Microbial consortium, method for producing a new alternative protein and alternative seaweed protein product

Abstract

The present invention relates to the biotechnology industry, and specifically to the development of a microbial consortium that is useful in the fermentation of seaweed. This invention further describes a method for producing a new alternative protein that combines the mycelium from the microbial consortium and the components of the seaweed used in the fermentation. The fermentation process allows the release and concentration of bioactive compounds present in the seaweed, such as proteins, amino acids, phenolic compounds and beta-glucans, which contribute to the functional properties of the final product. Furthermore, the invention comprises a new alternative protein product obtained using this specific method, characterised by the integration of the bioactive compounds of the seaweed with the mycelium of the consortium, which results in a food product with improved nutritional properties, functional bioactivity and better digestibility compared to seaweed.

Description

[0001]MICROBIAL CONSORTIUM, METHOD FOR PREPARING A NEW ALTERNATIVE PROTEIN AND PRODUCT ALTERNATIVE PROTEIN FROM SEAWEED FIELD OF THE INVENTION The present invention relates to the biotechnology industry, in particular to the development of a specialized microbial consortium for the fermentation of seaweed. More specifically, the invention relates to a method for the preparation of an alternative protein that integrates the mycelium of the microbial consortium with the bioactive components of the seaweed used in the fermentation. Finally, the invention relates to a product, an alternative protein obtained by this method, differentiated by its improved nutritional properties and functionality. STATE OF THE ART Rapid population growth has led to a growing interest in alternative protein sources that are sustainable and healthy to meet the needs of the population.The global food system is in need of transformation, as emerging data demonstrate a compelling link between diet, health, and the environment, which cannot be met due to existing planetary boundaries. Seaweeds are perceived as healthy by consumers and are rich in nutrients such as proteins, lipids, carbohydrates, vitamins, minerals, and biologically active compounds (polyphenols, fatty acids, and carotenes), which are nutritionally and health-relevant to the human diet. Seaweed production is more sustainable compared to land-based crops. Brown seaweed, in particular, is the fastest-growing linear organism, expanding up to 60 cm per day. Furthermore, multiple harvests can be produced per year, and they are good ecosystem builders, hosting hundreds of species of marine life and mitigating global warming.However, the main problem with using algae as food for humans and animals stems from the algal cell wall. This wall is a tightly integrated network of biopolymers, primarily polysaccharides (e.g., alginate, cellulose, fucoidan, hemicellulose), which are associated with proteins, phenols, and ions, including calcium and potassium. Therefore, pretreatment is necessary to release and / or concentrate these nutritional compounds, making them digestible or assimilated, in order to take advantage of these nutritional and / or nutraceutical properties. These pretreatments include physical-chemical, enzymatic, or biological processes, which can be used in conjunction. Seaweeds have low solubility and unpleasant flavors and odors, which is an impediment to the mass consumption or consumption of products made from algae.Currently, alternative proteins known as mycoproteins or single-cell proteins are produced through the fermentation of raw materials from plants, such as soybeans, legumes, cereals, and waste from the agricultural and food industries. Fermentation of these raw plant materials uses bacteria such as Lactobacilli, yeasts such as Saccharomyces cerevisiae, terrestrial ascomycete fungi such as Fusarium, and edible basidiomycete fungi such as Lentinula, Pleurotus, Agariscus, and Boletus as microorganisms. The use of raw materials from agricultural crops contains carbohydrates such as cellulose and starch. Therefore, it is necessary to use enzymes to break down these carbohydrates and improve fermentation.Additionally, it is important to highlight that fermentation processes involve maintaining specific parameters of pH, temperature, oxygen, and adequate carbon:nitrogen concentrations, and this is directly linked to the type of substrate and the type of microorganisms. This technology involves the production of an alternative protein using a fermentation process with an artificial consortium of fungi and using seaweed as a carbon source. Seaweed is a raw material considered sustainable and contains compounds considered nutraceuticals, such as polysaccharides, phenols, and flavonoids. To achieve this, an artificial consortium of marine and terrestrial fungi is used, which have different needs related to their growth conditions, pH, temperature, oxygen, and agitation. The process does not involve physicochemical pretreatment of the raw material, nor are enzymes added.The result is an alternative protein with at least 30 to 35% protein, with a digestibility close to 100% and a corrected digestibility called PDCAAs (protein digestibility-corrected amino acid scores) of 89%. It also includes components from the fermentation process, which allow the microbial consortium to produce enzymes capable of breaking polysaccharide bonds such as alginate (present in brown algae) and linked to proteins and phenolic compounds. This alternative protein contains these compounds free from seaweed along with the bioactive components of the medicinal and edible mushrooms that are part of the consortium.This product has a better flavor, texture, smell, viscosity, and