Method of production of biofuel (bioethanol) and probiotic microbial / cellular biomass with dairy raw materials and saccharomyces boulardii yeast

Abstract

“METHOD FOR PRODUCTION OF BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii” The present invention is intended for the production of biofuel (bioethanol) and probiotic microbial / cellular biomass with dairy raw materials, whether residual, by-products and / or co-products of the processing of dairy products and protein concentrates, commonly known as “whey”, milk whey, sweet whey, acid whey or even lactose whey, and when already free of the protein fraction, called “whey permeate, deproteinized whey, deproteinized acid whey, deproteinized sweet whey, deproteinized milk whey, and / or cheese whey permeate, together with the probiotic microorganism Saccharomyces boulardii, with the purpose of using the raw material in full, adding commercial value to the waste, developing new products for application in the most diverse areas (biofuels,food, supplements, among others), which consequently generates increased capital for industries, new industries, new jobs, savings in the cost of effluent treatment, implying a reduction in environmental effects caused by the incorrect disposal of dairy waste, and thus this invention patent has the precept of supporting eco-development.,

Description

/ 22 METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii Field of invention:

[001] This patent operates in the field of invention in the area of ​​biotechnology / fermentation processes and aims at the production of bioethanol biofuel and probiotic microbial / cellular biomass with the microorganism Saccharomyces cerevisiae Meyen ex EC Hansen var. cerevisiae (S. cerevisiae) Saccharomyces cerevisiae var. boulardii, or commonly referred to simply as Saccharomyces boulardii, in a reaction medium composed of dairy raw materials, whether these are residual, by-products and / or co-products of dairy processing and protein concentrates, commonly known as "whey", milk whey, sweet whey, acid whey or even whey, and when already free of the protein fraction, called "whey permeate", "deproteinized whey", "deproteinized acid whey", "deproteinized sweet whey", "deproteinized milk whey", and / or "cheese whey permeate".For the metabolic reactions of microorganisms to develop, it is necessary to carry out the hydrolysis of the lactose molecule into its monosaccharides glucose and galactose. The products generated by this inventive process include applications in biofuels, in this case bioethanol; food, acting as a protein source for human, animal and aquaculture consumption; functional products, salt substitute; condiment, source of vitamins and amino acids; supplements, with a considerable content of bioactive peptides; probiotic supplement; pharmaceutical and / or nutraceutical products; among other products derived from the cellular constitution of yeast or its metabolism. Any product described herein or derived from the methods disclosed herein may be used as is, or as a precursor or intermediate in the production of other products. Fundamentals of the invention and state of the art:

[002] The global volume of milk produced in the world is increasing and, according to the United States Department of Agriculture (USDA), is currently approximately 598.53 billion liters of bovine milk. Brazil is among the countries with high production, around 34.3 billion liters of milk. Approximately 32% (7.4 billion liters of milk) is destined for the cheese production industry, which produces with Petition 870250112683, dated 08 / 12 / 2025, page 8 / 47 / 22 this amount is equivalent to 770 thousand tons of cheese per year. Another 12.10% (2.8 billion liters of milk) are used for the production of casein, yogurts and fermented milks (USDA, 2017).

[003] “Whey”, cheese whey, lactosoro, sour or sweet cheese whey, is the liquid residue resulting from the production of cheese, casein and yogurt. Whey accounts for 80% to 90% of the total volume of milk initially used in the process, and retains on average 55% of the milk's nutrients. It consists of lactose (4.5% to 5% w / v), soluble proteins (0.6-0.8% w / v), lipids (0.4-0.5% w / v), vitamins (B complex and others), minerals (8-10% dry extract), lactic acid (0.05% w / v) and citric acid, non-protein nitrogenous compounds (urea and uric acid), bioactive compounds such as β-lactoglobulin, α-lactoglobulin, immunoglobulins, serum albumin and lactoferrin (CASAL et al., 2006; SISO, 1996).

[004] World whey production currently ranges from 160-180 million tons, with an estimated growth rate of 1 to 2% per year. Approximately 1.5 million tons of this amount are high biological value protein constituents, which drive a growing market for the production of protein concentrates, protein isolates, protein hydrolysates, among other products. Lactose, a carbohydrate present in whey, accounts for approximately 8.6 million tons of the total whey produced. The market for lactose application is more restricted than that for protein, due to the large number of people intolerant to this constituent (KHEZRI et al., 2016). The remainder of the total whey production consists of water (93-94%), lipids (0.05%), minerals (0.5%), the main ones being phosphates, citrates, chlorides, sulfates, carbonates, sodium bicarbonates, potassium, calcium and magnesium, and vitamins (BYLUND, 2003).

[005] The production of the liquid “whey permeate”, cheese whey permeate, deproteinized whey, deproteinized acid whey, deproteinized sweet whey, or deproteinized milk whey, occurs during the passage of milk or “whey” through an ultrafiltration membrane filtration system, used for the production of ultrafiltered cheese, concentrates, isolates, and protein hydrolysates. During the ultrafiltration process, the protein is retained in the membrane, and the liquid that permeates (passes) through the membrane originates the “whey permeate”. The volume of permeate generated is Petition 870250112683, dated 08 / 12 / 2025, page 9 / 47 / 22 practically the same as "whey", except for the protein content present in whey. Therefore, it is equivalent to an annual production of "whey permeate" of approximately 170 million liters.

[006] “Whey” and “whey permeate,” despite not containing non-biodegradable toxic compounds, are considered extremely polluting food industry waste, with high Biochemical Oxygen Demand (BOD) rates > 35,000 ppm and Chemical Oxygen Demand (COD) rates > 60,000 ppm (SISO, 1996; SMITHERS, 2008). Tunick (2008) estimated that 4000 L of whey could cause environmental damage equivalent to that caused by fecal waste produced by 1900 humans. The dumping of whey or permeate on the soil creates severe pollution concerns for the environment, as it affects physicochemical characteristics, resulting in decreased crop productivity. If the discharge occurs in bodies of water, the major consequence is the reduction of dissolved oxygen, thus hindering biodegradation, which represents a great risk to aquatic life, the environment, and also to human health (GHALY, AE; MAHMOUD, NS; RUSHTON, DG; ARAB, F.(2007). Potential environmental and health impacts of high land application of cheese whey. American Journal of Agricultural and Biological Science.v. 2, n.2, p.106-117.).

