BR102022006871A2 - PROCESS FOR OBTAINING PROTEIN EXTRACTS FROM PHOTOSYNTHETICIZING MICROORGANISMS, PROTEIN EXTRACTS FROM PHOTOSYNTHETICIZING MICROORGANISMS, USE OF PROTEIN EXTRACTS FROM PHOTOSYNTHETICIZING MICROORGANISMS, CULTURE MEDIUM OF HETEROTROPHIC ORGANISMS, USE OF HETEROTROPHIC ORGANISMS - Google Patents

Abstract

process for obtaining protein extracts from photosynthetic microorganisms, protein extracts from photosynthetic microorganisms, use of protein extracts from photosynthetic microorganisms, culture medium for heterotrophic organisms, use of heterotrophic organisms. the present invention refers to a process for obtaining protein extracts from photosynthetic microorganisms, namely microalgae and/or cyanobacteria, as well as their use as a source of nitrogen replacing the source of animal protein from bacterial culture media, yeasts, fungi and/or animal cells, called heterotrophic organisms. Additionally, the present invention proposes the use of the heterotrophic organisms produced, as well as the metabolites produced by them for industrial applications, such as in the production of vaccines, serums, pharmaceuticals and biopharmaceuticals, antibiotics for human and/or animal use, antitumor drugs, biomass, acids organic products, solvents, nucleic acids, biosurfactants, amino acids, cosmetics, biofertilizers, agricultural inoculants, phytohormones, foods, animal feed, among others.

Description

FIELD OF INVENTION

[001] The present invention falls within the field of Biology, more precisely in the area of Biotechnology, and describes a process for obtaining protein extracts from photosynthetic microorganisms, such as microalgae and/or cyanobacteria, as well as their use as a source of nitrogen replacing the source of animal protein from culture media of bacteria, yeast, fungi and/or animal cells, called heterotrophic organisms. Additionally, the present invention proposes the use of the heterotrophic organisms produced, as well as the metabolites produced by them for industrial applications, such as in the production of vaccines, serums, pharmaceuticals and biopharmaceuticals, antibiotics for human and/or animal use, antitumor drugs, biomass, acids organic products, solvents, nucleic acids, biosurfactants, amino acids, cosmetics, biofertilizers, agricultural inoculants, phytohormones, foods, animal feed, among others.

BASICS OF THE INVENTION

[002] As the world population grows, the demand for food also increases. In human nutrition, the protein fraction, more specifically, animal protein, is one of the most relevant. Currently, providing products with a high content of renewable and sustainable proteins is the food industry's greatest challenge (JONES, S. W., KARPOL, A., FRIEDMAN, S., MARU, B. T., TRACY, B. P. Recent advances in single cell protein use as a feed ingredient in aquaculture. Current Opinion in Biotechnology, v. 61, pp. 189-197, 2020).

[003] There is a tendency to replace protein of animal origin with other sources. Since 1950, microalgae have been studied because they contain a high concentration of proteins in their biomass. Because they are capable of consuming CO2, studies on optimizing growth conditions with the aim of increasing biomass production yield are carried out, as well as evaluating the contribution to reducing greenhouse gases (BUI, H. H.; TRAN, K. Q.; CHEN, W. H. Pyrolysis of microalgae residues - a kinetic study. Bioresource Technology, v. 199, pp. 362-366, 2016). Furthermore, interest in microalgae is growing, as optimizing cultivation conditions can induce the synthesis and accumulation of high levels of proteins (from 40% to 60% of biomass), carbohydrates, and lipids. (BARKA, A., BLECKER, C. Focus on: microalgae as a potential source of single-cell proteins. A review. Biotechnol. Agron. Soc. Environ., v. 20, pp. 427-436, 2016).

[004] Globally, the production of dry biomass from microalgae is around 20,000 tons.year-1. The gross sales value of Spirulina and Chlorella ranges from US$10,000 to US$30,000 per ton (BENEMANN, J. R., WOERTZ, I., LUNDQUIST, T. Autotrophic microalgae biomass production: from niche markets to commodities. Industrial Biotechnology, v. 14, pp. 3-10, 2018). The specific market for algae proteins was worth around 700 million dollars in 2019 and is estimated to grow 6% by 2026 (https://www.gminsights.com/methodology/detail/algae-protein-market, accessed on 05/20/2020, LARENS, L. M. L., MARKHAM, J., TEMPLETON, D. W., CHRISTENSEN, E. D., VAN WYCHEN, S., VADELIUS, E. W., PIENKOS, P. T. Development of algae biorefinery concepts for biofuels and bioproducts; a perspective on process-compatible products and their impact on cost-reduction. Energy & Environmental Science, v.10, pp.1716-1738, 2017).

[005] Many species of microalgae grow faster than terrestrial plants, making it possible to obtain a greater amount of biomass, as well as carbohydrates and lipids. Furthermore, its unicellular nature ensures a biomass with the same biochemical composition, which does not occur with terrestrial plants where different compounds are produced in specific parts such as fruits, leaves, seeds or roots, for example. (BAICHA, Z., SALAR-GARCIA, M. J., ORTIZ-MARTINEZ., HERNANDEZ- FERNANDEZ, F. J., LOS RIOS, A. P.; LABJAR, N., LOTFI, E., ELMAHI, M. A critical review on microalgae as an alternative source for bioenergy production: A promising low cost substrate for microbial fuel cells. Fuel Processing Technology, v.154, pp.104-106, 2016; RICHMOND, A. (Ed). CRC Handbook of microalgal mass culture. Florida: CRC, pp. 528, 1990).

STATE OF THE TECHNIQUE

[006] Some studies in the literature propose the use of extracts from photosynthetic microorganisms, as well as methods for obtaining them, such as:

[007] The document SINGH, R.N.; SHARMA, S.  Development of suitable photobioreactor for algae production - A review. Renewable And Sustainable Energy Reviews, vol. 16, no. 4, p. 2347-2353, 2012 comments on the types of photobioreactors used for microalgae cultivation, such as open and closed photobioreactors, presenting advantages and disadvantages of each of them. Regarding closed photobioreactors, they come in different shapes, such as flat plates and tubulars. These photobioreactors can be moved by “air lift” or using pumps. Tubular reactors can be arranged vertically or horizontally. There is also the possibility of working with mixture-type photobioreactors, commonly used in the cultivation of heterotrophic microorganisms. They present hybrid photobioreactors as promising, where different types of photobioreactors can be combined, in order to obtain the best conditions for microalgae production. Although this review article is important in the field of microalgae, the teachings provided in this article do not disclose the present invention, which particularly deals with extracts of photosynthetic microorganisms as a source of nitrogen in the cultivation of heterotrophic organisms.