solubility than seaweed and contains carbohydrates with a smaller molecular size than the polysaccharides found in seaweed. For example, alginate can be broken down into oligosaccharides and monosaccharides, which have a better texture and flavor. These oligosaccharides are even considered bioactive compounds with nutraceutical properties. SOLUTION TO THE TECHNICAL PROBLEM Recent research has shown that many strains of filamentous fungi can be grown in submerged liquid cultures. These fungi can use a variety of substrates to feed and grow. This process, known as submerged fermentation, is used to enhance and increase the nutritional content of the substrate in which the fungi grow. The substrates used come from agricultural crops or can be byproducts of the food industry.These fungi produce carbohydrase enzymes capable of degrading complex carbohydrates such as cellulose and hemicellulose. In particular, marine filamentous fungi associated with seaweed are capable of producing enzymes that can degrade the complex carbohydrates in seaweed into alginate. The use of these filamentous fungi allows for the valorization and increase of the nutritional content of seaweed. For example, the use of the marine fungus Paradendryphiella salina allows for increased concentrations of proteins, amino acids, phenols, and antioxidant activity, and decreased concentrations of complex carbohydrates such as alginate and cellulose in brown seaweed through submerged fermentation. However, it is observed that submerged fermentation of brown algae using the fungus P. salina is not efficient; approximately 50% of complex carbohydrates such as alginate and cellulose are not hydrolyzed.Thus, the result of this process is an alternative protein with low digestibility, unpleasant taste, and low solubility. Therefore, an algal fermentation method is proposed that uses an artificial consortium of marine filamentous fungi (Ascomycetes) and terrestrial filamentous fungi (Basidiomycetes) for fermentation. This solution takes advantage of the ability of filamentous fungi to cooperate and interact with each other in functional metabolic networks. Thus, terrestrial filamentous fungi (Basidiomycetes) (edible fungi) with the capacity to produce large amounts of carbohydrases are selected to assimilate the cellulose present in seaweed and produce chelating metabolites that enhance the enzymatic activity of the enzymes produced by marine fungi. These consortia have the ability to grow synergistically and cooperatively between terrestrial and marine fungi.In this context, an artificial consortium was designed, one that is not found naturally in nature. This consortium can perform complex functions that are impossible for a single organism. Microbial co-cultures can trigger the biosynthesis of new bioactive metabolites, increasing the diversity of secondary metabolites with activity or considered nutraceuticals found in both mushrooms and seaweed. Additionally, the use of edible basidiomycete fungi provides a unique flavor (umami), a meaty consistency, and bioactive compounds to the target product. Edible basidiomycete fungi are components that have the ability to stimulate the immune system, enhance cytokine secretion, regulate the immune response, protect against inflammation, and maintain intestinal health.SUMMARY DESCRIPTION OF THE INVENTION The present invention relates to an innovative biotechnological method for the production of a new alternative protein, using a microbial consortium specifically designed for the fermentation of seaweed. This method combines the capabilities of filamentous fungal mycelium with the bioactive biocompounds present in brown seaweed, achieving a unique product with nutritional, prebiotic and bioactive properties. The process is based on submerged fermentation (SSF), a sustainable and efficient bioprocess that uses microbial consortia formed by marine filamentous fungi (Ascomycetes, such as Paradendryphiella salina) and terrestrial filamentous fungi (Basidiomycetes, such as Pleurotus ostreatus, Lentinula edodes, Ganoderma lucidum and Trametes versicolor).This consortium is designed to maximize the transformation and valorization of seaweed biomass, particularly brown species such as Macrocystis pyrifera and Durvillaea spp. Advantages of the fermentation method and process: Efficient release of active biocompounds: During fermentation, the fungi in the consortium act synergistically to release bioactive components that, in the algae, are bound to complex carbohydrates, such as polysaccharides. The released biocompounds include oligosaccharides with prebiotic properties, phenolic compounds, proteins, and amino acids. These compounds not only increase the nutritional value of the product but also provide functional and bioactive properties, such as antioxidant activity and immunomodulatory effects.Integration of mycelium and algae compounds: The final product, the alternative protein, combines the mycelium produced by filamentous fungi, rich in protein and β-glucans, with the soluble biocompounds released from the algae during fermentation. This generates an enriched biomass with a balanced nutritional profile and unique characteristics not found in traditional protein sources. Accelerated and sustainable production: Unlike conventional protein sources, such as those of animal origin, which require months or years to produce, this method allows the generation of biomass rich in protein and bioactive compounds in a matter of hours. Furthermore, submerged fermentation minimizes the environmental impact, significantly reducing the use of water, land, and CO₂ emissions, positioning this invention as a sustainable alternative for the food industry.Final product properties: The resulting product, a novel alternative protein, is characterized by its high protein content (35–40% dry