Process for obtaining a soluble single-cell protein (PRO60) that can be used in various food matrices

Abstract

The present invention relates to a novel fermentation, extraction and purification process for obtaining a single-cell protein for use in the food industry, using a particular strain of Kluyveromyces marxianus referred to as KMPROTEO 1, which results in a product consisting of a single-cell protein powder with a protein content equal to or greater than 60%, that is highly bioavailable, which, since it is soluble, can be included in different food matrices, without changing the formulation or the profile of the product to which it is added, which we have named "PRO60". The process disclosed in this specification includes the description of the aerobic fermentation of the strain of Kluyveromyces marxianus referred to as KMPROTEO 1, as well as the use of the ingredient or final product obtained, by adding same to some preferred food matrices.

Description

[0001]PROCESS FOR OBTAINING A SOLUBLE SINGLE CELL PROTEIN (PRO60) THAT CAN BE USED IN VARIOUS FOOD MATRICES DESCRIPTION OBJECTIVE OF THE INVENTION The present invention relates to a novel fermentation, extraction and purification process for obtaining single cell protein powder for use in the food industry, using a particular strain of Kluyveromyces marxianus, called KMPROTEO 1, which results in an ingredient or final product consisting of the light phase (soluble in water), derived from the cell lysis of said yeast, containing a protein concentration equal to or greater than 60% (weight / weight%).The Kluyveromyces marxianus strain, called KMPROTEO 1, has been deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, at the National Center for Genetic Resources CNRG of the National Institute of Forestry, Agriculture and Livestock Research, which, after the corresponding viability certification, was assigned the access number CM-CNRG TB66. The process disclosed in terms of this descriptive report includes the description of the fed-batch aerobic fermentation of the Kluyveromyces marxianus strain, called KMPROTEO 1, as well as the unit operations of breaking, separating and drying the ingredient and its use as a final product, adding to some preferred food matrices.The present invention reveals a process for obtaining a soluble protein, the production of which comprises the following steps: i) scaling up of the fermentation culture for the production of biomass of the Kluyveromyces marxianus strain, called KMPROTEO 1; ii) continuous scaling up of culture in a ratio of 1:10 (inoculum: total fermentation volume) where the amount of medium from the first fermentation process corresponds to 10% of the final volume of the next; iii) fed-batch fermentation process, where the nutrient addition rate is based on the residual glucose concentration ^ ^ in the culture medium, which is maintained less than 0.5 g / L throughout the entire Fed-batch process; iv) cell concentration by centrifugation at 5,500 - 6,500 RPM, preferably at 6.000 RPM at room temperature, and removal of supernatant; v) exposure of 10% (weight / volume) yeast biomass to autolysis at 50-60 ° C, preferably at 55 ° C; vi) exposure of concentrated yeast to enzymatic lysis using an enzyme cocktail that includes at least one cellulase, a beta glucanase and a protease, at a temperature between 55 and 60 ° C; with a pH between 7 and 9, preferably at 7; vii) exposure of the yeast biomass to enzymatic lysis for 4-6 hours, preferably 5 hours; viii) after cell lysis, centrifugation is carried out to separate the soluble phase and the heavy phase at 5,500 - 6,500 RPM, preferably at 6,000 RPM, thus achieving the separation of soluble and insoluble fractions followed; ix) the soluble or light phase is subjected to a microfiltration process, with a membrane with a pore size of between 0.10 and 0.22, preferably 0.22 microns, to remove any heavy particles from the mixture; x) the soluble phase is concentrated by means of an evaporator, using temperatures between 60 and 70 °C, with a vacuum of between -0.07 to -0.08 MPa, preferably - 0.08 MPa, thus achieving an increase in the solids concentration to a range between 30 and 50 % (weight / volume) prior to drying; xi) the soluble phase is concentrated by spray drying with an inlet temperature between 180 and 190 °C and an outlet temperature between 80 and 90 °C, thus achieving a powder with a moisture content of less than 5%. At the end of the described process, we obtain an ingredient consisting of a novel single-cell protein powder with a protein content equal to or greater than 60%. It is highly bioavailable and, due to its soluble nature, allows its inclusion in different food matrices without altering the formulation or profile of the product to which it is added. We have named this ingredient "PRO60." BACKGROUND.^ ^ Proteins are essential compounds for good human nutrition, whose nutritional quality depends on their essential amino acid content. Due to the exponential increase in food demand derived from population growth, as well as the need for strategies for large-scale ethically sustainable food production, the quality of a protein currently depends largely on the efficiency and sustainability of its production. The search for sustainable, efficient, healthy, and high-quality nutritional alternative proteins that can organoleptically compete with plant and animal protein represents an area of ​​opportunity to meet market needs. Single cell protein (SCP), derived from microorganisms, is a viable strategy, as it uses less space, time, water, energy, and resources for its controlled large-scale production.The above has been reported by several authors in scientific journals. An example of this is the