A Novel Material for Selective Separation of Monogalactosyldiacylglycerols from Microalgae

JIANG Junpeng , YANG Miao CAO Xupeng WAN Huihui,LIU Shuqin, ZHONG Shijun, YAN Jingyu , and XUE Song,

1) Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

2) State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,Dalian 116023, China

3) Division of Solar Energy, Dalian National Laboratory of Clean Energy, Dalian 116023, China

4) University of Chinese Academy of Sciences, Beijing 100049, China

5) Faculty of Chemical, Environmental and Biological Science and Technology, Analytical Center,Dalian University of Technology, Dalian 116024, China

6) School of Bioengineering, Dalian University of Technology, Dalian 116024, China

7) Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics,Chinese Academy of Sciences, Dalian 116023, China

Abstract Monogalactosyldiacylglycerols (MGDGs) have potential applications in food products, cosmetics and pharmaceuticals.MGDGs from microalgae with high amounts of polyunsaturated fatty acids (PUFAs) have potential functions, which arise the interest of the researchers. MGDGs were prepared by silica gel column chromatography with the appropriate mobile phase, while due to the similarity to molecular structure of MGDGs, digalactosyldiacylglycerols (DGDGs) were the most difficult impurities to separate during the extraction process of MGDGs from Nannochloropsis oceanica IMET1 and Arthrospira platensis. In order to obtain MGDGs from microalgae using low toxic solvent system, a novel material Click thiol-ene cysteine (Click TE-Cys) was employed to achieve its selective separation by differentiation of the hydrophilic interaction. The mixture of MGDGs and DGDGs standards were separated from each other by Click TE-Cys solid phase extraction (SPE), which was further confirmed by the result of LC-MS. The molecular interaction of MGDGs and DGDGs with Click TE-Cys demonstrated that DGDGs had more hydrogen bonds with Click TE-Cys material, which might cause a higher hydrophilic interaction. In this study, the Click TE-Cys material exhibited higher hydrophilicity with DGDGs and effectively separated MGDGs from 4 species microalgae by ‘flow-through’ mode using ethanol as mobile phase.

Key words microalgae; MGDGs; DGDGs; selective separation; polarity

1 Introduction

During the last decades, glycolipids have received increasing attention due to its diverse applications (Hayashiet al., 2019; Kanget al., 2019; Leblondet al., 2019; Penget al., 2019). Monogalactosyldiacylglycerols (MGDGs)are a type of glycoglycerolipids, which contains one Dgalactosyl residue (predominantly in beta-form) linked to the sn-3 position of 1,2-diacylglycerols (Kimet al., 2018).They can then act as non-ionic emulsifiers in food products (Nakaeet al., 2000; Selmair and Koehler, 2010),cosmetics and pharmaceuticals (Wanget al., 2016). In addition, MGDGs from plants and microalgae exhibited antitumor, antiviral, anti-inflammatory and immunosuppressive activities (Morimotoet al., 1995; Maedaet al.,2007; Akasakaet al., 2016; de los Reyeset al., 2016; Leblondet al., 2019).

MGDGs are mainly found in the thylakoid membrane within the chloroplasts of higher plants and microalgae.They consititute half of the chloroplast lipids as the most abundant polar lipids in photosynthetic species (Dormann and Benning, 2002; Kimet al., 2018). The molecular structures of MGDGs in microalgae are similar to that of plants. The acyl groups of MGDGs in microalgae contained about 50% of long chain omega-3 polyunsaturated fatty acids (ω-3 PUFAs) (Menget al., 2017), such as EPA,DHA and ARA. Further, PUFAs proportions in MGDGs of spinach and rice grain were 30.5% and 30%, respectively, with C18:2 and C18:3 as the major PUFAs (Murakamiet al., 2003; Zhenget al., 2017). Omega-3 PUFAs are essential for human health and involved in blood lipids equilibrium (Fujino and Sakata, 1973). MGDGs from plants (spinach and rice), compared to that of microalgae would not be able to provide potential omega-3 PUFAs.In addition, microalgae can show higher photosynthetic efficiency comparatively. Thus, microalgae based MGDGs production have gained more attention.

