Abstract

Chlorella vulgaris is rich in secondary metabolites that defend against environmental stress and aid in detoxification. In particular, bioactive compounds extracted from C. vulgaris may enhance the growth of microorganisms and detoxify them in an ethanolic medium. We aimed to effectively extract and characterize bioactive compounds found in C. vulgaris and further test them for their beneficial effects on the growth of Saccharomyces cerevisiae cultured in an ethanolic medium.
Bioactive compounds in C. vulgaris were extracted using ultrasound and water as solvents. The extracts were analyzed for total phenol and flavonoid contents as part of their phytochemical composition. Their DPPH radical activity and Hydrogen peroxide scavenging activity were examined to determine their antioxidant properties and protective potential for S. cerevisiae in an ethanolic medium. Further, the extracts were added at 0.1, 0.5, 1, 2, 3, and 4% w/v concentrations into S. cerevisiae culture induced with 1% v/v ethanol for 23 days. The yeast cells’ density and viability were measured after 2, 5, 9, 13, 17, and 23 days.
The extracts of C. vulgaris were rich in phenols and flavonoids, which are important bioactive compounds. Higher concentrations of the extracts increased total phenols up to 47.67 GAE mg/L and total flavonoids up to 218.67 QE mg/L. The extracts’ antioxidant composition showed high DPPH activity (70.12%) and H₂O₂ scavenging activity (4.97%). After 23 days, the samples treated with C. vulgaris extracts maintained a high viability of the yeast cells. In particular, the samples with 2, 4, 0.1, and 1% of the extract had a cell viability of 95.75, 94.04, 89.15, and 74%, respectively. The positive control (1% ethanol alone) and negative control (yeast alone) had 47.71 and 21.01% viability, respectively. This drastic reduction in viability was due to lysis of the yeast cells caused by ethanol.
Ultrasound extraction with water as a solvent produced abundant beneficial secondary metabolites from C. vulgaris. The addition of C. vulgaris extract increased the viability and cell density of S. cerevisiae after 27 days, thereby protecting the yeast cells from the toxic effects of ethanol.

Keywords

Chlorella vulgaris, phytochemicals, antioxidants, microalgae, yeast, ultrasound-assisted extraction, Saccharomyces cerevisiae, viability, water extracts