[008] The documents BECKER, E. W., Microalgae as source of proteins, Biotechnology Advances, 25, p. 2017-210, 2007; BARKA, A., BLECKER, C. Focus on: Microalgae as a potential source of single-cell proteins. The review. Biotechnol. Agron. Soc. Environ., v. 20, pp. 427-436, 2016; KOYANDE, A. K., CHEW, K. W., RAMBABU, K., DINH-TOICHU, Y. T., SHOW. P.-L. Microalgae: A potential alternative to health supplementation for humans, Human Science and Human Wellness, 8, p.16-24, 2019; BHATTACHARYA, M., GOSWAMI, S.,  Microalgae - A green multi - product biorefinery for future industrial prospects, Biocatalysis and Agricultural Biotechnology, 25, 101580, 2020, describe that the production of proteins by microalgae varies quantitatively and qualitatively, depending on the cultivated species, being considered excellent sources of proteins and amino acids.

[009] “Single Cell Protein” products can be prepared from different microbial sources, including microalgae, yeast, fungi and bacteria (JONES, S. W., KARPOL, A., FRIEDMAN, S., MARU, B. T., TRACY, B. P. Recent advances in single cell protein use as a feed ingredient in aquaculture. Current Opinion in Biotechnology, v. 61, pp. 189-197, 2020).

[010] Products of plant origin are available for the preparation of nutrient-rich culture media that are efficient in in vitro cultures and in the production of bacterial biomass in a sustainable manner (MOURAD, E. F., SARHAN, M. S., DAANAA, H. S A ., ABDOU, M., MORSI, A. T., ABDELFADEEL, M. R., ELSAWEY, H., NEMR, R., EL-TAHAN, M., HAMZA, M. A., ABBAS, M., YOUSSEF, H. H., ABDELHADI, A. A., AMER , W. A., FAYEZ, M., RUPPEL, S., HEGAZI, N. A. Plant materials are sustainable substrates supporting new technologies of plant-only-based culture media for in vitro culturing of the plant. Microbes. Environ., v.33, pp 40-49, 2018).

[011] There are challenges and perspectives when cultivating microalgae for the production of nutraceuticals and food supplements, as well as the production of extracts and/or biomass, with interest in bioactive molecules with high added value. Particularly, the review article by Chaco-Lee &González-Marino (2010) (CHACON-LEE, T.; GONZÁLEZ-MARINO, G.  Microalgae for “Healthy” Foods — Possibilities and Challenges. Comprehensive Reviews in Food Science and Food Safety, v.9, pp. 655-675, 2010) seeks to highlight the components present in microalgae biomass, such as fatty acids, proteins and corresponding profiles of amino acids, polysaccharides, carotenoids and vitamins, in order to justify the proposal that the food industry food evaluate microalgae as a new source of health-promoting products.

[012] The article also presents classic processes for producing photosynthetic microorganisms, as well as how they are separated. However, nowhere in the aforementioned review article was there any mention found about obtaining protein extract from photosynthetic microorganisms, differentiating and, therefore, not revealing the present invention, which aims to obtain extracts from photosynthetic microorganisms and their use as a source of nitrogen in the cultivation of heterotrophic organisms.

[013] Regarding animal feed, the microalgae Chlorella vulgaris is a potential alternative to replace fish feed in aquaculture due to its similar protein composition. To obtain microalgal biomass, a photobioreactor was used in which it was possible to achieve a maximum biomass productivity of 699 mg.L-1.day-1 and 365 mg.L-1.day-1 of protein. Furthermore, the amino acid composition of microalgal protein was analyzed, in which Chlorella vulgaris showed that up to 70% of proteins consist of essential amino acids (CHEN, C.Y.; LEE, P. J.; TAN, C. H.; LO, Y. C.; HUANG, C. C. ; SHOW, P. L.; LIN, C. H.; CHANG, J. S. Improving protein production of indigenous microalga Chlorella vulgaris FSP-E by photobioreactor design and cultivation strategies. Biotechnology Journal, v. 10, n. 6, pp. 905-914, 2015). It is important to highlight that the work of Chen et al. (2015) does not mention anywhere in the text the possibility of working with extracts from microalgae, differentiating it from the present invention.

[014] Photosynthetic microorganisms may have a cell wall that is difficult to digest, resulting in a decrease in the bioavailability of their nutrients. Thus, aiming to improve the release of protein content for feeding fish and crustaceans, patent documents WO2019171293 and US2021/0000140A1 propose cell disruption in vibrating ball mills in a wet system, or other physical cell disruption processes, preferably wet, followed by of enzymatic hydrolysis with a mixture of at least two enzymes, such as lipase, pectinase, cellulase, hemicellulase, endo- and exo-arabanase, amylase or mixtures thereof, with at least one of the enzymes being lipase, pectinase or amylase.

[015] Thus, the present invention differs from documents WO2019171293 and US2021/0000140A1 as it provides for physical disruption simultaneously with enzymatic and/or chemical hydrolysis, which increases the disruption efficiency through the synergy of using both processes simultaneously. Furthermore, the present invention provides for the possible removal of lipids from cells, which in addition to allowing this fraction to be obtained for other uses, disrupts the cell membrane, allowing greater efficiency in cell disruption and the action of the hydrolases used in the cell disruption process. . Another improvement in the process proposed in the present invention is the treatment with proteases, which increases the bioavailability of the protein source for heterotrophic organisms, in addition to proposing possible treatment with nucleases and/or precipitations of peptides/proteins or genetic material to obtain protein extracts with low amount of genetic material. For the protein extract, whether purified or not, a concentration is still proposed for water removal and storage, with a further difference regarding the purpose of use of the extract of the present invention, which is the application in the cultivation of bacteria, yeasts, fungi and/or cells animals.

[016] Document US2020/0095176A1 describes a method of obtaining fertilizer and biostimulants for use in agriculture from microalgae and/or cyanobacteria biomass. To this end, the authors mention that the biomass of these microorganisms undergoes cell disruption followed by treatment with proteases, with pre-treatment with cellulase, until obtaining an extract rich in free amino acids, peptides and phytohormones. This extract is then added with nitrogen, phosphorus and/or potassium so that it can be used as a fertilizer. The cultivation media used for the purpose of document US2020/0095176A1 are residual media or media with clean water, but with the addition of fertilizers as nutrients, differentiating from the present invention, which does not use residual media, using salts of at least technical grade, preferably P.A, in order to avoid contaminants in the extracts that could interfere with the quality of the products produced by heterotrophic organisms that may grow in the protein extracts.