At present, MGDGs have been extracted from vegetables, such as spinach and bitter melon using silica or macro-porous resin column chromatography (Maedaet al.,2007; Matsuiet al., 2009; Akasakaet al., 2016). The purity of MGDGs from spinach using HP-20 and silica gel column chromatography with H2O/ethanol and chloroform/ethyl acetate system was determined at 98% (Akasakaet al., 2016). Similarly, the yield of MGDGs fromIsochrysis galbanapowder by silica gel column chromatography using less toxic solvents, such as hexane/ethanol system was 50% of content of MGDGs in microalgae(Menget al., 2017). This could be attributed to the interference of lipids, such as DGDGs (the structure shown in Fig.1) (de los Reyeset al., 2016) with similar structure in crude extracts of microalgae during the separation process,thus MGDGs separated by silica or macro-porous resin column chromatography is non-selective with low yield.

Fig.1 The structures of MGDG and DGDG. R1 and R2 are the acyl groups.

The aim of this study was to find a novel material for selective and rapid separation of MGDGs from DGDGs in microalgae using green solvents. Previous study reported that the binding of cysteine group to silica gel named Click thiol-ene cysteine (Click TE-Cys) can increase the hydrophilic ability (Shenet al., 2011, 2012),which can be used to separate MGDGs and DGDGs based on the difference in the numbers of galactosyl groups. The new material was introduced to efficiently separate MGDGs from DGDGs in microalgae crude extract.

2 Materials and Methods

2.1 Materials and Reagents

Standard MGDGs and DGDGs were procured from Avanti Polar Lipids, Inc. and dissolved in chloroform/methanol/water (4:1:0.1, v/v/v) to a final concentration of 1 mg mL−1. Silica solid phase extraction (SPE) cartridges were purchased from Waters Corporation Milford, Massachusetts USA. SPE cartridges and stainless-steel column packed with Click TE-Cys material were kindly donated by Separation Material Chemistry and Multi-component Chinese Medicine Group, China. Thin-layer chromatography (TLC) Silica Gel 60 F254 were purchased from Merck KGaA (Germany). Silica (200 – 300 mesh)was procured from Qingdao Mingyang Chemical Co. Ltd.(Qingdao, China). All other chemicals were of analytical reagent grade.

2.2 Microalgal Powder Preparation

Nannochloropsis oceanicaIMET1 was inoculated into modified 4-fold of f/2 medium (Fenget al., 2011) in a 100-L flat photobioreactor (100 × 10 × 120 cm) aerated with 2% CO2-enriched air at a flow rate of 0.2 m3h−1at 25℃. The cultures were illuminated with cool white fluorescent lamps providing an average irradiance of 150 μmol m−2s−1under 14 h light /10 h dark cycle (Menget al.,2015). After 7 d of cultivation, algal cells were collected and frozen pellets were lyophilized for 12 h. In addition,fresh powders ofPhaeodactylum tricornutumandChlorella sorokinianaGT1 were donated by SDIC, Microalgae Biotechnology Center (China) and that ofArthrospira platensiswas donatedby Ordos Lvfuyuan Biotechnology Corp. Ltd. (China).

2.3 Preparation of MGDGs Fractions from Microalgae by Silica Gel Column Chromatography

Dried microalgal cells (N. oceanicaIMET1 andA.platensis) were subjected to disruption by planetary ball mill (QM-3SP-2). The lipid-soluble substances were extracted from dried microalgal powders (10 g) with 100 mL of warm chloroform:ethanol (2:1, v/v) (60℃) for 4 h at 600 r min−1. The lipid-soluble substances fromN. oceanicaIMET1 andA. platensiswere subjected to silica gel column chromatography (200 mL) and eluted by 1200 mL of chloroform/methanol/H2O (4:1:0.1, v/v/v) to obtain MGDG fractions and other lipid fractions. The fractions from silica gel column chromatography were identified by TLC using chloroform/methanol/H2O (4:1:0.1, v/v/v).

