Process for obtaining an extract with a high lutein content from microalgae, extract with a high lutein content, and use of the obtained extract

Abstract

The present invention provides a process for obtaining an extract with a high lutein content from microalgae, comprising a microalgae cultivation process with high biomass and lutein production, as well as a process for extracting lutein and chlorophylls using green solvents. The microalgae belong to the genus Chlorella from Brazilian biodiversity, which produces metabolites comprising lutein and chlorophylls. The cultivation process for the microalgae of the genus Chlorella was optimized, and different carbon and nitrogen sources used in the culture medium were evaluated with respect to lutein production. The lutein extraction process employs solvents such as ethanol and glycerin, which are safe for cosmetic and nutraceutical applications. The obtained extract has a high lutein content and also contains chlorophylls and neoxanthin. Lutein assists in protecting the skin against photoinduced damage, and the extract with a high lutein content is suitable for use in cosmetic compositions, pharmaceutical compositions, and the food industry.

Description

[0001] PROCESS FOR OBTAINING AN EXTRACT WITH HIGH LUTEIN CONTENT FROM MICROALGAE, EXTRACT WITH HIGH LUTEIN CONTENT AND USE OF THE EXTRACT OBTAINED

[0002] FIELD OF THE INVENTION

[0003] [1] The present invention relates to the field of Biotechnology, more specifically to a process for cultivating microalgae and extracting bioactive compounds from microalgae biomass. The invention provides a process for obtaining an extract with a high lutein content from microalgae. To this end, the process includes different steps, such as the preparation of the culture medium, the cultivation of the microalgae, and the extraction of lutein, using green or environmentally friendly solvents. The extract with a high lutein content contains lutein and chlorophylls, and has applications in cosmetic compositions, pharmaceutical compositions, and the food industry.

[0004] DESCRIPTION OF THE STATE OF THE ART

[0005] [2] Lutein is industrially produced from Tagetes erecta flowers (family Asteraceae), which contain between 0.1 - 0.2% of dry matter carotenoids, of which 80% are lutein. The plant is cultivated for lutein production in Mexico, Peru, Ecuador, Spain, India and China (Sivel, M., Krácmar, S., Fisera, M., Klejdus, B., & Kubáh, V. (2014). Lutein content in marigold flower (Tagetes erecta L.) concentrates used for production of food supplements. Czech journal of food sciences). The lutein in T. erecta flowers is chemically bound to various types of fatty acids and, thus, through saponification of the extract, the fatty acid esters of lutein are converted into free lutein (Madhavi, DL, and Kagan, DI (2002). Process for the isolation of mixed carotenoids from plants. US6380442B1).

[0006] [3] Lutein production from microalgae biomass is very promising, since, in general, the lutein content in microalgae is higher than that found in T. erecta flowers. In addition, microalgae have a higher growth rate, photosynthetic efficiency and carbon mitigation potential than plants such as T. erecta (Zheng, H., Wang, Y., Li, S., Nagarajan, D., Varjani, S., Lee, DJ, & Chang, JS (2022). Recent advances in lutein production from microalgae. Renewable and Sustainable Energy Reviews, 153, 111795).

[0007] [4] To produce 1 kg of lutein, T. erecta needs more land and water, but fewer nutrients (N, P, K) and less energy than microalgae. Thus, lutein production by microalgae has a lower demand for land and water. In addition, microalgae can be cultivated on non-arable land in all seasons. However, among the obstacles to lutein production are the high harvesting costs and high energy demand for cell disruption and extraction (Lin, JH, Lee, DJ, & Chang, JS (2015). Lutein production from biomass: Marigold flowers versus microalgae. Bioresource Technology, 184, 421-428).

[0008] [5] Several microalgae species produce lutein, such as species of the genera Chlorella spp., Scenedesmus spp., and Muriellopsis spp. One of the most important ways to increase biomass and lutein production is related to optimizing the culture medium under mixotrophic or heterotrophic conditions (Muhammad, G., Butler, TO, Chen, B., Lv, Y., Xiong, W., Zhao, X., ... & Alam, MA (2022). Sustainable production of lutein — an underexplored commercially relevant pigment from microalgae. Biomass Conversion and Biorefinery, 1-22).

[0009] [6] There are methods for cultivating Chlorella aimed at producing lutein. In EP1808483B1, Chlorella sorokiniana SAG 211-32 is cultivated under mixotrophic conditions using Amon medium combined with nitrates in the amount of 40 mM and sodium acetate in the amount of 40 mM (0.04 M = 3.3 g / L). The lutein content obtained using 40 mM sodium acetate was 5.2 mg / g. Algal biomass is harvested by centrifugation. The algal biomass can be dried and used directly or mixed with other ingredients in food supplements. In addition, lutein can be concentrated by extraction for use in food supplements or pharmaceuticals.

[0010] [7] In EP2157167B1, the use of a medium supplemented with 20 - 60 mM sodium acetate (0.02 - 0.06 M = 1.6 - 4.9 g / L) and / or 100 mM glucose (0.1 M = 18 g / L) in the mixotrophic cultivation of C. sorokiniana SAG 211-32 and mutants of this species is revealed, aiming at obtaining lutein. In addition, photoautotrophic cultivation by bubbling air supplemented with 1% (v / v) CO2 through the cell suspension was also revealed. The lutein content obtained varied between 1.85 and 4.43 mg / g for the wild strain, and between 2.55 and 4.79 mg / g for the mutant strains.

[0011] [8] In another cultivation process, different factors were evaluated in the cultivation of C. sorokiniana aiming at high lutein production. Among the factors are sodium acetate and / or glucose in mixotrophic cultivation conditions. In mixotrophic cultures, Arnon medium, modified with K2HPO4 and NaNCE, and sodium acetate and / or glucose were used, either at the beginning of the culture or after 24 h (in the middle of the exponential phase). The use of sodium acetate (40 mM = 0.04 M = 3.3 g / L) with the addition of glucose (100 mM = 0.1 M = 18 g / L) during the exponential phase after 24 h of cultivation resulted in algal biomass with a lutein content of 2.6 mg / g of dry mass (Cordero, B. E, Obraztsova, I., Couso, L, Leon, R., Vargas, MA, & Rodríguez, H. (2011). Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis. Marine drugs, 9(9), 1607-1624).

[0012] [9] Lutein extraction processes from plants, such as Tagetes species, are carried out by conventional processes, such as maceration, and advanced processes, such as supercritical extraction and enzyme-assisted extraction (Manzoor, S., Rashid, R., Panda, BP, Sharma, V., & Azhar, M. (2022). Green lutein extraction from marigold flower petals, process optimization and its potential to improve the oxidative stability of sun oil. Ultrasonics sonochemistry, 85, 105994; Bhartia, US, & Reddy, GBS (2016).

[0013]

[0010] Lutein is a compound produced by microalgae, but it is not secreted by these microorganisms, remaining in the microalgae cell. Therefore, lutein extraction processes from microalgae involve a cell disruption or lysis step. For example, in the method for obtaining a lutein-rich composition from Chlorella vulgaris, described in US9315434B2, algal biomass is processed in a ball mill, followed by extraction with polar solvents, such as ethyl acetate, to obtain lutein-containing oleoresin, and supercritical extraction of the oleoresin using high-pressure CO2 to obtain an insoluble lutein-rich fraction (Looten, P., Patinier, S., Francais, E., Perrut, M., & Satre-Buisson, A. (2016). Method for preparing a composition rich in lutein produced by microalgae. US9315434B2).

[0014]

[0011] The use of petrochemical solvents in extraction processes, in addition to causing environmental problems, can impart unsuitable toxicity to cosmetic products. Thus, the use of ionic liquids for the extraction of lutein from microalgae has been proposed, without the need for a disruption step (Paliwal, C., Rehmanji, M., Shaikh, KM, Zafar, SU, & Jutur, PP (2022). Green extraction processing of lutein from Chlorella saccharophila in water-based ionic liquids as a sustainable innovation in algal biorefineries. Algal Research, 66, 102809). Another option is the use of supercritical fluids in carotenoid extraction processes from microalgae (Kitada, K., Machmudah, S., Sasaki, M., Goto, M., Nakashima, Y., Kumamoto, S., & Hasegawa, T. (2009). Supercritical CO2 extraction of pigment components with pharmaceutical importance from Chlorella vulgaris. Journal of Chemical Technology & Biotechnology: International Research in Process, Environmental & Clean Technology, 84(5), 657-661).However, although these alternatives are environmentally friendly, they add a high cost to the production process.