matter), high concentration of β-glucans (20 g / 100 g), and dietary fiber (20–25%). It also contains a combination of prebiotic oligosaccharides and bioactive compounds such as polyphenols, unsaturated fatty acids, and sterols (such as ergosterol). These properties make it ideal for applications in the food and nutraceutical industries, offering potential health benefits such as improving the gut microbiota, modulating the immune system, and reducing oxidative stress. Microbial consortium design and process optimization: The microbial consortium used in this method is developed through extensive screening of marine and terrestrial filamentous fungi.These microorganisms are able to degrade complex carbohydrates from brown algae, such as alginate and cellulose, and release associated biocompounds. Fermentation is carried out in a minimally supplemented medium, using algae as the primary carbon source, ensuring the sustainability and economic efficiency of the process. The bioprocess design also includes the optimization of physical conditions, such as dissolved oxygen, temperature, and agitation speed, to maximize consortium growth and biomass production. This approach combines principles of microbial physiology, ecology, and biotechnology to obtain a high-quality final product. Impact and Applications: This invention offers an innovative solution to address the growing demand for alternative protein sources, simultaneously addressing the challenges associated with environmental sustainability and global food security.The method is not only applicable to the production of alternative proteins for human consumption, but can also be adapted for the formulation of functional ingredients in foods, nutritional supplements, and pharmaceuticals. In summary, the present invention represents a significant advance in the use of renewable resources such as seaweed, combining them with advanced biological technologies to produce a functional, sustainable, and high-added-value food product. DESCRIPTION OF THE FIGURES Figure 1. Production of the alternative protein in a 5-liter bioreactor. A) Submerged fermentation, using the culture medium with seaweed, in a 5-liter bioreactor. B) Mycelium suspension of the artificial fungal consortium in the 5-liter bioreactor, after submerged fermentation. C) Mycelial biomass of the artificial fungal consortium cultivated, harvested, and filtered from the bioreactor. D) Mycelial biomass. Figure 2.A) Start of submerged fermentation (hour 0). At this initial point, 100 mL of unfermented medium was filtered using a 20-micron filter. The filtration process took approximately 12 minutes, reflecting the high viscosity and alginate concentration present in the initial medium. In the upper image, significant filter clogging is observed due to the high alginate content, while in the reactor (lower image), the medium presents a homogeneous color, with no visible presence of mycelium or suspended particles, indicating that the fermentation process has not yet begun. B) After 24 hours of fermentation, the microbial consortium begins to hydrolyze the complex carbohydrates present in the seaweed, mainly alginate. At this stage, the filtration time for the same 100 mL was drastically reduced to 3.2 minutes, indicating a significant decrease in the viscosity of the medium.In the filter, less material accumulation is observed, reflecting the initial degradation of the alginate. In the reactor, initial mycelial growth is observed, which begins to form small aggregates on the surface and in the liquid phase, indicating early colonization of the consortium. C) At 48 hours, the alginate hydrolysis process is more advanced, as evidenced by the decrease in filtration time to 2.1 minutes. In the filter, very little retained material is observed, confirming the transformation of alginate into more soluble compounds. In the reactor, the mycelium has grown significantly, forming a dense network that covers much of the medium. This growth reflects the efficient utilization of nutrients released by alginate degradation and the consumption of simple carbohydrates derived from seaweed. D) At 110 hours, fermentation is complete.The filtration time for the same 100 mL is further reduced to 30 seconds, indicating that almost all of the alginate has been degraded by the fungal enzymes. In the filter, virtually no retained material is observed, confirming the efficiency of the microbial consortium in bioconverting the medium. In the reactor, the mycelium has reached its maximum growth, forming a dense and compact biomass, while the liquid medium shows clear phase separation, reflecting the completion of the fermentation process. Figure 3. A) In the first stage of the culture (Hour 0), microscopic analysis at 40x magnification shows a high concentration of particles from seaweed (Durvillaea spp.), most of them larger than 50 µm. No fungal structures are observed, indicating that the inoculum has not yet begun to colonize the culture medium.These particles larger than 50 µm are characteristic of the initial algal matrix before the microbial consortium initiates its enzymatic activity. B) After 24 hours, the appearance of growing hyphae is observed, with diameters less than 50 µm, corresponding to the early stages of development of the microbial consortium (Pleurotus spp., Lentinula spp., and Paradendryphiella salina). The algal particles begin to fragment, with a progressive reduction in particles larger than 50 µm. This phenomenon suggests that the enzymes produced by the fungi are initiating the hydrolysis of complex carbohydrates, such as alginate, present in the algae. C) After 48 hours, microscopic analysis reveals significant growth of the hyphae, which now form a dense and well-developed network. The hyphae still have diameters smaller than 50 µm, but greater intertwining and coverage