Enhancing bioactivity of Chlorella vulgaris through enzymatic pretreatment and lactic acid fermentation

Keywords

Microalgae / Chlorella vulgaris / Fermentation / Lactic acid bacteria / Enzyme pretreatment / Antioxidant capacity

References

[1] Abdulhussain Kareem R, Razavi SH. Plantaricin bacteriocins: As safe alternative antimicrobial peptides in food preservation—A review. J Food Saf, 2020, 40: e12735

[2] Abedi E, Hashemi SMB. Lactic acid production – producing microorganisms and substrates sources-state of art. Heliyon, 2020, 6(10): e04974

[3] Aboobacker S, Kitrytė-Syrpa V, Šipailienė A, Rutkaitė R, Syrpas M. Fermentation-induced nutritional and in vitro antioxidant capacity changes in Arthrospira platensis (spirulina). Food Bioscience, 2025, 68: 106747

[4] Abou El-Souod GW, Morsy EM, Hassan LHS, El-Sheekh MM. Efficient Saccharification of the Microalga Chlorella vulgaris and its Conversion into Ethanol by Fermentation. Iran J Sci Technol Trans Sci, 2021, 45: 767-774

[5] Aguirre-Garcia YL, Nery-Flores SD, Campos-Muzquiz LG, Flores-Gallegos AC, Palomo-Ligas L, Ascacio-Valdés JA, Sepúlveda-Torres L, Rodríguez-Herrera R. Lactic Acid Fermentation in the Food Industry and Bio-Preservation of Food. Fermentation, 2024, 10: 168

[6] Al-Hammadi M, Güngörmüşler M. New insights into Chlorella vulgaris applications. Biotechnol Bioeng, 2024, 121(5): 1486-1502

[7] Alakomi H-L, Skyttä E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM. Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane. Appl Environ Microbiol, 2000, 66: 2001-2005

[8] Alhattab M, Kermanshahi-Pour A, Brooks MS-L. Microalgae disruption techniques for product recovery: influence of cell wall composition. J Appl Phycol, 2019, 31: 61-88

[9] Apak R, Güçlü K, Demirata B, Özyürek M, Çelik SE, Bektaşoğlu B, Berker KI, Özyurt D. Comparative Evaluation of Various Total Antioxidant Capacity Assays Applied to Phenolic Compounds with the CUPRAC Assay. Molecules, 2007, 12: 1496-1547

[10] Badrunanto, Purba LDA, Wikantyasning ER, Maryati, Prayitno J, Hidhayati N, Agustini NWS, Munawaroh HSH, Susanti H, Kusumawaty D, Rahman DY, Apriastini M, Prasetyadi, Admirasari R. Chlorella sp. as a promising natural antioxidant source: Research trends, bioactive components, and future perspectives. Algal Res, 2025, 92: 104341

[11] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-254

[12] Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci Technol, 1995, 28: 25-30

[13] Brányiková I, Maršálková B, Doucha J, Brányik T, Bišová K, Zachleder V, Vítová M. Microalgae—novel highly efficient starch producers. Biotechnol Bioeng, 2011, 108: 766-776

[14] Carreira-Casais A, Otero P, Garcia-Perez P, Garcia-Oliveira P, Pereira AG, Carpena M, Soria-Lopez A, Simal-Gandara J, Prieto MA. Benefits and Drawbacks of Ultrasound-Assisted Extraction for the Recovery of Bioactive Compounds from Marine Algae. Int J Environ Res Public Health, 2021, 18: 9153

[15] Chen P-T, Hong Z-S, Cheng C-L, Ng I-S, Lo Y-C, Nagarajan D, Chang J-S. Exploring fermentation strategies for enhanced lactic acid production with polyvinyl alcohol-immobilised Lactobacillus plantarum 23 using microalgae as feedstock. Bioresour Technol, 2020, 308: 123266

[16] Córdova O, Passos F, Chamy R. Enzymatic Pretreatment of Microalgae: Cell Wall Disruption, Biomass Solubilisation and Methane Yield Increase. Appl Biochem Biotechnol, 2019, 189: 787-797

[17] Damayanti A, Megawati, Winaningsih I, Al Taghna E, Supriyadi M, Zahra SA, Khusniyyah R, Adonai Tandirogang N, Avril Ervian Then N. Comparative study of pretreatment methods for the reverse enzymatic hydrolysis of Chlorella and Spirulina microalgae for bioethanol production. Biofuels, 2025, 16: 905-911