[0015]

[0012] Thus, the use of renewable solvents, or those derived from biomass, in lutein extraction processes from microalgae is a sustainable alternative to petrochemical solvents. Among the solvents derived from biomass are ethanol and glycerol. Water is also a sustainable or green solvent, as it has a low environmental impact, is safe for operators, and presents low health risks to consumers when compared to petrochemical solvents (Calvo-Flores, FG, Monteagudo-Arrebola, MJ, Dobado, JA, & Isac-García, J. (2018). Green and bio-based solvents. Topics in Current Chemistry, 376, 1-40).

[0016]

[0013] In document CN102976992A, ethanol was used as a solvent for the extraction of free lutein and its esters from Chlorella biomass by an ultrasonic method, followed by the addition of potassium hydroxide for saponification of the esters and extraction of free lutein with dichloromethane, resulting in 3.02 mg / g of lutein (dry weight). However, despite the use of ethanol in the first step, the process employs ultrasound, making it costly, and it has a saponification step and a final extraction with dichloromethane, which is not a renewable solvent.

[0014] In addition to lutein, microalgae also produce other bioactive compounds, such as chlorophylls, which are natural green pigments responsible for absorbing light energy and converting it into chemical energy through photosynthesis. Chlorophylls have antioxidant, anti-inflammatory, antimutagenic, and antimicrobial properties, and are widely used in the cosmetic, pharmaceutical, and food industries.There are different types of chlorophylls: A, B, C, D, and F, which have differences in their molecular structures based on cyclic tetrapyrrole (DA SILVA FERREIRA, Veronica; SANT' ANNA, Celso. Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications. World Journal of Microbiology and Biotechnology, v. 33, n. 1, p. 20, 2017).

[0017]

[0015] Microalgae of the genus Chlorella are also known as "emerald food" due to their high chlorophyll content. The main chlorophylls present in Chlorella microalgae are chlorophyll A and chlorophyll B. Chlorophylls can be extracted using organic solvents (DE ANDRADE, C. José; DE ANDRADE, L. Maria. An overview on the application of genus Chlorella in biotechnological processes. J. Adv. Res. Biotechnol, v. 2, p. 1-9, 2017).

[0018]

[0016] In the present invention, an optimized cultivation process for microalgae from Brazilian biodiversity was developed, with high production of biomass, lutein, and chlorophylls. The invention also includes a process for extracting lutein and chlorophylls using green solvents, which are environmentally sustainable and safe for application in cosmetic formulations.

[0019] SUMMARY OF THE INVENTION

[0020]

[0017] The present invention relates to a process for obtaining an extract with a high lutein content from the cultivation of microalgae from Brazilian biodiversity, which exhibits high biomass and lutein production, and the extraction of lutein and chlorophylls from the microalgae biomass using green solvents, i.e., solvents with low environmental impact, safe and with lower health risks.

[0018] In a first aspect, the invention relates to a process for obtaining an extract with a high lutein content from microalgae comprising: (a) preparation of culture medium; (b) inoculation of the microalgae; (c) cultivation of the microalgae; (d) harvesting of the microalgae to obtain microalgae biomass; (e) extraction of lutein and chlorophylls from the microalgae biomass, using green solvents.

[0021]

[0019] A second aspect of the invention relates to a high-lutein extract obtained by a process for obtaining a high-lutein extract from microalgae comprising: (a) preparation of culture medium; (b) inoculation of microalgae; (c) cultivation of microalgae; (d) harvesting of microalgae to obtain microalgae biomass; (e) extraction of lutein and chlorophylls from microalgae biomass using green solvents.

[0022]

[0020] Another aspect of the invention is the use of lutein-rich extract in cosmetic compositions, as lutein helps protect the skin from photo-induced damage due to its robust antioxidant action (Mitra, S., Rauf, A., Tareq, AM, Jahan, S., Emran, TB, Shahriar, TG, ... & Rengasamy, KR (2021). Potential health benefits of carotenoid lutein: An updated review. Food and Chemical Toxicology, 154, 112328). Furthermore, the lutein-rich extract contains chlorophylls, which are natural green pigments that can be used as colorants, as well as having bioactive properties such as antioxidant, antimicrobial, anti-inflammatory, and antimutagenic effects. Therefore, the chlorophylls present in the extract increase its applicability in cosmetic formulations.

[0023]

[0021] Additionally, due to the bioactive properties of lutein and chlorophylls, another use of lutein-rich extract is in pharmaceutical compositions.

[0024]

[0022] In addition, another use of lutein-rich extract is in the food industry.

[0025] DESCRIPTION OF THE FIGURES

[0026]

[0023] Figure 1. Focal point for collecting the microalga and micrograph obtained by optical microscope of the microalga C. sorokiniana strain deposited at NCMA (accession number 202112002).

[0024] Figure 2. Response surface and contour curve for the lutein response, in the culture of C. sorokiniana strain deposited at NCMA (accession number 202112002), for 14 days.

[0027]

[0025] Figure 3. Response surface for the lutein response, in the culture of C. sorokiniana strain deposited at NCMA (accession number 202112002), for 21 days.

[0028]

[0026] Figure 4. Observed effects of treatments T1 (BG-11 medium containing 2 g / l sodium acetate + 6 g / l glucose) and T2 (BG-11 medium containing 2 g / l sodium acetate + 3 g / l glucose) in relation to the control (BG-11 medium), in the cultivation of the microalga C. sorokiniana strain deposited in NCMA (accession number 202112002).

[0029]

[0027] Figure 5. Chromatograms of C. sorokiniana extracts using different solvents in the extraction.

[0030]

[0028] Figure 6. Response surface of the first design of the lutein extraction process from C. sorokiniana biomass using different solvent mixtures, in the smaller image are the reference points used (water, ethanol, glycerin).

[0031]

[0029] Figure 7. Validation of the use of different solvents in the extraction of lutein from C. sorokiniana (different letters indicate statistical difference by Tukey's test).

[0032]

[0030] Figure 8. Lutein extraction efficiency (mg / g of initial biomass) from C. sorokiniana using different extraction methods, in fresh / frozen biomass and in lyophilized / frozen biomass.

[0033]

[0031] Figure 9. Pareto chart of the standardized effects of the three variables (time, mass / solvent ratio, solvent change) and contour plot of the two variables selected as significant in the extraction efficiency of lutein from C. sorokiniana.

[0034]

[0032] Figure 10. Response surface of the 2-factor Central Composite Rotational design, considering the sum of the two lutein extractions from C. sorokiniana.

[0035]

[0033] Figure 11. Graphs of lutein extraction efficiency kinetics (mg / g) and lutein concentration (pg / mL) of C. sorokiniana using 80:20 solvent by dynamic maceration at 25 °C with orbital shaking at 200 rpm, in solid:liquid ratios of 1:6 and 1:16.

[0036]

[0034] Figure 12. Extraction yield (%) of lutein from C. sorokiniana using the solvent in 3 consecutive extractions, 1:16 ratio for 8 hours, for each solvent system. Highlighted are the percentages achieved in the first extraction.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038]

[0035] The term “green solvents” refers to solvents with low environmental impact, safe and with low health risk, and in the present invention includes water, ethanol, glycerol, 1-butanol, ethyl acetate or 1-propanol and a mixture of two or more of these.

[0039]

[0036] In this sense, the present invention provides a process for obtaining lutein-rich extract from microalgae comprising: (a) preparation of culture medium; (b) inoculation of microalgae; (c) cultivation of microalgae; (d) harvesting of microalgae to obtain microalgae biomass; (e) optionally, drying of microalgae biomass; (f) extraction of lutein and chlorophylls from microalgae biomass using green solvents.