are observed in the visual field.In parallel, the algal particles larger than 50 µm have decreased considerably, with the majority fragmented or transformed into more soluble compounds. This growth reflects a high metabolic activity of the microbial consortium. D) At 110 hours, a maximum density of hyphae is observed in the visual field, forming a dense and homogeneous three-dimensional structure, with diameters less than 50 µm. Seaweed particles larger than 50 µm are practically nonexistent, confirming that the hydrolysis process is almost complete. This advanced state of fungal growth and degradation of the initial substrate indicates that fermentation has reached its final stage, and the mycelium has completely colonized the medium. DETAILED DESCRIPTION OF THE INVENTION Recent research has shown that filamentous fungi can be grown in submerged liquid cultures, using various substrates to grow and enhance their nutritional content.These fungi produce enzymes that degrade complex carbohydrates, such as cellulose and hemicellulose, which increases the nutritional value of substrates such as agricultural by-products or seaweed. In particular, marine fungi such as Paradendryphiella salina have the ability to degrade carbohydrates present in algae, improving their protein and bioactive compound content. However, submerged fermentation of seaweed with this fungus has limitations in efficiency and quality of the final product. The present invention proposes an innovative biotechnological method for the production of a new alternative protein, taking advantage of the nutritional and bioactive properties of seaweed, specifically brown seaweed. Using a microbial consortium comprising a mixture of a marine fungus Paradendryphiella salina and at least one terrestrial fungus, selected from Pleurotus spp., Lentinula spp., Ganoderma spp. or Trametes spp., or a mixture thereof, in a ratio ranging from 1:1 to 4:1, specifically designed for the fermentation of brown algae, achieving a unique product with high nutritional, prebiotic and bioactive value. Using submerged fermentation (SMF), the method optimizes the transformation of algal biomass, reducing the environmental impact and accelerating the production of high-quality proteins in a sustainable environment. This offers a viable and ecological alternative for the food industry, with applications in both food products and nutraceutical and pharmaceutical supplements. Specifically, there is a microbial consortium for use in the fermentation of brown seaweed or seaweed waste, comprising a mixture of the marine fungus Paradendryphiella salina and at least one terrestrial fungus in a ratio ranging from 1:1 to 4:1, specifically the ratio between the marine fungus Paradendryphiella salina and the at least one terrestrial fungus is 2:1.The terrestrial fungus is selected from Pleurotus spp., Lentinula spp., Ganoderma spp. or Trametes spp., or a mixture thereof. There is also a method for producing a protein from brown seaweed, which comprises (a) incorporating into a bioreactor a volume of brown seaweed in water that is at a concentration ranging from 20 to 50 g / L; (b) incorporating into the bioreactor a nitrogen source; (c) incorporating into the bioreactor an amount of microbial consortium until achieving a concentration between 10 - 30% v / v, wherein the microbial consortium comprises a mixture of marine fungus Paradendryphiella salina and at least one terrestrial fungus in a ratio ranging from 1:1 to 4:1; and (d) fermenting the mixture that is in the bioreactor at a temperature equal to or greater than 25°C, where fermentation is a submerged fermentation (FSm).In step (a) of the method for producing protein from brown seaweed, the volume of brown seaweed in water comprises: - an amount of dried and ground brown seaweed, wherein the brown seaweed is selected from Macrocystis pyrifera and Durvillaea spp.; and - an amount of at least one salt, wherein the at least one salt is selected from the group consisting of KH₂PO₄, MgSO₄·7H₂O and combinations thereof, and is in an amount of between 0.5 and 3 grams per liter. Finally, the entire volume of brown seaweed in water is sterilized, said sterilization being carried out in an autoclave at a temperature between 100°C and 170°C. The nitrogen source used in the method for producing protein from brown seaweed is selected from yeast extracts, ammonium salts, or gaseous ammonium. The yeast extracts, where Candida spp., Saccharomyces spp., Pichia spp., Zygosaccharomyces spp.Wherein the ammonium salts are selected from ammonium chloride, ammonium sulfate or a mixture thereof. Likewise, the nitrogen source is sterilized, wherein said sterilization is carried out in an autoclave at a temperature between 100 ° C and 170 ° C. The microbial consortium used in the method of producing protein from brown seaweed comes from the cultivation of a marine fungus Paradendryphiella salina and at least one terrestrial fungus in a ratio ranging from 1: 1 to 4: 1, in a culture medium having a concentration between 15 and 30 g / L. The cultivation of this microbial consortium can be carried out in a culture medium, preferably sterile potato dextrose (PDB), for between 4 to 8 days, preferably for 5 days at a temperature between 22 - 27 ° C, at 100 - 150 rpm and at a pH 5.5 - 6; The at least one terrestrial fungus of the microbial consortium must be selected from Pleurotus spp., Lentinula spp., Ganoderma spp. or Trametes spp., or a mixture thereof. Similarly, the microbial consortium is sterilized, where said sterilization is carried out in an autoclave at a temperature between 100°C and 170°C. The fermentation of the protein production method is carried out for between 10 and 200 hours, for which it is necessary to adjust the pH between 4.5 and 5, and maintain the temperature between 25 and 27 °C, with stirring at 100 