[18] Dave AD, Bilgin H, Kitrytė-Syrpa V, Syrpas M. Optimisation of Pressurised Liquid Extraction and Subsequent Hydrolysate Fermentation by Lactiplantibacillus plantarum for Integrated Bioprocessing of Ulva sp. Mar Drugs, 2025, 23: 371

[19] de Carvalho Silvello MA, Gasparotto GA, Ferreira GF, Santos LO, Fregolente LV, Goldbeck R. Nutrient Optimisation Strategy to Increase the Carbohydrate Content of Chlorella vulgaris and Evaluation of Hydrolysis and Fermentation Performance. Bioenerg Res, 2023, 16: 2058-2067

[20] Dent T, LeMinh A, Maleky F. Comparison of Colorimetric Methods for Measuring the Solubility of Legume Proteins. Gels, 2024, 10(9): 551

[21] Effiom DI, Afegbua SL, Ado SA, Chia MA. Effect of single and combined pretreatment strategies applied to Chlorella vulgaris biomass on bioethanol production. Biofuels, 2023, 14: 165-172

[22] El-Naggar NE-A, Hussein MH, Shaaban-Dessuuki SA, Dalal SR. Production, extraction and characterisation of Chlorella vulgaris soluble polysaccharides and their applications in AgNPs biosynthesis and biostimulation of plant growth. Sci Rep, 2020, 10: 3011

[23] Esteghlalian A, Hashimoto AG, Fenske JJ, Penner MH. Modeling and optimisation of the dilute-sulfuric-acid pretreatment of corn stover, poplar and switchgrass. Bioresour Technol, 1997, 59: 129-136

[24] Fassi Fihri R, Ez-Zoubi A, Mbarkiou L, Amar A, Farah A, Bouchamma EO. Antibacterial and antioxidant activities of Chlorella vulgaris and Scenedesmus incrassatulus using natural deep eutectic solvent under microwave assisted by ultrasound. Heliyon, 2024, 10: e35071

[25] Ferreira AS, Ferreira SS, Correia A, Vilanova M, Silva TH, Coimbra MA, Nunes C. Reserve, structural and extracellular polysaccharides of Chlorella vulgaris: A holistic approach. Algal Res, 2020, 45: 101757

[26] Figueroa RHH, López-Malo A, Mani-López E. Antimicrobial activity and applications of fermentates from lactic acid bacteria – a review. Sustainable Food Technol, 2024, 2: 292-306

[27] Fortuin J, Leclercq CC, Silva RK, Shaplov AS, Contal S, Cambier S, Iken M, Fogliano V, Soukoulis C. Chlorella vulgaris protein isolate effectively protects Lacticaseibacillus rhamnosus GG viability during processing, storage, and in vitro digestion. Food Hydrocolloids, 2026, 172: 111999

[28] Garofalo C, Norici A, Mollo L, Osimani A, Aquilanti L. Fermentation of Microalgal Biomass for Innovative Food Production. Microorganisms, 2022, 10: 2069

[29] Gharehbeglou P, Sarabandi K, Akbarbaglu Z. Insights into enzymatic hydrolysis: Exploring effects on antioxidant and functional properties of bioactive peptides from Chlorella proteins. J Agric Food Res, 2024, 16: 101129

[30] Goel A, Halami PM. Structural and biosynthetic diversity of plantaricins from Lactiplantibacillus. Appl Microbiol Biotechnol, 2023, 107: 5635-5649

[31] Habeebullah K, Alagarsamy SF, Sattari S, Al-Haddad Z, Fakhraldeen S, Al-Ghunaim S, Al-Yamani A. Enzyme-assisted extraction of bioactive compounds from brown seaweeds and characterisation. J Appl Phycol, 2020, 32: 615-629

[32] Hayes M, Naik A, Mora L, Iñarra B, Ibarruri J, Bald C, Cariou T, Reid D, Gallagher M, Dragøy R, Galino J, Deyà A, Albrektsen S, Thoresen L, Solstad RG. Generation, Characterisation and Identification of Bioactive Peptides from Mesopelagic Fish Protein Hydrolysates Using In Silico and In Vitro Approaches. Mar Drugs, 2024, 22: 297