[0040]

[0037] In a preferred embodiment, the process for obtaining lutein-rich extract from microalgae comprises microalgae of the genus Chlorella. More preferably, the microalga is of the species Chlorella sorokiniana. Chlorella sorokiniana belongs to the group of green algae and corresponds to a microalga species occurring in aquatic environments that grows in free-living unicellular form. As shown in Figure 1, the strain used in the process of the present invention was isolated from a freshwater body on a private property called Chapada Imperial, located in the municipality of Brasília / DF, situated in the Brazilian Cerrado. The exact collection point corresponds to latitude 15°33'35.99” S and longitude 48°6'23.91” W.The collection of this strain was carried out under authorization number 39146-2 via the Biodiversity Authorization and Information System (Sisbio), granted by the Chico Mendes Institute for Biodiversity Conservation (ICMBio) (linked to the Ministry of the Environment of the Federative Republic of Brazil), in addition to authorization from the owner of Chapada Imperial. This microalga is conserved in the Collection of Microorganisms and Microalgae Applied to Agroenergy and Biorefineries of Embrapa Agroenergia (CMMAABio) and is registered under identification LBA39 in the Alelomicro System, Alelo Genetic Resources Portal (web platform of resources and tools for managing research and development data and information) of the Brazilian Agricultural Research Corporation (Embrapa). This microalga species does not correspond to an endangered or protected species.The use of this strain for Research and Technological Development purposes is registered under Access Registration number ABEE0A5 in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen), managed by the Genetic Heritage Management Council (CGen), which is also linked to the Ministry of the Environment of the Federative Republic of Brazil.

[0041]

[0038] The Chlorella sorokiniana strain was deposited at the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA), Bigelow Laboratories, which is an International Depositary Authority designated by the World Intellectual Property Organization (WIPO) for the deposit of biological material essential to the invention. Thus, in a more preferred embodiment of the invention, the process for obtaining lutein-rich extract from microalgae, the microalga Chlorella sorokiniana is of the strain deposited at the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) under accession number 202112002.

[0042]

[0039] In another preferred embodiment, the process for obtaining lutein-rich extract from microalgae is characterized by the culture medium in step (a) comprising BG-11 culture medium supplemented with a carbon source. In a further embodiment, the culture medium prepared in step (a) comprises sodium acetate, glucose, and glycerol as a carbon source. Preferably, the culture medium prepared in step (a) comprises sodium acetate and glucose as a carbon source. More preferably, the concentration of sodium acetate in the culture medium is in the range of 0 to 4 g / L. Even more preferably, the culture medium comprises sodium acetate at a concentration of 2 g / L. In another more preferred embodiment, the concentration of glucose in the culture medium is in the range of 0 to 12 g / L. Even more preferably, the culture medium comprises glucose at a concentration of 6 g / L.

[0040] In another preferred embodiment of the invention, the process for obtaining lutein-rich extract from microalgae is characterized by step (c) being carried out in a 12h / 12h photoperiod, with a light intensity of 4000 lux and aeration with a flow rate in the range of 0.4 to 0.6 L / min. More preferably, the microalgae cultivation in step (c) is carried out at a temperature in the range of 22 to 30 °C. Even more preferably, the microalgae cultivation in step (c) is carried out at a temperature of 25 °C. In another more preferred embodiment, the microalgae cultivation in step (c) is carried out for 4 to 22 days. Even more preferably, the microalgae cultivation in step (c) is carried out for 8 days.

[0043]

[0041] In yet another preferred embodiment, the process for obtaining lutein-rich extract from microalgae is characterized by step (d) being carried out by centrifugation.

[0044]

[0042] In another preferred embodiment, the process for obtaining lutein-rich extract from microalgae is characterized by the drying of the biomass in step (e) being carried out by freeze-drying, spray drying and vacuum drying. More preferably, the drying of the biomass in step (e) is carried out by freeze-drying.

[0045]

[0043] In another preferred embodiment, the process for obtaining lutein-rich extract from microalgae comprises extracting lutein and chlorophylls from microalgae biomass in step (f) by dynamic maceration. More preferably, the extraction of lutein and chlorophylls is carried out using ethanol, glycerol, mixtures of ethanol and glycerol, or mixtures of ethanol, glycerol, and water. Even more preferably, the extraction of lutein and chlorophylls is carried out using ethanol and glycerol. Much more preferably, the ethanol concentration is 40 to 100% w / w and the glycerol concentration is 0 to 60% w / w. In another more preferred embodiment, the extraction is carried out with wet biomass. In yet another more preferred embodiment, the extraction is carried out with dry biomass, preferably freeze-dried biomass. Even more preferably, the extraction is carried out in a solid / liquid ratio of 1:3 to 1:32.Preferably, the extraction is carried out in a solid / liquid ratio of 1:16. In another more preferred mode, the extraction is carried out for a time of 1 to 25 hours. In an even more preferred mode, the extraction is carried out for a time of 8 hours.

[0046]

[0044] A second aspect of the invention relates to lutein-rich extract obtained by the process of obtaining lutein-rich extract from microalgae as specified above. In a preferred aspect, the lutein-rich extract obtained by the process of the present invention contains lutein and chlorophylls.

[0047]

[0045] Lutein possesses several bioactive properties, such as antioxidant, anti-inflammatory, cardioprotective, and anticancer properties. Lutein is widely used to prevent the development of eye diseases. Lutein helps protect against macular diseases, such as age-related macular degeneration, cataracts, among others. Lutein can also be used to combat skin damage caused by radiation and prevent photoaging (SHEGOKAR, Ranjita; MITRI, Khalil. Carotenoid lutein: a promising candidate for pharmaceutical and nutraceutical applications. Journal of dietary supplements, v. 9, n. 3, p. 183-210, 2012). Thus, lutein and / or lutein-rich extracts are widely used in cosmetic compositions, pharmaceutical compositions, and in the food industry.

[0048]

[0046] Chlorophylls also have many benefits for human health, such as antioxidant, anti-inflammatory, anticancer and anti-obesity properties. Thus, chlorophylls have applications in cosmetic, pharmaceutical and food industries (EBRAHIML Peyman et al. Chlorophylls as natural bioactive compounds existing in food by-products: A critical review. Plants, v. 12, n. 7, p. 1533, 2023).

[0049]

[0047] Thus, another aspect of the invention relates to the use of lutein-rich extract obtained from microalgae in cosmetic compositions. Additionally, the use of lutein-rich extract obtained from microalgae is carried out in pharmaceutical compositions. Another use of lutein-rich extract obtained from microalgae is in the food industry.

[0050]

[0048] The following examples are intended to provide a better understanding of the invention, are given as illustrations only, and do not limit the invention in any way. As can be seen from the examples, the microalga Chlorella sorokiniana cultivated in an optimized culture medium results in microalgae biomass with a high lutein content. Furthermore, the examples show that extracting lutein from microalgae biomass using green solvents is technically feasible and a sustainable process. EXAMPLES OF EMBODIMENTS OF THE INVENTION

[0051]

[0049] The experiments were performed using the microalga C. sorokiniana strain deposited at the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) under accession number 202112002, from a cryopreserved sample. Cultures were performed using BG-11 medium (Rippka et al., 1979), supplemented with agar when semi-solid medium was used. Initially, the cryopreserved sample was thawed at room temperature and inoculated under sterile conditions in 350 ml of liquid BG-11 medium, followed by cultivation for seven days in a room with a temperature of 25 ± 2 °C, aeration with compressed air at a flow rate of 0.4 to 0.6 L / min and a 12h / 12h lighting cycle. From the liquid culture, plating was performed by streaking onto plates containing semi-solid BG-11 medium, incubated at 25 ± 2 °C with constant illumination.After 21 days of growth, a new culture was made in liquid medium from an isolated colony, under the same conditions mentioned above, from which the experiments described below were carried out.

[0052]

[0050] For the following experiments, an analytical method was implemented for the quantification of chlorophyll A, chlorophyll B, and lutein present in C. sorokiniana. The method is divided into two parts:

[0053]

[0051] (1) Lysis and extraction stage: in this stage the algal biomass is harvested, washed and lysed so that the substances present inside the cells are extracted and subsequently quantified.

[0054]

[0052] (2) Quantification step: The material extracted after cell lysis or the extracts produced in the extraction optimization experiments were analyzed by ultra-high performance liquid chromatography coupled to a diode array detector (UHPLC-DAD) using a Waters Acquity HSS T3 1.8 µm, 2.1 x 150 mm column.

[0055] Example 1: Selection of relevant variables in the cultivation of Chlorella sorokiniana for the production of biomass, chlorophylls, and lutein.

[0056]

[0053] This experiment was conducted with the objective of evaluating the effect of the variables sodium nitrate (NaNOs), glucose (C3H8O3), sodium acetate (C2H3NaO2), glycerol (C3H8O3), and urea (CH4N2O) on biomass production (evaluated as a function of dry weight) and the production of the dyes lutein, chlorophyll A, and chlorophyll B by the microalga. For this, the Variable Screening methodology was used, in which the effect of independent factors (variables under study) on the dependent variables (responses) is evaluated.

