Modular kits with a biomimetic carbohydrate block for oxygen-sensitive microorganisms

Abstract

The invention relates to a kit and a method for growing or fermenting oxygen-sensitive microorganisms with non-zero residual dissolved oxygen. The kit comprises a heat-stable, heat-sterilisable part A, having at least one source of nitrogen and / or peptides and at least one source of carbon; and a thermolabile part B, which can be added aseptically after cooling, providing a biomimetic carbohydrate block with an N-acetylhexosamine motif and additional components selected from fucosylated, sialylated, branched and / or sulphated structures. The method reconstitutes the medium by combining A and B, inoculates the microorganism and operates in a closed vessel without continuous purging of inert gas, maintaining dissolved oxygen below air saturation and, during part of the process, above zero and up to five milligrams per litre, in order to obtain viable biomass and / or cell-free supernatant for industrial fermentation.

Description

[0001] “Modular kits with biomimetic glucose block for oxygen-sensitive microorganisms”

[0002] Technical field

[0003] The present invention lies in the field of biotechnology, applied microbiology, and fermentation technology, particularly in culture media and operating methods for the cultivation or fermentation of microorganisms whose growth and / or viability is inhibited or compromised by the presence of oxygen. Specifically, it relates to a modular kit and a reconstituted culture medium composition incorporating a biomimetic carbohydrate block (BBG), as well as a cultivation / fermentation method that sustains microbial growth and / or viability under a dissolved oxygen (DO) regime below air saturation, maintaining a non-zero residual DO during at least part of the process, without requiring ultra-anaerobic conditions.

[0004] In a preferred embodiment, the invention employs an A / B architecture in which a heat-sterilizable, heat-stable basal fraction (Part A) is physically separated from a heat-labile fraction (Part B) that is aseptically added after cooling, preserving heat-sensitive components and minimizing degradation associated with heat treatments. Part B comprises the BBG, which includes at least one N-acetylhexosamine motif and, additionally, at least one carbohydrate component selected from fucosylated, sialylated, branched oligosaccharides and / or sulfated glycans, or combinations thereof, enabling a robust platform for monocultures and cocultures of oxygen-sensitive microorganisms under non-zero residual oxygen conditions.

[0005] The invention is applicable to the production of viable microbial biomass and / or cell-free supernatants (e.g., fermented / postbiotic ingredients), as well as their incorporation into food matrices, fermented beverages, and nutraceutical compositions or food supplements for non-therapeutic use, where the aim is to improve process reproducibility, operational stability, and microbial viability without resorting to strict control of absolute anaerobiosis, maintaining a single general inventive concept based on the functional combination of BBG, A / B medularity, and operation with non-zero residual OD.

[0006] 3.1 Technical context and industrial need

[0007] The industrial and semi-industrial production of microbial biomass, ferments, starter cultures, and fermentation products requires media and processes capable of maintaining the growth and viability of biologically valuable but oxygen-sensitive microorganisms. In many applications, these microorganisms exhibit inhibition, oxidative stress, loss of viability, or metabolic disturbances when exposed to dissolved oxygen above low thresholds, even when the process is carried out in closed equipment with standard sanitation, sterilization, and process control practices. In practice, the routine operation of fermentations and cultures involves micro-inputs of oxygen associated with sampling, connections, line transfers, inoculum preparation, replenishment of inputs, agitation, gas-liquid exchange, and temperature variations, as well as system permeability.These disturbances generate real-world conditions far from ideal anaerobiosis, especially during scale-up, where batch-to-batch stability and reproducibility are affected. 3.2 Limitations of conventional approaches based on strict anaerobiosis.

[0008] For oxygen-sensitive microorganisms, the traditional solution has been to impose strict or ultra-anaerobic conditions through continuous purging of inert gases, intensive use of anaerobic chambers, the use of oxygen scavengers or catalysts, highly sealed containers, and operational controls that minimize oxygen ingress. While these measures can reduce oxygen, they often increase costs, operational complexity, and dependence on specialized infrastructure, and do not completely eliminate the risk associated with micro-leaks and unavoidable handling or operational events. Furthermore, approaches focused solely on excluding oxygen tend to be fragile in terms of reproducibility.A small deviation can produce disproportionate effects on viability, latency time, yield, and metabolic profile, particularly in microorganisms with low tolerance to reactive oxygen species or with strict redox and nutritional microenvironment requirements.

