A simple and efficient method for extraction and quantification of capsaicin from pepper fruits through high performance thin layer chromatography

Bioprocess Biosyst Eng DOI 10.1007/s00449-017-1851-6 RESEARCH PAPER A simple and efficient method for the extraction and separation of menaquinone homologs from wet biomass of Flavobacterium Hongfei Wei1,2 · Genhai Zhao1 · Hui Liu1 · Han Wang1,2 · Wenfeng Ni1,2 · Peng Wang1 · Zhiming Zheng1 Received: 24 May 2017 / Accepted: 4 October 2017 © Springer-Verlag GmbH Germany 2017 Abstract Menaquinone homologs (MK-n), that is, MK-4, MK-5, and MK-6, can be produced by the fermentation of Flavobacterium. In this study, we proposed a simple and efficient method for the extraction of menaquinones from wet cells without the process of drying the biomass. Meanwhile, a rapid and effective solution for the separation of menaquinone homologs was developed using a single organic solvent, which was conducive to the recovery of the solvent. The results showed that the highest yield was obtained with pretreatment using absolute ethanol at a ratio of 6:1 (v/m) for 30 min and then two extractions of 30 min each using methanol at a ratio of 6:1 (v/m). The recovery efficiency of the menaquinones reached to 102.8% compared to the positive control. The menaquinone homologs were effectively separated using methanol as eluent at a flow rate of 0.52 mL/ min by a glass reverse-phase C18 silica gel column with a height-to-diameter ratio of 5.5:1. The recovery of menaquinones achieved was 99.6%. In conclusion, the methods for extraction and separation of menaquinone homologs from wet Flavobacterium cells were simple and efficient, which makes them suitable not only on a laboratory scale but also for application on a large scale. * Peng Wang pengwang@ipp.ac.cn * Zhiming Zheng zhengzhiming2014@163.com 1 Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China 2 University of Science and Technology of China, Hefei 230026, People’s Republic of China Keywords Wet cell extraction · Separation · Menaquinone homologs · Flavobacterium Introduction Menaquinone (MK)-n refer to a series of naphthoquinone derivatives, where n denotes the number of isoprene units in the side chain. It is generally presumed that menaquinones are produced during the fermentation of bacteria such as Escherichia coli, Flavobacterium, Deinococci, and Bacillus subtilis natto [1, 2]. The previous studies have shown that menaquinones are more effective clinically than phylloquinone with respect to osteoclastogenesis, hypocholesterolemic effects, and ability to slow atherosclerotic progression [3]. Bioassay data suggested that menaquinones as compared to phylloquinone were more efficient in reversing vitamin K deficiency, which, indeed, is due to their longer duration of biological response and slower hepatic turnover [4, 5]. However, these valuable compounds are not readily available at an affordable price, because their production involves several tedious and inefficient unit operations [6]. The extraction of menaquinones from the fermentation medium plays a critical role. Apart from the fermentation conditions, low extraction efficiency results in product loss and time consumption,even when high concentrations are achieved in the fermentation medium. Menaquinones are commonly recovered from microbial cells by a solid-phase extraction technique [7, 8], liquid–liquid extraction technique [2, 6, 9, 10], or supercritical fluid extraction technique [11–13]. However, all of these extraction methods are associated with some disadvantages as they involve complex procedures such as time-consuming sample pretreatment in the drying step and different organic solution proportions or expensive devices and high energy input. Therefore, it 13 Vol.:(0123456789) Bioprocess Biosyst Eng is necessary to develop a rapid and efficient method for the extraction of menaquinones from fermentation media. The characteristics of the side chain have a significant impact on the lipophilicity of a vitamer and may also result in substantial differences in properties such as intestinal absorption, transport, tissue distribution, and bioavailability [14]. There are several studies that correlate a high concentration of MK-4 with the levels of sphingomyelin and sulfatides in the brain, suggesting its role in the biosynthesis of this important class of lipids [15, 16]. On the other hand, some studies suggest that the bioavailability of menaquinone is related to the length of the side chain, such that medium-length