[017] In fact, considering that important applications of these extracts would be the production of vaccines and biopharmaceuticals, it is essential to have security of the origin of the inputs, with traceability guaranteed. Another difference between the processes of the present invention is that document US2020/0095176A1, as it works with wastewater, considers strains in mixture, expressed throughout the text as “microalgae, including cyanobacteria”. In other words, it does not present any type of restriction regarding the quantity and diversity of photosynthetic microorganisms, which is acceptable for application as a biofertilizer. In the case of the present invention, cultures without the presence of more than one photosynthetic microorganism are preferred, with limited presence of other species, with the need to guarantee that there is no presence of toxin-producing photosynthetic microorganisms, as the protein extracts obtained from these Cells, as mentioned above, will be destined for cultivation of heterotrophic organisms that produce compounds in which there is a need to guarantee the safety of there being no contaminants.

[018] Another difference between the processes of document US2020/0095176A1 and the present invention is that it proposes the cultivation of microorganisms in isolation and the preparation of protein extracts in isolation, considering the mixture of already processed extracts to form bases for the preparation of media cultivation of heterotrophic organisms. The present invention also considers obtaining protein and peptide extracts, as they are easier to purify and most heterotrophic organisms (bacteria, yeast, fungi) are protease producers. In specific cases in which it is desired to obtain free amino acids, particularly important in the case of animal cell cultivation, proteases are applied to extracts containing purified or partially purified proteins and peptides, differing from document US2020/0095176A1, where proteases are applied in crude extracts. Finally, what justifies the differences in the unit operations applied in the present invention and document US2020/0095176A1, is that the purposes of use are different, as evidenced above.

[019] Document US8486675B2 describes a process for removing fibers from microalgae and macroalgae for the production of low-fiber food/meal, that is, to obtain protein concentrates from algae, also removing antinutritional factors, using low g-force centrifugation , generating a protein content between 30 and 70%. Methods involving organic solvents, water and a mixture of solvents are cited for the differentiated precipitation of fiber and protein fractions. In addition to the application of centrifugation operations, sieving is also provided for phase separation. Enzymes normally used both to release phosphorus (phytase) and to hydrolyze fibers (cellulase) can be applied before separating the fiber and protein phases. From the separation of protein and fiber phases, specific enzymes can be used to obtain phase hydrolysates. These treatments can be applied to both microalgae and macroalgae in natura, as well as broken and/or degreased.

[020] The present invention differs from document US8486675B2 in that it does not seek to separate the protein fractions from the fiber fractions, since microalgae, microorganisms preferably used in the preparation of protein extracts, as well as cyanobacteria, growing in a medium with an adequate supply of nitrogen , have low fiber concentration values, and these do not harm the preparation of cultivation media. Treatment with hydrolases, although it can occur both in the present invention and in document US8486675B2, differs, as in the present invention these enzymes are preferentially added during the unitary cell disruption operation, which leads to simplification and reduction of process time. , and the presence of residual fibers and their hydrolysates, as they contain compounds normally present in the cell wall, is advantageous to the purpose for which the protein hydrolyzate is intended, which is to serve as a culture medium for heterotrophic organisms, which would be the case of cultivation of bacteria, yeast and fungi.

[021] In the present invention, unlike patent US8486675B2, there is provision for adding nucleases to the protein extract to remove nucleic acids and/or solubilize them. In the present invention, protein extracts are always obtained separately and the possible mixing of protein extracts occurs later, with them already prepared, concentrated or dried, so that they complement each other to provide adequate cell growth and/or formation of the desired product of the organism. heterotrophic. Additionally, the protein extract is not yet the final product, as it is only one of the components (nitrogen source) of the culture media to be prepared for the growth of heterotrophic organisms, although the contaminating compounds of the protein extract can contribute many nutrients to the cultivation of heterotrophic organisms.

[022] Document PT115535A describes a solution for the extraction of bioactive peptides of microalgal origin, from the species Nannochloropsis oceanica and Chlorella vulgaris, with antioxidant and antihypertensive action. The method of rupturing the cell wall of these microorganisms was used through the addition of acid, and by enzymatic hydrolysis using a mixture of cellulase and protease. The present invention differs from document PT115535A in that it uses physical cell disruption, associated with simultaneous treatment with enzymes, which can be hydrolases (cellulases, hemicellulases, pectinases, lipases, glucanases, mannases, chitinases, lysozyme, zymolase, isolated or in the form of a mixture, among others), with the possibility of also using nucleases, while the aforementioned patent uses a process in which only weak acid is used and, after this operation, a mixture of cellulase and protease is used to obtain a protein extract. Even in the case of chemical treatment to accelerate the cell disruption process, this is done simultaneously with the physical process. In this way, the efficiency of releasing the protein fraction and ease of separation is greatly increased in the present invention.

[023] The use of the protein extract of document PT115535A is for antihypertensive and antioxidant activity, being ready for use in this condition, while in the present invention, steps are provided for purification of the protein extract, concentration, combination of concentrated/dry extracts, as well as its incorporation into culture media for heterotrophic organisms, being, as previously mentioned, just one of their components.

[024] The development of animal protein-free media is important to increase the safety of the biological products produced, and thus, be used in pharmacological therapies and vaccines. The main disadvantage of using these animal-derived substances is the potential introduction of contaminants into the process and potentially into the final product (MERTEN, O.W. Safety issues of animal products used in serum-free media. Dev. Biol. Stand., v. 99, pp. 16780, 1999). Another risk associated with the use of animal serum includes the possibility of inter-batch variability and, to a lesser extent, intra-batch variability. Each of these risks can be reduced through the use of serum alternatives, especially the use of chemically defined medium (NIMS, R. W, HARBELL, J. W. Best practices for the use and evaluation of animal serum as a component of cell culture medium. In Vitro Cell.Dev.Biol., v.53, pp. 682-690, 2017).

[025] Document WO2017216818 presents a method for culturing obligatory or facultative anaerobic bacteria (Lactobacillus sp.) which comprises the inoculation of anaerobic bacteria in an extract of microalgae rich in omega-3 fatty acids, in which its use is proposed as the only component of cultivation medium, alternatively with the addition of only sugar, under anaerobic conditions. It mentions that a concentration of the microalgae Nannochloropsis gaditana lower than 10 g/L was sufficient to support the growth of Lactobacillus sp, but it indicated in its claim that the concentration of the microalgae to be used is sufficient to support the growth of anaerobic bacteria.

[026] The medium resulting from this growth, including cells and liquid fraction would be used without any treatment for nutritional purposes, as a food supplement and therapeutic and cosmetic uses. Considering that most industrial processes are aerobic, and, in this condition, the amount of biomass formed is much greater, a cultivation medium consisting only of microalgae extract would not be suitable, as there would be a need to use high concentrations of extract in order to support cell growth, which would make the process much more expensive. Additionally, there could be a lack of essential elements for the cellular growth of aerobic bacteria, such as sources of minerals, including phosphorus, potassium, magnesium, manganese, calcium, among others.