rpm and dissolved oxygen at 25% w / v. Specifically, it can be fermented between 12 and 120 hours, preferably 72 hours. To adjust the pH between 4.5 and 5, it is possible to use citric acid, sodium citrate, or a mixture of both. Furthermore, once the product has been fermented, it is possible to separate the liquid phase from the solid phase, and the solid phase obtained, which corresponds to the mycoprotein or single-cell protein, can then be freeze-dried or vacuum-dried.Finally, we have a protein that comes from the fermentation of brown seaweed prepared by the method indicated above, which comprises: - between 25% and 40%, preferably 35% w / w, of protein by dry weight; - between 15-25%, preferably 20% w / w of beta-glucans by dry weight; and - between 30-40%, preferably 35% w / w of total fiber by dry weight. The invention provides several differentiating and innovative elements: i) Unique microbial consortium: It uses marine filamentous fungi (Ascomycetes) and terrestrial fungi (edible Basidiomycetes), which work cooperatively to assimilate complex carbohydrates present in seaweed. ii) Efficient bioconversion process: The method releases and concentrates the bioactive compounds of seaweed, such as proteins, lower molecular weight polysaccharides (oligosaccharides), proteins and polyphenols, which are integrated into the final product.This approach combines the best of fungi and seaweed, generating a completely new product. iii) Improved food product: A product with superior sensory characteristics (better taste, smell, texture, viscosity, and solubility) and a more complete functional profile is obtained, with antioxidant, prebiotic, and bioactive properties. This product can be integrated into various food matrices to enrich them with protein, fiber (β-glucans), and bioactive compounds. The term "alternative protein" in this invention refers to a food product that combines an enriched base material with the cultivated mycelium of filamentous fungi. During the process, a significant increase in the biomass and nutritional quality of the final product is achieved, reaching desirable levels of protein and bioactive biocompounds.In a preferred embodiment, the final product has a high protein concentration along with advanced functional properties, such as: • Improved antioxidant activity. • Prebiotic properties derived from oligosaccharides and β-glucans. • Texture and flavor suitable for applications in the food industry. The digestibility and PDCAAS (Protein Digestibility Corrected Amino Acid Score) of the alternative protein developed by this method represent a significant improvement compared to the brown algae Macrocystis pyrifera and Durvillaea spp. These differences are attributed to the innovative submerged fermentation process with consortia of filamentous fungi, which optimizes the release and transformation of the structural components of the algae, increasing the bioavailability of proteins and essential amino acids.In terms of digestibility, the alternative protein obtained from the microbial consortium presented higher values ​​(1.01 ± 0.06) compared to Macrocystis pyrifera (0.47 ± 0.06) and Durvillaea spp. (0.55 ± 0.04). This significant increase is due to the consortium's ability to break down complex carbohydrates from algae into more easily assimilable compounds, such as oligosaccharides, which also possess prebiotic properties. The process eliminates or minimizes structural barriers that normally limit the digestibility of seaweed in its natural form. Furthermore, the PDCAAS of the mycelial protein (0.89 ± 0.08) significantly exceeds that of Macrocystis pyrifera (0.41 ± 0.02) and Durvillaea spp. (0.53 ± 0.08), indicating better protein quality.iv) Increase in essential and non-essential amino acids: The fermentation process also results in a significant increase in essential amino acids such as valine, leucine, isoleucine, phenylalanine, and methionine, which are crucial for human nutrition. These amino acids, which are limiting in many plant sources, are now present in higher concentrations due to the action of fungi in the bioconversion process. Furthermore, non-essential amino acids, such as glutamate, asparagine, and alanine, are responsible for the umami flavor and the improvement of organoleptic properties, such as taste and texture. Furthermore, the alternative protein offers versatility in terms of formulation, and can be adjusted to highlight or soften sensory characteristics according to market needs. For example, it is possible to enhance umami and salty flavors or reduce bitter and astringent flavors, adapting it to different food applications.In summary, this invention combines the efficiency of a sustainable biotechnological process with the generation of an innovative and highly functional food product that integrates the best properties of filamentous fungi and seaweed. The proposed method, the microbial consortium, and the resulting product have the potential to transform the food and nutraceutical industries by offering a sustainable, nutritious, and versatile solution to the growing global demand for alternative proteins. APPLICATION EXAMPLES EXAMPLE 1. Fungal inocula were prepared, and six 250-mL Erlenmeyer flasks were filled with 100 mL of a medium consisting of 25 g / L of potato dextrose (PBD). The flasks were wrapped with sterilizable overwrap and autoclaved (the sealed overwrap should make it easy to remove and place on the flask without losing shape). The flasks were autoclaved at 120 °C for 15 minutes.The flasks are placed in a HEPA laminar flow hood, previously sanitized with 70% ethanol and a 15-minute UV radiation treatment. The flasks are placed until the medium cools and is at room temperature. Subsequently, each flask is inoculated with 2 cm discs of Petri dish cultures of Paradendryphiella salina fungi, and consortia