2.4 MGDGs and DGDGs Standards Separated by Click TE-Cys SPE and Silica SPE

The Click TE-Cys SPE and Silica SPE were activated and equilibrated with 6 mL of ethanol before use. Then the standard mixture containing the MGDGs (10 μL) and DGDGs (10 μL) were dried under nitrogen and re-dissolved in 200 μL of ethanol. This solution was loaded onto Click TE-Cys SPE cartridge (1 g/6 mL) and Silica SPE cartridge (1 g/6 mL), respectively, and eluted with 10 mL of ethanol. The eluted fractions were identified by TLC with chloroform/methanol/H2O/triethylamine (50:40:8:50, v/v/v/v).

2.5 Analysis of the Separated MGDGs and DGDGs Standards by HPLC-MS

HPLC was performed on an ACCELA HPLC system(Thermo Scientific, San Jose, CA, USA) and equipped with Click TE-Cys column (5 μm, 150 mm × 4.6 mm i.d.).The column temperature was maintained at 30℃ with a flow rate of 1 mL min−1. The solution of ethanol (buffer A)and H2O (buffer B) was used as mobile phase. The gradient program was as follows: 0 –18 min, A/B (v/v): 100:0→ 100:0; 18 –28 min, 100:0 →50:50; 28 –38 min, 50:50→ 50:50; 38 –39 min, 50:50 → 100:0; 39 –45 min, 100:0→ 100:0. 5 μL of MGDGs and DGDGs standards (about 2 mg mL−1) was directly injected into mass spectrometer for analysis at split ratio of 1:1 (Huanget al., 2018). Mass monitoring was determined on a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Scientific,San Jose, CA, USA), equipped with a heated electrospray ionization source (HESI). Helium was used as collision gas and the sheath and nitrogen was used as auxiliary gas.The operating conditions of mass spectrometer were: capillary voltage of 3000 V, capillary temperature of 300℃,vaporizer temperature of 250℃, sheath gas pressure of 35 and aux gas pressure of 10. Data acquisition and processing were analyzed using Xcalibur 2.2 software (Thermo Scientific, USA) (Huanget al., 2018).

2.6 Enrichment of MGDGs from Microalgae by Click TE-Cys SPE

Crude extracts from about 25 mg of microalgal biomass were obtained using Bligh and Dyer method (Bligh and Dyer, 1959) and were dissolved in 400 μL of ethanol before loading onto Click TE-Cys SPE cartridge (1 g/6 mL). Then, MGDGs fractions were eluted and enriched with 6 mL of ethanol and identified by TLC as described in section 2.4.

2.7 Simulation of the Molecular Interaction of MGDG and DGDG with Click TE-Cys

The simulation of their molecular interaction with Click TE-Cys was designed by Gauss View 6. The theoretical structure of Click TE-Cys was designed using Gauss View 6 based on the carbon content in the material and synthetic process. The difference between MGDGs and DGDGs in acyl groups was ignored. These acyl groups were linolenic acids in both MGDGs and DGDGs.

3 Results and Discussion

3.1 MGDGs Fractions Separated from DGDGs Using Conventional Method

The separation process of MGDGs from the crude lipids from microalgae by silica gel column chromatography is based on the difference in polarity of lipid structures(Maedaet al., 2007; Matsuiet al., 2009; Akasakaet al.,2016). As shown in Fig.2, DGDGs partially mixed with MGDGs during the separation process by silica column chromatography. Based on the structures (de los Reyeset al., 2016) shown in Fig.1, DGDGs had similar structures to MGDGs due to their similar polarities. Results showed that MGDGs and DGDGs were not easy to separate by silica gel column chromatography due to its low separation efficiency.