document SINGLE CELL PROTEIN STATE-OF-THE-ART, INDUSTRIAL LANDSCAPE AND PATENTS 2001–2016, published by Ritala and collaborators in 2017, which carried out a bibliographic review of developments and patents from 2001 to 2016 related to the production of single-cell protein using various substrates, processes and organisms, inferring its potential over other conventional protein sources. Likewise, in the document R. ECENT TO DVANCES IN S INGLE C ELL P ROTEIN USE AS AFEED INGREDIENT IN AQUACULTURE, published by Shawn W Jones et al., compared the biomass conversion efficiency and protein content of plants, animals, insects, and microorganisms such as algae, bacteria, and yeast, demonstrating the potential of single-cell protein over conventional protein sources. In the publication INNOVATIONS AND DEVELOPMENTS IN SINGLE CELL PROTEIN: BIBLIOMETRIC REVIEW AND PATENTS ANALYSIS, Gislane Oliveira Ribeiro et al. conducted a bibliometric analysis of publications and patents addressing innovations and developments in the production of single-cell protein. In this 2023 document, the sustainable potential and increasing use of this protein for feed production are recognized, as well as the importance of investing in research and development of technologies for its production.^ ^ Single-cell protein presents a good nutritional profile, depending on the microorganism used; for example, yeasts used for commercial production contain between 20% and 60% of total protein at the cellular level. Additionally, protein extracts derived from yeast are usually a source of important vitamin groups and essential amino acids, making this protein source an attractive and innovative alternative that competes with traditional sources. The development, discovery and creation of new high-quality protein sources for the production of healthier foods and in good quantity is a challenge, not only for the industry, but also for technological and scientific development aimed at the production of protein with highly efficient processes and with an appropriate amino acid profile from yeast biomass, thus obtaining a balanced ingredient with a high concentration of available nitrogen as protein.Single-cell protein obtained from yeast biomass is a bioavailable product obtained from conventional raw materials, such as mono- and disaccharides and inexpensive nitrogen sources from industrial food or agri-food waste. The production and consumption of yeast-derived protein involves the use of sustainable processes and inputs to provide consumers with high-quality, high-value nutrients. In general, single-cell protein production includes: i) preparation of the nutrient medium for the proper and efficient growth of the yeast or microorganism in question; ii) fermentation culture, either in solid or liquid state; iii) separation and concentration (in some cases, only concentration); and iv) processing into an ingredient and food product. The term "single-cell protein," or "protein from microorganisms," has two connotations.The first of these refers to the use of the entire cell, which contains the desired protein within the confines of the cell wall, which is less available or functional; the second refers to the protein that has been isolated or separated from the cellular entity of the microorganism, which is of better quality and higher concentration. In the latter scenario, cell wall degradation, if necessary for the characteristics of the final ingredient, is performed to make the intracellular protein of the yeast more accessible (or bioavailable). ^ ^ Traditionally, several methods have been used for yeast cell disruption, including: i) mechanical force; ii) chemical disruption with detergents; enzymatic; and; iii) autolytic, from the induction of cellular autolysis; or a combination of the above methods.Cell autolysis has been used for the commercial production of single-cell proteins, as is the case with Marmite® and Vegemite®, edible spreads made from yeast extract. Induction is achieved by heating yeast cells to between 45 and 50°C for long enough for intracellular enzymes to partially hydrolyze the cell wall. It is worth mentioning that in this process, proteins are also reduced to small peptides, altering the quality and quantity of the original protein. Several processes for extracting cellular protein from yeast have already been patented. An example of this is European patent EP3670646A1, entitled FUNCTIONAL YEAST PROTEIN CONCENTRATE, which presents a mechanical lysis method at a pH of 6.5-8.5 to extract protein from a cell suspension of the yeast S. cerevisiae.Cell lysis is performed at a temperature below 40°C; the soluble fraction is filtered to reduce the content of molecules smaller than 30 kDa; and this fraction is dried. The protein concentrate, which has a mild flavor, comprises a large amount of folded proteins, which are capable of aggregating to form a solid protein matrix when heated. These characteristics allow the product to be added to various food matrices. Similarly, several methods have been proposed to improve the non-mechanical lysis of yeast creams, which commonly involve the autolytic capacity of these microorganisms to produce enzymes that degrade the cell wall itself.Such is the case of US patent US4810509A, called METHOD FOR PRODUCING YEAST EXTRACT (KANEGAE Yukihiro, SUGIYAMA Yoshio, MINAMI Kanshiro), where the yeast extract of good flavor is obtained from heating a biomass suspension to 55° - 70° C, allowing the cells to break at a pH between 8 and 10. Some more current methods have proposed the use of agents that help improve the autolytic process, such as chitosan from Japanese patent EP0466922A1, called YEAST EXTRACT PRODUCTION (ORIGANE, Akira; SATO, Takasi), in