[027] Document PI 1107211-3 B1 uses the freshwater microalgae Scenedesmus sp, whose cultivation is carried out in a chamber with a closed bioreactor system, in an appropriate medium. To extract the substances of interest, the biomass is separated from the cultivation medium through centrifugation, followed by freezing and freeze-drying. Thus, lyophilized biomass is used in the preparation of extracts, through sequential extraction with solvents in order of polarity, or using the biomass in the preparation of suspensions. Through the bioassays carried out, the plant biostimulant effect was identified, that is, the plant growth-promoting effect was evidenced, through the use of extracts and/or suspensions obtained from the freshwater microalgae Scenedesmus sp.

[028] Document EP0049632B1 provides for the use of microalgae extract in the cultivation of animal cells, in which the aqueous extract is used at a maximum concentration of 0.05%. To obtain the extract, the suspension of microalgae in an aqueous medium, with or without addition of alkali or acid, is maintained at room temperature or, preferably, heated at high temperatures, the preferred temperature being 100 °C according to this document. The liquid fraction is separated and optionally purified by suitable techniques such as gel filtration or dialysis. The use of room temperature in contact with microalgae is not efficient for their rupture and release of metabolites. If acid or base are added to carry out this procedure, there is future difficulty in neutralizing and consequent salinization of the extract, which leads to the need for dialysis to remove the salts. The increase in temperature involves an increase in cost and, per se, and without the use of an associated physical cell abrasion disruption agent, it does not constitute an efficient method of cell disruption, reducing the efficiency of organic nutrient removal.

[029] Document EP0067697B1 proposes a method for culturing human cells (fibroblasts) using a culture medium containing microalgae extract, such as Chlorella, Scenedesmus or Spirulina, so that there is no morphological or genetic change over subsequent generations. In this process, a fraction (fraction A1) of patent EP0049632B1 is used, which is a microalgae extract, with the addition of fetal bovine serum. The inventors mention that the effect of stimulating fibroblast metabolism by these extracts is not clear, presenting the hypothesis that they may be compounds with a high molar mass, such as glycoprotein and polysaccharide. In the present invention, for each liter of medium, an aqueous microalgae extract of 0.3 to 400 mg is used.

[030] Patent EP0067697B1, differently from the present invention, uses material of animal origin (fetal bovine serum) in the composition of the cultivation medium, so that, when using the microalgae extract, the intention was to provide the fibroblasts with molecules that stimulate cellular metabolism. The concentration of microalgae extract used (maximum of 0.04%) highlights this effect of stimulating cellular metabolism and not being used as a source of nitrogen, since a cultivation medium consisting of free amino acids was used as a source of nitrogen, which It is common in animal cell cultures, as they are deficient in the production of extracellular proteases.

[031] In the same vein as patents EP0067697B1 and EP0049632B1, it was suggested the use of microalgae extracts, such as Chlorella vulgaris, enriched with a growth factor, as a serum substitute in the cultivation of mammalian cell lines of biomedical importance in 2D and 3D cell culture in vitro, with the subsequent increase in the yield of protein production and maintenance of the stem cell phenotype (NG et al., 2020). In the case of serum replacement in animal cell cultures, we are not thinking about a source of nitrogen, but rather different compounds that contribute to cellular homeostasis. Thus, the present invention also differs from this article, as the aim is to use microalgae extract as a source of nitrogen in the cultivation of heterotrophic organisms.

[032] In culturing animal cells to obtain cultured meat, OKAMOTO and collaborators (2019) applied nutrients extracted from microalgae to the culture medium for the cultivation of mammalian cells. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, while 18 of the 20 amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. The best hydrolysis results were at temperatures greater than or equal to 100°C. In the case of sugars, excess hydrolysis under extreme conditions can lead to sugar degradation, in addition to low amounts of glutamine and tryptophan being observed in the case of acid hydrolysis. Applying alkaline hydrolysis to obtain amino acids, the amino acids arginine, cystine and histidine were absent in the extracts.

[033] This work, which focused on the release of both glucose and free amino acids through acidic or alkaline treatment, since animal cells are not efficient producers of proteases, requiring the use of free amino acids in formulations, which is not the case of the present invention, where the aim is not to release free glucose, nor is the aim to release free amino acids, although in obtaining protein extracts, with the formation of peptones, there may eventually be the release of amino acids in the extracts.

[034] In the article by Okamoto and collaborators (2019), the physical disruption process was not used, which also greatly limits the process of releasing proteins so that they can undergo the hydrolysis process. In a different way, in the present invention, it is proposed to use physical disruption in the presence of hydrolases, with the possibility of adding nucleases, so that the proteins can be easily separated from the extract, by precipitation, for example, and then undergo treatment with proteases to obtain peptones.

[035] Additionally, isolated acid hydrolysis, which in the case of this article takes longer, although it is efficient for obtaining sugars and amino acids, leads to operational difficulties in purifying the monomers so that they can be used in the process.

[036] Document WO2011013707 describes a method for producing L-amino acids that comprises the preparation of processed microalgae products for use as carbon sources for bacteria that produce these, such as fatty acids, starch, glucose and glycerol, obtaining these by chemical treatments and/or with lipases and amylases. However, there is no prediction of the protein content of microalgae biomass as a source of nitrogen.

[037] Document US 7955833 refers to a cell-free animal protein culture medium comprising a combination of peptides derived from soy and yeast hydrolysates. The invention also provides an animal protein-free culture process, in which cells are grown and propagated without components of animal origin. This process is useful for cultivating cells, recombinant or infected with a virus, and for producing biological products by culturing cells in conditions devoid of protein components of animal origin. Considering that soybeans have essentially the same protein composition, with only some variations depending on the variety used in planting and production conditions, their use would be somewhat limited compared to photosynthetic microorganisms, which, as they are of Several different genera and species have different protein compositions.

[038] In this sense, different from the teachings of the prior art, the present invention proposes an extract from cyanobacteria and/or microalgae to replace animal protein in culture media, with the possibility of making mixtures of different extracts from different species, to meet the nutritional need of the microorganism(s) to be cultivated.

SUMMARY OF THE INVENTION

[039] The present invention aims to propose a process for obtaining protein extracts from photosynthetic microorganisms, namely microalgae and/or cyanobacteria, as well as their use as a source of nitrogen replacing the source of animal protein from culture media. heterotrophic organisms, which may be bacteria, yeast, fungi or animal cells. Additionally, the present invention aims to propose the use of the heterotrophic organisms produced, as well as the metabolites produced by them, for industrial applications, such as in the production of vaccines, serums, drugs and biopharmaceuticals, antibiotics for human and/or animal use, antitumor, biomass, organic acids, solvents, nucleic acids, biosurfactants, amino acids, cosmetics, biofertilizers, agricultural inoculants, phytohormones, foods, animal feed, among others.

BRIEF DESCRIPTION OF FIGURES

[040] To obtain a total and complete visualization of the objective of this invention, figures are presented, to which reference is made, as follows.