of Pleurotus spp., and Lentinula spp. Each consortium or monoculture in three triplicate flasks (6 flasks total). These plate cultures must be between 5 and 7 days old. The inoculated flasks are placed in a shaker or shaking incubator at 100 rpm and 25 °C, for 7 days. A 2-liter bioreactor was filled with 1 liter of medium consisting of 30 g / L of Durvillaea spp., 1 gram of KH2PO4, and 0.5 grams of MgSO47H2O. Any open ports in the bioreactor were wrapped with aluminum foil and sterilized in an autoclave at 120°C for 30 minutes.The bioreactor was transferred to a clean, previously sanitized bench, installed, and cooled for 12 hours. The bioreactor was inoculated with 100 mL of the inoculum from each consortium and from the previously grown monoculture. The bioreactor had a 1 vvm air supply and was maintained at 25°C. 20 uL / L of antifoam was added. After 48 hours, the culture pH rises approximately 2 points; this is because the fungi are hydrolyzing the alginate (from the seaweed) and consuming the product of this hydrolysis. Samples are taken every 24 hours for microscopic observations of the culture, and a microbiological analysis is performed through total coliform counts to ensure that the cultures are not contaminated and to verify the presence and growth of mycelium. Finally, the liquid medium is separated from the solid portion, or mycoprotein, using vacuum filtration. Up to 15 g / L dry weight of alternative protein is obtained.The alternative protein is dried through freeze-drying and this product can be used as a food ingredient. The product has approximately 35% protein (dry weight), 20% beta-glucans, 35% total fiber, and a higher concentration of total phenolic compounds as seen in Table 1. The table presented shows the comparison of total phenolic compounds and antioxidant activity between the alternative protein batches obtained through fermentation duplicates and the initial substrate (Durvillaea spp.), used as a reference brown seaweed. Batches 1 and 2, which represent duplicates of the fermentation process, present a significant increase in total phenolic compounds compared to Durvillaea spp. Although the antioxidant activity of Durvillaea spp. is moderately high, Batch 2 shows a significant improvement.This suggests that, in addition to releasing phenolic compounds, the fermentation process may generate new antioxidant metabolites that contribute to the increased antioxidant activity in the final products. The final product was observed to have a mild umami aroma, distinct from the taste and smell of seaweed. Table 1 Samples Total phenolic compounds (mg GAE / g) Antioxidant activity (µmol TE / g) L. ote 1 1,05 21,5 L ote 2 1,32 25,5 D urvillaea spp. 0,83 22,7EXAMPLE 2 A 5-L bioreactor was filled with 3 L of a growth medium consisting of 30 g / L of Durvillaea spp. (dry weight), 1 g / L of KH₂PO₄, 0.5 g / L of MgSO₄ 7H₂O, and 20 μL / L of antifoam. The medium was autoclaved at 120 °C for 30 minutes. The bioreactor was subsequently transferred to a clean benchtop previously sanitized with 70% ethanol and allowed to cool for 12 hours. Once at room temperature, the bioreactor was inoculated with 300 mL of inoculum previously prepared as in Example 1, which included consortia of Pleurotus spp., Lentinula spp., and Paradendryphiella salina. The culture was maintained at 25–27°C with an air supply of 1 vvm and shaking at 50 rpm for 48 hours. Thereafter, to promote mycelial growth and alginate hydrolysis, shaking was increased to 100 rpm and the air supply to 1.5 vvm.These modifications optimized oxygen transfer, favoring the metabolic activity of the microbial consortium. Daily microscopic monitoring and microbiological analyses were performed to ensure culture purity and the absence of bacterial contamination. The pH of the culture increased approximately 2 points due to the hydrolysis of the seaweed alginate by enzymes secreted by the fungi. The culture was harvested after 96 hours by vacuum filtration to separate the liquid medium from the solid mycelium. A mycoprotein was obtained with a yield of 14–15 g / L dry weight. This product was dried by lyophilization, resulting in an alternative protein with approximately 33–36% protein by dry weight. The nutritional characteristics of the final product (batches 1 and 2 are duplicates of the fermentation process) are shown in Table 1 and Table 2.Compared to the results obtained for the alternative protein, Durvillaea spp. presents inferior nutritional characteristics. In terms of total protein, Durvillaea spp. reaches only 10.79 mg / g in dry weight. Furthermore, the amino acid profile shows that the sum of essential amino acids in Durvillaea spp. is 58.04 mg / g, while the sum of nonessential amino acids amounts to 77.04 mg / g. These differences reflect the limitations of Durvillaea spp. as a protein source, highlighting the need for innovative processes such as fermentation with microbial consortia to improve its nutritional and functional quality. Furthermore, Durvillaea spp. contains a high ash content (26.06%), reflecting a higher presence of salts and minerals, which are significantly reduced in the alternative protein as a result of the fermentation process.This indicates that bioconversion not only improves the protein and amino acid profile, but also decreases the levels of inorganic compounds, making the product more suitable for food and functional applications TABLE 2 Nutritional composition (% dry weight) Moisture Protein Total carbohydrates Fatty acids Batch 1 12.3 10.2 36.5 38.2 3.2 Batch 2 13.5 11.2 33.8 39.1 2.2 TABLE 3 Amino acids. (mg / g of product on a dry weight basis) 2 Histidine 10.98 9.57 Threonine + Arginine 36.91 33.27 Tyrosine 11.37 11.97 Essential amino acids Alanine 21.26 15.20 Methionine 13.02 14.80 Valine 16.83 14.88 Phenylalanine 14.66 14.06 Leucine 20.92 20.20 Isoleucine 19.91 19.00 