[0009] 3.3 Limitations of standard media and non-biomimetic supplements

[0010] Standard culture media and their variants, even when "rich," do not always provide cues and substrates that functionally replicate the physiological microenvironment experienced by many oxygen-sensitive microorganisms in their natural niches. In particular, the availability of certain complex carbohydrates, carbohydrate motifs, and glycans associated with mucins or biological matrices can influence adhesion, metabolism, membrane stability, biofilm formation, interaction with other microorganisms, and tolerance to oxidative stress.

[0011] Adding generic supplements can improve growth under ideal conditions, but it often fails to address the core problem: maintaining growth and / or viability when non-zero residual dissolved oxygen is present for at least part of the process. In these cases, performance may depend on factors that are difficult to control, such as the precise carbohydrate profile, consumption kinetics, local redox state, and the integrity of heat-labile components.

[0012] 3.4 Stability problems due to sterilization and handling of thermolabile components

[0013] In industrial processes, it is common to sterilize the entire medium or most of its components by heat. However, many ingredients relevant to the performance of oxygen-sensitive microorganisms can be heat-labile, degrade through Maillard reactions, oxidize, hydrolyze, or lose functionality during heating and storage. This affects both the effectiveness of the medium and its consistency between batches.

[0014] Some procedures separate components to add them after sterilization; however, this practice is usually ad hoc, lacks a defined modular architecture, and does not systematically integrate a biomimetic carbohydrate block designed to sustain viability under non-zero residual dissolved oxygen.

[0015] 3.5 Need for a more robust technical solution

[0016] Therefore, there is a need for media, kits, and methods that, without relying on strict anaerobiosis and without requiring complex infrastructure, allow the cultivation or fermentation of oxygen-sensitive microorganisms under realistic operating conditions where residual dissolved oxygen may be present. Such a solution must be reproducible, scalable, and compatible with industrial sterilization, while preserving temperature-sensitive components and providing biomimetic carbohydrate signals / substrates capable of supporting growth and / or viability in a dissolved oxygen regime below the air saturation of the medium at the culture temperature.

[0017] In particular, a platform is required that combines: (i) a modular formulation architecture that separates a heat-sterilizable, thermostable basal fraction from an aseptically additive, temperature-labile fraction, and (ii) a biomimetic carbohydrate block incorporating N-acetylhexosamine motifs along with additional carbohydrate components selected from fucosylated, sialylated, branched and / or sulfated structures, so that the process can operate with non-zero residual dissolved oxygen during at least part of the culture or fermentation, maintaining growth and / or viability without ultra-anaerobiosis.

[0018] 4. SUMMARY OF THE INVENTION

[0019] 4.1 Overview and special technical contribution

[0020] The present invention provides a unitary technical solution for the cultivation or fermentation of oxygen-sensitive microorganisms under realistic operating conditions where non-zero residual dissolved oxygen exists during at least part of the process, without requiring ultra-anaerobic conditions. The special technical contribution lies in the functional combination of: (i) a biomimetic carbohydrate block designed to support growth and / or viability under residual oxygen, (ii) a modular formulation architecture that separates a heat-sterilizable, thermostable basal fraction (Part A) from a temperature-labile fraction that can be aseptically added after cooling (Part B), and (iii) a process operating mode under a dissolved oxygen regime below the air saturation of the medium at the culture temperature, maintaining non-zero residual dissolved oxygen during at least part of the cultivation or fermentation.

[0021] In preferred embodiments, the invention allows the process to be carried out in closed containers without the need for continuous purging of inert gas, tolerating typical operating disturbances (sampling, inoculation, transfers, mixing variations and gas-liquid transfer), and maintaining batch-to-batch reproducibility.

[0022] 4.2 Modular Kit (Part A / Part B)

[0023] In a first embodiment, the invention is implemented by means of a modular kit for preparing a culture medium. Part A is heat-stable, physically separate, and comprises at least one nitrogen and / or peptide source and at least one carbon source, and may include buffers and / or salts to adjust pH and ionicity conditions prior to sterilization. Part A is heat-sterilized and cooled. Part B is heat-labile, physically separate, and intended to be incorporated by aseptic addition after cooling Part A. Part B comprises the biomimetic carbohydrate block, which includes (a) at least one N-acetylhexosamine motif selected from N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, or functionally equivalent derivatives, and (b) at least one additional carbohydrate component selected from fucosylated oligosaccharides, sialylated oligosaccharides, branched oligosaccharides, and / or sulfated glycans, or combinations thereof.In preferred embodiments, the biomimetic carbohydrate block further includes galacto-oligosaccharides and / or an oligosaccharide fraction with a degree of polymerization of 3 to 5. In preferred embodiments, Part B is provided in various formulations of complexity (low, intermediate, and high), so that the user selects the level of complexity of the biomimetic carbohydrate block based on the microorganism, the target physiological niche, or the operating conditions, without altering the general inventive principle.