[33] Hiltunen M, Kannangara R, Nakandalage B, Söderena O, Timilsina H, Pirhonen J, Pulkkinen K. Growth and biomass composition of Chlorella vulgaris using nutrient-rich water and CO2 from a recirculating aquaculture system. Aquaculture, 2026, 610: 742956

[34] Ibrahim SA, Ayivi RD, Zimmerman T, Siddiqui SA, Altemimi AB, Fidan H, Esatbeyoglu T, Bakhshayesh RV. Lactic Acid Bacteria as Antimicrobial Agents: Food Safety and Microbial Food Spoilage Prevention. Foods, 2021, 10: 3131

[35] Ijaola AO, Akamo DO, George TT, Sengul A, Adediji MY, Asmatulu E. Algae as a potential source of protein: A review on cultivation, harvesting, extraction, and applications. Algal Res, 2024, 77: 103329

[36] Kim N-J, Li H, Jung K, Chang HN, Lee PC. Ethanol production from marine algal hydrolysates using Escherichia coli KO11. Bioresour Technol, 2011, 102: 7466-7469

[37] Kim KH, Choi IS, Kim HM, Wi SG, Bae H-J. Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilised yeast fermentation. Bioresour Technol, 2014, 153: 47-54

[38] Kitada K, Machmudah S, Sasaki M, Goto M, Nakashima Y, Kumamoto S, Hasegawa T. Antioxidant and Antibacterial Activity of Nutraceutical Compounds from Chlorella vulgaris Extracted in Hydrothermal Condition. Sep Sci Technol, 2009, 44: 1228-1239

[39] Kovaleski G, Ventura SPM. Benefits, challenges and future of incorporation of algae into dairy products such as cheese and yogurt. J Food Compos Anal, 2025, 147: 108045

[40] Kusmiyati K, Hadiyanto H, Fudholi A. Treatment updates of microalgae biomass for bioethanol production: A comparative study. J Clean Prod, 2023, 383: 135236

[41] Labbafi S, Zahedi A, Kanani B. Optimising bioethanol production from Chlorella vulgaris and Dunaliella salina using integrated renewable energy systems. Biomass Bioenergy, 2025, 202: 108186

[42] Lee C-G, Kang D-H, Lee D-B, Lee H-Y. Pretreatment for Simultaneous Production of Total Lipids and Fermentable Sugars from Marine Alga, Chlorella sp. Appl Biochem Biotechnol, 2013, 171: 1143-1158

[43] Lee SY, Show PL, Ling TC, Chang J-S. Single-step disruption and protein recovery from Chlorella vulgaris using ultrasonication and ionic liquid buffer aqueous solutions as extractive solvents. Biochem Eng J, 2017, 124: 26-35

[44] Liu H, Ru G, Zhang Z, Li Y, Xia C, Lu C, Zhang Q. Experimental study on optimisation of initial pH for photo-fermentation bio-hydrogen under different enzymatic hydrolysis of chlorella vulgaris. Bioresour Technol, 2021, 338: 125571

[45] Lorente E, Farriol X, Salvadó J. Steam explosion as a fractionation step in biofuel production from microalgae. Fuel Process Technol, 2015, 131: 93-98

[46] Mahdy A, Mendez L, Ballesteros M, González-Fernández C. Enhanced methane production of Chlorella vulgaris and Chlamydomonas reinhardtii by hydrolytic enzymes addition. Energy Conv Manag, 2014, 85: 551-557

[47] Md Nadzir S, Yusof N, Nordin N, Kamari A, Yusoff MZM. A review of microalgal cell wall composition and degradation to enhance the recovery of biomolecules for biofuel production. Biofuels, 2023, 14: 979-997

[48] Meireles Mafaldo Í, de Medeiros VPB, da Costa WKA, da Costa Sassi CF, da Costa Lima M, de Souza EL, Eduardo Barão C, Colombo Pimentel T, Magnani M. Survival during long-term storage, membrane integrity, and ultrastructural aspects of Lactobacillus acidophilus 05 and Lacticaseibacillus casei 01 freeze-dried with freshwater microalgae biomasses. Food Res Int, 2022, 159: 111620

[49] Mendes AR, Spínola MP, Lordelo M, Prates JAM. Chemical Compounds, Bioactivities, and Applications of Chlorella vulgaris in Food, Feed and Medicine. Appl Sci, 2024, 14: 10810