[007] Since lactose is the largest fraction of solids present in both whey and whey permeate, several biotechnological processes have been applied to produce industrially important and valuable products from these two liquid process residues (PRAZERES et al., 2012). Whey and whey permeate have been used as fermentation substrates for the production of biological products, since the heterogeneous mixture of lactose, residual proteins and lipids, vitamins, minerals, etc., is an excellent source of nutrients for the growth of microorganisms. Some of the products derived from the fermentation of whey and whey permeate include bioethanol (SISO, 1996) and single-cell protein (BABU et al., 2014).

[008] The fermentation of “whey” and “whey permeate” for the production of bioethanol biofuel has proven to be a lucrative option, as it is a beneficial process for the reuse and bioremediation of these highly processed industrial byproducts. Petition 870250112683, dated 08 / 12 / 2025, page 10 / 47 / 22 pollutants, as it implies a reduction in costs incurred due to the need for complex and cost-intensive effluent treatment processes required for disposal.

[009] Furthermore, there are driving factors in the global economy that make it necessary to increase the production of biofuel (bioethanol), one of which refers to the global warning about the future scarcity of fossil fuels, which has been estimated to last a maximum of 40 to 50 years. Linked to this energy deficiency of fossil fuels, there is also the appeal from governmental institutions regarding the reduction of environmental pollution and global warming, factors that have driven the market in the search for non-polluting fuels, such as bioethanol.Given this, the conversion of "whey" and "whey permeate" into bioethanol is desirable, since the raw material to be used is secondary, or even residual from the dairy industry processing, is generated in high volume, and does not compromise the main production of dairy products, and can be directed to the production of biofuel on a large scale, thus aligning with the aforementioned objective of increasing biofuel production without compromising the environment, and using the raw material in its entirety, i.e., zero waste emissions.

[010] The growing global demand for food and dietary protein, particularly in underdeveloped countries, has stimulated the search for non-conventional protein sources in recent years, whether for human consumption or animal feed. This is due to official projections regarding the population expected to reach around 9.8 billion individuals in 2050 (UN DESA, 2017), which consequently necessitates a 60% increase in the energy efficiency of food compared to current levels to meet the demand of this new population (UNBR, 2018).

[011] One of the unconventional sources of protein is of microbial origin. The term single cell protein (SCP) emerged in the 1960s to specify microbial biomass produced by fermentation, and according to the Massachusetts Institute of Technology, it refers to the dried cells of edible unicellular microorganisms such as yeasts, fungi, bacteria, and microalgae cultivated in large-scale culture systems for use as protein sources in food for humans or animal feed (SCRIMSHAW, Petition 870250112683, dated 08 / 12 / 2025, page 11 / 47 / 22 1968).

[012] Yeast SCP production technologies have emerged as a promising way to solve the problem of global protein scarcity (ASHY et al., 1982). And over the years they have evolved into bioconversion processes of low-value by-products (waste) into products with high nutritional value and market price (UGALDE & CASTRILLO, 2002).

[013] Other terms such as “microbial biomass”, “cellular biomass” and “microbial biomass protein” are currently used to refer to proteins or protein concentrates obtained from microbial cells.

[014] Microbial protein can be consumed directly, i.e., intact cell, particularly in animal feed formulations, or it can be lysed, and its internal and external parts extracted and processed, and can be used as an ingredient or a substitute for protein-rich foods, suitable for both human consumption or as animal feed, and / or in the form of supplements, additives (flavor enhancers) and agents that provide texture (UGALDE & CASTRILLO, 2002).

[015] Microbial biomass production has become desirable due to the rapid growth of microorganisms, i.e., extraordinary reproductive capacity in a short period of time, for example, algae: 2-6 hours, yeasts 1-3 hours, bacteria 0.5-2 hours; the possibility of using a diverse number of substrates and foods not consumed by humans, carrying out recycling and contributing to the preservation of the environment; high protein content, reaching 30 to 70% of the desiccated microbial content; the amino acid profile of many SCP microorganisms generally has excellent nutritional quality, comparable to a chicken egg; continuity of the microbial protein production process independently; production independent of seasonal and climatic variations, and can be easily protected from extreme weather events; they are independent of light, growing 24 hours a day;In addition to the possibility of controlling nutritional composition through genetic and environmental manipulations (ARAÚJO et al., 2009; NASSERI et al., 2011).;

[016] Developing countries, such as Brazil, are favorable for the production of microbial biomass, as they are rich in agro-industrial waste, such as Petition 870250112683, dated 08 / 12 / 2025, page 12 / 47 / 22 of dairy processing waste, “whey” and “whey permeate”, which, when used in the cultivation of microorganisms, can be a viable alternative for bioconversion into protein-rich microbial biomass, reducing around 90-95% of its constituents in terms of biochemical oxygen demand (BOD) (GRUBB & MAWSON, 1988), resulting in high value-added bio-ingredients for the food industry (BELEM et al., 1997).

[017] Yeasts are classified according to the processes involved in sugar metabolism and energy generation, and can be called aerobic microorganisms when their metabolism is exclusively respiratory, or facultative when they metabolize glucose through fermentation. Many authors classify yeasts as facultative aerobes because, depending on the conditions imposed for their growth, the availability of oxygen for their metabolism, and the type and concentration of sugars present in the reaction medium, they may exhibit both respiratory and fermentative metabolism. Yeasts that exhibit the Crabtree effect, that is, those that perform alcoholic fermentation under aerobic conditions, are called Crabtree-positive, resulting in lower biomass production, since the greater amount of fermentable sugars is converted into ethanol. Crabtree-negative yeasts, on the other hand, produce more biomass from the glucose available in the medium.Yeasts of the genus Saccharomyces that are Crabtree positive perform respiration under aerobic conditions, but if a certain amount of glucose is added to the medium, the yeast automatically reverses its metabolism and begins to perform alcoholic fermentation, and therefore are called short-term Crabtree, as is the case of the yeast that was used in this invention patent (DASHKO et al., 2014).