[0024] 4.3 Composition of reconstituted culture medium

[0025] In a second embodiment, the invention comprises a reconstituted culture medium composition incorporating a basal fraction with nitrogen and / or peptide sources and carbon sources, optionally with buffer and / or salts, and the described biomimetic carbohydrate block. The composition is configured to support the growth and / or viability of the microorganism under a dissolved oxygen regime below the air saturation of the medium at the culture temperature, maintaining non-zero residual dissolved oxygen during at least part of the process. In preferred embodiments, the biomimetic carbohydrate block is provided at a selected total concentration within an operating range, and the N-acetylhexosamine motif represents a mass fraction of the block, allowing modulation of the signal / biomimetic substrate intensity without loss of inventive unity.

[0026] 4.4 Cultivation or fermentation method with residual dissolved oxygen

[0027] In a third embodiment, the invention provides a method for growing or fermenting at least one oxygen-sensitive microorganism comprising: heat-sterilizing a heat-stable basal fraction, cooling it, aseptically adding a heat-labile fraction containing the biomimetic carbohydrate block, inoculating the microorganism, and growing or fermenting under a dissolved oxygen regime below the air saturation of the medium at the culture temperature, maintaining non-zero residual dissolved oxygen during at least part of the culture or fermentation.

[0028] In preferred embodiments, the heat-labile fraction is sterilized by filtration prior to aseptic addition and is incorporated when the basal fraction is below a temperature threshold that preserves heat-sensitive components. The method can be performed in monoculture and / or co-culture and can be applied to selected microorganisms from strict anaerobes, microaerotolerant organisms, or facultative anaerobes with oxygen-inhibited growth, including intestinal microorganisms and those of probiotic interest.

[0029] 4.5 Preferred operating conditions: DO and redox windows

[0030] In preferred embodiments, the method operates with dissolved oxygen at selected moderate, reduced, or variable regimes, maintaining non-zero residual dissolved oxygen during at least part of the process. In further preferred embodiments, the process is carried out under a redox potential compatible with the growth and / or viability of the oxygen-sensitive microorganism, using a redox conditioner included in Part A or supplied in a separate container for aseptic addition.

[0031] In preferred embodiments, at least two selected process parameters of pH, dissolved oxygen, redox potential, turbidity or DO, and temperature are monitored, and at least one selected operating condition of stirring, gas-liquid exchange, headspace volume and / or addition of redox conditioner is adjusted to maintain a functional operating window.

[0032] 4.6 Products obtained and commercial applications

[0033] In another embodiment, the method produces a obtained or obtainable microbiological composition comprising viable biomass and / or a cell-free fermentation supernatant, optionally in combination with carriers, excipients, cryoprotectants and / or stabilizing agents, and may be presented in stabilized forms such as lyophilized, spray-dried, microencapsulated, granulated or concentrated.

[0034] In preferred commercial applications, the obtained or obtainable microbiological composition is incorporated as a culture, ferment, functional ingredient and / or biological additive in food products or beverages, and / or formulated as a nutraceutical composition or food supplement for non-therapeutic use, maintaining in all cases the same general inventive concept based on the biomimetic carbohydrate block, the A / B medularity and operation with non-zero residual dissolved oxygen.

[0035] 5. OPERATIONAL DEFINITIONS

[0036] Oxygen-sensitive microorganism: A microorganism whose growth and / or viability is inhibited, reduced, or destabilized by dissolved oxygen, requiring controlled DO conditions for reproducible performance.

[0037] Dissolved oxygen (DO): Concentration of dissolved oxygen in the culture medium, typically expressed in mg / L or as a percentage relative to saturation at the culture temperature.

[0038] Non-zero residual dissolved oxygen: Condition in which DO remains measurable and greater than 0 during at least part of the process, even below air saturation.

[0039] DO regime below air saturation: Operation with DO below the air saturation level of the medium at the culture temperature, including moderate, reduced, or variable regimes. Ultra-anaerobiosis: Operating condition intended to maintain DO practically at zero in a strict and sustained manner; the invention does not require it, since it can operate with residual DO other than zero (below air saturation) during at least part of the process, and there may be transient phases of very low DO due to biological consumption.