[041] Figure 1 presents a flowchart showing all the modalities addressed in the present invention.

[042] Figure 2 presents a graph of the cellular concentration of C. vulgaris as a function of time in cultivation in a tubular photobioreactor using a semi-continuous process, with a cutoff fraction of 80%. Cuts were made at 7, 14 and 18 days.

[043] Figure 3 shows a graph of the growth of B. atrophaeus ATCC 9372 in TSB medium with inoculum previously grown in TSB liquid medium (TSB1), inoculum previously grown in solid medium, in a Roux bottle, with PCA medium (TSB2) and in modified TSB medium, using microalgae protein extract as a nitrogen source.

DETAILED DESCRIPTION OF THE INVENTION

[044] The present invention refers to a process for obtaining protein extracts from photosynthetic microorganisms, mainly microalgae and/or cyanobacteria, as well as their use as a source of nitrogen replacing the source of animal protein from organism culture media heterotrophs, which may be bacteria, yeast, fungi or animal cells, as represented in Figure 1.

[045] In general, said process of the present invention comprises the following steps: (a) Cultivation of photosynthetic microorganisms; (b) Separation of collected cells; (c) Cellular Rupture; (d) Purification of the protein extract, optionally; and (e) Drying/lyophilization, optionally.

[046] The cultivation of photosynthetic microorganisms, whether genetically modified or not, can be in closed photobioreactors, such as tubular or flat plate, or open, such as tanks, for example, but not limited to these types of photobioreactors, with different systems of circulation of cells, under sterile and/or aseptic conditions, and the cultivation may not be axenic, however, preferably without the presence of other photosynthetic microorganisms or with quantities that do not exceed 10% of their total cells, not admitting the presence of microorganisms producing toxins.

[047] Photobioreactors are illuminated with natural or artificial light, preferably natural, and conducted under photoautotrophic, mixotrophic or heterotrophic conditions, preferably photoautotrophic. Temperatures can be from 20°C to 45°C, preferably from 25°C to 35°C, for different types of processes, such as batch, fed-batch and its variations, semi-continuous process and/or continuous process under non-continuous conditions. limited nitrogen, preferably with media with nitrogen concentrations greater than 0.5 mM, more preferably greater than 2 mM. The pH value is preferably controlled in the optimal growth range of the photosynthetic microorganism to be cultivated, which depending on the species, may have a growth pH value between 4.0 and 11.0.

[048] In the case of microalgae, such as Chlorella vulgaris, for example, the optimum pH value is between 6.5 and 8.0, and in the case of cyanobacteria, such as Arthrospira platensis, for example, the optimum pH value is between 9.0 and 10.0, although narrower ranges of pH value can be used to optimize cell growth conditions. Thus, depending on the species, there will be a range of optimal pH value to be maintained in the growing medium, or the pH value can be maintained at a fixed value for growth to occur under optimal conditions.

[049] pH control can be done with alkalis, or with acids, organic or inorganic, preferably with the use of carbon dioxide, from different industrial sources, including power plants, thermoelectric plants, cement industries, but not limited to these, preferably through a device that allows automatic pH control. The cultivation medium can be made up of mineral media, with different salinities and pH values, with or without the presence of organic compounds and/or the addition of these, with the presence of nutrients for cell growth, including vitamins and microelements. The salts used in the preparation of the cultivation media must be at least technical grade, preferably P.A. grade.

[050] Once the cultivation of photosynthetic microorganisms has been carried out, the cells collected from the culture, preferably from its final phase, can be separated by physical and/or chemical processes, including filtration, with or without the use of vacuum, filtration tangential, centrifugation, flocculation, sedimentation, and there may be a combination of these techniques, with the possibility of washing the cells. Cells recovered while still wet, preferably, or that have undergone a drying process, are resuspended in water, for example, deionized, distilled, potable, not limited to these types of water, or in saline media, preferably buffered, to be treated by cell disruption processes, which may be mechanical, such as disruption in ball mills or reactors, using glass, ceramic, stainless steel, or other materials spheres, disruption in high-pressure homogenizers, disruption by ultrasound, operated by continuous or discontinuous form.

[051] Alternatively, other physical methods can be used, such as using high temperature, preferably above 40°C, freezing and thawing, chemical methods, including the use of alkaline, acid, or detergent compounds, and/or enzymatic compounds for disruption cell phone. These cell disruption processes can be used together, preferably, alone, or in sequence, in different orders. Solvents can still be used to extract lipids prior to the cell disruption process.

[052] The synergy of physical treatment of cells and chemical or enzymatic treatment comes from reducing the size of particles (“cellular debris”), increasing the surface of action for the action of enzymes or acids/bases used in the hydrolysis of polysaccharides and/or or nucleic acids, facilitating the separation of the protein fraction. To this end, in cases of physical disruption associated with chemical treatments, equipment for physical disruption of cells and their components that will come into contact with the reaction medium must be constructed with materials resistant to acids and bases, such as stainless steel 316, preferably added with other metals in the metal alloy, such as titanium and niobium, titanium alloys, silicon, zirconium, for example, ceramics, not limited to these materials.

[053] In the case of enzymatic treatments associated with physical disruption, thermostable enzymes must be used, preferably resistant to temperatures above 40°C, more preferably resistant to temperatures above 50°C, with the possibility of using equipment with temperature control . Although the use of wet biomass, which can be recently collected from the cultivation process, or from biomass kept under refrigeration, frozen or not, is the preferred way of obtaining the extract of photosynthetic microorganisms, alternatively dry, degreased or no, which can go through the same processes used to obtain the wet biomass extract.

[054] The use of degradation enzymes (hydrolases) such as cellulases, hemicellulases, pectinases, lipases, glucanases, mannases, chitinases, lysozyme, zymolase, isolated or in the form of a mixture, among others, can be carried out to increase the release of proteins free in the cultivation medium. Alternatively, to obtain purer protein suspensions, in the cell disruption process, in addition to the addition of hydrolases, nucleases can be added, facilitating the separation of proteins by precipitation through the addition of salts or by adjusting the pH to different isoelectric points.

[055] Hydrolases and nucleases should preferably be added during the physical disruption process. Surfactants, of synthetic or biological origin, preferably of biological origin, can be added to the physical cell disruption process, as well as physical cell disruption treatment simultaneously with the chemical or enzymatic treatment for this purpose. The proteins released into the culture medium from the cell disruption step can be used with or without purification, and, alternatively, proteases can be added, preferably of microbial and/or vegetable origin, which act at a pH value of 1. 0 to 11, for release of peptides and amino acids.

[056] Purification can be carried out by means of precipitation with the aid of the addition of salts, such as ammonium sulfate or sodium chloride, or by pH correction for the isoelectric point of proteins, peptides and/or amino acids, not limited to these methods of precipitation of protein materials.