[018] Yeasts are of fundamental importance to the biofuel production industry, the scientific community, the production of food supplements, medicine, and also in agriculture. Since ancient times, yeasts have been used in the production of beverages and food. During the First and Second World Wars, yeasts such as Cyberlindnera jadinnii, formerly known as Candida utilis, were reported as food, that is, a direct source of protein that was distributed to soldiers, civilians, and even prisoners, replacing animal protein. In more recent years Petition 870250112683, dated 08 / 12 / 2025, page 13 / 47 / 22 yeasts are being used as additives in PetFoods and animal feed in general, when used for the purpose of human food the term from the English single-cell protein, or from the Portuguese proteina unicelular is used to denote the use of yeasts (UGALDE & CASTRILLO, 2002).

[019] Over the last few decades, yeasts have been used in the production of bioethanol, as hosts for heterologous expression of proteins of interest, in the production of enzymes, and as a model for the biological study of eukaryotic organisms (CADETE et al., 2014). Yeasts are also used as probiotics.The term defined by Guarner and Schaafsma in 1988 (GUARNER & SCHAAFSMA, 1998), accepted by the FAO / WHO in 2002 and currently used to define probiotics is, "live microorganisms that, when consumed in adequate amounts, confer a health effect on the host," that is, in its broadest sense, probiotic organisms are those that, after passing through the digestive system to the large intestine, are able to remain active, that is, they resist the enzymes that act in the gastrointestinal tract, and while the transient process is triggered by the large intestine, they manage to colonize it, being able to adhere to the intestinal mucosa, characteristics that confer health benefits to the host (NAGPAL et al., 2012).

[020] The literature by Kechagia et al. (2013) entitled Health Benefits of Probiotics: A Review, reports benefits such as modulation of the immune system, protection against pathogenic organisms, improvement in the digestive system, rebalancing it in cases of diarrhea caused by foodborne infections, or even by the excessive use of antibiotics.

[021] Saccharomyces boulardii was discovered by the French microbiologist Henri Boulard in 1920, during a visit to Indochina in search of new yeast strains that could be used in fermentation processes. During his stay in the country, a cholera outbreak occurred, and the researcher noticed that some people who drank a special tea did not develop cholera. This tea was made by boiling lychee and mangosteen peel. Henri Boulard then decided to isolate the agent responsible for this effect, which is when he discovered a special variety of yeast, which he named "Saccharomyces boulardii". The patent for this yeast was then purchased by Biocodex Laboratories in 1947, which began researching and marketing this microorganism as a supplement. Petition 870250112683, dated 08 / 12 / 2025, page 14 / 47 / 22 dietary (McFARLAND, 2010).

[022] The yeast S. boulardii is classified as non-pathogenic, meaning it is safe for use in food as it does not present a risk of toxicity. Saccharomyces cerevisiae and Saccharomyces cerevisiae var boulardii are the only yeast strains marketed for human use (CZERUCKA et al., 2010). Saccharomyces boulardii is currently found on the market in the form of dietary supplements in capsules or sachets.

[023] Studies by McFarland et al. (1995) and Guslandi et al. (2000) showed its effectiveness in the prevention and / or treatment of intestinal disorders, including antimicrobial-associated diarrhea, recurrent disease caused by the bacterium Clostridium difficile, acute diarrhea in adults and children induced by a variety of enteric pathogens, traveler's diarrhea, and recurrent cases of Crohn's disease or ulcerative colitis.

[024] Yeasts of the genus S. cerevisiae have the status “Generally Recognized As Safe” (GRAS) by the Food and Drug Administration (FDA, USA), and by the European Union where five strains of S. cerevisiae are considered probiotics (NCYC SC 47, NCYC 1026, CNCM I-1077, CNCM I-1079 and MUCL 39885) for applications in animal feed (BÜCHL et al., 2010).

[025] The document that considers Saccharomyces boulardii as a safe biological product is under FDA numbering, Docket No. FDA-2004-N-0063, Document Number: E9-25803 published in Federal Register / Vol. 74, No. 206 / Tuesday, October 27, 2009 / Notices. The strain used in this invention is marketed under the name Saccharomyces cerevisiae Meyen ex EC Hansen ATCC® MYA-796™ CCT 4308 and / or Saccharomyces boulardii Reference UFPEDA 1176 from the Tropical Cultures Collection of the André Tosello Foundation.

[026] In terms of biosafety, Saccharomyces boulardii is classified as a microorganism belonging to risk group 1 (BSL-1), that is, it is characterized as an organism that does not cause disease in healthy adult humans by the NIH Guidelines for Research Involving Recombinant DNA Molecules (2002), or a microorganism that does not cause disease in humans or animals (with no or little individual and community risk) by the World Health Organization Laboratory Biosafety Manual 3rd Edition, 2004; both definitions are described in the document Biosafety in Petition 870250112683, dated 08 / 12 / 2025, page 15 / 47 / 22 Microbiological and Biomedical Laboratories (BMBL) (2009). 5thEdition of the US Public Health Service Guidelines.

[027] The advantage of using Saccharomyces boulardii is due to the fact that it is not sensitive to antibacterials (BUZZINO & VAUGHAN-MARTINI, 2006).

[028] From a biotechnology perspective, fermentation processes involving yeasts stand out for their versatility in the use of industrial waste, i.e., renewable raw materials for obtaining new products.

[029] In 2008 and 2009, Sachs published in his books the emergence and definition of the term sustainable development, which was conceived at the Stockholm Conference in 1972, being defined as that in which objectives greater than the mere multiplication of wealth act, that is, it has a social purpose as its main objective, permeated by respect for environmental conditions and, finally, based on solutions that are economically viable. Over the years, this 1972 definition has evolved in its understanding, and is currently based on 7 dimensions of sustainability: social, cultural, ecological, environmental, territorial, economic and political, conferring a systemic concept of eco-development.

[030] It is observed that the world's consumption of renewable natural resources currently exceeds the planet's carrying capacity. Thus, producing a biofuel from a renewable source and also producing a co-product with probiotic potential (microbial / cellular biomass) that can be applied in food, supplements, medicines, pharmaceuticals, among others, offers society and companies a strategic vision that includes the sustainable development of the planet. In addition, production using dairy industrial waste directly impacts the reduction of environmental problems (inadequate disposal of waste) and economic problems (reduction in biofuel costs, generation of new industries, employment, new product).