[0040] Glycidic biomimetic block (BBG): Mixture of carbohydrates and / or glycans, preferably aseptically added as a thermolabile fraction (Part B), that mimics glycic signals and substrates from biological niches; includes an N-acetylhexosamine motif and at least one additional fucosylated, sialylated, branched and / or sulfated component, optionally with GOS.

[0041] Functional operating window: The range of dissolved oxygen (DO) and / or redox potential within which the microorganism maintains reproducible growth and / or viability under non-zero residual dissolved oxygen. Redox potential (Eh / ORP): A measure of the oxidizing / reducing state of the medium; it can be expressed in mV and used to infer compatibility between the environment and the microorganism.

[0042] Modular kit: Set of components supplied in physically separate parts to reconstitute a culture medium, including at least a thermostable Part A and a thermolabile Part B.

[0043] Part A thermostable: Heat-sterilizable basal fraction comprising nitrogen and / or peptide sources and carbon sources, optionally with buffers and / or salts.

[0044] Thermolabile Part B: Separate fraction intended for aseptic addition after cooling of Part A, comprising the biomimetic glucide block and optionally other thermolabile components. Aseptic addition: Incorporation of Part B into the cooled medium under conditions that prevent microbial contamination, including sterile filtration where applicable.

[0045] N-acetylhexosamine motif: Unit or motif selected from N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), N-acetylmannosamine (ManNAc) and functionally equivalent derivatives.

[0046] Fucosylated component: Carbohydrate or glycan that includes fucose or a fucosylated motif, including fucosylated oligosaccharides and functionally equivalent derivatives.

[0047] Sialylated component: Carbohydrate or glycan that includes sialic acid or a sialylated motif, including sialylated oligosaccharides and functionally equivalent derivatives.

[0048] Branched component: Oligo / polysaccharide with branched architecture, including arabinogalactans, fructans, pectins or functionally equivalent fragments.

[0049] Sulfated glycan: Polysaccharide or glycan with sulfate groups, including chondroitin sulfate, dextran sulfate, heparan sulfate or functionally equivalent fragments.

[0050] Galacto-oligosaccharides (GOS): Oligosaccharides derived from galactose, capable of cooperating with the N-acetylhexosamine motif in supporting growth / viability under residual oxygen. Degree of polymerization (DP): Number of monomeric units in an oligosaccharide; DP 3-5 refers to oligosaccharides with three to five units.

[0051] Pareto-type biomimetic signature: Compositional profile of the glucide biomimetic block in which a major fraction of GOS is complemented by minor fractions of N-acetylhexosamine and fucosylated, sialylated and branched / sulfated components.

[0052] Redox conditioner: A compound added to promote a redox potential compatible with growth / viability, including cisterna, ascorbate, thioglycolate, glutathione, or combinations thereof. Monoculture: A culture or fermentation in which a single, intentionally dominant species or strain is inoculated and grown.

[0053] Co-culture: Cultivation or fermentation with two or more microorganisms intentionally inoculated or maintained as a consortium, under the same functional operating window.

[0054] Microbiological composition obtained or obtainable: Product resulting from the method comprising viable biomass and / or cell-free fermentation supernatant, optionally with carriers, excipients, cryoprotectants and / or stabilizers.

[0055] Cell-free supernatant (postbiotic): Liquid fermentation fraction with no detectable viable cells, which may contain bioactive metabolites derived from the culture.

[0056] Stabilized form: Presentation of biomass and / or supernatant in formats such as freeze-dried, spray-dried, microencapsulated, granulated or concentrated, optionally reconstitutable.

[0057] 6. DETAILED DESCRIPTION OF THE INVENTION

[0058] 6.1 Inventive principle and technical unity

[0059] The invention is based on a unitary technical solution for sustaining the growth and / or viability of oxygen-sensitive microorganisms when the process operates with non-zero residual dissolved oxygen during at least part of the culture or fermentation, without requiring ultra-anaerobiosis. The special technical contribution is embodied in the functional combination of: (i) a biomimetic carbohydrate block, (ii) a modular formulation architecture with physical separation of a thermostable basal fraction (Part A) and a thermolabile fraction that can be aseptically added (Part B), and (iii) operation under a dissolved oxygen regime below the air saturation of the medium at the culture temperature, maintaining non-zero residual dissolved oxygen during at least part of the process.