REFERENCES

1. Rani K, Sandal N, Sahoo PK. A comprehensive review on chlorella – its composition, health benefits, market and regulatory scenario. The Pharma Innovation Journal. 2018;7(7):584–589.
2. Okechukwu QN, Adepoju FO, Hassani MI, Kovaleva EG, Rao AR, Ravishankar GA. Suitability of microalgae and fungi in meat analogs: an overview. In: Ravishankar GA, Rao AR, Tahergorabi R, Mohan A, editors. Handbook of plant-based meat analogs. Innovation, technology and quality. Academic Press; 2024. pp. 121–146. https://doi.org/10.1016/B978-0-443-21846-0.00017-4
3. Dolganyuk V, Belova D, Babich O, Prosekov A, Ivanova S, Katserov D, et al. Microalgae: A promising source of valuable bioproducts. Biomolecules. 2020;10(8):1153. https://doi.org/10.3390/biom10081153
4. Kumar N, Goel N. Phenolic acids: Natural versatile molecules with promising therapeutic applications. Biotechnology Reports. 2019;24:e00370. https://doi.org/10.1016/j.btre.2019.e00370
5. Plaza M, Santoyo S, Jaime L, Avalo B, Cifuentes A, Reglero G, et al. Comprehensive characterization of the functional activities of pressurized liquid and ultrasound-assisted extracts from Chlorella vulgaris. LWT – Food Science and Technology. 2012;46(1):245–253. https://doi.org/10.1016/j.lwt.2011.09.024
6. Okechukwu QN, Yama I, Kovaleva EG. Enzymatic extraction of growth factor in Chlorella and possible protective effects of Chlorella extracts on yeast growth. AIP Conference Proceedings. 2020;2280(1):030013. https://doi.org/10.1063/5.0018029
7. Ścieszka S, Gorzkiewicz M, Klewicka E. Innovative fermented soya drink with the microalgae Chlorella vulgaris and the probiotic strain Levilactobacillus brevis ŁOCK 0944. LWT. 2021;151:112131. https://doi.org/10.1016/j.lwt.2021.112131
8. Dantas DMM, Cahú TB, Oliveira CYB, Abadie-Guedes R, Roberto NA, Santana WM, et al. Chlorella vulgaris functional alcoholic beverage: Effect on propagation of cortical spreading depression and functional properties. PLoS ONE. 2021;16(8):e0255996. https://doi.org/10.1371/journal.pone.0255996
9. Chlorella – the most exciting nutritional discovery on planet earth. Abeille d’Or Corporation; 2014. 56 p.
10. Pantoja Munoz L, Purchase D, Jones H, Raab A, Urgast D, Feldmann J, et al. The mechanisms of detoxification of As(III), dimethylarsinic acid (DMA) and As(V) in the microalga Chlorella vulgaris. Aquatic Toxicology. 2016;175:56–72. https://doi.org/10.1016/j.aquatox.2016.02.020
11. Laopaiboon L, Suporn S, Klanrit P, Phukoetphim N, Daengbussadee C, Laopaiboon P. Novel effective yeast strains and their performance in high gravity and very high gravity ethanol fermentations from sweet sorghum juice. Energies. 2021;14(3):557. https://doi.org/10.3390/en14030557
12. Coffman RE, Kraichely KN, Kreutzberger AJB, Kiessling V, Tamm LK, Woodbury DJ. Drunken lipid membranes, not drunken SNARE proteins, promote fusion in a model of neurotransmitter release. Frontiers in Molecular Neuroscience. 2022;15:1022756. https://doi.org/10.3389/fnmol.2022.1022756
13. Okechukwu QN, Kanwugu ON, Adadi P, Okpala COR, Kovaleva EG. Potential of Chlorella vulgaris powder to enhance ethanol-cultured Saccharomyces cerevisiae. Journal of Taibah University for Science. 2023;17(1):2187602. https://doi.org/10.1080/16583655.2023.2187602
14. Karabín M, Jelínek L, Kotrba P, Cejnar R, Dostálek P. Enhancing the performance of brewing yeasts. Biotechnology Advances. 2018;36(3):691–706. https://doi.org/10.1016/j.biotechadv.2017.12.014
15. Samakkarn W. Ratanakhanokchai K, Soontorngun N. Reprogramming of the ethanol stress response in Saccharomyces cerevisiae by the transcription factor Znf1 and its effect on the biosynthesis of glycerol and ethanol. Applied and Environmental Microbiology. 2021;87(16):e00588-21. https://doi.org/10.1128/AEM.00588-21
16. Kupina S, Fields C, Roman MC, Brunelle SL. Determination of total phenolic content using the Folin-C assay: Single-laboratory validation, first action 2017.13 Journal of AOAC International. 2018;101(5):1466–1472. https://doi.org/10.5740/jaoacint.18-0031
17. Huang R, Wu W, Shen S, Fan J, Chang Y, Chen S, et al. Evaluation of colorimetric methods for quantification of citrus flavonoids to avoid misuse. Analytical Methods. 2018;10(22):2575–2587. https://doi.org/10.1039/C8AY00661J
18. Adadi P, Kovaleva EG, Glukhareva TV, Barakova NV. Production and investigations of antioxidant rich beverage: Utilizing Monascus purpureus IHEM LY2014-0696 and various malts. Agronomy Research. 2018;16(S2):1312–1321. https://doi.org/10.15159/AR.18.028
19. Essiedu JA, Adadi P, Kovaleva EG. Production and characterization of beer supplemented with Hibiscus sabdariffa (Malvaceae). Food Frontiers. 2021;3(2):328–338. https://doi.org/10.1002/fft2.127
20. Haida Z, Hakiman M. A comprehensive review on the determination of enzymatic assay and nonenzymatic antioxidant activities. Food Science and Nutrition. 2019;7(5):1555–1563. https://doi.org/10.1002/fsn3.1012
21. Kitada K, Machmudah S, Sasaki M, Goto M, Nakashima Y, Kumamoto S, et al. Antioxidant and antibacterial activity of nutraceutical compounds from Chlorella vulgaris extracted in hydrothermal condition. Separation Science and Technology. 2009;44(5):1228–1239. https://doi.org/10.1080/01496390902729056