menaquinones are more bioavailable than short-chain ones [17]. Obviously, menaquinones with side chains of different lengths could have different functions in the body. Therefore, it is necessary to separate the menaquinone homologues so as to match the remedy to the case and improve the efficiency of menaquinones. The present study proposed a rapid and effective method for the extraction of menaquinones from wet biomass of Flavobacterium fermentation, in which the time and energy consumed in drying the cells was omitted. We designed different experimental conditions that involved varying the solvent, processing time, and water percentage of the wet microbes for optimization purposes. Meanwhile, we developed a simple method for separating menaquinone homologues produced by Flavobacterium using a single organic solvent. Several separation conditions, including the choice of organic solvent, flow rate, height-to-diameter ratio, recovery ratio, and loading quantity of the menaquinones were evaluated to optimize the process. Materials and methods Reagents and standards Chromatographic quality methanol and dichloromethane obtained from Sigma–Aldrich (USA) were used as eluents in the mobile phase of HPLC. All other chemicals and organic solvents purchased from Sinopharm Chemical Reagent Co. Ltd. (China) were used to extract menaquinones from wet microbes and for the culture substrate. The menaquinone standard, MK-4, was purchased from Sigma-Aldrich (USA). The stock solutions were individually prepared in methanol (Sigma) and protected from light exposure and evaporation during storage. HPLC conditions and statistical analysis The extracted menaquinones were analyzed with an LC-16 HPLC system (Shimadzu, Kyoto, Japan) equipped with a UV–Vis detector (SPD-16, Shimadzu) and column oven 13 (CTO-16, Shimadzu). The analytical column was a reversephase C18 (VP-ODS, 4.6 mm I.D. × 250 mm, Shimadzu), and the temperature of the column was maintained at 35 °C. The UV–Vis detector was operated at 248 nm for menaquinones. A mixture of methanol and dichloromethane (4:1, v/v) was used as the mobile phase at a flow rate of 1.0 mL/ min. Aliquots of 20 μL were injected manually using a loop injection valve (Shimadzu). Menaquinone species were identified and quantified according to their retention times and the UV spectrum of each peak was observed in UV detector. Menaquinone-4 (MK-4) was used as the quantitative standard for the menaquinones. The amounts of menaquinones were calculated from the peak areas based on the standard curves of MK-4. All extraction experiments were repeated at least three times and the data were analyzed for comparison by the software SPSS 19 and reported as mean ± standard deviation (SD). The yield was expressed as mg menaquinones/g dry biomass. Cell cultures and harvesting The media consisted of 2% glycerol, 1% peptone (fish), 0.15% yeast extract, 0.45% K2HPO4, 0.3% NaCl, and 0.03% MgSO4·7H2O were inoculated with 10% Flavobacterium seed solution. Fermentation was conducted for 6 days aerobically at 37 °C and 200 rpm in a rotary shaker in a 30-L fermenter containing 18 L of identical media. Sample preparation At the end of cultivation, the cells in 25 mL of fermentation liquor were placed in a 50-mL centrifuge tube and centrifuged at 12,000 rpm for 15 min. The weight of the isolated wet cells was calculated as: wet cells weight (WCW) = total weight of the centrifuge tube containing wet cells − weight of the empty centrifuge tube. A few of the tubes containing wet cells were lyophilized for 24 h. And then, the weight of the dry cells was calculated as: dry cells weight (DCW) = total weight of the centrifuge tube containing dry cells − weight of the empty centrifuge tube. The water content in the cells was calculated from the following formula: water content = (WCW − DCW)/WCW × 100%. Extraction of menaquinones from dry cells The dry samples were crushed and then treated according to the following procedures: Various organic solvents, including methanol, absolute ethyl alcohol, acetone, n-butanol, trichloromethane, isopropyl alcohol, acetonitrile, n-hexane, and petroleum ether were individually added to the centrifuge tubes at a ratio of 10:1 (v:m) and mixed with the bacterial sludge followed by quiescent extraction for 1 h. Then, the samples were centrifuged at Bioprocess Biosyst Eng 12,000 rpm for 10 min and each supernatant was collected as the first extraction solution. The foregoing extraction steps were repeated twice, and the second and third