verification step during the MK extraction. Purified MKs were easily identified. However, if the MK concentration is low, the MK band (Rf ≈ 0.7) on the TLC plate will be weak and not easily observed. As a result, minor MK components would not be detected by UPLC/MS or HPLC/MS. Accordingly, the MK intensity can be increased by concentrating the extract or increasing the injection volume. Better separation of MKs can be obtained using the ACQUITY UPLC® HSS C18 column (1.8 μm 2.1 × 100 mm) with the UPLC system. The elution time is important for MK analysis. In order to avoid invalid elution, it is better to use an MK-15 standard as a reference for the analysis of UPLC or HPLC results, especially when the strain contains MK-12 to MK-15. In addition, MKs are delicate components that are easily degraded. To prevent photo-oxidation, strong light should be avoided during the MK extraction process.
Overall, the results obtained using the LCM method were comparable in quality to those obtained using the standard freeze-dried approach. All tested strains extracted via the LCM method showed higher concentrations of MKs when compared with those extracted by the Collins method. Due to the high efficiency of MK extraction, the LCM method may serve as a general method for MK identification and screening of vitamin K 2 -producing Actinomycetes strains.

Strain cultivation and collection
All strains used in this research were cultured on 2216E agar plates. A single colony was incubated in test tube containing 5 mL of 2216E medium at 28°C with 150 rpm shaking until an optical density (OD at 600 nm) of 0.6 was reached. Next, 2 mL of seed culture was added into 200 mL of 2216E medium in 1-L Erlenmeyer flasks and incubated for 3 to 4 days on a rotary shaker at 150 rpm and 28°C. Cells were divided into two equal parts for different MK extraction methods and then collected in 50-mL centrifugal tubes using an Eppendorf 5804R centrifuge at 6000×g for 15 min. Three independent replicates of strain cultivation and collection were carried out.

Lysozyme-chloroform-methanol extraction method
Since the thick cell wall of Actinomycetes can resist organic solvent extraction, an improved method named the lysozyme-chloroform-methanol (LCM) method was used to extract MKs. For this method, lysozyme was used to break the cell walls, followed by a chloroformmethanol (2:1 v/v) extraction. After concentration using a rotary evaporator and purification via thin-layer chromatography (TLC), MKs were observed using a UV analyzer at 254 nm and eluted using isopropanol. Three replicates were carried out for the LCM method in this study. Extraction steps were as follows: 1. Wet cellular mass (0.7-1.0 g) was acquired for each strain as described above. 2. Cells were washed twice with 20 mL of 10 mM Tris-HCl buffer (pH 7.4) to avoid media contamination, and then they were suspended in 50 mL of 10 mM Tris-HCl buffer (pH 7.4) with 50 mg of lysozyme. The solution was mixed well by shanking for 1 min and incubated in a 37°C water bath for 1 h (with 1 min of shaking every 10 min) to digest the cell wall. Next, the mixture was centrifuged for 15 min at 6000×g to collect the lysozyme-treated cells.
(Note: the cell walls of some Actinomycetes are easy digested by lysozyme, forming a colloidal solution that is hard to centrifuge; thus, the next water removal step is important.) 3. Lysozyme-treated cells were washed with 5 mL of methanol (or ethanol) to remove water. The methanol (or ethanol) should be collected because it can dissolve some MKs. (Note: for easily-digested Actinomycetes, remove the water as much as possible after centrifugation and then add an equal volume of methanol (or ethanol) to the remaining solution. Shake several times and then centrifuge for 15 min at 6000×g. Cells can then be collected and washed with 5 mL of methanol (or ethanol). The top water-methanol solution should also be collected.) 4. Chloroform/methanol (10 mL, 2:1 v/v) was added to the cells and then shaken for 1 min to extract MKs. The chloroform-methanol extraction was repeated three times. Approximately 30 mL of crude extract was acquired. 5. The methanol (or ethanol) and chloroformmethanol extracts were collected and dried using a rotary evaporator at 35°C. The dry product was then dissolved with 500 μL of chloroform-methanol (2:1 v/v) three to four times. 6. The chloroform-methanol-dissolved crude extract was purified by TLC, which was performed using 0.

Quantification of menaquinones
Three replicates were carried out to compare the LCM method and the Collins method. For each replicate, cells of each strain were divided into two equal parts, one for the LCM method and the other for the Collins method. Tubes containing wet cells for the Collins method were freeze-dried for 12 h. The dry cell weight (DCW) was calculated as: DCW = the total weight of the centrifuge tube containing dry cells -weight of the empty centrifuge tube. The DCW used in the LCM method was based on that used for the Collins method. MK concentrations were measured via the UPLC system, using MK-9 as the standard, as previously described [28]. The yield was expressed as mg menaquinones/g dry biomass. The data were analyzed for comparison using SPSS 19 software and values are reported as mean ± standard deviation (SD) (n = 3). One-way analysis of variance (ANOVA) was used to test the significance of the two methods.