[057] In the case of obtaining extracts rich in free amino acids, the action of proteases should preferably be on previously purified protein extracts. Subsequently, the proteins, peptides and/or amino acids can optionally undergo dialysis to remove salts and be purified by different methods, including tangential filtration, aqueous two-phase system, ion exchange chromatography, gel chromatography and/or affinity chromatography.

[058] Furthermore, a step of precipitation of genetic material from photosynthetic microorganisms can also be included using, for example, precipitation processes with solvents, such as ethanol, or other substances that promote their precipitation, or the genetic material can undergo treatment with enzymes (nucleases) or other compounds, such as alkalis, for example, that lead to their hydrolysis, so that the separation of the hydrolyzed genetic material and proteins by tangential filtration separation processes is facilitated. These procedures can occur before or after the purification process of the protein fraction itself. Nucleic acids, after purification, can be used in food formulations, including applications as flavor enhancers in foods.

[059] The protein extracts obtained preferably have a low amount of nucleic acids, preferably less than 3%, more preferably less than 1%, more preferably even less than 0.5%, free of toxins, with protein contents greater than 30%, more preferably above 40%, most preferably still above 50% in relation to total solids. However, depending on the heterotrophic microorganism that will be cultivated and the purpose of the product to be produced, crude extracts can be used, without purification of the proteins and their extracts or with partial purification of these compounds.

[060] It is also expected that protein extracts from more than one species of photosynthetic microorganism can be mixed, with or without genetic modification, so that the mixture of extracts from these microorganisms can be the same or similar in amino acid composition, as well as the amino acid sequence of proteins and their derivatives of animal or vegetable origin, aiming to supply amino acids and their proportions, in the form of peptides/proteins, with or without the presence of amino acids, as well as with or without added amino acids, that meet specific demands of heterotrophic organisms.

[061] The proportion between the extracts of photosynthetic microorganisms, with or without genetic modification, can vary from 1% to 99%, with the addition of a maximum of 50% of free amino acids in the mixture, obtained from other sources, preferably not of natural origin. animal. To the protein extracts, coming from a photosynthetic microorganism or more than one photosynthetic microorganism, protein extracts of plant origin can be added, with proportions that can vary from 1 to 99%, so that the mixture can meet the specific demands of heterotrophic organisms .

[062] In the case of using microorganisms with genetic modification, nucleotide sequences can be constructed, incorporated into the DNA of the cell or organelles, such as chloroplast, for example, or in plasmids, which produce proteins, as well as peptides, with a composition of amino acids that meet the specific demands of heterotrophic organisms, with an enrichment of amino acids in these molecules, as well as with the same or similar composition to proteins of animal origin and their derivatives.

[063] In particular, it is expected that the compounds produced by these microorganisms will have compositions similar to the peptones of milk protein origin, and different peptones may be produced by different genetically modified photosynthetic microorganisms or by just one.

[064] In any condition in which the protein material from photosynthetic microorganisms, called protein extract, is obtained, it may undergo processes to increase its concentration or not, and subsequent drying processes, natural and/or artificial, in drums may be applied, drying tunnels, trays, with or without reduced pressure, with or without air circulation, inert gases and/or heated steam.

[065] Additionally, the protein extract can be dried by freeze-drying or spray drying, preferably, and other drying processes can be used. The protein extract can go through a grinding and screening process to standardize the particle size.

[066] When purifying the protein extract, there is the possibility of separating the nucleic acids from the biomass. The separation of the nucleic acid fraction from the protein fraction can be done by precipitation of the protein fraction or the nucleic acid fraction, preferably the protein fraction, or by the addition of nucleases to release nucleic acids and nucleotides from the hydrolysis of DNA and RNA from the photosynthetic microorganisms, which can be easily separated from the protein fraction by precipitation or through treatment with filter membranes. These nucleic acids, after purification, can be used in food formulations, including applications as flavor enhancers in foods.

[067] Once obtained, the protein extract is used as a source of nitrogen in the cultivation of heterotrophic organisms, whether genetically modified or not, and may be the only source of nitrogen in the cultivation medium, whether from a single photosynthetic microorganism or from a mixture of more of a photosynthetic microorganism, in different proportions.

[068] The cultivation of heterotrophic organisms may include the addition of other sources of nitrogen in order to supply one or more amino acids, nucleic acids, which may, alternatively, come from complex nitrogen sources, such as bacterial extract, yeast extract, water corn maceration, purified or not. Urea and/or inorganic nitrogen sources can complement the protein extract in the growth of heterotrophic organisms. Particularly, in the case of cell cultivation to obtain vaccines and biopharmaceuticals, preferably, the complementary complex nitrogen sources are not of animal origin.

[069] The extracts can be added in levels, considering their dry basis, in values from 0.01% to 30%, more preferably, in the range of 0.1% to 15%, more preferably still in the range of 1% to 10%.

[070] Other nutrients, such as organic carbon sources, preferably monosaccharides and disaccharides, more preferably monosaccharides, vitamins and mineral salts, such as phosphorus sources, in the form of phosphoric acid or salts of phosphorus, calcium, potassium, among others, and micronutrients they can be constituents of the cultivation medium, regardless of the type of cultivation process used (batch, fed-batch, semi-continuous, continuous and their variations).

[071] The culture medium containing the protein extract can be sterilized with it already in its composition, or there can be a separate sterilization of the nitrogen source and other constituents of the culture medium, particularly an organic carbon source, to avoid a chemical reaction. Maillard.

[072] The cultivation processes can preferably be under aseptic or sterile conditions, although there may be the possibility of cultivation with the presence of other organisms, be aerobic or anaerobic, with or without the addition of antifoams, with positive or negative pressure in the bioreactors, with or without bubbling of gases, such as nitrogen, air, oxygen, mixtures of oxygen and air, with pH values of 2.5 to 11.0, preferably between 4.0 and 9.0, more preferably between 5.5 and 8.0. Growth will be at temperatures of 20°C to 65°C, more preferably between 25°C and 45°C, most preferably between 28°C and 37°C. These pH and temperature values must meet conditions that allow the optimal growth of heterotrophic organisms.

[073] During cultivation, preferably at the end of it, the heterotrophic organism will preferably be separated from the residual culture medium, and the cells produced and/or culture medium will be used to recover the product of interest, different processes may be used to this purpose, including physical, chemical and/or enzymatic processes, in different combinations and stages. The molecules of interest may be purified and/or modified, depending on the purpose of use.

[074] Finally, the present invention proposes the use of the heterotrophic organisms produced, as well as the metabolites produced by them for industrial applications, such as in the production of vaccines, serums, drugs and biopharmaceuticals, antibiotics for human and/or animal use, antitumor , biomass, organic acids, solvents, nucleic acids, biosurfactants, amino acids, cosmetics, biofertilizers, agricultural inoculants, phytohormones, foods, animal feed, among others.