extraction solutions were collected. All extractions were conducted at room temperature. The volume of the three extracts supernatant was quantified, and the content of menaquinones was measured and calculated with HPLC for future analysis. The organic solvent that gave the maximum extraction yield of menaquinones from the dry cells was designated as the positive control. Extraction of menaquinones from wet microbes The extraction procedure from the wet microbes was the same as that from the dry cells except for the sample pretreatment by lyophilization. The organic solvent that gave the maximum extraction yield of menaquinones from the wet cells was designated as the negative control. Improved extraction procedures for wet cells An appropriate volume of absolute ethyl alcohol was added to the wet microbes contained in the centrifuge tubes, so that the ratio of the volume of ethanol to the weight of wet cells was ranged from 2:1 to 7:1 (v:m). At the same time, an appropriate volume of methanol was added to the positive control (dry cells) and negative control (wet cells) at a ratio of 6:1 (v:m). And then, the samples were mixed, followed by quiescent extraction for 1 h. Each supernatant was collected as the first extraction solution after centrifuging at 12,000 rpm for 10 min. The same proportion of methanol as used for the first extraction of the controls was added to the samples and controls, mixed, and extracted quiescently for 1 h. After centrifugation, the supernatants were designated the second extracts. This step was repeated once to obtain a set of third extracts. The three supernatant of each sample were quantified with HPLC and the total content of menaquinones was calculated. All samples were conducted in triplicate. Separation of menaquinone homologs The extracts of menaquinones were evaporated to dryness under reduced pressure and re-dissolved in a single organic solvent such as methanol, absolute ethyl alcohol, or acetonitrile. A glass column filled with reverse-phase C18 silica gel was made up according to the manufacturer’s instructions. Different organic solvents were added to elute the menaquinones. The flow rates, height-to-diameter ratios, and recovery ratios of the menaquinones were evaluated for optimization purposes. The menaquinone homologs were determined by their absorptions and mass spectra with an LC–MS system equipped with a 6200 series TOF/6500 series analyzer (Agilent Technologies, Santa Clara, CA, USA). Results Effect of extractions of menaquinones from dried and wet microbes The extractions of menaquinones from dry Flavobacterium cells were initially carried out using different organic solvents at room temperature without any modifier, and the extraction yields are shown in Fig. 1. Obviously, the extraction yields obtained by using methanol, absolute ethyl alcohol, acetic acid, and n-butanol were higher than those of the other solvents. The highest extraction yield was obtained using methanol (P < 0.05) and was set as the positive control in the following experiments. The extraction yields of menaquinones from wet microbes using different organic solvents are shown in Fig. 2. The Optimization of pretreatment time for the wet biomass The optimal ratios of absolute ethyl alcohol and methanol to wet cells were used for extractions. Firsts, the samples in centrifuge tubes were pretreated with ethanol quiescently for 10, 20, 30, or 40 min, and then, they were extracted with methanol twice for 30 min each time. The three supernatant of each sample were quantified with HPLC and the total contents of menaquinones were calculated. All samples were conducted in triplicate. Fig. 1 Concentration of menaquinones obtained from dry cells using different extractants. Extraction was carried out three times with 5 mL of organic solvent for 1 h each time at room temperature 13 Bioprocess Biosyst Eng Fig. 2 Concentration of menaquinones obtained from wet cells using different extractants. Extraction conditions were the same as in Fig. 1 maximum extraction yield from wet microbes was obtained with methanol, followed by absolute ethyl alcohol, isopropanol, acetic acid, and acetonitrile. Obviously, the extraction amounts obtained from dichloromethane, n-hexane, and petroleum ether were significantly lower than those obtained from the other organic solvents (P < 0.05). However, each of the extraction yields from wet cells was less than that of the control. This may be because the presence of water reduced the extraction rate. Effect of water