[0057]

[0054] A fractional factorial design 2 5 ' 1 The experiment, consisting of 19 trials, was used to evaluate the effect of the 5 variables (factors) listed in Table 1, with each variable being evaluated at two levels (in addition to intermediate levels). Table 2 presents the different combinations of variables under study for each of the 19 experiments.

[0058] Table 1. Levels of the factors studied in the fractional factorial design 2 5 ' 1 .

[0059] * The BG-11 culture medium already contains 1.5 g / L of sodium nitrate. For the experiments, when appropriate, sodium nitrate was added to the flasks in quantities that reached the values ​​shown in the table.

[0060]

[0055] For the cultivations, 19 500ml Erlenmeyer flasks were identified, and 350 ml of BG11 medium and the corresponding amount of each reagent, except glucose, were added to each. Each Erlenmeyer flask was closed with a cotton swab and gauze, and a glass pipette, used for aeration, with a filter at the outer end, was used. The entire assembly was autoclaved for 20 min at 121 °C and 1 atm. In order to avoid the occurrence of the Maillard reaction, the glucose was weighed on aluminum foil, which was folded, identified, and placed inside a beaker for autoclaving. After autoclaving, within the laminar flow hood, each aluminum foil bag containing glucose was opened and the contents added to the corresponding Erlenmeyer flask.

[0061] Table 2. Fractional factorial design 2 5 ' 1 .

[0056] The microalga C. sorokiniana strain deposited at NCMA (accession number 202112002) was inoculated under sterile conditions, so that in each Erlenmeyer flask the initial OD was 0.05 (À=445nm). The cultures were maintained in a room with a temperature of 25 ± 2°C, aeration with compressed air at a flow rate of 0.4 to 0.6 L / min and a 12h / 12h photoperiod, with a light intensity of 4000 lux. Sampling for evaluation of response variables was performed 14, 21, and 28 days after inoculation. From the samples obtained, cell lysis, dye extraction, and chromatographic analyses were applied. The evaluation of the results was performed using Statistica software version 12.0, with a 90% confidence interval.

[0062]

[0057] The effects of these independent variables on the dependent variables dry weight (mg / ml) and chlorophyll A and B (pg / ml) and lutein (pg / ml) contents are presented in Tables 3, 4 and 5, where it is possible to verify the coded independent variables, as well as the dependent (or response) variables obtained for each assay performed after 14 days, 21 days and 28 days of cultivation, respectively.

[0063]

[0058] Tables 6, 7, 8, and 9 show the p-values ​​obtained for all independent variables in relation to the observed response variables, as well as the R2 values ​​obtained. Observing the p-values ​​obtained for each independent variable evaluated, which show the significance of these factors in the effect obtained for each of the response variables, it was not possible to verify significant effects with the addition of glycerol (5 g / L and 10 g / L) and urea (1 g / L and 2 g / L), nor with an increase in the concentration of sodium nitrate (1.5 g / L and 3 g / L) in any of the evaluated cultivation intervals (14, 21, and 28 days). The factor that showed the best overall response, considering all response variables in the experiment, was sodium acetate, which presented p < 0.1 for dry weight (with 14 and 21 days of cultivation), lutein (with 14 and 21 days of cultivation), chlorophyll A (14 days of cultivation) and chlorophyll B (14 and 21 days of cultivation).Glucose showed a significant effect only on dry weight after 21 days of cultivation.

[0064]

[0059] By evaluating the results of each response variable (dry weight, chlorophylls, and lutein) in relation to each independent variable (factors), it is possible to assume which factors may have a greater or lesser effect on the observed responses. Considering that the only independent variables that showed a significant effect at a 90% confidence level (p < 0.1) were sodium acetate and glucose, these factors were selected for the full central composite rotatable design.

[0065] Table 3. Dry weight (mg / mL), chlorophyll A (Δg / mL), chlorophyll B (Δg / mL) and lutein (Δg / mL) after 14 days of cultivation, for each trial of the fractional factorial design. 5 ' 1 . Table 4. Dry weight (mg / mL), chlorophyll A (Δg / mL), chlorophyll B (Δg / mL) and lutein (Δg / mL) after 21 days of cultivation, for each trial of the fractional factorial design. 5 ' 1 .

[0066] Table 5. Dry weight (mg / mL), chlorophyll A (Δg / mL), chlorophyll B (Δg / mL) and lutein (Δg / mL) after 28 days of cultivation, for each trial of the fractional factorial design. 5 ' 1 .

[0067]

[0068] Table 6. p-values ​​obtained for the independent variables in relation to dry weight, in each trial in the 2-part fractional factorial design. 5 ' 1 .

[0069] Table 7. p-values ​​obtained for the independent variables in relation to chlorophyll A, in each trial in the 2-part fractional factorial design. 5 ' 1 . Table 8. p-values ​​obtained for the independent variables in relation to chlorophyll B, in each trial in the 2-part fractional factorial design. 5 ' 1 .

[0070] Table 9. p-values ​​obtained for the independent variables in relation to lutein, in each trial in the 2-part fractional factorial design. 5 ' 1 .

[0071]

[0060] The results presented in Table 6 show that there was an increase in the amount of biomass produced (evaluated as dry weight) between 21 and 28 days of cultivation. However, a decrease in chlorophyll A (Table 7), chlorophyll B (Table 8), and lutein (Table 9) levels was observed during the same period. Since factorial designs do not allow for replicates in the experiments, in this experiment only the central points were evaluated in triplicate trials. However, due to the observed decrease in the response variables of interest, it was decided to perform the RCCD only at 14 and 21 days.

[0072] Example 2: evaluation of the effects of selected factors on biomass, chlorophyll, and lutein production.

[0073]

[0061] Based on the factors selected from the screening performed in example 1, which were sodium acetate and glucose, a set of trials was carried out, based on a central composite rotatable design (CCRD) 2 2 The experimental design was complete, with 4 axial points and 3 central points, resulting in a total of 11 trials. These trials evaluated the effects of the independent variables sodium acetate (0 to 4 g / L) and glucose (0 to 6 g / L) on the dependent variables dry weight (mg / ml) and lutein content (pg / ml), 14 and 21 days after inoculation. The actual values ​​of the independent variables used in the trials are presented in Table 10, while the actual and coded variables used in each trial of the experimental design are in Table 11.

[0074] Table 10. Levels of independent variables used in the central composite rotatable design (CCRD) 2 2 full.

[0075]

[0062] For the cultivations, 11 Duran flask-type reactors of 500 ml with lids, with one air inlet and one air outlet, both with filters, were identified and, in each one, 350 ml of BG-11 medium and the corresponding amount of sodium acetate shown in Table 13 were added. The sodium acetate was added in salt form. The entire set was autoclaved for 20 min at 121 °C and 1 atm. For glucose, a 180 g / L (1M) solution was made, which was autoclaved separately. Under sterile conditions, the volume of the 180 g / L glucose solution was added to each flask so that the final glucose concentration in the medium was as indicated in Table 11.

[0076] Table 11. Central composite rotatable design (CCRD) 2 2 full.

[0077]

[0063] To ensure that all cultures had the same final volume, autoclaved distilled water was added to the flasks that did not receive or received a smaller volume of glucose solution. Therefore: for level -1, 1.7 ml of glucose solution and 9.9 ml of distilled water were added; for level 0, 5.8 ml of glucose solution and 5.8 ml of distilled water were added; for level +1, 9.9 ml of glucose solution and 1.7 ml of distilled water were added; for level +1.41, 11.6 ml of glucose solution were added.

[0064] The microalga C. sorokiniana strain deposited at NCMA (accession number 202112002) was inoculated under sterile conditions so that in each reactor the initial OD was 0.05 (À=445nm). The cultures were maintained in a room with a temperature of 25 ± 2°C, aeration with compressed air at a flow rate between 0.4 and 0.6 L / min, and a photoperiod of 12h / 12h, with a light intensity of 4000 lux.Sampling for evaluation of response variables was performed between 14 and 21 days after inoculation. From the samples obtained, cell lysis, dye extraction, and chromatographic analyses were applied as previously described.

[0078]

[0065] The evaluation of the results was performed using Statistica software (StatSoft, Tulsa, USA), version 12.0, with a 90% confidence interval. After obtaining and evaluating the coefficients of the effects of the variables, an analysis of variance (ANOVA) was performed, which consists of evaluating the coefficients of determination (R²). 2 ) and the F-test, verifying whether the model provides an adequate fit to the experimental data.