which the autolytic method for S. cerevisiae and ^ ^ P. pastoris was improved by adding chitosan at 0.01-3% weight / weight, at a pH of 2.5 - 7.5, at 30-54°C for 10-20 hours. Another similar case is the addition of enzymes that aid in the hydrolysis of carbohydrates and proteins in the yeast cell wall.This is addressed in US patent US11602156B2, entitled YEAST PROTEINS (Rudy Menin, Pauline Spolaore, and Isabelle Mouly; 2018), which provides a method in which a yeast cream is exposed to thermal plasmolysis at a temperature between 70 and 95 °C, for a period between 30 seconds and 4 hours; subsequently, the insoluble fraction and the soluble fraction are separated, and finally the insoluble fraction is subjected to the activity of at least one ribonuclease enzyme and one glucanase, sequentially or simultaneously, at a temperature between 40 and 65 °C, for a period of 8 to 24 hours. Furthermore, in the cited patent, the insoluble fraction, which is flavorless, has a nucleotide content of less than 3% and a protein content of at least 72%, which ensures its quality and safety for human consumption. (This patent corresponds to application US17049897).Food safety is a very important issue, as it aims to protect the well-being of consumers of the final product. To this end, various regulatory bodies determine whether an ingredient or product poses health risks. These bodies include the Food and Agriculture Organization of the United Nations (FAO) and the Food and Drug Administration (FDA), as well as various government agencies specific to each country. In the case of single-cell protein, an important step to ensure its consumption is the removal of nucleic acids, since the rapid production of biomass implies a high content of nucleic acids: cellular DNA and RNA. This content depends on cell growth conditions, such as the nutrients in the fermentation culture or even the carbon / nitrogen ratio.Specifically, high concentrations of nucleic acids and purines represent a problem for human health, since they increase the concentration of uric acid in the blood plasma and therefore, promote the appearance of kidney stones and gout. In commercially available products, some methods to decrease the concentration of purines in yeast-derived products involve increasing the temperature (60 °C - 100 °C) to induce the degradation of nucleic acids by endogenous ribonucleases or the use of immobilized exogenous ribonucleases. The increase in temperature also induces the diffusion of degraded DNA and RNA to the cell exterior, so the cost of this process falls on the increase in temperature.Currently, most of the state of the art, which presents a solution to this problem, uses changes in temperature to induce nucleotide lysis, as is the case of US patent US3947605A, called PROCESS FOR PREPARING HIGH YIELDS OF SINGLE-CELL PRODUCTS THAT HAVE A REDUCED PURINES CONTENT AND A HIGH NUTRITIONAL VALUE (CHAO, Kwei C). Other patents propose pH changes in an aqueous medium, to carry out cell lysis or to precipitate proteins from the protein fraction, as is the case of US patents: US3887431, called YEAST PROTEIN ISOLATE WITH REDUCED NUCLEIC ACID CONTENT AND PROCESS FOR PRODUCTION THEREOF (Ernest Aleck Robbins et al.); and patent US3991215A called MANUFACTURING PROTEIN ISOLATE WITH LOW NUCLEIC ACID CONTENT FROM A THERMAL PROCESS (Ernest Aleck Robbins).Strains belonging to the species Kluyveromyces marxianus have been isolated from a wide variety of habitats (fruits, rotting sisal leaves, sugar factory wastewater, etc.). This species was first described in 1888 by EC Hansen, after being isolated and named Saccharomyces marxianus. The genus was first described as Kluyveromyces in 1956, and was divided into six clades in 1998. Kluyveromyces marxianus is a widely diverse species, both metabolically and polymorphically speaking, that presents relevant characteristics for the food industry from the perspective of sustainability and biotechnology.Among its characteristics are its high growth rates, due to its small size, which allows them to grow faster than other organisms; they have a wide range of carbohydrate consumption, such as residual whey lactose, molasses and inulin; its ability to produce compounds of industrial interest, such as enzymes for the hydrolysis of carbohydrates and proteins; as well as its ability to glycosylate therapeutic proteins, or its capacity to produce ethanol at a temperature of 40 ° C - 45 ° C. ^ ^ Some strains have shown to have the capacity for protein secretion and biomass production, resulting in their use for the production of enzyme preparations and their possible use for the expression of recombinant proteins, such as the Kluyveromyces marxianus strain CGMCC10621, described in the Chinese patent CN105087403A called KLUYVEROMYCES MARXIANUS AND ITS APPLICATIONS (LYU HONG, et al.), Another important metabolic feature is that, unlike yeasts of the Saccharomyces species, Kluyveromyces marxianus have a strong “negative Crabtree effect”, since no ethanol is produced after applying a glucose pulse to the cells during aerobic growth. That is, when the oxidoreductive metabolism is activated in Kluyveromyces marxianus, as a function of the increase in glycolytic flux, the maximum reproductive capacity of the cells is not achieved. Some studies have demonstrated the potential of Kluyveromyces marxianus biomass as an excellent alternative to replace Saccharomyces cerevisiae for the production of yeast autolysates, glucans insoluble in alkaline pH, glucans and natural bio-emulsifiers. Paul J. Anderson, in his scientific publication THERMOTOLERANT SINGLE CELL PROTEIN PRODUCTION BY KLUYVEROMYCES MARXIANUS VAR. MARXIANUS, and Gisela Paez, in their technical publication “Continue Production of Single C.ELL P ROTEIN OFKLUYVEROMYCES MARXIANUS VAR. MARXIANUS FROM DILUTED CHEESE WHEY, demonstrated that various strains of Kluyveromyces marxianus grown in molasses present a good amino acid profile for the production of single-cell protein, with a balanced distribution compared to other yeasts and international reference standards (FAO), as well as with conventional protein sources such as egg and wheat. The above suggests the potential use of this single-cell protein. Even, as described in the Chinese patent CN105707436A called APPLICATIONS OF KLUYVEROMYCES YEAST (LYU HONG; MO WENJUAN; ZHOU JUNGANG; YU YAO), some strains of Kluyveromyces marxianus have already been applied as safe additives in animal feed where, in addition, it improves their digestion.Application WO2011140649A1 entitled FERMENTATION PROCESS OF A SUBSTRATE USING A MIXED CULTURE FOR THE PRODUCTION OF AN EDIBLE BIOMASS FOR ANIMAL AND / OR HUMAN CONSUMPTION (LEWANDOWSKI, Raymond [CA]), is directed to a process comprising a combination of yeasts and bacterial strains for the production of consumable biomass, from substrates comprising a simple sugar. More particularly, the claimed object includes the use of Lactobacillus fermentum, Kluyveromyces marxianus and Saccharomyces unisporus. Many strains of Kluyveromyces marxianus have already been accepted as GRAS, as have other strains commonly used in the food industry such as Saccharomyces cerevisiae and Kluyveromyces lactis. Compared to its predecessors, Kluyveromyces marxianus has better characteristics of industrial interest, even without genetic improvement (GMO-free).Among them, its natural ability to produce extracellular enzymes, such as pectinases and cellulases, which facilitate and reduce the cost of its concentration and separation processes for release during protein production, presenting lower viscosity than its fungal counterparts. Kluyveromyces marxianus, has proven to be an emerging, non-pathogenic yeast, with great potential for use in the food and biomedical industries, particularly for the production of single-cell protein or the expression of recombinant proteins; however, there is little information on biomass scaling systems for it at an industrial level. Some patents show Batch or Fed-batch systems for the production of Kluyveromyces marxianus biomass. The Chinese patent WO2017036294A1 called KLUYVEROMYCES MARXIANUS AND ITS APPLICATIONS (LV HONG et al.), shows a Fed-batch system for producing biomass from the Kluyveromyces marxianus strain (with deposit number CGMCC 10621), a hyper-secretor of recombinant proteins, in which its growth is proposed from 50 g / L of glucose and 40 g / L yeast extract, at a temperature between 28 and 35 °C; pH of 7.0, DO of 30%, where, during the Fed-batch process, a residual glucose concentration in the medium is maintained constant between 5 and 15 g / L. In the state of the art, fermentative methods have been proposed from cheap sources of nutrients (derived from the food industry) for the production of Kluyveromyces marxianus var. biomass. lactis, as is the case of patent application BG48706A1 called METHOD FOR PREPARING PROTEIN (SIMOVA, ^ ^ Emilina et al.), which proposes using de-proteinized, ultrafiltered, and improved whey, from the addition of 0.5-0.9% (NH4)2HPO4 and 10-20% corn extract, at a pH of 5.0, to cultivate, for 24 hours, the strain Kluyveromyces marxianus var. lactis NBPMCC1502 with aeration, at a speed of 0.008 s^¹, at 52.3 rad / s and a temperature of 28 to 32ºC. The processes available in the state of the art do not synergistically resolve the consecutive process of release of intracellular proteins and the consecutive steps for the generation of products for the food industry. The Kluyveromyces marxianus strain, called KMPROTEO 1, protected under Mexican patent MX 402824 B, has an increased capacity for obtaining unicellular biomass and subsequently unicellular protein, from the fermentation of whey.The fermentation process of said patent uses cheese whey as a propagation medium and allows the production of a high protein product, which will be in the range of 50 to 90%. The KMPROTEO 1 strain in particular is the one used in the novel process of the invention revealed in terms of the present specification. BRIEF EXPLANATION OF THE FIGURES.The novel aspects that are considered characteristic of the fermentation, extraction and purification process for obtaining single-cell protein for use in the food industry, using a particular strain of Kluyveromyces marxianus, called KMPROTEO 1, which are disclosed herein, will be set forth with particularity in the accompanying claims; however, the invention itself, both for its structural organization, together with other objects and advantages thereof, will be better understood in the following detailed description of certain preferred embodiments when read in relation to the accompanying figures, where: Figure 1 shows the flow diagram of the soluble single-cell protein production process revealed by the present invention.Figure 2 shows the macroscopic morphological characterization of the Kluyveromyces marxianus KMPROTEO 1 strain, in solid PDA medium at 24 and 96 hours of growth, at 35 °C, under aerobic conditions. ^ ^ Figure 3 shows the microscopic morphology of Kluyveromyces marxianus KMPROTEO 1, after having grown in solid PDA medium, at 24 and 96 hours, at 35 °C under aerobic conditions. Figure 4 shows the kinetics of the exponential growth of the yeast Kluyveromyces marxianus KMPROTEO 1, in medium C (described below) during the fermentation process in Fed-batch, under the conditions mentioned in this specification. Figure 5 is a graph showing the exponential growth phase of the fermentation process