[031] During the prior art search, it was found that there are ethanol productions with various substrates and yeasts, such as US patent 2013 / 008416 A1 entitled “Method for producing ethanol and yeast protein feed from whey permeate”, which aims at the production of ethanol and biomass with whey permeate, with different amounts of Kluyveromyces marxianus or different strains of yeast culture, this patent is dated from Petition 870250112683, dated 08 / 12 / 2025, page 16 / 47 / 22 2013. Meanwhile, patent PI 0903048-4 A2, entitled "Ethanol production with whey / cheese," aims at producing ethanol from whey or cheese with different yeast species (Saccharomyces, Rodotorula, Pichia, Schizocaccharomyces, Zygossacharomyces, Hansenula), and its process was patented in Brazil in 2009. Or, patent WO2014184189A3, entitled "Process for simultaneous saccharification and fermentation of whey permeate," which invents a process for producing ethanol from substrates containing lactose, where the medium is simultaneously saccharified, i.e., the lactose molecule is hydrolyzed into its monosaccharides to produce ethanol at a pH between 3.5 and 5.5, using microorganisms for fermentation of the Saccharomyces sp. type.

[032] In the literature, the abstract presented at the 2012 AIChE Annual Meeting by MALEK, RA; EL SAYED, S.; EL BADRY, I.; OTHMAN, NZ; SARMIDI, MR; AZIZ, RA; EL ENSHASY, HA was found, entitled Semi-Industrial Production of Probiotic / Biotherapeutic Yeast Saccharomyces Boulardii in High Cell Density Culture Using Different Cultivation Strategies, in which the authors developed cultures in a fed-batch system with a reactor volume of 15 liters, with a semi-defined medium (basal medium) and feeding with a solution composed of glucose, amino acids and vitamins, obtaining in its best condition a maximum production of cellular biomass in dry weight of 80 gL-1e without the significant presence of ethanol in the culture.

[033] The article entitled Bioprocess optimization for biomass production of probiotic yeast Saccharomyces boulardii in semi-industrial scale by CHIN, TS; OTHMAN, NZ; MALEK, RA; ELMARZUGI, Na.; LENG, OM; RAMLI, S.; MUSA, NFF; AZIZ, R.; El ENSHASY, H., published in Journal of Chemical and Pharmaceutical Research, 2015, v. 7, n.3, p.122-132, presents assays aimed at improving the cell growth kinetics of the yeast Saccharomyces boulardii for the production of biotherapeutic cell biomass on a semi-industrial scale. In this study, the optimized medium was composed of: 20 g L-1 of Glucose, 15 g L-1 of corn liquor, 1 g L-1 of NaNÜ3, 6 g L-1 of KH2PO4, 3 g L-1 of MgSO4.7H2O; 0.002 g L-1 of CuSO4.5H2O; 0.001 g L-1 of FeSO4.7H2O and 0.01 g L-1 of ZnSO4.7H2O, and was carried out in a 16-liter bioreactor with and without controlled pH. The highest biomass production in this study was 8.2 g L-1 under the pH-controlled cultivation condition. Petition 870250112683, dated 08 / 12 / 2025, p. 17 / 47 / 22

[034] The article entitled Statistical Optimization of Critical Components for High Cell Mass Production of Saccharomyces Boulardii: A Potential Probiotic Yeast, authored by OTHMAN, NZ; ISHAK, A. .; ABD MALEK, R.; RAMLI, S.; AZIZ, RA; EL ENSHASY, H. (2014) presented at the 2014 AIChE Annual Meeting, aimed at high cell biomass production of S. boulardii under optimal growth conditions. To this end, the authors grew the yeast in different culture media in order to find those that best presented growth conditions for the S. boulardii strain. Optimization was performed by varying three independent parameters (glucose, meat extract, and NaNO3). The optimal culture performed in a shaken flask consisted of 40 g L-1 of glucose; 4.94 g L-1 of meat extract and 1.38 g L-1 of NaNO3, and had a production of 11.19 g L-1 of cell mass, implying an increase of 167.06% when compared to the non-optimized medium.The optimized culture was then carried out in a 16 L (semi-scale) bioreactor, with one assay performed at a controlled pH of 5.5 and another at an uncontrolled pH. The culture under uncontrolled pH conditions showed a biomass production of S. boulardii of 10.08 g L-1a more than the value obtained in the culture with controlled pH, impacting a 25% increase in biomass production.

[035] Finally, there is also the article published in the literature “Medium optimization and kinetics modeling for the fermentation of hydrolyzed cheese whey permeate as a substrate for Saccharomyces cerevisiae var. boulardii”, by the authors: Trigueros, DEG; Fiorese, ML; Kroumov, AD; Hinterholz, CL; Nadai, BL; Assunção, GM. In this work, the objective was to optimize the culture medium using the central composite rotatable design (CCRD) tool coupled with nonlinear mathematical programming (NLP), with the aim of investigating the growth of Saccharomyces cerevisiae var. boulardii in cheese whey permeate. The assays comprised lactose hydrolysis followed by anaerobic glucose-galactose fermentation in a batch system.The authors evaluated the influence of hydrolyzed permeate concentration and mineral supplementation such as nitrogen, magnesium, and potassium, with the final result of this optimization being the growth of the yeast in question. The culture medium was determined empirically using a statistical methodology and was also based on the elemental composition and stoichiometry of the anaerobic yeast. The proposed kinetic mathematical model considered... Petition 870250112683, dated 08 / 12 / 2025, page 18 / 47 / 22, regarding the metabolization of carbon sources, with glucose metabolization being rapid and galactose metabolization slow, also considered inhibition by the ethanol product, catabolic repression by glucose, and the formation of secondary metabolic products. As a final answer, the authors obtained an optimal overall biomass yield value using the mathematical model employed of Yx / s = 0.50, where the cellular biomass was estimated at 8.20 g L-1 using 40 g L-1 of hydrolyzed permeate.