[0079]

[0066] Table 12 shows the actual and coded values ​​of the independent variables used in this study, as well as the responses obtained for each trial of the experimental design.

[0080]

[0067] Based on the DCCR results, the influence of the independent variables on the dependent variables was observed by evaluating the effects, standard error, and statistical significance (p-value). In this study, a 90% confidence interval was chosen. After eliminating non-significant factors, the significance of the regression and lack of fit was verified at a 90% confidence level using an Analysis of Variance (ANOVA) with the F-test for the studied design.

[0081]

[0068] Statistical evaluation of the data obtained showed that the linear and quadratic terms of the sodium acetate variable had a negative effect on the dry weight response after 14 days of cultivation (Table 13). Similarly, the interaction between the two variables under study also had a negative effect on the response, at the 90% confidence level. The linear factor of glucose showed a positive effect, indicating that an increase in glucose content resulted in an increase in lutein content. Table 12. Values ​​of dry weight and lutein responses, for 14 and 21 days of cultivation, in each DCCR assay.

[0082] Table 13. Estimated effect, error, t-value, and degree of statistical significance (p) for each factor in the model coded for lutein content obtained from the 14-day culture.

[0083]

[0084]

[0069] After eliminating non-significant factors (quadratic term of the glucose variable), the significance of the regression and lack of fit was verified at a 90% confidence level. An Analysis of Variance (ANOVA) was performed using the F-test for the studied design. The R value 2 The results obtained show that regression explains 87.89% of the variations in the results. Fcal (10.89) was greater than FTab (3.18), indicating that the mathematical model is explained by regression and not by residuals. It can therefore be said that the mathematical model obtained (Eq. 1) is predictive.

[0085]

[0070] Lutein 14 days = 8.28 - 1.036* AC - 1.34* AC 2 + 1.79*G1 - 1.255*AC*G1 (Eq. 1)

[0086]

[0071] In Eq. 1: AC = sodium acetate; G1 = glucose.

[0087]

[0072] Figure 2 shows the response surface and contour curve, both constructed from the obtained model. The maximum response value is obtained with glucose at the higher concentration and sodium acetate at the lower concentration. That is, using higher glucose concentrations, it is possible to see an increase in lutein content when lower concentrations of sodium acetate are used.

[0088]

[0073] The effects of linear and quadratic factors and their interaction on the 21-day lutein response can be observed in Table 14. Table 14. Estimated effect, error, t-value, and degree of statistical significance (p) for each factor in the model coded for lutein content obtained from the 14-day culture.

[0089]

[0074] Based on the results obtained, it can be seen that the linear and quadratic terms of the variables sodium acetate and glucose significantly affect, at the 90% confidence level, the production of lutein by 21-day culture (21-day lutein). The linear term of glucose was the only one that showed a positive effect, indicating that an increase in glucose content results in an increase in lutein production.

[0090]

[0075] After eliminating non-significant factors (interaction term between variables), the significance of the regression and lack of fit was verified at a 90% confidence level. An Analysis of Variance (ANOVA) was performed using the F-test for the studied design. The R value 2The results obtained show that regression explains 89.90% of the variations in the results. Fcal (13.36) was greater than FTab (3.18), indicating that the mathematical model is explained by regression and not by residuals. These results show that the model obtained (Eq. 2) can be considered predictive.

[0091]

[0076] Lutein 21 days = 11.21 - 1.15*AC - 2.42*AC2 + 2.23G1 - 1.40G12 (Eq. 2)

[0092]

[0077] In Eq. 2: AC = sodium acetate; G1 = glucose.

[0093]

[0078] Figure 3 presents the response surface constructed from the obtained model.

[0079] The glucose variable had a significant positive effect on the lutein response at 21 days. It can be observed that, regardless of the sodium acetate concentration, an increase in glucose content results in an increase in the lutein content obtained. For the sodium acetate variable, it is observed that the highest dry weight values ​​are obtained when concentrations close to the central point are used - concentrations close to the minimum or maximum have a negative effect on the response.

[0094] Example 3: Validation of selected growing conditions in the DCCR

[0095]

[0080] Based on the DCCR results, triplicate cultures of the microalga C. sorokiniana strain deposited in NCMA (accession number 202112002) were performed, altering the medium as follows: control: BG11 medium (triplicate); treatment 1 (T1): BG11 medium + 2g / l Sodium Acetate + 6g / l Glucose (triplicate); treatment 2 (T2): BG11 medium + 2g / l Sodium Acetate + 3g / l Glucose (triplicate).

[0096]

[0081] The cultures were carried out in photobioreactors assembled with 2 L Duran parts, with one air inlet and one air outlet, both with filters, and filled with 1 L of the respective culture medium. In order to avoid the occurrence of the Maillard reaction, the glucose solution was autoclaved separately and then added to the medium. A glucose stock solution of 200 g / L was prepared. For the triplicate with 6 g / L of glucose, 30 ml of the stock solution were added, and for the triplicate containing 3 g / L of glucose, 15 ml of the stock solution + 15 ml of autoclaved distilled water were added. For the triplicate of the control condition, 30 ml of distilled water were added in order to maintain the final volume of all flasks equal.

[0097]

[0082] The microalga C. sorokiniana strain deposited at NCMA (accession number 202112002) was inoculated under sterile conditions, so that in each reactor the initial OD was 0.05 ( = 445 nm). The cultures were maintained in a room with a temperature of 25 ± 2 °C, aeration with a flow rate in the range of 0.4 to 0.6 L / min, and a photoperiod of 12h / 12h, with a light intensity of 4000 lux. Sampling for evaluation of the effects of the variables was carried out 8, 15 and 22 days after inoculation. From the samples obtained, the methods of cell lysis, lutein extraction, chlorophyll A and chlorophyll B, and analyses by ultra-high performance liquid chromatography coupled to a diode array detector (UHPLC-DAD) were applied, as described previously. The results were evaluated by comparing means using Tukey's test in Statistica 12.0 software (StatSoft, Tulsa, USA), with a 90% confidence interval.

[0098]

[0083] After 8 days of cultivation, the treatments differed from the control and from each other for dry weight, chlorophyll B, and lutein, at a significance level of 90%. After 15 days of cultivation, both treatments diverged from the control for all response variables. However, only for dry weight was there a statistically significant difference between treatments at the 90% significance level, with the other responses being statistically equal to each other.

[0099]

[0084] After 22 days of cultivation, for dry weight and lutein, the treatments diverged from the control, but were equal to each other. For chlorophyll A and chlorophyll B, treatment 2 was equal to treatment 1 and the control, and treatment 1 differed from the control. Figure 4 illustrates the effects observed in the dependent variables (dry weight, chlorophyll A, chlorophyll B and lutein) from the treatments, in relation to the control (C).

[0100]

[0085] The comparison of means between treatments, performed using Tukey's test and assuming a significance level of 90%, showed that the dry weight of TI is significantly higher than T2 and C in 8- and 14-day cultures, but not in 22-day cultures, where it is equal to T2. After 8 days of culture, the production of chlorophyll B and lutein in TI is significantly higher than T2 and C. Treatment TI shows advantages in dry weight at 8 and 15 days of culture, and in lutein, chlorophyll B, and chlorophyll A at 8 days of culture.

[0101]

[0086] It was also found that dry weight is considerably reduced (p less than 0.1) with increasing cultivation time from 8 to 22 days in T2, showing no significant difference between TI and the control. Lutein content did not show significant variation in cultivation time greater than 8 days, indicating that time has no positive or negative effect on the production of this carotenoid.

[0102]

[0087] Although there was a significant increase in lutein in the control group between 8 and 22 days, the total lutein is lower in control cultures (without sodium acetate and glucose), indicating that these two carbon sources favor carotenoid production. In treatment 2 (T2), a significant reduction in chlorophyll A content can be observed in 22-day cultures compared to 8-day cultures.

[0103]

[0088] It is possible to verify that an increase in cultivation time did not result in a significant increase in the response variables of interest. In the case of chlorophyll A, there was even a reduction in production at T2. From the point of view of cultivation cost, considering the time of use of the production structure, the best option would be to carry out cultivation for 8 days, since increasing the cultivation time does not result in an increase in the production of dyes. However, it is necessary to estimate biomass productivity on a larger scale, considering the production system to be used, in order to verify, based on the production volume, whether the gain in production in chlorophyll and lutein levels compensates for using twice the amount of glucose in the production process.