in Fed-batch in natural logarithm (Ln) over time.From the slope, the specific growth rate of the yeast Kluyveromyces marxianus KMPROTEO 1 in medium C was calculated during the fermentation process in Fed-batch, under the conditions mentioned in this document. Figure 6 shows the process diagram for the production of the soluble single-cell protein disclosed by the present invention, illustrating the relevant equipment per unit operation. Figure 7 shows the drying process for the production of the soluble single-cell protein disclosed by the present invention, resulting in a soluble powder. Figure 8 shows the powdered product resulting from the production process of single-cell protein from Kluyveromyces marxianus KMPROTEO 1. DETAILED DESCRIPTION OF THE INVENTION.In this specification, the process for obtaining an ingredient is revealed, consisting of a novel single-cell protein powder, with a protein content equal to or greater than 60%, highly bioavailable, which being soluble, allows its inclusion in different food matrices, without changing the formulation or the profile of the product to which it is added, which we have named "PRO60". The revealed process involves the standardization of four stages after fermentation and washing of the biomass: i) cell rupture; ii) separation of soluble and insoluble cellular phases; iii) concentration of soluble phase, and; iv) drying of soluble phase. The product resulting from the novel process revealed in this specification is a food grade additive, very rich in proteins, amino acids and oligopeptides, as well as in some organic acids, nucleic acids and mineral salts (in lesser proportion).^ ^ The powdered product resulting from the novel process disclosed in the present specification has a flavor ranging from acidic to neutral and a pleasant characteristic odor. Due to its organoleptic, functional and nutritional attributes, the product resulting from the process disclosed in the present specification can be added to various food matrices, such as sausages, ice cream, pasta, sauces, breads and fried foods, adding nutritional properties to said matrices. In the food industry, protein extracts, such as the one disclosed in the present invention, are usually added to improve the organoleptic characteristics, such as flavor, smell and texture of various conventional products, adding a high nutritional value to them compared to other food additives. The main characteristics of the single-cell protein of the present invention are shown in the table below.Table 1 Main characteristics of the single-cell protein. The final product, resulting from the process disclosed in this specification, presents functional stability and safety for consumption. The good yield of biomass production and the extraction of the phase rich in soluble and highly available protein, allows the production of the product to be scalable to industrial volumes in an easy and replicable manner. The production process of the single-cell protein powder, disclosed in this specification, whose flow diagram is shown in Figure 1, has production and scalability advantages over other protein sources directed to the food industry. The yeast strain Kluyveromyces marxianus KMPROTEO 1, used in the process disclosed in this specification, is a eukaryotic microorganism, ^ ^ with a high cell growth rate in a medium rich in dextrose, as the main carbon source, and yeast extract and ammonium sulfate as the main nitrogen source.This strain belongs to the Fungi kingdom, Ascomycota phylum, Saccharomycotina subphylum, Saccharomycetes class, Saccharomycetales order, Saccharomycetaceae family, Kluyveromyces genus and Kluyveromyces marxianus species. The KMPROTEO 1 strain presents a colonial morphology in YPD medium (yeast extract, peptone and dextrose), spherical ovoid, creamy, white. At a microscopic level, it measures about 1.9-2.5 ^m wide and 2.8-4 ^m long, whose reproduction is given by polar budding and presents ascospore formation, as shown in Figure 2. The KMPROTEO 1 strain, used in the process disclosed in this specification, was selected for its high growth rate of ^ = 0.1 / h under the conditions described herein, at a temperature of 35 °C and 200 rpm; its biomass production corresponds to 60 g / L / h and a protein content in the finished product greater than 50%, as shown in Figure 3.The preferred fermentation, cell disruption, concentration and drying processes will be described below. 1. FERMENTATION PROCESS This specification first discloses an optimized process for producing yeast biomass on a commercial scale, starting from a culture fed under aerobic conditions, with a preferred fermentation time of 48-96 hours, to reach a cell concentration of 60 g / L of biomass, using the Kluyveromyces marxianus KMPROTEO 1 strain. This fermentation process for producing single-cell protein consists of the following steps: i) Yeast reactivation: To reactivate the KMPROTEO 1 strain, 200 microliters of cryopreserved yeast (concentration of 1x10^^¹^ CFU / mL) are inoculated into a flask with a nominal volume of 1 L mL, containing 400 mL of medium A (described in Table 2 below).The inoculated flasks are incubated at 35 °C for 12 hours, at a shaking speed of 300 RPM. ^ ^ Table 2. Medium A, for the activation of strain KMPROTEO 1. ii) Scale-up: After the incubation time, the 400 mL from the previous step are inoculated into 3.6 L of medium B (described in Table 3 below) in a stirred tank fermenter, to form a total volume of 4 liters, where the inoculum concentration corresponds to 10% (volume / volume) of the culture. The inoculated reactor is placed at an air flow of 1 vvm (aeration volume per volume of medium per minute), at a propeller stirring speed of 200 RPM, at a