3.2 Click TE-Cys Material with Amino and Carboxyl Groups to Separate MGDGs and DGDGs

Fig.2 Fractions separated from crude lipids of A. platensis and N. oceanica IMET1 by silica gel column chromatography and detected by thin-layer chromatography (TLC) in (a) and (b), respectively. The positions of MGDG and DGDG were confirmed by MGDGs and DGDGs standards (data not shown).

As shown in Fig.2, silica exhibited low separation efficiency of MGDGs and DGDGs, so it was imperative to develop a new material by binding hydrophilic groups on silica gel to achieve their separation. The thiol group in cysteine and vinyl silica by ‘thiol-ene’ click reaction can preserve amino and carboxyl groups, thus maintaining its zwitterionic property, making the new material highly hydrophilic compared to silica gel (Shenet al., 2011,2012). Previous study showed that the Click TE-Cys material exhibited high hydrophilicity and column efficiency could be used to efficiently separate oligosaccharides with different degree of polymerizations using H2O: acetonitrile system (Shenet al., 2011). As MGDGs and DGDGs contain one and two galactosyl groups (Refer Fig.1 for their structures), respectively, which showed hydrophilic interaction with Click TE-Cys material.

In order to investigate whether Click TE-Cys SPE could separate MGDGs from DGDGs, their standards were loaded onto Click TE-Cys SPE and Silica SPE (used as control). Since, the application of Click TE-Cys stationary phase was performed in hydrophilic interaction liquid chromatography (HILIC) mode, polar solvent was selected as eluent. MGDGs, DGDGs and other polar lipids can be dissolved in ethanol, which is regarded as an environment-friendly solvent. As shown in Fig.3(a), the mixture of MGDGs and DGDGs standards subjected onto Click TE-Cys SPE and eluted with 10 mL ethanol (number 3 in Fig.3a) showed only one spot through TLC analysis, which was confirmed as MGDG based on the standard (number 1 in Fig.3a). This demonstrated that Click TE-Cys material exhibited higher hydrophilic interaction with DGDGs due to higher number of hydroxyl groups to form hydrogen-bond interaction, when compared to MGDGs.

In control, the mixture of MGDGs and DGDGs standards subjected onto Silica SPE and eluted with 6 mL and 10 mL ethanol (number 2 and 3 in Fig.3b) showed two spots in TLC analysis, which matched the position of MGDGs and DGDGs standards (number 1 in Fig.3b).This showed that MGDGs and DGDGs could not be separated by Silica SPE eluted by ethanol. In addition, this was confirmed that MGDGs fractions were combined with DGDGs during the separation process (Fig.2). This might be attributed to the weak hydrophilic interaction of silica, which could not differentiate MGDGs and DGDGs.

Fig.3 The comparison of MGDGs and DGDGs separated by Click TE-Cys SPE and Silica SPE. In Fig.3(a), 1–3 represented MGDGs standard, DGDGs standard and the mixture of MGDGs and DGDGs standards subjected onto Click TE-Cys SPE eluted with 10 mL ethanol, respectively. In Fig.3(b), 1–3 represented the mixture of MGDGs and DGDGs standards, the mixture of MGDGs and DGDGs standards subjected onto Silica SPE eluted with 6 mL ethanol and 10 mL ethanol, respectively.

Fig.4 Standard total ion chromatogram (TIC, a) and extracted ion chromatogram (EIC, b, c) of MGDGs and DGDGs standards mixture were detected by HPLC-MS and mass spectra (d, e) were the fractions showed in b and c, respectively.