[0104] Example 4: Selection of the extractor system

[0105]

[0089] In this experiment, the maceration process was adopted to select the most efficient extraction mixture in relation to lutein. For the extractions, a solid:liquid ratio of 1:100 was used, and the tubes were left in the dark in an orbital shaker at 200 rpm for 10 hours at 25°C. The solvent systems used and the lutein content determined by chromatographic analysis are described in Table 15. The results were analyzed using Statistica software.

[0106] Table 15. Conditions tested in the simplex centroid design with internal points for lutein extraction from C. sorokiniana.

[0107]

[0108]

[0090] Analyzing the chromatograms (Figure 5), it was possible to verify that whenever water was present in the extraction medium, there was degradation of chlorophyll. This fact was evidenced by the appearance of peaks not previously present in the initial region of the chromatogram (from 0 to 5 minutes). These peaks presented absorption spectra in the UV-Vis region similar to that of chlorophyll, suggesting that it may have reacted in a water-rich medium, generating substances that eluted at the beginning of the chromatographic run.

[0109]

[0091] Statistical analysis of the centroid simplex results revealed that the model obtained was not mathematically significant. However, based on the contour plot (Figure 6), a trend of increasing lutein extraction efficiency was observed with increasing ethanol content in the extraction system, and the presence of glycerin did not interfere either positively or negatively. Whereas, the presence of water was detrimental to the extraction and integrity of the dyes.

[0110]

[0092] Thus, in the validation experiment for the selection of the extraction system, 3 conditions were selected: A - ethanol (EtOH), B - ethanol:glycerin (EtOFLGli 80:20, v / v), C - water:ethanol:glycerin (H2O:EtOH:Gli 20:60:20, v / v / v). The tests were performed in triplicate at a solid:liquid ratio of 1:14, the tubes were left in the dark in an orbital shaker at 200 rpm for 6 hours at 25 °C.

[0093] The results of this experiment (Figure 7) showed that conditions A and B did not present statistical differences between them and showed lutein extraction efficiency greater than 70% when compared with the analytically determined lutein value of the control (CTL).

[0111]

[0094] Therefore, the ethanol:glycerin mixture (80:20) was selected as the extraction system. This mixture was preferred over 95% ethanol because it proved more suitable for cosmetic formulations.

[0112] Example 5: Selection of a method for extracting lutein from C. sorokiniana

[0113]

[0095] The experiments in this stage aimed to evaluate the dynamic maceration process in relation to the analytical extraction method and whether the inclusion of auxiliary agents in cell disruption would bring any benefit to the efficiency of the extraction.

[0114]

[0096] The processes were performed in triplicate, at a solid:liquid ratio of 1:370, on both fresh and lyophilized Chlorella sorokiniana samples, as described below:

[0115]

[0097] (1) control (CTL): 200 p L of microalgae suspension + 400 p L of phosphate buffer (0.05 pH 7) + 200 p L of bead; cell lysis by “bead-beating” for 5 min, 200 p L of lysed suspension + 800 p L of pure acetone, 2 min agitation, 15 min stand in the dark;

[0116]

[0098] (2) control with water (CTL + H2O): 200 p L of microalgae suspension + 400 p L of distilled water + 200 p L of bead, cell lysis by “bead-beating” for 5 min, 200 p L of lysed suspension + 800 p L of pure acetone, 2 min agitation, 15 min stand in the dark;

[0117]

[0099] (3) dynamic maceration (MD): 1 mL of microalgae suspension + 400 p L distilled water + 7.6 mL absolute ethanol + 2 mL glycerin; stirring at 25°C, 250 rpm, dark, for 24h;

[0118]

[0100] (4) cell lysis followed by dynamic maceration (Lyse-MD): 1 mL of microalgae suspension + 400 p L distilled water + 200 p L bead; cell lysis by bead-beating for 5 min; 200 pL of lysed suspension + 3.8 mL absolute ethanol + 1 mL glycerin, agitation at 25°C, 250 rpm, dark, for 24h;

[0119]

[0101] (5) dynamic maceration with bead (MD+bead): 1 mL of microalgae suspension + 400 pL of distilled water + 7.6 mL absolute ethanol + 2 mL of glycerin + 400 pL of bead; stirring at 25°C, 250 rpm, dark, for 24h;

[0120]

[0102] (6) dynamic maceration with diatomaceous earth (MD+earth): 1 mL of microalgae suspension + 400 p L distilled water + 7.6 mL absolute ethanol + 2 mL glycerin + 400 p L diatomaceous earth; agitation at 25°C, 250 rpm, dark, for 24h.

[0121]

[0103] In experiments conducted with fresh samples (Figure 8), mean comparison analysis (Tukey p<0.05) showed that the MD+bead and Lyse-MD processes had the highest lutein extraction efficiency, showing no difference from the controls (CTL and CTL+H2O). The MD process had the lowest lutein extraction efficiency. Even so, this process showed more than 80% efficiency, which commercially could be sufficient due to its lower cost.

[0122]

[0104] In experiments with lyophilized biomass, basically, no difference was observed between the controls and the processes involving dynamic maceration (Figure 8), with only the MD+soil process being different.

[0123]

[0105] When comparing the difference in extraction efficiency between fresh and lyophilized samples (T-test p<0.05), with the exception of the MD process, all others were less efficient on lyophilized biomass. In this experiment, an excess of solvent was used in relation to the initial biomass (1:370) and, therefore, the water present in the biomass did not represent a significant contribution to the composition of the solvent used and, therefore, appears not to have had an effect on the degradation of the dyes.

[0124]

[0106] After evaluating the tested processes, the dynamic maceration extraction process is the simplest among those studied, does not use any additives besides the biomass and the extraction system itself, and can be used with fresh or lyophilized biomass. Therefore, the MD process has better technical and economic viability and, for this reason, it was selected for the optimization experiments. Example 6: optimization of the maceration process

[0125]

[0107] The dynamic maceration process with the extracting mixture ethanol glycerol (80:20, v / v)), although not proving to be the most efficient in lutein extraction, was selected for the optimization stage due to its technical and economic viability in relation to the other methods.

[0126]

[0108] In the optimization of the dynamic maceration process, lyophilized biomass of Chlorella sorokiniana was used, using an ethanol:glycerin mixture (80:20) as solvent. An experimental design of the Central Composite Rotational Design type with 3 factors was carried out using the operating conditions described in Table 16.

[0127] Table 16. Matrix of the Central Composite Rotational Design with 3 factors for lutein extraction from C. sorokiniana.

[0128]

[0109] Statistical analysis of the results showed that the model was significant and presented R 2 of 0.875. The significant effects (p<0.05) were: number of extractions (X1) quadratic, time (X2) linear and quadratic, and ratio (X3) linear. From these results and the analysis of the contour plots presented in Figure 9, it was found that another factorial design, with the variables time and solid:liquid ratio, was necessary.

[0129]

[0110] Thus, an experimental design of the Central Composite Rotational Design type with 2 factors was carried out in two stages, using lyophilized biomass of Chlorella sorokiniana, ethanol and glycerin (80:20) as the extraction mixture, the conditions used in each stage are described in Table 17.

[0130]

[0111] After the time of each test, the samples were centrifuged and the liquid extracts were collected and analyzed for lutein content; these results refer to the first extraction performed in this design. Ethanol:glycerin (80:20) was added to the remaining pellets, again reproducing the conditions described in Table 17.

[0131] Table 17. Matrix of the Central Composite Rotational Design with 2 factors for lutein extraction from C. sorokiniana, performed in two stages.

[0132]

[0112] Statistical analysis of the results from the first extraction showed that only the linear time effect (X2) was statistically significant with a 5% significance level. The statistically significant model presented an R2 of 0.765, and is represented below by the equation:

[0133]

[0113] Y = 2.64 + 0.58 - X2

[0134]

[0114] Where, Y = lutein extraction efficiency (mg / g of dry biomass) and X2 = time (minutes), the coefficients presented in the equation refer to the coded variables.

[0135]

[0115] After the second extraction, the extracts produced were collected and analyzed. Thus, the sum of the values ​​obtained in the first and second extraction was considered as the response variable of the total extraction performed under the conditions described in Table 18. Table 18. Matrix of the Central Composite Rotational Design with 2 factors used in the evaluation of the total lutein extraction from C. sorokiniana.