[0060] The desired technical effect includes, but is not limited to, greater robustness against micro-oxygen ingress, greater batch-to-batch reproducibility, preservation of thermolabile components, and stabilization of crop performance under realistic operating windows.

[0061] 6.2 Modular architecture of the kit and the reconstituted medium (Part A / Part B)

[0062] In one embodiment, the invention is implemented as a modular kit for preparing a culture medium, wherein the components are supplied physically separate to allow for differentiated sterilization treatments and subsequent aseptic addition.

[0063] Part A is a heat-sterilizable, thermostable base fraction comprising at least one nitrogen and / or peptide source and at least one carbon source. In preferred embodiments, Part A further comprises one or more buffers and / or salts, being adjusted to a pre-sterilization pH between, for example, 5.5 and 7.5, and having an ionic strength compatible with the growth of the target microorganism.

[0064] Part B is a physically separated, heat-labile fraction intended for aseptic addition after sterilizing and cooling Part A. Part B comprises the biomimetic carbohydrate block. In further embodiments, the kit comprises a third container holding one or more additional heat-labile components, such as vitamins, trace elements, and / or a redox conditioner for aseptic addition. In an alternative embodiment, the invention is implemented as a reconstituted culture medium composition comprising the basal fraction (functional equivalent of the reconstituted Part A) and the biomimetic carbohydrate block (functional equivalent of the added Part B), configured to operate with non-zero residual dissolved oxygen under subsaturation conditions.

[0065] 6.3 Glucose biomimetic block: composition, functions and preferred realizations

[0066] The biomimetic carbohydrate block is designed to emulate carbohydrate signals and / or substrates associated with biological microenvironments and to cooperate with process operation under non-zero residual dissolved oxygen. In one embodiment, the block comprises: (a) at least one N-acetylhexosamine motif selected from N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, or functionally equivalent derivatives; and (b) at least one additional carbohydrate component selected from fucosylated oligosaccharides, sialylated oligosaccharides, branched oligosaccharides, and / or sulfated glycans, or combinations thereof.

[0067] In preferred embodiments, the biomimetic carbohydrate block further comprises galacto-oligosaccharides, optionally including a fraction with a degree of polymerization of 3 to 5. In additional embodiments, the block includes branched components selected from arabinogalactans, fructans, pectins or functionally equivalent fragments, and / or sulfated glycans selected from chondroitin sulfate, dextran sulfate, heparan sulfate or functionally equivalent fragments.

[0068] In one embodiment, Part B is formulated to provide a total concentration of the glucose biomimetic block in the reconstituted medium between 0.05 g / L and 20 g / L. In embodiments, the N-acetylhexosamine motif represents between 5% and 95% by mass of the block, allowing modulation of the “biomimetic intensity” depending on the microorganism and operation.

[0069] In a preferred embodiment, the block exhibits a Pareto-type biomimetic signature, where, as a mass percentage of the total block, galacto-oligosaccharides represent 60% to 80%, the N-acetylhexosamine motif represents 10% to 25%, the fucosylated component represents 2% to 10%, the branched and / or sulfated glycan component represents 2% to 10%, and the sialylated component represents 2% to 10%.

[0070] 6.4 Block complexity levels and selection by use (low / medium / high)

[0071] In preferred embodiments, Part B is provided in a plurality of formulations of varying complexity, maintaining the same general inventive principle and allowing for pragmatic selection based on cost, availability of inputs, or required robustness:

[0072] (i) low complexity: N-acetylhexosamine motif and galacto-oligosaccharides;

[0073] (i) intermediate complexity: motif of N-acetylhexosamine, galacto-oligosaccharides and at least one fucosylated and / or sialylated component;

[0074] (iii) high complexity: N-acetylhexosamine motif, galacto-oligosaccharides, at least one fucosylated and / or sialylated component, and at least one sulfated glycan and / or branched polysaccharide.

[0075] These embodiments allow the biomimetic block to be adapted to microorganisms with different microenvironmental requirements, without introducing a second general inventive concept. 6.5 Preparation of the medium: differential sterilization and aseptic addition

[0076] In one embodiment, Part A is reconstituted in an aqueous vehicle, optionally adjusted for pH and salts, and then heat-sterilized. A representative heat regime comprises, for example, treatment equivalent to 115°C to 125°C for 10 to 30 minutes. The sterilized Part A is then cooled before adding Part B.