[036] With the exception of this last work which used the yeast in question used in this invention patent, there is no other mention in the prior art search regarding the production of bioethanol and probiotic microbial / cellular biomass from dairy waste, i.e., residual raw materials, by-products and / or co-products of dairy processing and protein concentrates, commonly known as “whey”, whey, sweet whey, acid whey or even whey, deproteinized whey, deproteinized acid whey, deproteinized sweet whey, deproteinized whey, cheese whey permeate, or “whey permeate” with the yeast Saccharomyces boulardii. No patent documents or articles were found, much less products existing on the market that mention the production of the yeast Saccharomyces boulardii with dairy raw material originating from waste, by-products and / or co-products of dairy processing and / or concentrates. proteinaceous. Description of the approach to the technical problem and innovative differentiator:

[037] Aiming to solve the problems discussed above, and in line with eco-development, was the objective of developing this invention patent.

[038] The invention in question consists of producing biofuel (bioethanol) and probiotic microbial / cellular biomass, using dairy raw materials, whether these are residual, by-products and / or co-products of dairy processing and protein concentrates, commonly known as “whey”, milk whey, sweet whey, acid whey or whey, and when already free of the protein fraction, called “whey permeate”, deproteinized milk whey, deproteinized acid whey, deproteinized sweet whey, deproteinized milk whey, and / or cheese whey permeate and the probiotic yeast Saccharomyces boulardii.

[039] Bioethanol has emerged as an important alternative fuel and Petition 870250112683, dated 08 / 12 / 2025, page 19 / 47 / 22, is favorable for the preservation of the environment for future generations, as it does not produce toxic emissions during combustion, making it effective in reducing pollution and global warming, and therefore emerging as a greener fuel.

[040] In 2017, the total amount of bioethanol produced in the world was approximately 117.2 billion liters, of which 98.9 billion liters were used as fuel. The largest producer of bioethanol in the world is the United States with approximately 60 billion liters, followed by Brazil with approximately 26.2 billion liters (USDA, 2017).

[041] In Brazil, the largest source of substrate used for large-scale bioethanol production is sugarcane, with approximately 321,500 thousand metric tons used in 2017, and on a smaller scale, corn and sugarcane bagasse, with usage amounts of 1,171 thousand tons of corn and 0.0444 thousand tons of bagasse (USDA, Brazil Biofuels Annual, 2017).

[042] The production of bioethanol from sugarcane currently faces a complicating factor: the agricultural area available for planting has remained the same since 2013 (USDA, Brazil Biofuels Annual, 2017), meaning there has been no significant increase, directly impacting the efficiency of bioethanol production per hectare, which consequently points to a future crisis in the production of this biofuel.

[043] Although new production alternatives are being explored, such as the production of bioethanol from corn and sugarcane bagasse, it is noteworthy that the amount is very small. In the case of corn crops, this is due to the fact that this crop is primarily used for human and animal feed, and there is not yet a significant amount to be applied to bioethanol production.

[044] Sugarcane bagasse is an alternative to increase efficiency in bioethanol production; however, on average only 40% of sugarcane bagasse, an organic material, is made up of cellulose and can be hydrolyzed to generate glucose monosaccharides for use in the yeast fermentation process; the other 60% of constituents in bagasse do not contain fermentable carbohydrates.

[045] In recent years, the focus has been on the industrial use of agro-industrial waste as raw material for the production of bioethanol, as a means of reuse and bioremediation as well as to avoid the scarcity of food crops or fertile soils, Petition 870250112683, dated 08 / 12 / 2025, page 20 / 47 / 22 among other agricultural resources. Thus, the production of bioethanol from waste sources has become a profitable process, which is the purpose of this invention patent.

[046] The problem solved by this invention patent is related to the full use of raw materials, preservation of the environment, and reduction in wastewater treatment costs.

[047] With regard to the full use of dairy raw materials, the cultivation process described in this patent has potential for industrial applicability, since the composition and high concentration of essential nutrients necessary for the metabolism of the yeast Saccharomyces boulardii are found in the dairy raw materials in question (cheese whey permeate, whey permeate, deproteinized whey, whey, acid whey, sweet whey, whey), such as a high concentration of carbon source from lactose; minerals, the main ones being: calcium, sodium, phosphorus, manganese, potassium, zinc, iron, copper; organic protein; lipids; in addition to vitamins such as vitamin A, riboflavin (B2), pyridoxine (B6), thiamine, cobalamin, vitamin C, calcium pantothenate (B5) and biotin (B8), thus incurring low financial expenditure on supplementing the culture medium necessary for the growth of S. boulardii.

[048] From an environmental point of view, the present invention acts as a cost-effective and ecologically sound solution, since dairy raw materials (residues, by-products, co-products) have a high polluting potential, caused by the high quantity of nutrients, i.e., organic and inorganic matter, which makes the parameters for measuring this polluting potential extremely high and worrying, as they present a high rate of chemical oxygen demand (COD) and biochemical oxygen demand (BOD), which correspond to the amount of oxygen consumed in the degradation of organic matter through chemical reactions or by the action of microorganisms, respectively. And, if disposed of inappropriately, i.e., without proper treatment, it becomes highly damaging to the environment.

[049] From an economic point of view, this invention patent reduces or even eliminates the costs of effluent treatment and also promotes profit for companies. Dairy effluents, when properly treated for subsequent discharge into the environment, do not require considerable financial amounts for their complete treatment. Petition 870250112683, dated 08 / 12 / 2025, page 21 / 47 / 22, becoming extremely costly for the generating companies. With the application of this innovation, the treatment costs of these residual effluents from the dairy industry are eliminated, and new commercial products are also generated from this inventive process.

[050] The purpose of this document is to demonstrate that the main constituent (largest quantity) of the culture medium is lactose, which is a residual byproduct of the dairy industry. This makes the production process of biofuel (bioethanol) and probiotic microbial / cellular biomass economically viable, profitable, and with high potential for application in the industrial sector. This is because the volume of raw material generated is high, the cost of the raw material is low, and the need for supplementation when required by the microorganism Saccharomyces boulardii is minimal. In other words, it promotes the rational use of natural resources without impacting the environment.