[50] Miller GL. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem, 1959, 31: 426-428

[51] Nagybákay NE, Sarapinaitė L, Syrpas M, Venskutonis PR, Kitrytė-Syrpa V. Optimisation of pressurised ethanol extraction for efficient recovery of hyperoside and other valuable polar antioxidant-rich extracts from Betula pendula Roth leaves. Ind Crops Prod, 2023, 205: 117565

[52] Nasirpour N, Ravanshad O, Mousavi SM. Ultrasonic-assisted acid and ionic liquid hydrolysis of microalgae for bioethanol production. Biomass Conv Bioref, 2023, 13: 16001-16014

[53] Nicolotti C, Cirlini M, Del Vecchio L, Hadj Saadoun J, Bernini V, Gatti M, Bottari B, Martelli F. Lactic Acid Fermentation of Chlorella vulgaris to Improve the Aroma of New Microalgae-Based Foods: Impact of Composition and Bacterial Growth on the Volatile Fraction. Foods, 2025, 14: 1511

[54] Park C, Lee JH, Yang X, Yoo HY, Lee JH, Lee SK, Kim SW. Enhancement of hydrolysis of Chlorella vulgaris by hydrochloric acid. Bioprocess Biosyst Eng, 2016, 39: 1015-1021

[55] Paterson S, Majchrzak M, Alexandru D, Di Bella S, Fernández-Tomé S, Arranz E, de la Fuente MA, Gómez-Cortés P, Hernández-Ledesma B. Impact of the biomass pretreatment and simulated gastrointestinal digestion on the digestibility and antioxidant activity of microalgae Chlorella vulgaris and Tetraselmis chuii. Food Chem, 2024, 453: 139686

[56] Pérez-Alva A, MacIntosh AJ, Baigts-Allende DK, García-Torres R, Ramírez-Rodrigues MM. Fermentation of algae to enhance their bioactive activity: A review. Algal Res, 2022, 64: 102684

[57] Postma PR, Suarez-Garcia E, Safi C, Yonathan K, Olivieri G, Barbosa MJ, Wijffels RH, Eppink MHM. Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates. Bioresour Technol, 2017, 224: 670-679

[58] Rahman MM, Hosano N, Hosano H. Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies. Molecules, 2022, 27: 2786

[59] Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorisation assay. Free Radic Biol Med, 1999, 26: 1231-1237

[60] Rezvankhah A, Yarmand MS, Ghanbarzadeh B, Mirzaee H. Generation of bioactive peptides from lentil protein: degree of hydrolysis, antioxidant activity, phenol content, ACE-inhibitory activity, molecular weight, sensory, and functional properties. Food Measure, 2021, 15: 5021-5035

[61] Safi C, Zebib B, Merah O, Pontalier P-Y, Vaca-Garcia C. Morphology, composition, production, processing and applications of Chlorella vulgaris: A review. Renew Sustain Energy Rev, 2014, 35: 265-278

[62] Saha S, Maji S, Ghosh SK, Maiti MK. Engineered Chlorella vulgaris improves bioethanol production and promises prebiotic application. World J Microbiol Biotechnol, 2024, 40: 271

[63] Sanjeewa KKA, Herath KHINM, Kim Y-S, Jeon Y-J, Kim S-K. Enzyme-assisted extraction of bioactive compounds from seaweeds and microalgae. TRAC Trends Anal Chem, 2023, 167: 117266

[64] Ścieszka S, Klewicka E. Influence of the Microalga Chlorella vulgaris on the Growth and Metabolic Activity of Lactobacillus spp. Bacteria Foods, 2020, 9: 959

[65] Siezen RJ, van Hylckama Vlieg JE. Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer. Microb Cell Fact, 2011, 10: S3

[66] Singleton VL, Orthofer R, Lamuela-Raventós RM (1999) [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In: Methods in enzymology. Academic Press, pp 152–178

[67] Sivaramakrishnan R, Kanwal S, Incharoensakdi A, Nirmal N, Srimongkol P. Exploring the nutraceutical and functional food potential of microalgae: Implications for health and sustainability. J Agric Food Res, 2025, 22: 102148

[68] Spain O, Funk C. Detailed Characterisation of the Cell Wall Structure and Composition of Nordic Green Microalgae. J Agric Food Chem, 2022, 70: 9711-9721