Total Sum Essential Amino Acids 165.09 152.99 Serine 17.62 13.67 Aspartic acid 34.65 25.26 Non-essential amino acids Glutamic acid 15.54 13.65 Glycine 12.45 9.30 Total Essential and non-essential amino acids 246.10 214.90 EXAMPLE 3 A 20-L bioreactor was filled with 12 L of a culture medium consisting of 35 g / L of Durvillaea spp. (dry weight), 1.5 g / L of KH₂PO₄, 0.8 g / L of MgSO₄ 7H₂O, and 30 μL / L of antifoam. The medium was autoclaved at 121 °C for 1 hour. Subsequently, the bioreactor was transferred to a clean bench previously sanitized with 70% ethanol and allowed to cool for 10 hours.Once at room temperature, the bioreactor was inoculated with 1.2 liters of previously prepared inoculum, following the method described in Example 1, which included consortia of Pleurotus spp., Lentinula spp., and Paradendryphiella salina. The culture was maintained at a temperature of 25–27 °C with an air supply of 0.5 vvm and an initial agitation of 50 rpm for the first 48 hours. From this point on, to promote mycelial growth and efficient alginate hydrolysis, agitation conditions were increased to 70 rpm and the air supply to 1 vvm. These modifications promoted greater oxygen transfer, optimizing the metabolic activity of the microbial consortium. Monitoring was carried out consisting of filtration analysis (to detect the decrease in the concentration of alginate present in Durvillaea spp) and microscopic analysis of the medium and the fermentation products at 24 hours, 48 ​​hours, and 110 hours.See Figures 2 and 3, and microbiological analysis to verify the purity of the culture and ensure the absence of bacterial contamination. The pH at the beginning of fermentation was 5.5, and after 110 hours of fermentation, the pH of the culture increased approximately 2.5 points, evidencing the hydrolysis of alginate from the seaweed and its consumption by the fungi in the consortium. The culture was harvested at 110 hours by vacuum filtration to separate the liquid medium from the solid mycelium. Table 4 summarizes the results of amino acid and PDCAAS digestibility, compared to Durvillaea spp. TABLE 4 Digestibility of Samples PDCAAS amino acids In Vitro Alternative protein 1.01 0.89 Durvillaea spp. 0.55 0.53 In conclusion, a significant decrease in alginate concentration was observed during the fermentation process, supported by microscopic observations and the efficiency of the filtration process.This finding is consistent with the action of the microbial consortium, whose enzymatic activity broke down the complex carbohydrates present in the seaweed, generating a more accessible substrate for mycelial growth and protein production. Furthermore, this decrease in alginate is directly associated with the improved digestibility of the final product. This increase highlights how the bioconversion performed not only transforms the original algal matrix but also increases the bioavailability of nutrients such as amino acids, as evidenced by the PDCAAS of the alternative protein. These results confirm that the reduction in alginate concentration, combined with the optimized fermentation process, not only improves the nutritional properties of the product but also establishes an efficient and sustainable approach for the production of alternative proteins with potential applications in the food industry. EXAMPLE 4.To evaluate the effectiveness of the Durvillaea spp. bioconversion process mediated by the microbial consortium, composed of filamentous fungi of the genera Pleurotus, Lentinula, Hericium, Ganoderma, and Paradendryphiella, a comprehensive analysis of the content of β-glucans and ergosterol, two bioactive compounds of particular interest, in the resulting mycelial biomass was performed. This biomass was obtained by submerged fermentation in a controlled bioreactor, where the culture medium contained Durvillaea spp. as the sole carbon source. The process was carried out under constant stirring and aeration conditions to promote a dense and highly dispersed suspension of mycelial biomass, which was subsequently harvested, washed, filtered, freeze-dried, and ground into a fine flour.The quantification of β-glucans was performed using a rigorous analytical procedure that began with the treatment of the ground samples with 12M sulfuric acid at 4°C for 2 hours to solubilize the glucans, followed by additional hydrolysis in 2M sulfuric acid at 100°C for 2 hours. The specific determination was carried out using the Megazyme Yeast and Mushroom (K-YBGL) kit, which employs a mixture of exo-1,3-β-glucanase and β-glucosidase to completely hydrolyze the residual glucan fragments. The final quantification was performed using the glucose oxidase-peroxidase reagent (GOPOD), calculating the β-glucan content by difference after determining the contribution of α-glucans.In parallel, ergosterol content was analyzed by hexane extraction (1:60 w / v) and subsequent high-performance liquid chromatography (HPLC) analysis using a reversed-phase C18 Onyx monolithic column and photodiode array detection. Quantification was performed using a calibration curve in the range of 0.1–0.3 mg / mL of ergosterol standard. The results revealed a significant increase in the β-glucan content in the bioconversion products, reaching values ​​of 22.79 ± 0.87 g / 100 g dry weight in mycelial biomass, compared to 10.73 ± 2.2 g / 100 g dry weight in Durvillaea spp. , representing an increase of up to 112%. Regarding ergosterol, levels of 1.20 ± 0.16 mg / g were detected in the mycelial biomass, while none were detected in the original algal biomass, confirming its fungal origin.These results demonstrate the effectiveness of the bioconversion process in generating products with an improved nutraceutical profile, demonstrating the ability of the selected fungi to transform algal biomass into products with greater functional value.