[051] In view of this, the technique, the raw material, and the Saccharomyces boulardii yeast are part of the biotechnological transformation process and the inventive technology that is intended to be patented, this being the difference in relation to patents already existing in research databases. Description of the drawings:

[052] The invention is described below in one embodiment, and for better understanding, reference will be made to the attached drawing, which is represented by Figure 1. FIGURE 1: General view of the method according to the invention for the production process of biofuel (bioethanol) and probiotic microbial / cellular biomass with the yeast Saccharomyces boulardii. Detailed description of the invention:

[053] The objective of this invention patent concerns the production of biofuel (bioethanol) and probiotic microbial / cellular biomass with the yeast Saccharomyces boulardii in a reaction medium composed of dairy raw materials, whether these are residual, by-products and / or co-products of dairy processing and protein concentrates, commonly known as "whey", milk whey, sweet whey, acid whey or even whey, and when already free of the protein fraction, called "whey permeate", deproteinized whey, deproteinized acid whey, deproteinized sweet whey, Petition 870250112683, dated 08 / 12 / 2025, page 22 / 47 / 22 deproteinized whey, and / or cheese whey permeate.

[054] For the process to be effective, it is necessary to break down the lactose present in the raw materials mentioned above into its monomers, glucose and galactose, through a hydrolysis process that can be enzymatic or chemical-physical.

[055] The growth process of Saccharomyces boulardii and the production of bioethanol develop through a combination of biochemical and synthetic conversions that result in the production of bioethanol, with concomitant production of high value-added co-products such as probiotic microbial / cellular biomass, and also in smaller quantities secondary organic acids.

[056] Depending on the process conditions employed for the growth of S. boulardii, and the desired final product quantity, i.e., higher concentration of bioethanol or probiotic cell biomass, supplementation of the reaction medium with macro or micro elements such as hydrogen (H), oxygen (O), carbon (C), nitrogen (N), phosphorus (P), sulfur (S) may or may not be necessary, which together with selenium (Se) are considered essential for all classes of microorganisms, the first four being considered macronutrients, and the last three micronutrients, or even trace elements (metals), such as: vanadium (V), molybdenum (Mo), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cofactors (biotin, riboflavin), growth promoters, such as some minerals, vitamins and amino acids.

[057] The production process may or may not require aeration of the medium, and this aeration may be by direct injection of oxygen, compressed air, or by mechanical agitation.

[058] All these considerations are necessary in the development of the process and constitution of the culture medium, and directly influence the maximum assimilation of the dairy raw material used, and consequently in the production of bioethanol and probiotic microbial / cellular biomass.

[059] The production process, as illustrated in Figure 1, comprises the following steps. Form of presentation of the dairy raw material - (1). The dairy raw material can be used in liquid or solid form. When in liquid form, it must be kept refrigerated, between 4 and 7 °C, in order to avoid the degradation of nutrients and the proliferation of biological contaminants. Petition 870250112683, dated 08 / 12 / 2025, page 23 / 47 / 22 When in solid form, it must be stored in a clean, dry, moisture-free environment at room temperature. Preparation of dairy raw material - (2). When the dairy raw material is in solid form, the lactose content must be verified by appropriate analytical methodologies, such as colorimetric, spectrophotometric methods, high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UHPLC), Brix meter or other conventional methodologies that allow lactose quantification. Then, water is added in sufficient quantity to obtain the desired lactose concentration for the inoculum or for the reaction medium (fermentation process), as well as to allow the enzyme to act adequately on the lactose molecule. When the dairy raw material is in liquid form, the lactose concentration must be determined by the same methodologies.If the concentration found is higher than that intended for the inoculum or reaction medium, dilution with water is carried out until the desired concentration is reached; if it is lower, the dairy raw material must be concentrated in an evaporator-type equipment until the lactose concentration suitable for the inoculum or reaction medium is reached. In both situations, the amount of water must be sufficient to allow the development of the enzymatic hydrolysis reaction, varying according to the desired concentration of hydrolyzed lactose. Enzymatic hydrolysis of dairy raw material - (3). Hydrolysis of the dairy raw material is necessary because the microorganism used in this process, the yeast Saccharomyces boulardii, does not directly assimilate disaccharides in its metabolism, so the lactose molecule present in the dairy raw materials must be hydrolyzed to release the monosaccharides glucose and galactose.Enzymatic hydrolysis of lactose is carried out using the enzyme β-galactosidase (β-Gal; β-D-galactoside galactohydrolase; EC 3.2.1.23), also called lactase, derived from microorganisms, plants and / or animals. The hydrolysis process consists of adding the enzyme to an aqueous solution of the dairy raw material, adjusted to pH 6.5, under agitation of at least 100 rpm and a controlled temperature of 30 °C, conditions favorable to the enzyme's action. The hydrolysis time is variable, depending on the agitation and temperature control system employed, and the hydrolysis kinetics should be monitored until at least 90% lysis of the lactose molecules present in the dairy raw material used is obtained. Supplementation of the reaction medium (inoculum or fermentation / culture process) - (4). Supplementation of the medium may be necessary depending on the origin and prior processing of the dairy raw material, as well as the desired quantity of final product. Supplements of the macro, micro, or trace element type may be used, such as monobasic potassium phosphate, Petition 870250112683, dated 08 / 12 / 2025, page 24 / 47 / 22 dibasic potassium phosphate, urea, ammonium sulfate, yeast extract, meat peptone, soy tryptone, inactivated cane yeast extract, brewer's yeast extract, magnesium sulfate, amino acids, vitamins, among others. Cellular activation of Saccharomyces boulardii - (5). The strain used in this invention is marketed under the name Saccharomyces cerevisiae Meyen ex EC Hansen ATCC® MYA-796™ CCT 4308 and / or Saccharomyces boulardii, reference UFPEDA 1176, from the Tropical Cultures Collection - André Tosello Foundation, and may be supplied in lyophilized form or in an angled tube.Cell activation can be performed in media such as YEPD (yeast extract peptone dextrose), YMA (yeast malt extract), YNB (yeast nitrogen base), MA (Mannitol Agar), glucose and salts, sucrose and salts, maltose and salts, fructose and salts, lactose and salts, lactose broth, and undefined media, which must be previously sterilized at 121 °C for 15 minutes, followed by cooling to the desired use temperature. The activation process should be conducted in a variable agitation system between 10 and 150 rpm, in a pH range of 3 to 7, with or without buffering, and a temperature of 20 to 45 °C. Activation time is variable, and the process must be monitored in order to identify the moment preceding the end of the exponential phase of cell growth, in order to avoid a high proportion of dead or defective cells that could compromise the efficiency of the bioethanol and probiotic microbial / cellular biomass production process. Inoculum - (6).The inoculum, also called pre-vat, is used to reduce the adaptation time of the yeast to the reaction medium. It consists of a medium prepared with dairy raw material, with or without supplementation, in concentrations and conditions identical or similar to those of the reaction medium used in the production of bioethanol and probiotic microbial / cellular biomass. The medium intended for the inoculum must be subjected to a process to reduce microbiological contaminants, no less than pasteurization. Pasteurization can be carried out by a slow process (with constant agitation, at 65 °C, maintaining this temperature for 30 minutes) or by a rapid process (heating to 71-75 °C for 15 seconds, followed by cooling with ice water to 2-3 °C). The yeast previously activated in step (5) is added to the inoculum culture.The inoculum development time is variable and must be monitored in order to identify the moment preceding the end of the exponential growth phase of the yeast Saccharomyces boulardii, avoiding compromising the process due to the presence of dead or defective cells. Reaction medium (inoculum, fermentation / culture or must) - (7). The reaction medium is composed of dairy raw material in varying concentrations from 5 g^L-1 to non-inhibitory concentrations, prepared as described in steps (2) and (3), with or without. Petition 870250112683, of 08 / 12 / 2025, page 25 / 47 / 22 supplementation according to step (4), the supplementation being dependent on the type of procedural raw material used. The pH of the reaction medium (inoculum, fermentation / culture or must) is variable, ranging from 3 to 7, and can be adjusted with or without buffering. The reaction medium must be subjected to a process to reduce microbiological contaminants, with at least one pasteurization, which can be carried out by a slow process (with constant agitation, at 65 °C, maintaining this temperature for 30 minutes) or a fast process (heating to 71-75 °C for 15 seconds, followed by cooling with ice water at 2-3 °C). Reaction system (reactor and / or bioreactor) - (8). The reaction system must have variable agitation control between 10 and at least 900 rpm, manual or automatic pH control, and automatic temperature control within the process development temperature range, which occurs between 20 and 45 °C.It must also have a sterile system for exhausting gases, such as carbon dioxide, and / or for relieving internal pressure in the reactor or bioreactor, as well as a sterile system for the intake of oxygen or compressed air. The oxygen or compressed air intake must have equipment for flow control and a device for releasing microbubbles of air or oxygen into the reaction medium. The reaction system must undergo a sanitization and sterilization or pasteurization process to eliminate chemical and microbiological contaminants before being used in the production of bioethanol and probiotic microbial / cellular biomass. Production process of bioethanol and probiotic microbial / cellular biomass - (9). The production process comprises steps (1) to (8). The inoculum prepared according to step (6) is added to the reaction medium. The process time is variable and depends on the substrate concentration and the desired final product.During the process, the production of probiotic microbial / cellular biomass, bioethanol formation, nutrient concentration, and the presence of possible contaminants must be monitored. Products formed by the process - (10). The products formed from steps (1) to (9) include, as main products, bioethanol and probiotic microbial / cellular biomass. Other compounds may also be formed on a smaller scale, such as secondary organic acids, esters, ketones, and aldehydes. Recovery and purification of the products formed - (11 A and B). After steps (1) to (10) are carried out, the reaction medium is centrifuged to separate the probiotic microbial / cellular biomass from the other components of the medium. The recovered biomass (11 A) can be reused in a new production process or marketed, in its active or inactive whole form, or lysed.The yeast-treated reaction must (11 B) is subjected to a distillation process to separate the bioethanol from the other components. Petition 870250112683, dated 08 / 12 / 2025, page 26 / 47 / 22 components of the medium, making it suitable for commercialization.