fermentation temperature of 35 °C ± 2 °C; pH between 5 and 5.5. The culture is fermented for 4 to 6 hours, until reaching an optical density (OD 600 nm) greater than 1. Table 3. Medium B, for the production of KMPROTEO 1 biomass. The 4 liters obtained from the previous culture are inoculated into 36 liters of medium B (Table 3) in a 100-liter nominal stirred-tank bioreactor, maintaining the same medium ratio described above, giving a total of 40 L of work. The fermentation conditions remain the same as the previous step. After the fermentation time, the 40 liters are pumped to the production reactor, which must have a nominal capacity of 1,000 liters, where the Batch fermentation (closed batch) begins, with a volume of 360 liters. For the Batch process, the same operating parameters mentioned above are maintained, as well as the same concentration of the previous culture medium. Once 400 liters of fermentation are obtained in Batch mode at an optical density of >1.0 (OD 600 nm), the fed-batch fermentation begins, where the continuous addition of nutrients found in medium C (described in Table 4, below) takes place; at the end of the process, 200 liters of medium C (Table 4) will have been added, completing a total volume of 600 liters at a concentration of 60 grams per liter and a total biomass of 36 kilos. The addition of medium begins when the pH of the previous step begins to drop and reaches 3; the optical density of the culture is greater than or equal to 10 OD; and the biomass concentration is above 4 grams per liter. Table 4. Concentration of medium C inputs in Fed-batch fermentation. During the Fed-batch fermentation model, the following parameters are maintained in operation (^ = 0.10 h-1 depends on the volume): ^^^ The continuous addition of medium C (Table 4) is carried out so that the residual glucose concentration in the fermentation medium is always less than 0.5 g / L and the ethyl alcohol concentration less than 1 g / L ^^^ A pH is maintained in a range between 5 and 5.5 by the constant addition of sterile 1 N sodium hydroxide ^^^ The oxygenation of the culture must be constant greater than 50% ^^^ The foam concentration must be controlled by adding a previously prepared and sterilized antifoam at a concentration of 70% (volume / volume) ^^^ The respiratory quotient (RQ) of the fermentation between 1 and 1.5 ^ ^ The culture is released when 60 grams per liter of biomass are achieved in the production reactor.Figure 4 shows the kinetics of exponential growth of the yeast Kluyveromyces marxianus KMPROTEO 1 in grams per liter (g / L) over time in hours (h), in medium C (Table 4) during the fermentation process in Fed-batch, under the conditions mentioned in this document, where the maximum peak of biomass production is given between 25 and 30 hours, being 60 g / L. Figure 5 is the graph that shows the exponential growth phase of the fermentation process in Fed-batch in natural logarithm (Ln) over time. From the slope, the specific growth rate of the yeast Kluyveromyces marxianus KMPROTEO 1 in medium C (Table 4) was calculated during the fermentation process in Fed-batch, under the conditions described in this document. 2.CELL DISRUPTION Once the required cell concentration is obtained, the culture is pumped into a flow-through tank, which feeds a rotating disc centrifuge, which is required to concentrate the cellular biomass, separating it from the rest of the supernatant. The biomass is centrifuged at a speed of 5,500 - 6,500 RPM, preferably 6,000 RPM, with 4-minute discharges; 1-s opening; and 360 L / h pumping. The biomass is resuspended in an equal volume of process water and centrifuged again under the same conditions, to remove residual compounds from the culture medium. Finally, the biomass is dissolved in water, to obtain a 10% (weight / volume) solution. After concentrating the biomass by centrifugation, it is resuspended in water, bringing it to a solids percentage between 10% and 20% (weight / volume).Subsequently, the mixture is pumped to an enzymatic reactor, where enzymatic cell lysis is carried out using an enzyme cocktail, which includes cellulases, beta glucanases and proteases, in a percentage (weight / weight) of 1.0, 1.0 and 0.2% respectively, with the following control parameters: temperature between 55 ° C and 60 ° C, preferably 55 ° C; with a pH between 7 and 9, and; with a hydrolysis time between 4 and 6 hours. These enzymes allow to improve cell lysis and protein release under the same conditions in which cell autolysis is carried out. ^ ^ For the process of intracellular protein release of the Kluyveromyces marxianus strain KMPROTEO 1, a disruptive cell lysis method composed of two foundations is used: i) autolysis, and; ii) enzymatic lysis. This method, standardized based on conditions previously described in the literature.The rupture obtained by enzymatic hydrolysis coupled to cell autolysis, under the aforementioned conditions, is the optimal method for the process disclosed in this specification, because it releases a significant amount of protein into the soluble medium, in a range between 25% and 30% of solids solubilized in the medium, with a protein concentration between 60% and 62%, as well as a low concentration of nucleic acids generated during fermentation. The suspended biomass was placed under different conditions, as described in the following Table: Table 5. Effect of varying TEMPERATURE and pH on cell rupture of K. marxianus KMPROTEO 1. VARIABLES: temperature of 55 °C; pH of 5.6 and 8; lysis time of 6 h. RUPTURE METHOD: enzymatic lysis, autolysis; mechanical lysis at 7,000 psi (two passages). For the release of intracellular protein, the rupture by enzymatic lysis revealed in the present specification is a novel method, because it releases a significant amount of protein to the soluble medium, in a range between 28% and 33% of solids solubilized to the medium, thus achieving a high protein concentration in the final powder product, in a range of 60-63%, while reducing the content of nucleic acids generated during fermentation. 