content on extraction yield Extraction from wet cells was conducted using methanol and ethanol solutions containing different water content, respectively. Figure 3 shows that the extraction amounts of menaquinones extracted gradually increased with a reduction in water content in both methanol and ethanol systems. The extraction yields obtained using methanol were significantly less than those for ethanol when the content of organic solvents was less than 95%, while they were slightly higher than those of ethanol when the content was exceeded 95% (P < 0.05). However, each of the menaquinone yields obtained from wet cells was less than that of the positive control. Fig. 3 Effect of different water contents on extraction yields of menaquinones using methanol and ethanol ratio of 6:1 (v/m) each time. Compared to that of the positive control, the highest recovery efficiency of menaquinones reached 102.8%, which was much higher than that of the negative control (93.3%) (P < 0.05). As shown previously, the water content had a significant effect on the extraction yield from wet cells. Moreover, the amount of time required to extract the maximum amount of menaquinones from wet cells is an important parameter to consider. Wet cells samples were pretreated with absolute ethyl alcohol for 10, 20, 30, and 40 min, and extracted twice with methanol for 30 min (Fig. 5). The results showed that pretreatment with absolute ethyl alcohol from 10 to 40 min gave a significant improvement with the highest extraction efficiency occurring at 30 min (P < 0.05). The extraction rates were almost equal from 30 Effect of improved extraction procedures for wet cells To improve the extraction yields of menaquinones directly from wet biomass, we pretreated the wet microbes using absolute ethyl alcohol at different ratios (v/m). The results indicated that the pretreatment could significantly enhance the extraction yields of menaquinones (Fig. 4). The highest yield was obtained by extracting twice using methanol at a 13 Fig. 4 Effect of pretreatment of wet cells with different volume-tomass ratios on the recovery of menaquinones (pretreatment once with ethanol and extraction twice with methanol) Bioprocess Biosyst Eng Discussion Effect of extraction of menaquinones from dry and wet microbes Fig. 5 Effect of different pretreatment times using ethanol on the recovery of menaquinones to 40 min and stabilized after 30 min. Therefore, a 30-min pretreatment time was an optimal preprocessing time. Separation of the menaquinone homologs The polarity of the organic solvent was an important consideration for the separation of the menaquinone homologs. Several factors including the flow rate of the eluent, the ratio of height to diameter, and the recovery rate of menaquinones played important roles in the separation. In the present study, the menaquinone homologs, that is, MK-4, MK-5, and MK-6, were effectively separated using methanol, absolute ethyl alcohol, or acetonitrile in a glass reverse-phase C18 silica gel column (Table 1). The results showed that the flow rate and recovery ratio using methanol were higher than those for both absolute ethyl alcohol and acetonitrile, but the content of menaquinones per mass of silica gel was lowest. Taking the high value of the menaquinones and the reusability of the silica gel into account, methanol was selected as the optimum eluent. Mass spectra yielded m/z values of 467, 535, and 603 for MK-4, MK-5, and MK-6, respectively, indicating that all of them were detected as Na+-associated ion species and corresponded to the menaquinone homologs (Fig. 6). Table 1 Optimization of separation of menaquinone homologs using different organic solvent in a glass reverse-phase C18 silica column In this study, we identified a single organic solvent for extracting menaquinones from dry cells. The highest extraction yield was obtained using methanol among the selected organic solvents, most likely due to the fact that methanol has the highest polarity index among the solvents tested in accordance with a previous study [15]. However, the pretreatment process required drying equipment and was time-consuming. The results of extraction from wet cells showed that the extraction yields obtained using methanol, absolute ethyl alcohol, and isopropanol were significantly higher than those obtained using dichloromethane, n-hexane, and petroleum ether, indicating that the polarity of the organic solvent has a substantial effect on the extraction efficiency. On the other hand, the