[060] From the examples it is shown that the present invention process, in relation to other existing processes, is highly energy efficient, since none of the raw materials used are wasted, nor does any of the products formed lack a noble commercial field of application.

[061] This invention process for producing bioethanol using dairy feedstock and Saccharomyces boulardii yeast has a high conversion rate from raw material to final product. Considering the amount of dairy feedstock produced worldwide annually, 160-180 million tons, with an estimated growth rate of 1 to 2% per year, this would mean an increase in the bioethanol market of around 30 to 40%, which consequently reduces the costs of this biofuel. Furthermore, since milk production fluctuates much less than sugarcane cultivation, as the latter is more affected by seasonality and has also had a stagnant harvest in recent years, this invention process presented here would lead to greater availability of the product on the market, resulting in a price for this biofuel throughout the year, unlike what happens today, where the price drop only occurs during the sugarcane harvest season.Furthermore, linked to this inventive process, there is not only the large-scale production of biofuel, but also the production of a probiotic food, which can be used by humans and animals, helping to prevent diseases and thus reducing healthcare costs for both animals and humans.

[062] This inventive process enables the creation of new industries, and consequently new jobs. It opens up new export markets, as biomass can be applied to animal feed in an active form (lyophilized, encapsulated), which causes the consuming organism to improve its performance, i.e., increase its mass gain, which consequently reduces its production time.

[063] This invention process generates a new source of protein for human and animal nutrition, which is nutritionally rich and easily digestible, contributing to the reduction of hunger in developing countries, or even promoting an alternative to the inefficiencies that plague our food systems, and which cause us to still have 800 million people who go hungry, and at the same time more than 2 Petition 870250112683, dated 08 / 12 / 2025, page 27 / 47 / 22 billion people suffer from nutrient deficiencies, whether due to insufficient macronutrients or excess, as is the case with overweight (obesity). If the probiotic biomass produced by this invention is hydrolyzed, it can still be used as a protein food source for those suffering from allergies to animal proteins, or in the nutrition of athletes for muscle mass gain, dietary supplementation for the elderly, or also in the food industry as an effective functional agent, since it has excellent interfacial properties, that is, it has the potential to serve as an emulsifying ingredient in a variety of products, for example, sauces, extracts, dehydrated soup, broths, among others, it also has antioxidant action, increasing the shelf life of products, which can boost a new market for ingredients.

[064] This inventive process directly impacts the transformation and industrialization of new products, thus contributing to the protection of the ecosystem. It creates new opportunities for small businesses, such as cheese factories, to expand their production, which is currently limited by the difficulties in disposing of the waste generated in their production processes. Therefore, in addition to broadening the horizons of small industries, it also significantly impacts the segments of protein concentration from dairy materials, becoming an important means of adding value to raw materials for the food industry and a solution for the environment.