3. CONCENTRATION ^ ^ After hydrolysis, the mixture is returned to the centrifuge feed tank and the mixture is centrifuged, thus separating the product into 2 flows and obtaining a soluble (or light) phase and an insoluble (or heavy) phase, thus separating soluble and insoluble fractions.Centrifugation is carried out at a speed of 5,500 - 6,500 RPM, preferably 6,000 RPM, with 4-minute discharges; 1-s opening; and a pumping rate of 360 L / h. Subsequently, the soluble phase is subjected to microfiltration using a ceramic filter with a pore opening of between 0.10 and 0.22, preferably 0.22 microns, at a filtration rate of 20 Ml / min (10-30% pump power), a transmembrane pressure less than 0.5 bar, and a constant pumping pressure between 0.25 and 0.30 bar, thus ensuring that the product is free of insoluble particles larger than 0.22 micrometers. The permeate product passes to a balance tank prior to the next unit operation. During this process, a loss of 2-10% of total protein is obtained. The soluble phase is concentrated by means of an evaporator at a temperature between 50 °C and 70 °C (preferably at 60 °C) and a vacuum between -0.07 and -0.08 MPa, preferably -0.08 and -0.1 Kpa, thus evaporating water from the extract and therefore raising the solids concentration of the mixture to a range between 30 and 50% (weight / volume) prior to drying. 4. DRYING Once the protein extract has been concentrated to 30% - 50% solids (weight solids / volume water), the drying process begins (preferably at 30%), where the soluble phase is spray dried at an inlet temperature between 180 ° C and 190 ° C and an outlet temperature between 70 ° C and 90 ° C (preferably at 80 ° C), at a constant flow rate of 8.5 L / h, at a solids concentration of 30%; and an atomization pressure of 5 Kgf / cm². Atomizer speed: 550 Hertz - 30,000 RPM. Air flow speed: 13 m / s. The product obtained will be a fine powder, light amber brown / cream yellow in color with a humidity equal to or less than 5%.The powdered ingredient obtained from the novel process disclosed in this specification is added to various food matrices, such as cheeses, yogurt or spreadable dips, achieving a homogeneous incorporation and a finished product with superior sensory qualities, while maintaining a vegan profile. CHARACTERISTICS AFTER DRYING: Light amber brown to creamy yellow color Appearance: Fine powder Particle size passed >99% in mesh #60 Total N: 9-10% Protein close to >60%, between 60 and 62% INDUSTRIAL APPLICATION.The novel fermentation, extraction and purification process for obtaining single-cell protein for use in the food industry, using the Kluyveromyces marxianus strain called KMPROTEO 1, derived from the cell lysis of said yeast, containing a protein concentration equal to or greater than 60% (weight / weight%) and the product directly obtained from the process, which results in a water-soluble ingredient or final product, are concepts that clearly meet the requirement for industrial application. It is noted that, as of this date, the best method known to the undersigned for putting the aforementioned invention into practice is the one that is clear from reading this description.Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that, in light of the teachings of this invention, changes and modifications may be made thereto without departing from the spirit or scope of the disclosure, including the appended claims. ^ ^.

Claims

CLAIMS.

1. A fermentation, extraction and purification process for obtaining single-cell protein with a protein content greater than or equal to 60%, for use in the food industry, wherein said process uses a particular strain of the yeast Kluyveromyces marxianus, called KMPROTEO 1, deposited under the terms of the Budapest Treaty, with accession number CM-CNRG TB66. 2.The process according to claim 1, which results in obtaining a soluble single-cell protein, wherein said process comprises the following steps: i) scaling up of the fermentation culture for the production of biomass of the Kluyveromyces marxianus strain, called KMPROTEO 1; ii) continuous scaling up of culture in a ratio of 1:10 (inoculum: total fermentation volume) where the amount of medium from the first fermentation process corresponds to 10% of the final volume of the next; iii) fed-batch fermentation process, where the nutrient addition rate is given based on the residual glucose concentration in the culture medium, which is maintained less than 0.5 g / L throughout the entire Fed-batch process; iv) cell concentration by centrifugation at 6.000 RPM at room temperature, and removal of supernatant; v) exposure of the 10% (weight / volume) yeast biomass to autolysis at a temperature between 50°C - 60°C, preferably at 55°C; vi) exposure of concentrated yeast to enzymatic lysis using an enzyme cocktail that includes at least one cellulase, one beta glucanase and one protease, at a temperature between 55°C and 60°C; with a pH between 7 and 9 (preferably 7); vii) exposure of the yeast biomass to enzymatic lysis for 4-6 hours, preferably 5 hours; viii) after cell lysis, centrifugation is carried out to separate the soluble phase and the heavy phase at 6,000 RPM, thus achieving the separation of soluble and insoluble fractions followed; ^ ^. ix) the soluble, or light, phase is subjected to a microfiltration process, with a membrane with a pore size between 0.10 and 0.22, preferably 0.22 