[0136]

[0116] Statistical analysis of the total extraction results (sum of the two extractions) showed that only the interaction between ratio (X1) and time (X2) was not significant (p<0.10). The statistically significant model presented R 2 The value of 0.960 is represented by the following equation:

[0137]

[0117] Y= 3.76 + 0.17 XI - 0.32 XI 2 +0.71 X2 -0.17 X2 2

[0138]

[0118] Where, Y = lutein extraction efficiency (mg / g of dry biomass), Xl = time (minutes) and X2 = solid:liquid ratio, the coefficients presented in the equation refer to the coded variables.

[0139]

[0119] In the response of the model generated for total extraction, the maximum point calculated by the software for the solid:liquid ratio was 1:32, while the time was 1006 minutes (16.8 hours), that is, outside the studied range (maximum value was 914 minutes). The behavior of the total extraction parameters is shown in the contour plot in Figure 10.

[0140]

[0120] Since the optimum time predicted by the model is outside the studied range, to find it, an extraction kinetics experiment was carried out at a solid-liquid ratio of 1:16 with only one extraction. And, for the production of a more concentrated lutein extract where the extraction efficiency of the substance in question was not considered, the extraction kinetics at a solid:liquid ratio of 1:6 was studied. In this experiment, the extraction time was extrapolated up to 72 hours, beyond the limits studied so far. The results obtained in the extraction kinetics experiments are shown in Figure 11.

[0121] The behavior of the suspensions at a 1:16 ratio, in terms of the amount of lutein extracted per gram of biomass, increases with time and then establishes a plateau. The extraction points at 8 and 12 hours showed no statistical difference, the points at 12 and 24 hours also did not differ, and from 24 hours onwards no significant increase in lutein content was observed.The points above 12 hours were statistically equal to the control, indicating that all lutein would have been extracted within 12 hours.

[0141]

[0122] The behavior of the suspensions in the 1:6 ratio was more complex, the amount of lutein extracted increases up to 8 hours of extraction and then a drop can be observed up to 72 h. Thus, at the 8 h point, the maximum extraction efficiency (75%) was reached and the content of lutein extracted from that moment on was statistically lower than the control (Table 19).

[0142]

[0123] The 1:16 solid / liquid ratio showed the highest extraction efficiency (lutein per gram of initial algal biomass); however, the lutein concentration in the extract obtained from the 8-hour extraction at the 1:6 ratio was 543 pg / mL, a concentration 2.6 times higher than that of the extract (211 pg / mL) obtained from the 1:16 ratio at the same extraction time. Therefore, although the 1:6 ratio does not extract all the lutein present, the smaller amount of solvent present results in a more concentrated extract in less time.

[0143]

[0124] In conclusion, to achieve the maximum lutein concentration in the extract, it would be better to use extraction with a solvent ratio of 1:6, as a concentration of 546 pg / mL was achieved after 8 hours of extraction, while a ratio of 1:16 would lead to a maximum concentration of 283 pg / mL after 72 hours of extraction. However, considering the kinetics of extraction efficiency (mg / g), when using the 1:6 ratio, we saw that the maximum extraction to be achieved is 75% of the value present in the biomass (Table 19) after 8 hours of extraction. Increasing the extraction time, in this case, causes the degradation of lutein (Figure 11), for unidentified reasons. Table 19. Extraction efficiency (mg / g) of lutein from C. sorokiniana by dynamic maceration with solvent 80:20 at 25 °C with orbital shaking at 200 rpm (different letters in the columns indicate a statistically significant difference p<0.05) in the ratio 1:6 and 1:16.

[0144]

[0125] Therefore, after analyzing the results, we conclude that the most suitable lutein extraction method is carried out for 8 hours, if a solvent / mass ratio of 1:6 or 1:16 is used, achieving 75 to 77% extraction efficiency, using ethanol / glycerin solvent 80:20, using lyophilized biomass of C. sorokiniana, at room temperature, with agitation by dynamic maceration and without lysis (cell rupture).

[0145]

[0126] Subsequently, the extraction method with algal biomass was validated. Thus, in this step, the validation was performed with fresh / frozen C. sorokiniana biomass and also with lyophilized / frozen biomass, in a 1:16 ratio (dry mass), in an orbital shaker at 25°C, 200 rpm for 8 hours. In this step, extraction with fresh or lyophilized biomass was considered, with EtOH extraction solvent or an ethanol / glycerin mixture 80:20, and the need for a pre-washing step for the fresh sample to remove excess water still present.

[0146]

[0127] The treatments performed in the validation were: 1) Fresh EtOH: fresh / frozen biomass with ethanol as extraction solvent; 2) Fresh 80:20: fresh / frozen biomass with ethanol:glycerin (80:20) as extraction solvent; 3) Fresh with EtOH washing: fresh / frozen biomass with prior washing with ethanol in a 1:1 ratio (wet mass) for 5 min, ethanol was added to the remaining pellet as extraction solvent; 4) Fresh with 80:20 washing: fresh / frozen biomass with prior washing with ethanol in a 1:1 ratio (wet mass) for 5 min, ethanol:glycerin (80:20) was added to the remaining pellet as extraction solvent; 5) Freeze-dried EtOH: freeze-dried / frozen biomass with ethanol as extraction solvent; 6) Lyophilized 80:20: lyophilized / frozen biomass with ethanol: glycerin (80:20) as extraction solvent.

[0147]

[0128] The first extraction was performed under the conditions described above, and the lutein content of the resulting extracts was quantified. The remaining pellets were subjected to two more extractions under the same conditions as the first extraction, and the lutein content of the resulting extracts was quantified. After three consecutive extractions, the remaining pellets were resuspended in their respective extraction systems, beads were added, and they were placed in a bead-b eating system for 5 minutes. The lutein content of the extracts from this fourth extraction was also quantified. The sum of all four extractions was considered the control value.

[0148]

[0129] In order to circumvent the problem of the presence of water in the fresh sample, we suggest a pre-washing of the algal biomass with 95% ethanol before the start of the actual extraction. The post-wash liquid fraction was analyzed by UHPLC to verify possible lutein losses and it was found that only 0.32% of the lutein was lost during this step. The best extraction efficiencies were achieved in treatment 3 (Fresh with EtOH washing) and 6 (Lyophilized 80:20) as observed in Figure 12.

[0149]

[0130] The washing step in treatments 3 and 4 extracted only 0.3% of the total lutein, and increased the extraction efficiency to 60% for the ethanol treatment and 38% for the ethanol:glycerin (80:20) treatment in the first extraction.

[0131] The best extraction yield for treatments 3, 4, 5 and 6 was obtained in the first extraction, while for treatment 2 the best yield occurred in the second extraction and for treatment 1 in the third extraction. In treatments 1 and 2, the first extraction functioned as the 5-minute pre-wash used in treatments 3 and 4.

[0150]

[0132] Based on the analysis of the results obtained in the validation and kinetic assay, in general for microalgae biomass, an extraction for 8 hours is recommended if a 1:16 solid:liquid ratio is used, employing an ethanol / glycerin 80:20 solvent, for the extraction of lyophilized biomass at room temperature, with agitation by dynamic maceration. With just one extraction, it is possible to extract approximately 51 to 75% of the lutein, eliminating the need for pre-washing with ethanol.

[0151]

[0133] For fresh algal biomass, pre-washing with ethanol in a 1:1 ratio for 5 minutes is recommended. The extraction method employs an ethanol / glycerin mixture of 80:20, and to achieve an extraction efficiency above 70%, 2 extractions of 8 hours are necessary.

[0152]

[0134] Thus, from the solvent mixture selection and extraction optimization experiments, working ranges were obtained in relation to the evaluated parameters that allow flexibility of choice, depending on the desired result. However, for the production of a lutein-rich extract using a sustainable, low-cost method and with the use of green solvents, the following conditions were selected: lyophilized sample, ethanol / glycerin mixture 80:20, solid / liquid ratio 1:16, dynamic maceration of 8h.

[0153] Example 7: Stability study

[0154]

[0135] The glycerinated extract containing lutein prepared from C. sorokiniana biomass by dynamic maceration was subjected to a stability experiment, determining the half-life of lutein. The extract was obtained from C. sorokiniana biomass by dynamic maceration for 8 h, using an extraction system consisting of an ethanol / glycerin mixture 80:20, biomass / solvent ratio of 1:16, only one extraction, with orbital shaking at 200 rpm at 25°C. The stability study of the extract was carried out by incubating the extract in triplicate at 4, 25 and 50°C, for 15 days in the dark. The calculation of the half-life (r) was based on the following equations: ln(C / Co) = - kter = ln(2) / k, where C = final concentration of lutein; Co = initial concentration of lutein; t = time in hours; k = first-order degradation constant; r = half-life time.

