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Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC–ELSD to reveal accumulation differences of sugar and lipid

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Abstract

Objectives

To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process.

Results

The extraction of mycelia with HCl following a photometric phenol–sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1–3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze–thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass.

Conclusions

This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

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References

  • Arnold WN, McLellan MN (1975) Trehalose and glycogen levels during the initial stages of growth of Candida albicans. Physiol Chem Phys 7(4):369–380

    PubMed  CAS  Google Scholar 

  • Bajpai PK, Bajpai P et al (1991) Production of arachidonic acid by Mortierella alpina ATCC 32222. J Ind Microbiol 8(3):179–185

    Article  PubMed  CAS  Google Scholar 

  • Ball SG, Morell MK (2003) From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Annu Rev Plant Biol 54:207–233

    Article  PubMed  CAS  Google Scholar 

  • Barzen-Hanson KA, Wilkes RA et al (2018) Quantitation of carbohydrate monomers and dimers by liquid chromatography coupled with high-resolution mass spectrometry. Carbohydr Res 468:30–35

    Article  PubMed  CAS  Google Scholar 

  • Beever RE, Laracy EP (1986) Osmotic adjustment in the filamentous fungus Aspergillus nidulans. J Bacteriol 168(3):1358–1365

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cassani L, Quintana G et al (2018) Relationship between carbohydrate composition and fungal deterioration of functional strawberry juices preserved using non-thermal treatments. J Sci Food Agric 98(9):3271–3279

    Article  PubMed  CAS  Google Scholar 

  • Charoensiddhi S, Lorbeer AJ et al (2016) Enzyme-assisted extraction of carbohydrates from the brown alga Ecklonia radiata: effect of enzyme type, pH and buffer on sugar yield and molecular weight profiles. Process Biochem 51:1503

    Article  CAS  Google Scholar 

  • Chen Y, Vaidyanathan S (2013) Simultaneous assay of pigments, carbohydrates, proteins and lipids in microalgae. Anal Chim Acta 776:31–40

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Hao G et al (2015) Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi. Sci Rep 5:11247

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Dourou M, Mizerakis P et al (2017) Storage lipid and polysaccharide metabolism in Yarrowia lipolytica and Umbelopsis isabellina. Appl Microbiol Biotechnol 101(19):7213–7226

    Article  PubMed  CAS  Google Scholar 

  • Dourou M, Aggeli D et al (2018) Critical steps in carbon metabolism affecting lipid accumulation and their regulation in oleaginous microorganisms. Appl Microbiol Biotechnol 102(6):2509–2523

    Article  PubMed  CAS  Google Scholar 

  • Dvořáčková E, Šnóblová M (2014) Carbohydrate analysis: from sample preparation to HPLC on different stationary phases coupled with evaporative light-scattering detection. J Sep Sci 37(4):323–337

    Article  PubMed  Google Scholar 

  • François J, Parrou JL (2001) Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 25(1):125–145

    Article  PubMed  Google Scholar 

  • Ghebregzabher M, Rufini S et al (1979) Improved thin-layer chromatographic method for sugar separations. J Chromatogr 180(1):1–16

    Article  PubMed  CAS  Google Scholar 

  • Hallsworth JE, Magan N (1994) Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi. Microbiology 140(10):2705–2713

    Article  CAS  Google Scholar 

  • Hao G, Chen H et al (2014) Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina. Appl Environ Microbiol 80(9):2672–2678

    Article  PubMed  PubMed Central  Google Scholar 

  • Hao G, Chen H et al (2015) Metabolic engineering of Mortierella alpina for arachidonic acid production with glycerol as carbon source. Microb Cell Fact 14:205

    Article  PubMed  PubMed Central  Google Scholar 

  • Hernández D et al (2015) Saccharification of carbohydrates in microalgal biomass by physical, chemical and enzymatic pre-treatments as a previous step for bioethanol production. Chem Eng J 262:939

    Article  Google Scholar 

  • Ikegami T et al (2008) Highly efficient analysis of underivatized carbohydrates using monolithic-silica-based capillary hydrophilic interaction (HILIC) HPLC. Anal Bioanal Chem 391(7):2533–2542

    Article  PubMed  CAS  Google Scholar 

  • Kang Q, Chen S et al (2019) Comparison on characterization and antioxidant activity of polysaccharides from Ganoderma lucidum by ultrasound and conventional extraction. Int J Biol Macromol 124:1137–1144

    Article  PubMed  CAS  Google Scholar 

  • Laurentin A, Edwards CA (2003) A microtiter modification of the anthrone-sulfuric acid colorimetric assay for glucose-based carbohydrates. Anal Biochem 315(1):143–145

    Article  PubMed  CAS  Google Scholar 

  • Lin CY, Wu H et al (2007) Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics. Metabolomics 3(1):55–67

    Article  CAS  Google Scholar 

  • Lu H, Chen H et al (2019) Evaluation of metabolome sample preparation and extraction methodologies for oleaginous filamentous fungi Mortierella alpina. Metabolomics 15(4):50

    Article  PubMed  Google Scholar 

  • Luard EJ (1982) Accumulation of intracellular solutes by two filamentous fungi in response to growth at low steady state osmotic potential. Microbiology 128(11):2563–2574

    Article  CAS  Google Scholar 

  • Ma C, Sun Z et al (2014) Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC–ELSD. Food Chem 145:784–788