In order to further confirm that Click TE-Cys material had a higher hydrophilicity than silica gel, chromatographic retention behavior of the mixture of MGDGs and DGDGs standards were detected by mass spectrometry (Fig.4). As shown in Fig.4d, the ion fragments at m/z 781, 782 and 809 had a higher abundance, which comprised the molecular weight of MGDGs. The peak of extracted ion chromatogram (EIC, b) was defined as MGDGs. Similarly, the ion fragments at m/z 937, 939,951 and 960 showed higher abundance, which consisted the molecular weight of DGDGs and the peak of EIC (c)was defined as DGDGs (Fig.4e). Thus, the retention times of MGDGs and DGDGs were 3.05 min and 16.30 min of total ion chromatogram (TIC, a), respectively, which was separated by Click TE-Cys material. This result revealed that Click TE-Cys material could better retain DGDGs compared to MGDGs. The molecular interaction of MGDGs and DGDGs with Click TE-Cys were shown in Fig.5. It was observed that in the interaction between MGDG and Click TE-Cys, there were 5 to 6 possible positions required for the formation of hydrogen bonds (Fig.5a). Similarly, there were 6 to 7 possible positions of hydrogen bonds between DGDGs and Click TE-Cys (Fig.5b). These results demonstrated that Click TE-Cys had higher number of hydrogen bonds with DGDGs, thus, it could retain DGDGs than MGDGs.

Fig.5 The interaction of Click TE-Cys with MGDGs (a) and DGDGs (b) were designed by Gauss View 6. The positions of the imaginary lines were used for the formation of hydrogen bonds. The atoms in red, white, light gray, dark gray, yellow and blue in the picture represented oxygen atom, hydrogen atom, silicon atom, carbon atom, sulfur atom and nitrogen atom,respectively.

3.3 MGDGs and DGDGs in Microalgae Could Be Separated by Click TE-Cys SPE

In order to investigate the separation behavior of MGDGs and DGDGs in microalgae by Click TE-Cys SPE, crude lipids extracted fromN. oceanicaIMET1,C.sorokinianaGT1,A. platensisandP. tricornutumwere subjected to separation by Click TE-Cys SPE. DGDGs and lipids exhibiting higher polarity than DGDGs were completely absorbed by Click TE-Cys as no spot was observed with crude lipids at the same position (Fig.6).However, MGDGs were not absorbed by Click TE-Cys material, and the size of spots, representing MGDGs, did not change between eluted samples and original samples in four species of microalgae. This showed that Click TE-Cys material could selectively enrich MGDGs in different species of microalgae.

Click TE-Cys material could be the new material for selective separation of MGDGs from corresponding high polar compounds in microalgae, plants and other microorganisms. Compared to silica column chromatography,Click TE-Cys material was an efficient, rapid and selective method in a flow through mode, in which MGDGs flowed through the solid phase and interfering substance,such as DGDGs were absorbed on the solid phase.Moreover, since ethanol was used to separate MGDGs from the extracts, this would expand the application of MGDGs in cosmetics, pharmaceuticals, food and fine chemical products.

Fig.6 TLC analysis of the crude lipids and fractions separated by Click TE-Cys SPE. 1–4 represented crude lipids extracted from N. oceanica IMET1, C. sorokiniana GT1,A. platensis and P. tricornutum and 1’-4’ represented crude lipids eluted from Click TE-Cys SPE with ethanol,respectively. Standard represented MGDGs and DGDGs standards.

4 Conclusions

In this study, Click TE-Cys material with amino and carboxyl groups exhibited higher hydrophilicity and column efficiency compared to silica gel and was able to efficiently separate MGDGs from DGDGs. Compared with MGDGs, the more hydroxyl groups in DGDGs would form more hydrogen bonds with Click TE-Cys material, which may generate a higher hydrophilic interaction. The Click TE-Cys material could be a potential material to enrich MGDGs from microalgae selectively in a flow through mode. Ethanol was the only mobile phase,which almost had no disadvantages on the application of MGDGs in cosmetics, pharmaceuticals, food and fine chemical products. Click TE-Cys material could be used to prepare MGDGs from natural products by means of reducing the price of the material or increasing the stability of the material in order to reuse for several times.

Acknowledgements

This work was supported by the National High Technology Research and Development Program ‘863’ (No.14AA022004), the Key Research and Development Plan in Shandong Province (No. YYSP016), and the National Natural Science Foundation of China (No. 21505131).