extraction yields of the organic solvents containing an –OH group, such as methanol, ethanol, and isopropanol, were higher than those of other solvents except for n-butanol. This was probably because the –OH group could form a covalent bond with the water contained in the wet cells, thereby facilitating the infiltration of organic solvents. Moreover, the yields of menaquinones extracted from the wet cells directly using organic solvents were lower than that of the control. It is probable that the water contained in the samples reduced the capacities of the organic solvents to break down or dissolve the cell walls, thus leading to low extraction yields of menaquinones. Obviously, this method was able to extract menaquinones from wet microbes, but with lower yields than that of the control for 3 h at least. In addition, tedious purification and separation steps were required to obtain high recoveries of menaquinones from the fermentation media. Therefore, it is necessary to develop a rapid and efficient extraction method for menaquinones. Effect of water content on extraction A previous study showed that several variables, including the percentage of solids in the cell slurry, the polarity of the organic solvent, and the ratio of solvent to cell slurry volume, have a substantial effect on extraction efficiency [18]. Organic solvent Content of menaquinones/ mass of silica gel (μg/g) Flow rate (mL/min) Height–diameter ratio (cm: cm) Recovery ratio of menaquinones (%) Methanol Absolute ethyl alcohol Acetonitrile 54.5 203.6 131.5 0.52 0.16 0.69 5.5: 1 14.0: 1 5.4: 1 99.6 98.7 98.9 13 Bioprocess Biosyst Eng Our results showed that the extraction yield obtained using absolute ethyl alcohol was higher than that obtained using methanol when the water content was more than 5%, indicating that absolute ethyl alcohol had a better dewatering effect on the wet cells. However, the extraction yields obtained using both methanol and ethanol appeared to be equal when the water content was less than 5%, indicating that methanol afforded a better permeability of the cell wall or membrane. Therefore, absolute ethyl alcohol was applied as a dehydrating agent, and methanol was used as the extractant in the following experiments. Effect of improved extraction procedures for wet cells The objective of this study was to develop an efficient solvent extraction method for recovering menaquinones from wet cells of Flavobacterium. To reduce the energy input, all extraction operations were carried out at room temperature without agitation. Two variables, the ratio of solvent to cell slurry mass and extraction time, were optimized. Compared to a conventional solid-phase extraction technique [7, 8], a liquid–liquid extraction technique [2, 6, 9, 10], or a supercritical fluid extraction technique [11–13], our method offers three distinct advantages (1) a rapid and simple pretreatment procedure; (2) a single and simplified organic solvent that is conveniently recovered and cyclically used; (3) no requirement of considerable energy input or expensive devices. Apparently, simplicity, speed, efficiency, environmental friendliness, and inexpensiveness make our method suitable not only for laboratory-scale but also for industry-scale processes. Separation of the menaquinone homologs Fig. 6 Mass spectra of menaquinones isolated from Flavobacterium. a MK-4; b MK-5; c MK-6. The conditions of mass spectrometry were as follows: ESI capillary voltage at 3.5 kV; mass spectrometry temperature: 350 °C; MK-4, MK-5, and MK-6 concentrations: 100, 100, and 300 mg/L; injection volume: 2 μL 13 Several methods have been reported for separating menaquinone homologs, including thin-layer chromatography methods [7, 19] and high-performance liquid chromatography (HPLC) methods [15, 17, 20–22]. However, theses separation methods are associated with several problems. The quantity of menaquinone sample obtained by thin-layer chromatography is too little to meet the needs of downstream experiments. Similarly, a menaquinone sample obtained with HPLC is dissolved in several organic solvents and this increases the difficulty of recovering each kind of organic solvent recovery. Moreover, these separation methods for menaquinones are merely suitable for experimental analysis in the laboratory rather than for an industrial production process in a factory. Obviously, our method has overcome such drawbacks and can be conveniently applied to separating menaquinone homologs. This separation method could also be used as one of the