Claims

1. A microbial consortium for use in the fermentation of brown seaweed or seaweed waste, comprising a mixture of the marine fungus Paradendryphiella salina and at least one terrestrial fungus in a ratio ranging from 1:1 to 4:

1.

2. The microbial consortium according to claim 1, wherein the terrestrial fungus is selected from Pleurotus spp., Lentinula spp., Ganoderma spp. or Trametes spp., or a mixture thereof.

3. The microbial consortium according to claim 1, wherein the ratio between the marine fungus Paradendryphiella salina and at least one terrestrial fungus is 2:

1. 4.A method for producing a protein from brown seaweed, comprising (e) incorporating into a bioreactor a volume of brown seaweed in water at a concentration ranging from 20 to 50 g / L; (f) incorporating into the bioreactor a nitrogen source; (g) incorporating into the bioreactor a quantity of microbial consortium until a concentration of between 10-30% v / v is achieved, wherein the microbial consortium comprises a mixture of the marine fungus Paradendryphiella salina and at least one terrestrial fungus in a ratio ranging from 1:1 to 4:1; (h) fermenting the mixture in the bioreactor at a temperature equal to or greater than 25°C.

5. The method for producing a protein according to claim 4, wherein the volume of brown seaweed in water comprises: a quantity of dried and ground brown seaweed; and a quantity of at least one salt. 6.The method of preparing a protein according to claim 5, CHARACTERIZED in that the brown seaweed is selected from Macrocystis pyrifera and Durvillaea spp.

7. The method of manufacturing a protein according to claim 5, characterized in that at least one salt is selected from the group consisting of KH₂PO₄, MgSO₄ 7H₂O, and combinations thereof.

8. The method of manufacturing a protein according to claim 7, characterized in that at least one salt is present in an amount of between 0.5 and 3 grams per liter.

9. The method of manufacturing a protein according to claims 5 to 8, characterized in that the volume of brown seaweed in water is sterilized.

10. The method of manufacturing a protein according to claim 4, characterized in that the nitrogen source comprises is selected from yeast extracts, ammonium salts, or gaseous ammonium.

11. The method of manufacturing a protein according to claim 10, characterized in that the nitrogen source is sterilized. 12.

13. The method for producing a protein according to claim 4, CHARACTERIZED in that the microbial consortium is obtained from the cultivation of the marine fungus Paradendryphiella salina and at least one terrestrial fungus in a ratio ranging from 1:1 to 4:1, in a culture medium having a concentration of between 15 and 30 g / L.

14. The method for producing a protein according to claim 12, CHARACTERIZED in that the at least one terrestrial fungus of the microbial consortium is selected from Pleurotus spp., Lentinula spp., Ganoderma spp. or Trametes spp., or a mixture thereof.

14. The method for producing a protein according to claims 12 and 13, CHARACTERIZED in that the microbial consortium is sterilized.

15. The method of preparing a protein according to any of claims 4 to 14, CHARACTERIZED in that the sterilization is carried out in an autoclave at a temperature between 100°C and 170°C. 16.The method of preparing a protein according to claim 4 CHARACTERIZED in that the fermentation is a submerged fermentation (FSm).

17. An alternative protein prepared by the method claimed in claim 4, CHARACTERIZED in that it comprises between 30% to 40% protein, between 15% to 25% beta-glucans and between 30% to 40% total fiber.

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