microns, to remove any heavy particles from the mixture; x) the soluble phase is concentrated by means of an evaporator, using temperatures between 60 °C and 70 °C, with a vacuum of -0.08 MPa, thus achieving an increase in the solids concentration to a range between 30 and 50 % (weight / volume) prior to drying; xi) the soluble phase is concentrated by spray drying with an inlet temperature between 180 °C and 190 °C and an outlet temperature between 80 °C and 90 °C, thus achieving a powder with a moisture content of less than 5%; 3.The method for scaling up the biomass of the Kluyveromyces marxianus KMPROTEO 1 strain described in claim 2, wherein the fermentation culture consists of the following steps: (i) yeast reactivation: To reactivate the KMPROTEO 1 strain, by inoculating 200 microliters of cryopreserved yeast (concentration of 1x10^^¹^ CFU / mL), in a flask with a nominal volume of 1 L mL containing 400 mL of medium "A" (described in Table 2 below), and subsequent incubation of the inoculated flasks at 35 ° C for 12 hours at a shaking speed of 300 RPM; Table 2 Medium A, for the activation of KMPROTEO 1 strain. (ii) scale-up comprising the following steps: • after the incubation time, the 400 mL from the previous step are inoculated into 3.6 L of medium "B" (described in Table 3, inserted below) in a stirred tank fermenter, to form a total volume of 4 liters, where the inoculum concentration corresponds to 10% (volume / volume) of the culture, where the inoculated reactor is placed at a ^ ^ air flow of 1 vvm (aeration volume per volume of medium per minute), a propeller agitation speed of 200 RPM, at a fermentation temperature of 35 °C ± 2 °C; pH between 5 and 5.5 and where the culture is fermented between 4 and 6 hours, until reaching an optical density (OD 600 nm) greater than 1; Table 3 Medium B, for the production of KMPROTEO 1 biomass. • the 4 liters obtained from the previous culture are inoculated into 36 liters of medium B (Table 3), in a 100-liter nominal stirred-tank bioreactor, maintaining the same medium ratio described above, giving a total of 40 L of work, keeping the fermentation conditions the same as those of the previous step; • after the fermentation time, the 40 liters are pumped to the production reactor, which must have a nominal capacity of 1,000 liters, where the Batch (closed batch) fermentation begins, with a volume of 360 liters, maintaining the same operating parameters mentioned above, as well as the same concentration of the previous culture medium; • once 400 liters of fermentation are obtained in Batch mode at an optical density of >1.0 (DO 600 nm) the fermentation is started in Fed-Batch (fed batch), where the continuous addition of nutrients found in medium C (described in Table 4 below) is carried out; at the end of the process, 200 liters of medium "C" will have been added to complete a total volume of 600 liters at a concentration of 60 grams per liter and a total biomass of 36 kilos, where the addition of medium starts when the pH of the previous step begins to drop and reaches 3, the optical density of the culture is greater than or equal to 10 DO, and the biomass concentration is above 4 grams per ^ ^. liter, according to the concentration of inputs described in the following table: Table 4 Concentration of inputs of medium C in Fed-batch fermentation. where during the Fed-batch fermentation model the following parameters are maintained in the operation (^ = 0.10 h-1 depends on the volume): a) the continuous addition of medium "C" (table 4) is carried out so that the concentration of residual glucose in the fermentation medium is always less than 0.5 g / L and the concentration of ethyl alcohol less than 1 g / L; b) a pH is maintained in a range between 5 and 5.5 by the constant addition of 1 N sodium hydroxide, sterile; c) oxygenation of the culture must be constant greater than 50%; d) the foam concentration must be controlled by adding previously prepared and sterilized antifoam at a concentration of 70% (volume / volume); e) the respiratory quotient (RQ) of the fermentation between 1 and 1.5; and where the culture is released when 60 grams per liter of biomass are achieved in the production reactor. 4.The fermentation method of the Kluyveromyces marxianus strain called KMPROTEO 1 described in claim 2, is carried out in Fed-batch (fed batch), which includes the kinetic and control parameters, optimized for the production at any scale of single-cell protein with the Kluyveromyces marxianus strain called KMPROTEO1:

5. The fermentation method in Fed-batch (fed batch) of the Kluyveromyces marxianus strain called KMPROTEO 1, described in claim ^ ^. 2, includes the formulation of medium "C", optimized for the production of single-cell protein at any scale.

6. The product obtained as a result of the process described in claim 2, consisting of a single-cell protein powder, with a protein content equal to or greater than 60%, highly bioavailable and with a nitrogen content greater than or equal to 9.6%.

7. The powdered product obtained as a result of the process described in claim 2, which, being soluble, allows its inclusion in different food matrices, without changing the formulation or the profile of the product to which it is added.

8. The product obtained as a result of the process described in claim 2 is a fine powder with a particle size less than 250 microns. 9.The product obtained directly from the process according to claim 6 is a water-soluble ingredient, which allows its inclusion in different food matrices, without changing the formulation or nutritional profile of the product to which it is added, improving its organoleptic characteristics. ^ ^.

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