steps in the purification of menaquinone homologs. Bioprocess Biosyst Eng Conclusion The extraction method from wet cells demonstrated here represents a process that can greatly decrease the energy input and the time required for sample pretreatment. Of the solvents screened, methanol had the highest extraction efficiency, approaching 102.8%, at a ratio of 6:1 (v/m) for 30 min each time, with pretreatment using absolute ethanol at a ratio of 6:1 (v/m) for 30 min. The separation of menaquinone homologs, MK-4, MK-5, and MK-6, was carried out by using a single organic solution. Obviously, this method is advantageous for the recycling of reagents on a large scale. Acknowledgements This work was financially supported by the Key 863 Fund of China (2014AA021704) and Natural Science Foundation of Anhui Province (1308085MA07, 1608085QC46). Compliance with ethical standards Conflict of interest interest. All authors declare that they have no competing References 1. Nowicka B, Kruk J (2010) Occurrence, biosynthesis and function of isoprenoid quinones. BBA-Biomembranes 1797:1587–1605 2. Berenjian A, Mahanama R, Talbot A, Regtop H, Kavanagh J, Dehghani F (2014) Designing of an intensification process for biosynthesis and recovery of menaquinone-7. Appl Biochem Biotechnol 172:1347–1357 3. Kurosu M, Begari E (2010) Vitamin K2 in electron transport system: are enzymes involved in vitamin K2 biosynthesis promising drug targets. Molecules 15:1531–1553 4. Shearer MJ (1992) Vitamin K metabolism and nutriture. Blood Rev 6:92–104 5. Suttie JW (1995) The importance of menaquinone in human nutrition. Annu Rev Nutr 15:399–417 6. Berenjian A, Mahanama R, Kavanagh J, Dehghani F (2015) Vitamin K series: current status and future prospects. Crit Rev Biotechnol 35:199–208 7. Dunphy PJ, Phillips PG, Brodie AF (1971) Separation and identification of menaquinones from microorganisms. J Lipid Res 12:442–449 8. Hammond RK, White DC (1969) Separation of vitamin K2 isoprenologues by reversed-phase thin-layer chromatography. J Chromatogr A 45:446–452 9. Sato T, Yamada Y, Ohtani Y, Mitsui N, Murasawa H, Araki S (2001) Production of menaquinone (vitamin K2)-7 by Bacillus subtilis. J Biosci Bioeng 91:16–20 10. Berenjian A, Mahanama R, Talbot A, Biffin R, Regtop H, Valtchev P, Kavanagh J, Dehghani F (2011) Efficient media for high menaquinone-7 production: response surface methodology approach. New Biotechnol 28:665–672 11. Irvan AY, Saeki T, Daimon H, Fujie K (2006) Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge. J Chromatogr A 1113:14–19 12. Hanif M, Atsuta Y, Fujie K, Daimon H (2012) Supercritical fluid extraction and ultra performance liquid chromatography of respiratory quinones for microbial community analysis in environmental and biological samples. Molecules 17:2628–2642 13. Irvan HU, Faisal M, Daimon H, Fujie K (2006) Application of modified supercritical carbon dioxide extraction to microbial quinone analysis. Appl Microbiol Biotechnol 69:506–509 14. Skattebol L, Aukrust IR (2010) Process for the preparation of vitamin K2. European Patent 2346806 A1 15. Denisova NA, Booth SL (2005) Vitamin K and sphingolipid metabolism: evidence to date. Nutr Rev 63:111–121 16. Ferland G (2012) Vitamin K and the nervous system: an overview of its actions. Adv Nutr 3:204–212 17. Manoury E, Jourdon K, Boyaval P, Fourcassié P (2013) Quantitative measurement of vitamin K2 (menaquinones) in various fermented dairy products using a reliable high-performance liquid chromatography method. J Dairy Sci 96:1335–1346 18. Wollis RM, McCurdy AT, Ogborn MK, Wahlen BD, Quinn JC, Pease LF, Seefeldt LC (2014) Improving energetics of triacylglyceride extraction from wet oleaginous microbes. Bioresour Technol 167:416–424 19. Matschiner JT, Amelotti JM (1968) Characterization of vitamin K from bovine liver. J Lipid Res 9:176–179 20. Wakabayashi H, Onodera K, Yamato S, Shimada K (2003) Simultaneous determination of vitamin K analogs in human serum by sensitive and selective high-performance liquid chromatography with electrochemical detection. Nutrition 19:661–665 21. Gao M, Liu H, Yang M, Hu J, Shao B (2004) Indirect identification of isoprenoid quinones in Escherichia coli by LC-MS with atmospheric pressure chemical ionization in negative mode. J Basic Microbiol 44:424–429 22. Martano C, Mugoni V, Dal Bello F, Santoro MM, Medana C (2015) Rapid high performance liquid chromatography-high resolution mass spectrometry methodology for multiple prenol liquids analysis in zebrafish embryos. J Chromatogr A 1412:59–66 13