[065] In addition to all the advantages mentioned, production by the process in question shows its joint role in the search for a balance between environmental protection and economic growth, producing today without depleting resources so that it is possible to produce again tomorrow, that is, a process of sustainable eco-development. Illustrative example:

[066] The full use of dairy raw materials adds commercial value to industries with the new products formed, reduces the environmental effects caused by incorrect disposal of dairy raw materials in the environment, as well as reducing the cost of treating these effluents, by-products and / or co-products.

[067] First example of reaction condition in which the reactor was fermented at 37°C, with pH 6.0 and a concentration of 300 gL-1 of whey permeate, obtained 69.43 gL-1 of ethanol and 13.73 gL-1 of microbial / cellular biomass, in 45 h of fermentation. Petition 870250112683, dated 08 / 12 / 2025, page 28 / 47 / 22

[068] Second example of reaction condition in which the reactor was fermented at 26°C, pH 6.0 and a concentration of 300 gL-1 of whey permeate, 57.79 gL-1 of ethanol and 23.45 gL-1 of microbial / cellular biomass were obtained in 45 h of fermentation.

[069] Third example of reaction condition in which the reactor was fermented at 37°C, pH 6.0 and 100 gL-1 of whey permeate, obtained 24.44 gL-1 of ethanol and 11.41 gL-1 of microbial / cellular biomass, in 45 h of fermentation.

[070] Although the present invention process has been described in detail and some examples have been demonstrated, it is evident that modifications and adaptations of these embodiments will occur to those skilled in the art. However, it is understood that such modifications and adaptations are within the spirit and scope of the present inventive process. Petition 870250112683, dated 08 / 12 / 2025, page 29 / 47

Claims

1 / 4 CLAIMS 1. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii characterized by comprising the steps of: presentation and storage of the dairy raw material (1); preparation of the dairy raw material (2); enzymatic hydrolysis of the dairy raw material (3); supplementation of the reaction medium (4); cell activation of Saccharomyces boulardii (5); inoculum preparation (6); preparation of the reaction medium (7); use of a reaction system in a reactor and / or bioreactor (8); process for producing bioethanol and probiotic microbial / cellular biomass (9); obtaining the products formed by the process (10); recovery and purification of bioethanol and probiotic microbial / cellular biomass (11); 2. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by using, as dairy raw materials, residues, by-products or co-products of the dairy industry, including sweet or acid whey, whey, whey protein, deproteinized whey, deproteinized whey, sweet or acid deproteinized whey, and preferably, whey or cheese permeate; 3. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by employing the probiotic yeast Saccharomyces cerevisiae Meyen ex EC Hansen var. boulardii, commonly referred to as Saccharomyces boulardii, marketed under the designations Saccharomyces cerevisiae Meyen ex EC Hansen ATCC® MYA796™ CCT 4308 and / or Saccharomyces boulardii reference UFPEDA 1176 from the Tropical Cultures Collection - André Tosello Foundation, preserved in lyophilized form or in inclined maintenance tubes; Petition 870260024344, dated 03 / 16 / 2026, page 5 / 22 2 / 4 4. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by carrying out the hydrolysis of lactose present in the dairy raw material via enzymatic means, employing β-galactosidase enzymes (β-Gal; β-D-galactoside galactohydrolase; EC 3.2.1.23), which are added to an aqueous solution of dairy raw material at a concentration ranging from 0.00001 to 15%, in a pH range ranging from 1 to 10, with or without buffering, and a temperature ranging from 10 to 50 °C; 5. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by being conducted with inoculum prepared from previously activated Saccharomyces boulardii yeast, according to the cell activation step, in a medium consisting of dairy raw material with or without supplementation, subjected to a slow or rapid pasteurization process to reduce microbiological contaminants, under conditions of agitation, pH and temperature adjusted to favor cell growth until near the end of the exponential phase; 6. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by carrying out the fermentation in a reaction medium composed of dairy raw material according to claim 2, in concentrations ranging from 5 g <-1 to non-inhibitory concentrations, prepared from hydrolyzed raw material according to claim 4, with or without supplementation according to claim 7, at a pH ranging from 3 to 7, with or without buffering, the medium being previously subjected to a slow or rapid pasteurization process, and conducting the fermentation under temperature conditions within the range of 20 to 45 °C, as per Petition 870260024344, of 16 / 03 / 2026, page 6 / 22 3 / 4 variable agitation from 10 rpm until necessary for homogenization of the medium; 7. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by employing in the supplementation of the reaction medium (inoculum or fermentation / cultivation process), supplements of the macro, micro or trace element type, including monobasic potassium phosphate, dibasic potassium phosphate, urea, ammonium sulfate, yeast extract, meat peptone, soy tryptone, inactivated cane yeast extract, brewer's yeast extract, magnesium sulfate, amino acids and vitamins; 8. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by being conducted in a reaction operating system comprising a reactor and / or bioreactor with variable agitation control between 10 and at least 900 rpm, manual or automatic pH control and automatic temperature control within the range of 20 to 45 °C, equipped with a sterile system for gas exhaust and / or internal pressure relief, as well as a sterile system for oxygen or compressed air intake with flow control and a device for releasing microbubbles of air or oxygen into the reaction medium, wherein the reaction operating system is capable of being sanitized and sterilized or pasteurized; 9. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized by allowing the recovery of probiotic microbial / cellular biomass by centrifugation of the reaction medium, separating it from the other components of the medium, and the recovery of bioethanol by conventional, fractional or purified distillation process; Petition 870260024344, dated 03 / 16 / 2026, page 7 / 22 4 / 4 10. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized in that the bioethanol obtained can be used as a biofuel in any applications compatible with its physicochemical properties; 11. METHOD FOR PRODUCING BIOFUEL (BIOETHANOL) AND PROBIOTIC MICROBIAL / CELLULAR BIOMASS WITH DAIRY RAW MATERIALS AND THE YEAST Saccharomyces boulardii according to claim 1, characterized in that the probiotic microbial / cellular biomass of Saccharomyces boulardii can be used in whole, active or inactive forms, dried or in natura, cell-free, lysed, microbial protein, cell extract or possibly microencapsulated, for food, nutraceutical, pharmaceutical or zootechnical applications. Petition 870260024344, dated 03 / 16 / 2026, page 8 / 22