[075] The following examples are intended only to illustrate, without limiting the scope of the present invention. Any variations capable of being carried out by a person skilled in the art must be considered to be within the scope of the present invention.

EXAMPLES OF THE INVENTION Strain maintenance

[076] The microalgae Chlorella vulgaris, used as an example of a photosynthetic microorganism, was maintained in standard Bold liquid culture medium.

[077] Bacillus atrophaeus ATCC 9372, used as an example of a heterotrophic organism, was maintained in a 0.2 M calcium acetate solution, in the form of a matrix suspension.

Standard Cultivation Medium

[078] Standard Medium (Bold) (BISCHOFF, Harry William, BOLD, Harold C. Some Soil Algae from Enchanted Rock and Related Algal Species. Austin, Tex: University of Texas, Phycological Studies, n. IV, 1963) was used for the growth of Chlorella vulgaris.

[079] Standard Tryptic Soy Broth Medium (TSB) was used for the cultivation of Bacillus atrophaeus ATCC 9372. The TSB medium contains peptones of animal origin.

Modified medium for the cultivation of Bacillus atrophaeus ATCC 9372

[080] The modified TSB Medium, with microalgae protein extract, instead of the peptones from the standard TSB medium (casein and soy peptones), for the cultivation of Bacillus atrophaeus ATCC 9372.

Obtaining C. vulgaris biomass Inoculum preparation

[081] The preparation of Chlorella vulgaris inoculum was carried out in Erlenmeyer flasks with a capacity of 500 mL, adding 20 mL of the inoculum fixed at 4000 mg.L-1 of microalgae in 180 mL of Bold medium, incubating at 25 °C at 110 rpm for up to 10 days, with a light intensity of 80 (± 10) μmol photons.m-2.s-1.

Cultivation in tubular photobioreactor

[082] The cultivation of Chlorella vulgaris was carried out in a tubular photobioreactor with a capacity of 35 L at 25° C, with the inoculum obtained under the conditions indicated in the preparation of the inoculum. The culture was moved by an air lift system, the pH was maintained at 7.5 ± 0.5 and light intensity of 40 (± 10) μmol photons m-2.s-1, for up to 50 days in a semi- continuous, using Bold medium.

Preparation of microalgal extract

[083] After cultivating the microalgae, the biomass was centrifuged at 4000 rpm for 30 minutes and the biomass was washed with deionized water to remove salts. Deionized water was added again, and the cells in this suspension were disrupted with 1 mm glass beads and vigorously shaken for 50 minutes. The pearls were removed and the suspension was dried in a forced circulation oven at 60 °C, and finally, the biomass was crushed with the aid of a mortar and pestle.

Experiments carried out in standard Bacillus atrophaeusATCC9372 medium (TSB medium) and in modified TSB medium, using microalgae protein extract

[084] Two cultures were carried out in standard Bacillus atrophaeus medium, at 37 °C, at 150 rpm, for 24 hours, and in the first culture the bacteria also previously grew in TSB liquid medium and in the second culture the bacteria previously grew in PCA solid medium, with the spore suspension obtained by removing the cells with a glass bead and resuspension in physiological solution. The cultures, in triplicate, were carried out in Erlenmeyer flasks with a capacity of 250 mL. In the case of the modified medium, 100 mL of test culture medium was prepared with a composition similar to that of TSB, but with the protein source replaced by Chlorella vulgaris extract, in an equivalent amount of organic nitrogen, incubating at 37 ° C, at 150 rpm, for 24 hours, and compared to standard cultures (TSB medium).

[085] Bacterial counts were carried out by serial dilutions in 0.9% sodium chloride solution in which 1 mL aliquots were transferred to Petri dishes and then 20 mL of PCA were added, with the plates were incubated in ovens at 37 °C for 48 hours, after the medium solidified. After 48 hours, the count was carried out considering the plates that presented up to 300 UFC.mL-1. RESULTS OBTAINED

[086] Chlorella vulgaris cultivations were carried out using a semi-continuous process in a 35 L photobioreactor, with a cutoff fraction of 80%. The cell concentration values during the cycles of the semi-continuous process varied between 0.22 g.L-1 and 1.46 g.L-1 (Figure 2), and the process proved to be stable, with no tendency for cell concentration to drop between one cycle and other.

[087] After the cell growth stage, the cells were recovered to obtain protein extracts from microalgae, as previously reported (item “Preparation of the microalgal extract”). The protein content of the dry microalgal protein extract was 14.03% ± 0.16% and of the dry TSB medium was 49.10% ± 2.61%, so that in the modified TSB medium the concentration of microalgal extract was included. 104.8 g.L-1 to supply the same protein concentration as the TSB medium.

[088] The growth of Bacillus atrophaeus ATCC 9372 was tested in a culture medium with Chlorella vulgaris extract (microalgal protein extract), using as a comparison parameter the growth of this bacteria in TSB medium, whether from an inoculum previously grown in TSB liquid medium. (TSB1) or inoculum previously grown on solid medium, in a Roux bottle, with PCA medium (TSB2) (Figure 3).

[089] In TSB1, a standard medium in which Bacillus atrophaeus ATCC 9372 was pre-adapted to the TSB medium, when inoculating a 102CFU.mL-1 population, the bacteria immediately grew exponentially up to 8 h of cultivation, reaching a maximum population of 3. 7 x 108CFU.mL-1 in 24 hours, obtaining a maximum specific growth rate (μm) of 1.57 h-1. However, when replacing peptones with Chlorella vulgaris extract, the bacteria needed 6 hours to adapt, in which it grew exponentially up to 12 hours of cultivation, and reached a maximum population of 1.9 x 107CFU.mL- 1 in 24 hours. Its μm value was 1.25 h-1.

[090] On the other hand, the experiments carried out indicate that replacing the peptones in the TSB medium with microalgal proteins is a good alternative, as growth with cells not previously adapted in TSB medium (TSB2 experiment) and growth with Chlorella extract vulgaris led to equivalent cell growth (Figure 3) (p = 0.513). In both cases, there was a lag phase, which was more pronounced in the medium with microalgae extract, which could be avoided with prior growth of the cells in this medium.

[091] When the Bacillus atrophaeus inoculum was previously cultivated in TSB medium (TSB1 experiment), there was no lag phase of growth, which is probably due to the fact that the cells were previously adapted to the medium before their growth, which can have provided greater cell growth compared to growth from cultivation with microalgal extract (p = 0.000).

[092] However, in comparison of the final cellular concentrations obtained with the microalgal protein extract and in TSB media, there was no statistical difference (p = 0.121), reinforcing the idea that the microalgal protein extract could be used as a source of proteins, which would be particularly important in the production of vaccines and biopharmaceuticals, where it is convenient to replace animal proteins with other protein sources.