[0155]

[0136] In the stability study at 4°C, the lutein levels at baseline and after 15 days of incubation showed no statistical difference; therefore, it was not possible to estimate the half-life at this temperature. The half-life was calculated for temperatures of 25 and 50°C as shown in Table 20, being 12.47 days at 25°C and 10.19 days at 50°C. In addition to the half-life, it was possible to monitor the degradation of lutein in relation to its initial concentration (Table 21).

[0156] Table 20. Calculation of the half-life of the glycerinated extract incubated at 25 and 50 °C for 15 days.

[0157] Table 21. Lutein degradation, in percentage, relative to the initial concentration in the glycerinated extract.

[0158]

[0159] * Data not obtained.

[0160] Example 8: Characterization of the extract

[0161]

[0137] The glycerinated extract of example 7, obtained by an optimized extraction process defined in example 6, was characterized in terms of the composition of the major compounds responsible for its color, i.e., pigments, and in terms of the presence of undesirable compounds in dermocosmetic formulations. Thus, the extract was characterized in terms of lutein, chlorophyll A, and chlorophyll B content by HPLC-PAD. In addition, a mass spectrometry analysis was performed to ensure that no undesirable substance was produced in dermocosmetic formulations.

[0162]

[0138] After comparing the experimentally obtained data with those available in the databases used, it was possible to identify the compounds found in the C. sorokoniana extract (Table 22).

[0163]

[0139] In addition to lutein, the presence of neoxanthin, another xanthophyll, was detected. Chlorophylls A and B were detected, and the presence of pheophorbide A and pyropheophorbide A, both chlorophyll degradation products, was also detected. These products appear in quantities below the quantification limit. Thus, only the major compounds were quantified: lutein: 238.58 pg / mL, chlorophyll A: 864.93 pg / mL, chlorophyll B: 303.93 pg / mL. Furthermore, no undesirable components were detected in significant concentrations. Table 22. Major compounds identified in the C. sorokoniana extract.

[0164] * Most likely candidate based on a comparison of the data obtained with those available in the literature databases.

[0165] ** The concentration was low and it was not possible to obtain the absorption spectrum on the UPCL-DAD, but it was possible to detect this substance by mass spectrometry.

Claims

CLAIMS 1. Process for obtaining an extract with a high lutein content from microalgae, characterized by comprising: (a) preparation of culture medium; (b) microalgae inoculation; (c) microalgae cultivation; (d) harvesting microalgae to obtain microalgae biomass; (e) optionally, drying of the microalgae biomass; (f) extraction of lutein and chlorophylls from microalgae biomass using green solvents.

2. Process for obtaining an extract with a high lutein content from microalgae according to claim 1, characterized in that the microalgae are of the genus Chlorella.

3. Process for obtaining an extract with a high lutein content from microalgae according to claim 2, characterized in that the microalgae is of the species Chlorella sorokiniana.

4. Process for obtaining an extract with a high lutein content from microalgae according to claim 3, characterized in that the microalga Chlorella sorokiniana is of the strain deposited at the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) under accession number 202112002.

5. Process for obtaining a high-lutein extract from microalgae according to any one of claims 1 to 4, characterized in that the culture medium prepared in step (a) comprises BG-11 culture medium supplemented with a carbon source.

6. Process for obtaining a high-lutein extract from microalgae according to any one of claims 1 and 5 characterized in that the culture medium prepared in step (a) comprises sodium acetate, glucose and glycerol as a carbon source.

7. Process for obtaining a high-lutein extract from microalgae according to claim 6, characterized in that the culture medium prepared in step (a) preferably comprises sodium acetate and glucose as a carbon source.

8. Process for obtaining an extract with a high lutein content from microalgae according to claim 7, characterized in that the culture medium prepared in step (a) comprises sodium acetate at a concentration in the range of 0 to 4 g / L.

9. Process for obtaining an extract with a high lutein content from microalgae according to claim 8, characterized in that the culture medium prepared in step (a) comprises sodium acetate, preferably at a concentration of 2 g / L.

10. Process for obtaining an extract with a high lutein content from microalgae according to any one of claims 7 to 9, characterized in that the culture medium prepared in step (a) comprises glucose at a concentration in the range of 0 to 12 g / L.

11. Process for obtaining an extract with a high lutein content from microalgae according to claim 10, characterized in that the culture medium prepared in step (a) preferably comprises glucose at a concentration of 6 g / L.

12. Process for obtaining an extract with a high lutein content from microalgae according to claim 1, characterized in that the microalgae cultivation in step (c) is carried out in a 12h / 12h photoperiod, with a light intensity of 4000 lux and aeration with a flow rate in the range of 0.4 to 0.6 L / min.

13. Process for obtaining an extract with a high lutein content from microalgae according to claim 12, characterized in that the microalgae cultivation in step (c) is carried out at a temperature in the range of 22 to 30 °C.

14. Process for obtaining an extract with a high lutein content from microalgae according to claim 13, characterized in that the microalgae cultivation in step (c) is preferably carried out at a temperature of 25 °C.

15. Process for obtaining an extract with a high lutein content from microalgae according to claim 12, characterized in that the microalgae cultivation in step (c) is carried out for a period of 4 to 22 days.

16. Process for obtaining an extract with a high lutein content from microalgae according to claim 15, characterized in that the microalgae cultivation in step (c) is preferably carried out over 8 days.

17. Process for obtaining an extract with a high lutein content from microalgae according to claim 1, characterized in that the microalgae harvest in step (d) is carried out by centrifugation.

18. Process for obtaining an extract with a high lutein content from microalgae according to claim 1, characterized in that the drying of the biomass in step (e) is carried out by lyophilization, spray drying and vacuum drying.

19. Process for obtaining an extract with a high lutein content from microalgae according to claim 18 characterized in that the drying of the biomass in step (e) is preferably carried out by lyophilization.

20. Process for obtaining an extract with a high lutein content from microalgae according to claim 1, characterized in that the extraction of lutein and chlorophylls from microalgae biomass in step (f) is carried out by dynamic maceration.

21. Process for obtaining an extract with a high lutein content from microalgae according to claim 20 characterized in that the extraction of lutein and chlorophylls from microalgae biomass in step (f) is carried out using ethanol, glycerol, mixtures of ethanol and glycerol or mixtures of ethanol, glycerol and water.

22. Process for obtaining an extract with a high lutein content from microalgae according to claim 1 characterized in that the extraction of lutein and chlorophylls from microalgae biomass in step (f) is carried out using a mixture of ethanol and glycerol.

23. Process for obtaining an extract with a high lutein content from microalgae according to claim 22, characterized by the ethanol concentration being from 40 to 100% v / v and the glycerol concentration being from 0 to 60% v / v.

24. Process for obtaining an extract with a high lutein content from microalgae according to claim 22, characterized in that step (f) is carried out with wet biomass.

25. Process for obtaining an extract with a high lutein content from microalgae according to claim 22, characterized in that step (f) is carried out with dry biomass.

26. Process for obtaining an extract with a high lutein content from microalgae according to claim 25, characterized in that step (f) is preferably carried out with lyophilized biomass.

27. Process for obtaining an extract with a high lutein content from microalgae according to claim 21 characterized in that step (f) is carried out in a solid / liquid ratio of 1:3 to 1:

32.

28. Process for obtaining an extract with a high lutein content from microalgae according to claim 27 characterized in that step (f) is carried out in a solid / liquid ratio of preferably 1:

16.

29. Process for obtaining an extract with a high lutein content from microalgae according to any one of claims 1 to 28, characterized in that step (f) is carried out for a time of 1 h 25 h.

30. Process for obtaining an extract with a high lutein content from microalgae according to claim 29 characterized in that step (f) is preferably carried out for a period of 8 h.

31. Lutein-rich extract characterized by being obtained by a process defined in any one of claims 1 to 30.

32. Extract with a high lutein content according to claim 31, characterized by containing lutein and chlorophylls.

33. Use of extract with high lutein content defined in claims 31 and 32 characterized by being in cosmetic compositions.

34. Use of the extract with a high lutein content defined in claims 31 and 32, characterized by being in pharmaceutical compositions.

35. Use of the extract with a high lutein content defined in claims 31 and 32, characterized by being in the food industry.

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