[0077] In a preferred embodiment, Part B is prepared as a heat-labile solution and sterilized by filtration through a filter with a pore size of 0.45 µm or smaller, optionally 0.22 µm or smaller. Part B is incorporated by aseptic addition when the cooled basal fraction is at 45 °C or lower, reducing thermal degradation of heat-labile components and preserving the functionality of the biomimetic block.

[0078] 6.6 Method of cultivation or fermentation under non-zero residual dissolved oxygen

[0079] In one embodiment, the method comprises: (i) providing the heat-stable basal fraction; (ii) heat-sterilizing and cooling; (iii) aseptically adding the heat-labile fraction comprising the biomimetic carbohydrate block; (iv) inoculating at least one microorganism; and (v) culturing or fermenting under a dissolved oxygen regime below the air saturation of the medium at the culture temperature, maintaining non-zero residual dissolved oxygen during at least part of the process.

[0080] In preferred embodiments, the process is carried out in a closed vessel without continuous purging of inert gas, so that oxygen control is achieved by a combination of vessel configuration, headspace volume, mixing conditions, and limitation of gas-liquid exchange, accepting the presence of non-zero residual oxygen within a functional window. The method can be carried out in monoculture and / or coculture. In coculture, the biomimetic carbohydrate block and the operational window are configured to support a microbial consortium under conditions where strict oxygen exclusion is impractical or not reproducible at scale.

[0081] 6.7 Operating windows for DO and redox conditioning (Eh / ORP)

[0082] In preferred embodiments, the culture or fermentation is carried out under a dissolved oxygen (DO) regime below air saturation, by operating in a closed vessel and adjusting the gas-liquid exchange, mixing conditions, and headspace volume, without requiring continuous purging of inert gas. The DO regime comprises at least one of: (a) moderate DO (>0.5 mg / L and up to 5 mg / L), (b) reduced DO (>0 mg / L and <0.5 mg / L), and (c) variable DO (fluctuating between moderate and reduced), which may start transiently at moderate and transition to reduced due to biological consumption. In representative embodiments, the process operates under microaerobic conditions (e.g., 0.05–0.50 mg / L during at least part of the process). In co-cultivation, combined consumption can temporarily bring the DO to very low or near zero values, which may be compatible with performance, without implying the need to impose ultra-anaerobiosis through external actions.The DO is measured in mg / L using a calibrated sensor; additionally, the process can be controlled by redox potential (Eh / ORP) measured in mV, adjusting when necessary using compatible redox conditioners (e.g., cysteine, ascorbate, thioglycolate or glutathione), incorporated in Part A or supplied for aseptic addition.

[0083] 6.8 Monitoring and Operational Adjustments (Optional Implementation) During culture or fermentation, at a minimum, DO (mg / L), Eh / ORP (mV), pH, and temperature are monitored and recorded at the beginning (t=0) and at reproducible control points (e.g., 12, 24, and 48 h; preferably every 4–6 h during the first 24 h and then every 12–24 h), with aseptic sampling via a septum or port to maintain the closed vessel character. The operation is adjusted to maintain a functional operating window compatible with the microorganism and the process objective, using the following control variables: (i) mixing conditions (static or limited agitation, modulating gas-liquid transfer), (ii) working volume / headspace ratio, and (iii) redox conditioning by aseptic addition of compatible agents when the ORP deviates from the defined operating range.If the DO rises above the desired range, gas-liquid transfer is reduced (e.g., by decreasing effective mixing and / or adjusting headspace). If the DO falls to very low or near-zero values, this condition is maintained, or oxygen availability is moderately increased according to the process objective, without requiring continuous purging of inert gas. Adjustments are documented along with DO / ORP / pH and correlated with performance indicators (e.g., CFU / mL and / or metabolic product) to ensure batch-to-batch repeatability.

[0084] 6.9 Target microorganisms and breadth by niche (without limitation to a single strain)

[0085] In embodiments, the oxygen-sensitive microorganism comprises a strict, microaerotolerant, or facultative anaerobe whose growth is inhibited by oxygen. In preferred embodiments, it is selected from intestinal microorganisms / probiotics, including lactic acid bacteria, bifidobacteria, short-chain fatty acid-producing bacteria, and mucinolytic bacteria, or combinations thereof. In a preferred embodiment, the method comprises Akkermansia and / or Bifidobacterium microorganisms, optionally co-cultured with at least one lactic acid bacterium, without limiting the applicability of the medium / kit / method to other oxygen-sensitive microorganisms, while maintaining the inventive unity anchored to the same specific technical contribution.