[69] Sriyod K, Reungsang A, Plangklang P. One-step multi enzyme pretreatment and biohydrogen production from Chlorella sp. biomass. Int J Hydrog Energy, 2021, 46: 39675-39687

[70] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol, 2002, 83: 1-11

[71] Sylwia Ś, Elżbieta K. Algae Chlorella vulgaris as a factor conditioning the survival of Lactobacillus spp. adverse Environ conditions LWT, 2020, 133: 109936

[72] Syrpas M, Bukauskaite J, Paskauskas R, Basinskiene L, Venskutonis P. Recovery of lipophilic products from wild cyanobacteria (Aphanizomenon flos-aquae) isolated from the Curonian Lagoon by means of supercritical carbon dioxide extraction. ALGAL RESEARCH-BIOMASS BIOFUELS Bioprod, 2018, 35: 10-21

[73] Syrpas M, Bukauskaitė J, Ramanauskienė K, Karosienė J, Majienė D, Bašinskienė L, Venskutonis PR. Ultrasound-Assisted Extraction and Assessment of Biological Activity of Phycobiliprotein-Rich Aqueous Extracts from Wild Cyanobacteria (Aphanizomenon flos-aquae). J Agric Food Chem, 2020, 68: 1896-1909

[74] Syrpas M, Valanciene E, Augustiniene E, Malys N. Valorization of Bilberry (Vaccinium myrtillus L.) Pomace by Enzyme-Assisted Extraction: Process Optimisation and Comparison with Conventional Solid-Liquid Extraction. Antioxidants, 2021, 10: 773

[75] da Teixeira RSS, da Silva AS, Ferreira-Leitão VS, Silva Bon EP. Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements. Carbohydr Res, 2012, 363: 33-37

[76] Tomassi E, Arouna N, Caruso MG, Girgenti A, Picone P, Nuzzo D, Pucci L. Fermentation of Chlorella vulgaris and Aphanizomenon flos-aquae biomass improves the antioxidant profile. LWT, 2025, 215: 117183

[77] Tu W-L, Huang J-M, Ou C-M. Lactic acid production from green microalgal biomass hydrolysates via dilute acid pretreatment and fermentation. BioResources, 2026, 21: 535-546

[78] Verni M, Verardo V, Rizzello CG. How Fermentation Affects the Antioxidant Properties of Cereals and Legumes. Foods, 2019, 8: 362

[79] Wang C, Onyeaka H, Miri T, Soltani F. Chlorella vulgaris as a food substitute: Applications and benefits in the food industry. J Food Sci, 2024, 89: 8231-8247

[80] Xia D, Qiu W, Wang X, Liu J. Recent Advancements and Future Perspectives of Microalgae-Derived Pharmaceuticals. Mar Drugs, 2021, 19: 703

[81] Yaghoubzadeh Z, Safari R. Extraction of Bioactive Peptides from Chlorella vulgaris Using Enzymatic Hydrolysis: A Green Natural Antioxidant. Int J Pept Res Ther, 2025, 31: 33

[82] Yang J, Cai D, Liu X, Zhu L, Zhang C, Peng Q, Han Y, Liu G, Yang M. Glucose Conversion for Biobutanol Production from Fresh Chlorella sorokiniana via Direct Enzymatic Hydrolysis. Fermentation, 2023, 9: 284

[83] Yin L-J, Jiang S-T, Pon S-H, Lin H-H. Hydrolysis of Chlorella by Cellulomonas sp. YJ5 Cellulases and Its Biofunctional Properties. J Food Sci, 2010, 75: H317-H323

[84] Yuan Q, Li H, Wei Z, Lv K, Gao C, Liu Y, Zhao L. Isolation, structures and biological activities of polysaccharides from Chlorella: A review. Int J Biol Macromol, 2020, 163: 2199-2209

[85] Zhang Y, Vadlani PV, Kumar A, Hardwidge PR, Govind R, Tanaka T, Kondo A. Enhanced D-lactic acid production from renewable resources using engineered Lactobacillus plantarum. Appl Microbiol Biotechnol, 2016, 100: 279-288

[86] Zhou N, Zhang Y, Wu X, Gong X, Wang Q. Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2. Bioresour Technol, 2011, 102: 10158-10161

Funding

Lietuvos Mokslo Taryba(S-MIP-23-78)

RIGHTS & PERMISSIONS

The Author(s)