[093] Although the invention has been extensively described, it is obvious to those skilled in the art that various changes and modifications can be made without said changes falling within the scope of the invention.

Claims (24)

1. Process for obtaining protein extracts from photosynthetic microorganisms characterized by the fact that it comprises the following steps: (a) Cultivation of photosynthetic microorganisms; (b) Separation of collected cells; (c) Cell rupture; (d) Purification of the protein extract, optionally; and (e) Drying/lyophilization, optionally. 2. Process, according to claim 1, characterized by the fact that the photosynthetic microorganisms are selected from the group consisting mainly of microalgae and cyanobacteria, preferably Chlorella vulgaris and Arthrospira platensis, respectively. 3. Process, according to claim 1, characterized by the fact that in step (a) the cultivation is carried out in photobioreactors preferably under sterile and aseptic conditions, preferably without the presence of other photosynthetic microorganisms or with quantities that do not exceed 10% of total cells of these. 4. Process, according to claim 3, characterized by the fact that the photobioreactors are illuminated with natural or artificial light, preferably natural, conducted under photoautotrophic, mixotrophic or heterotrophic conditions, preferably photoautotrophic, at temperatures ranging from 20°C to 45° C, preferably from 25°C to 35°C, conducted by different types of process, preferably batch, fed batch and its variations, semi-continuous process and continuous process in conditions not limited to nitrogen, preferably with media with higher nitrogen concentrations than 0.5 mM, more preferably, greater than 2 mM. 5. Process, according to claim 4, characterized by the fact that the growth pH value of the photosynthetic microorganism varies between 4.0 and 11.0, where for microalgae, the optimum pH value is preferably in the range between 6.5 and 8.0, and for cyanobacteria, the optimum pH value is preferably in the range between 9.0 and 10.0. 6. Process, according to claim 1, characterized by the fact that in step (b) the collected cells are separated by physical and/or chemical processes selected from the group consisting mainly of tangential filtration, centrifugation, flocculation, sedimentation, and combinations thereof , in which subsequently, the dry or wet cells, preferably wet, are resuspended in water, mainly deionized, distilled and potable water or saline media, preferably in buffered saline media. 7. Process, according to claim 1, characterized by the fact that in step (c) cell disruption is preferably carried out by mechanical, physical, chemical, enzymatic processes, and combinations thereof. 8. Process according to claim 7, characterized by the fact that the mechanical processes are selected from the group consisting mainly of ball mills or reactors, glass spheres, ceramics, stainless steel, high pressure homogenizer and ultrasound. 9. Process, according to claim 7, characterized by the fact that the physical processes are selected from the group consisting mainly of the use of high temperature, preferably above 40°C, freezing and thawing. 10. Process according to claim 7, characterized by the fact that the chemical processes are selected from the group consisting mainly of alkaline compounds, acids and detergents. 11. Process according to claim 7, characterized by the fact that the enzymatic processes are selected from the group consisting mainly of degradation enzymes selected from the group consisting preferably of hydrolases and nucleases, mainly cellulases, hemicellulases, pectinases, lipases, glucanases, mannases , chitinases, lysozyme, zymolase, isolated or in the form of a mixture. 12. Process, according to claim 7, characterized by the fact that in physical processes associated with chemical processes, the components of the physical cell disruption equipment comprise materials resistant to acids and bases, mainly 316 stainless steel, added with other metals in the metallic alloy, preferably titanium and niobium, titanium alloys, silicon, zirconium and ceramics. 13. Process, according to claim 7, characterized by the fact that in enzymatic processes associated with physical processes, the enzymes are thermostable, preferably resistant to temperatures above 40°C, more preferably resistant to temperatures above 50°C, in which optionally uses equipment with temperature control. 14. Process, according to claim 1, characterized by the fact that in step (d) purification is carried out preferably by means of precipitation with the aid of the addition of salts, mainly ammonium sulfate or sodium chloride, and by pH correction to the isoelectric point of proteins, peptides and/or amino acids. 15. Process, according to claim 14, characterized by the fact that optionally the proteins, peptides and/or amino acids are subjected to dialysis and are purified by methods selected from the group consisting mainly of tangential filtration, aqueous two-phase system, chromatography of ion exchange, gel chromatography and affinity chromatography. 16. Protein extracts from photosynthetic microorganisms obtained according to the process defined in any one of claims 1 to 15, characterized by the fact that they have a low amount of nucleic acids, preferably less than 3%, more preferably less than 1%, more preferably even less than 0.5%; are free of toxins; and have protein contents greater than 30%, more preferably above 40%, more preferably even above 50% in relation to total solids. 17. Protein extracts, according to claim 16, characterized by the fact that the proportion between the extracts of photosynthetic microorganisms varies between 1% and 99%. 18. Protein extracts, according to claim 16 or 17, characterized by the fact that a maximum of 50% of free amino acids obtained from other sources, preferably not of animal origin, is optionally added to the extract. 19. Protein extracts, according to any one of claims 16 to 18, characterized by the fact that protein extracts of vegetable origin are optionally added, with proportions that may vary from 1 to 99%. 20. Use of protein extracts from photosynthetic microorganisms, as defined in any one of claims 16 to 19, characterized by the fact that it is a source of nitrogen in the cultivation of heterotrophic organisms. 21. Use, according to claim 20, characterized by the fact that the extracts are added in levels, considering their dry basis, of 0.01% to 30%, more preferably, in the range of 0.1% to 15%, more preferably still in the range of 1% to 10%. 22. Culture medium for heterotrophic organisms, characterized by the fact that it comprises protein extracts of photosynthetic microorganisms as defined in any one of claims 16 to 19, other nitrogen sources selected from the group consisting mainly of bacteria extract, yeast extract, water corn maceration, urea and inorganic nitrogen sources, organic carbon sources, preferably monosaccharides and disaccharides, more preferably monosaccharides, vitamins and mineral salts, phosphorus sources, mainly in the form of phosphoric acid or phosphorus, calcium and potassium salts. 23. Culture medium, according to claim 22, characterized by the fact that it has pH values of 2.5 to 11.0, preferably between 4.0 and 9.0, more preferably between 5.5 and 8, 0, and temperatures of 20°C to 65°C, more preferably between 25°C and 45°C, most preferably between 28°C and 37°C. 24. Use of heterotrophic organisms and their metabolites produced in the culture medium, as defined in claim 22 or 23, characterized by the fact that it is mainly in the production of vaccines, serums, drugs and biopharmaceuticals, antibiotics for human and/or animal use, antitumors, biomass, organic acids, solvents, nucleic acids, biosurfactants, amino acids, cosmetics, biofertilizers, agricultural inoculants, phytohormones, food and animal feed.