[0086] 6.10 Performance and functional equivalence criteria

[0087] In practical applications, the cultivation / fermentation produces biomass with a viable concentration of at least 10 7CFU / mL. Under comparable conditions and for a predetermined time, the medium with the biomimetic carbohydrate block provides a viability increase of at least 1 10⁻⁹ compared to a control lacking said block. For the purposes of the invention, “functionally equivalent derivatives / fragments” are compounds that, in the context of the method and under non-zero residual OD, contribute comparably to sustaining growth and / or viability within the functional operating window.

[0088] 6.11 Composition of microbiological compounds obtained or obtainable and stabilized forms

[0089] In embodiments, the method produces a obtained or obtainable microbiological composition comprising: (i) viable biomass of at least one oxygen-sensitive microorganism and / or (ii) cell-free fermentation supernatant, optionally with carrier, excipient, cryoprotectant, and / or stabilizing agent. In embodiments, it is presented in a selected stabilized form of lyophilized, spray-dried, microencapsulated, granulated, or concentrated form, optionally reconstitutable in an aqueous or food-grade vehicle, facilitating logistics, storage, dosage, and reconstitution. In embodiments, the composition is incorporated as a culture / ferment / functional ingredient / biological additive in foods or beverages (including fermented products), or formulated as a nutraceutical / food supplement for non-therapeutic use, optionally combined with prebiotics, fibers, proteins, minerals, vitamins, and / or acceptable excipients.

[0090] 7. EXAMPLES / REALIZATIONS (DATA AND VALIDATION)

[0091] The following examples are non-limiting and are presented to demonstrate enableability, reproducibility, and the technical effect of the inventive core: (i) biomimetic glucide block, (ii) modular Part A / Part B architecture (thermostable fraction + thermolabile fraction), and (iii) operation under a dissolved oxygen regime below air saturation while maintaining non-zero residual dissolved oxygen during at least part of the process, without requiring ultra-anaerobiosis.

[0092] 7.0 Measurement and validation methods (applicable to all examples)

[0093] In all examples, the method's performance is validated by standardized measurements of viability / growth, physicochemical process conditions, and, where applicable, metabolic products. Viability is quantified as CFU / mL by plate counting on appropriate media (selective, differential, or non-selective depending on the microorganism) or by an equivalent validated method; dilutions, incubation conditions, and counting criteria are kept constant between assay and control. Dissolved oxygen (DO) is measured in mg / L using a previously calibrated electrochemical or optical sensor (including temperature correction where applicable) and recorded along with pH and temperature; redox potential (Eh / ORP) is measured in mV using a calibrated ORP electrode.For comparison, an illustrative control is run in parallel, maintaining the same inoculum, Part A, vessel, working volume / headspace, temperature, and sampling schedule, but whose Part B lacks the biomimetic carbohydrate block, replacing it with a simple carbohydrate and / or a low-complexity, non-biomimetic prebiotic. When metabolites are evaluated, they are quantified in the cell-free supernatant (sterile filtrate) using an appropriate analytical method and reported in concentration units (e.g., mg / L). Results are expressed as absolute values ​​and, comparatively, as an increase relative to the control (e.g., difference in 100 CFU / mL) and / or compliance with defined performance thresholds for the process.

[0094] 7.0.1 Table / Matrix of Preferred Realizations

[0095] The following matrix summarizes preferred (non-limiting) operating combinations of the modular kit Part A / Part B and the culture / fermentation method under a dissolved oxygen regime below air saturation, maintaining non-zero residual dissolved oxygen during at least part of the process.

[0096] Table 7-0. Preferred Realizations Matrix (non-limiting). Realization Axis Preferred Option 1 Preferred Option 2 Preferred Option 3

[0097] Part A thermoset and

[0098] Architecture of the Equal Equal kit

[0099] Part B thermoladil

[0100] Part A heat sterilizable; Part B

[0101] Sterilization sterilizable by

[0102] Equal differential sterile filtration and aseptic incorporation after cooling

[0103] Aseptic addition at compatible temperature

[0104] Thermal window with stability for addition of components Equal Equal Part B thermolabiles (e.g., equal to or less than 45 °C)

[0105] Reason for N- Reason for N-

[0106] Composition Motif of N-acetylhexosamine and to the acetylhexosamine and to the functional of acetylhexosamine and less one less one component Part B (BBG) oligosaccharides of additional component branched and / or fucosylated and / or sialylated sulfated