    Article  PubMed  CAS  Google Scholar 

  • Masuko T, Minami A et al (2005) Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 339(1):69–72

    Article  PubMed  CAS  Google Scholar 

  • Muir JG, Rose R et al (2009) Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC). J Agric Food Chem 57(2):554–565

    Article  PubMed  CAS  Google Scholar 

  • Patel A, Karageorgou D et al (2020) An overview of potential oleaginous microorganisms and their role in biodiesel and omega-3 fatty acid-based industries. Microorganisms 8(3):434

    Article  PubMed Central  CAS  Google Scholar 

  • Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51

    Article  PubMed  CAS  Google Scholar 

  • Roach PJ (2002) Glycogen and its metabolism. Curr Mol Med 2(2):101–120

    Article  PubMed  CAS  Google Scholar 

  • Ruiz-Matute AI et al (2011) Derivatization of carbohydrates for GC and GC-MS analyses. J Chromatogr B Analyt Technol Biomed Life Sci 879(17–18):1226–1240

    Article  PubMed  CAS  Google Scholar 

  • Sakuradani E (2010) Advances in the production of various polyunsaturated fatty acids through Oleaginous Fungus Mortierella alpina breeding. Biosci Biotechnol Biochem 74(5):908–917

    Article  PubMed  CAS  Google Scholar 

  • Schulze C, Strehle A et al (2017) Carbohydrates in microalgae: comparative determination by TLC, LC-MS without derivatization, and the photometric thymol-sulfuric acid method. Algal Res 25:372–380

    Article  Google Scholar 

  • Shanmugavelan P, Kim SY et al (2013) Evaluation of sugar content and composition in commonly consumed Korean vegetables, fruits, cereals, seed plants, and leaves by HPLC–ELSD. Carbohydr Res 380:112–117

    Article  PubMed  CAS  Google Scholar 

  • Shen H, Zhang X et al (2017) Compositional profiles of Rhodosporidium toruloides cells under nutrient limitation. Appl Microbiol Biotechnol 101(9):3801–3809

    Article  PubMed  CAS  Google Scholar 

  • Sun S, Wang H et al (2016) Simultaneous determination of rhamnose, xylitol, arabitol, fructose, glucose, inositol, sucrose, maltose in jujube (Zizyphus jujube Mill.) extract: comparison of HPLC-ELSD, LC-ESI-MS/MS and GC-MS. Chem Cent J 10:25

    Article  PubMed  PubMed Central  Google Scholar 

  • Tchakouteu SS, Chatzifragkou A et al (2015) Oleaginous yeast Cryptococcus curvatus exhibits interplay between biosynthesis of intracellular sugars and lipids. Eur J Lipid Sci Technol 117(5):657–672

    Article  CAS  Google Scholar 

  • Thompson SN (2003) Trehalose—The Insect “Blood” Sugar. Adv Insect Physiol 31:205–285

    Article  CAS  Google Scholar 

  • Vial J, Jardy A (1999) Experimental comparison of the different approaches to estimate LOD and LOQ of an HPLC method. Anal Chem 71(14):2672

    Article  CAS  Google Scholar 

  • Wang H, Yang B et al (2011) Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina. Microbiology (Reading) 157(Pt 11):3059–3070

    Article  CAS  Google Scholar 

  • Wang Y, Wang H et al (2013) Lentinan extracted from shiitake mushrooms (Lentinus edodes) improves the non-specific immunity of sea cucumber (Apostichopus japonicus). Aquacult Int 21(6):1261–1277

    Article  CAS  Google Scholar 

  • Wynn JP, Hamid A et al (1999) The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology (Reading) 145(Pt 8):1911–1917

    Article  CAS  Google Scholar 

  • Zhao G, Chen X et al (2013) Ultrasound assisted extraction of carbohydrates from microalgae as feedstock for yeast fermentation. Bioresour Technol 128:337–344

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31722041, 31901659), the National First-class Discipline Program of Food Science and Technology (JUFSTR20180102), the Jiangsu Province “Collaborative Innovation Center for Food Safety and Quality Control”.

Supplementary Information

Supplementary Table 1—Components of intracellular fatty acids produced in Mortierella alpina.

Supplementary Fig. 1—HPLC–ELSD chromatograms of five carbohydrate standards peaks on Sugar-Pak I column (300 mm × 6.50 mm, 5 μm): (a) frucose; (b) glucose; (c) galactose; (d) lactose; (e) trehalose. Solvent: water, the flow rate was 0.4 mL·min-1 and the column held at 85 °C. A drift tube temperature of 60°C and a gas flow rate of 2.5 L·min-1 were used.

Supplementary Fig. 2—HPLC–ELSD chromatograms of five carbohydrate standards peaks on a XBridge® BEH amide column

Supplementary Fig. 3—Chromatograms of intracellular carbohydrates extracted from 50mg dried powder by six methods separating on Sugar-Pak I column.

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Correspondence to Haiqin Chen.

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Chen, H., Chen, H., Lu, H. et al. Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC–ELSD to reveal accumulation differences of sugar and lipid. Biotechnol Lett 43, 1289–1301 (2021). https://doi.org/10.1007/s10529-021-03120-2

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  • DOI: https://doi.org/10.1007/s10529-021-03120-2

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