22. Scieszka S, Klewicka E. Influence of the microalga Chlorella vulgaris on the growth and metabolic activity of Lactobacillus spp. bacteria. Foods. 2020;9(7):959. https://doi.org/10.3390/foods9070959
23. Csatlos N-I, Simon E, Teleky B-E, Szabo K, Diaconeasa ZM, Vodnar D-C, et al. Development of a fermented beverage with Chlorella vulgaris powder on soybean-based fermented beverage. Biomolecules. 2023;13(2):245. https://doi.org/10.3390/biom13020245
24. Krishnamoorthy A, Rodriguez C, Durrant A. Sustainable approaches to microalgal pre-treatment techniques for biodiesel production: A review. Sustainability. 2022;14(16):9953. https://doi.org/10.3390/su14169953
25. Kulkarni S, Nikolov Z. Process for selective extraction of pigments and functional proteins from Chlorella vulgaris. Algal Research. 2018;35:185–193. https://doi.org/10.1016/j.algal.2018.08.024
26. Okechukwu QN, Adadi P, Kovaleva EG. Production and analysis of beer supplemented with Chlorella vulgaris powder. Fermentation. 2022;8(11):581. https://doi.org/10.3390/fermentation8110581
27. Dantas DMM, Costa RMPB, Carneiro-da-Cunha MG, Galvez AO, Drummond AR, Bezerra RS. Bioproduction, antimicrobial and antioxidant activities of compounds from Chlorella vulgaris. Research and Reviews: Journal of Botanical Sciences. 2015;4(2):12–18.
28. Vieira MV, Turkiewicz IP, Tkacz K, Fuentes-Grünewald C, Pastrana LM, Fuciños P, et al. Microalgae as a potential functional ingredient: Evaluation of the phytochemical profile, antioxidant activity and in-vitro enzymatic inhibitory effect of different species. Molecules. 2021;26(24):7593. https://doi.org/10.3390/molecules26247593
29. Munteanu IG, Apetrei C. Analytical methods used in determining antioxidant activity: A review. International Journal of Molecular Sciences. 2021;22(7):3380. https://doi.org/10.3390/ijms22073380
30. Baliyan S, Mukherjee R, Priyadarshini A, Vibhuti A, Gupta A, Pandey RP, et al. Determination of antioxidants by DPPH radical scavenging activity and quantitative phytochemical analysis of Ficus religiosa. Molecules. 2022;27(4):1326. https://doi.org/10.3390/molecules27041326
31. Christodoulou MC, Palacios JCO, Hesami G, Jafarzadeh S, Lorenzo JM, Domínguez R, et al. Spectrophotometric methods for measurement of antioxidant activity in food and pharmaceuticals. Antioxidants. 2022;11(11):2213. https://doi.org/10.3390/antiox11112213
32. Pak VV, Ezeriņa D, Lyublinskaya OG, Pedre B, Tyurin-Kuzmin PA, Mishina NM, et al. Ultrasensitive genetically encoded indicator for hydrogen peroxide identifies roles for the oxidant in cell migration and mitochondrial function. Cell Metabolism. 2020;31(3):642–653.e6. https://doi.org/10.1016/j.cmet.2020.02.003
33. Collin F. Chemical basis of reactive oxygen species reactivity and involvement in neurodegenerative diseases. International Journal of Molecular Sciences. 2019;20(10):2407. https://doi.org/10.3390/ijms20102407
34. Bhuvana P, Sangeetha P, Anuradha V, Syed Ali M. Spectral characterization of bioactive compounds from microalgae: N. oculata and C. vulgaris. Biocatalysis and Agricultural Biotechnology. 2019;19:101094. https://doi.org/10.1016/j.bcab.2019.101094
35. Goiris K, Muylaert K, Voorspoels S, Noten B, de Paepe D, Baart GJE, et al. Detection of flavonoids in microalgae from different evolutionary lineages. Journal of Phycology. 2014;50(3):483–492. https://doi.org/10.1111/jpy.12180
36. Zakaria SM, Kamal SMM, Harun MR, Omar R, Siajam SI. Subcritical water technology for extraction of phenolic compounds from Chlorella sp. microalgae and assessment on its antioxidant activity. Molecules. 2017;22(7):1105. https://doi.org/10.3390/molecules22071105
37. Liu J, Chen F. Biology and industrial applications of Chlorella: Advances and prospects. In: Posten C, Chen SF, editors. Microalgae biotechnology. Cham: Springer; 2016. pp. 1–35. https://doi.org/10.1007/10_2014_286
38. Michalak M, Pierzak M, Kręcisz B, Suliga E. Bioactive compounds for skin health: A review. Nutrients. 2021;13(1):203. https://doi.org/10.3390/nu13010203
39. Postaru M, Tucaliuc A, Cascaval D, Galaction A-I. Cellular stress impact on yeast activity in biotechnological processes – A short overview. Microorganisms. 2023;11(10):2522. https://doi.org/10.3390/microorganisms11102522
40. Kubota S, Takeo I, Kume K, Kanai M, Shitamukai A, Mizunuma M, et al. Effect of ethanol on cell growth of budding yeast: Genes that are important for cell growth in the presence of ethanol. Bioscience, Biotechnology, and Biochemistry. 2004;68(4):968–972. https://doi.org/10.1271/bbb.68.968
41. Flatt T, Partridge L. Horizons in the evolution of aging. BMC Biology. 2018;16:93. https://doi.org/10.1186/s12915-018-0562-z
42. Li S, Vazquez JM, Sudmant PH. The evolution of aging and lifespan. Trends in Genetics. 2023;39(11):830–843. https://doi.org/10.1016/j.tig.2023.08.005
43. Kumari R, Jat P. Mechanisms of cellular senescence: Cell cycle arrest and senescence associated secretory phenotype. Frontiers in Cell and Developmental Biology. 2021;9:645593. https://doi.org/10.3389/fcell.2021.645593
44. Lutchman V, Medkour Y, Samson E, Arlia-Ciommo A, Dakik P, Cortes B, et al. Discovery of plant extracts that greatly delay yeast chronological aging and have different effects on longevity-defining cellular processes. Oncotarget. 2016;7:16542–16566. https://doi.org/10.18632/oncotarget.7665