[0107] Reduced tariff scheme with phases

[0108] Variable (fluctuating) dissolved oxygen, very low transient levels

[0109] ° . Moderate or reduced between moderate and

[0110] (lower than due to biological consumption, reduced air saturation) without continuous purging

[0111] Additional Adjustment Combination of the

[0112] Mixing / transfer strategies using previous gas-liquid control and conditioning documented by headspace redox when applicable process variables

[0113] Variables of DO, ORP, pH and DO, ORP, pH and DO, ORP, pH and temperature monitoring temperature temperature Implementation axis Preferred option 1 Preferred option 2 Preferred option 3

[0114] Microorganisms

[0115] Compatible strains of the genus Cocultivos

[0116] Strict anaerobic microorganisms, Akkermansia and / or under the same target window (examples) microaerotolerant or oxygen-inhibited Bifidobacterium

[0117] Viable biomass and

[0118] Products Free of supernatant

[0119] Viable biomass free of resulting cells

[0120] Measurable increase in viability

[0121] Quantified viability

[0122] Criteria regarding the control as CFU / mL and performance... Equally illustrative (e.g., comparison against (indicative) on the order of at least one illustrative control, a logarithmic magnitude)

[0123] 7.0.2 Operational measurement definitions and validation criteria

[0124] For the purposes of reproducibility and comparison between test and control, the following definitions and operational criteria apply:

[0125] 1. Dissolved oxygen (DO): is expressed as a concentration in mg / L and is measured with a calibrated electrochemical or optical sensor, applying temperature corrections where appropriate. “Non-zero residual DO” means a measurable DO greater than zero during at least part of the process.

[0126] 2. Redox potential (ORP / Eh): expressed in mV and measured with a calibrated ORP electrode. 3. pH and temperature: recorded using a probe or calibrated instrument standard for use in bioprocesses.

[0127] 4. Recording of variables: DO, ORP, pH and temperature are recorded at the beginning and at reproducible points throughout the process (e.g., at intervals of hours during the first 24 h and subsequently at intervals of 12-24 h), maintaining equivalent sampling conditions between trial and control.

[0128] 5. Viability / growth: quantified as CFU / mL by plate count on appropriate media, using consistent dilutions, incubation, and counting criteria between assay and control. 6. Illustrative control: defined as a process under equivalent conditions of vessel, working volume, headspace, temperature, inoculum, and sampling schedule, in which Part B lacks BBG and is replaced by a simple carbohydrate and / or a less complex, non-biomimetic prebiotic.

[0129] 7. Cell-free supernatant: obtained by biomass separation and sterile filtration where applicable; it is considered “cell-free” when no viable cells are detected under the applied control method or when it has been sterilely filtered through a suitable pore membrane.

[0130] 8. Metabolites (when applicable): are quantified in the cell-free supernatant by an appropriate analytical method and reported in concentration units.

[0131] 7.1 Example 1 - Pareto-type biomimetic block without sustained ultra-anaerobiosis

[0132] 7.1.1 Objective

[0133] To demonstrate that a Pareto-type biomimetic carbohydrate block, implemented in a Part A / Part B modular architecture, allows for high cell densities and viability under non-zero residual OD, without requiring sustained ultra-anaerobic infrastructure.

[0134] 7.1.2 Representative microorganism

[0135] An oxygen-sensitive microorganism is selected for its susceptibility to transient increases in oxygen concentration (OD) during preparation, sampling, or transfer. As a non-limiting example, a fastidious intestinal microorganism, e.g., Bifidobacterium longum subsp. infantis (strain M-63), may be used, although this strain is not required; other oxygen-sensitive strains and / or microorganisms may be used in accordance with the claims.

[0136] 7.1.3 Formulation design (modular architecture Part A / Part B)

[0137] Part A (thermostable; heat sterilizable): basal medium with at least one source of nitrogen and / or peptides, at least one source of carbon, buffer and salts; optionally includes a redox conditioner compatible with heat sterilization.

[0138] Part B (thermolabile; aseptic addition): biomimetic carbohydrate block with Pareto-type signature configured, in relative mass of the total block, as: galacto-oligosaccharides -70%; N-acetylhexosamine motif -15%; fucosylated component -5%; branched component -5%; sialylated component -5%.

[0139] Illustrative control: formulation with the same Part A, but in Part B it lacks the biomimetic carbohydrate block (BBG), replacing it with a simple carbohydrate and / or a low-complexity non-biomimetic prebiotic.

[0140] 7.1.4 Summary procedure

[0141] Part A (thermostable) of the medium is prepared in a closed container (e.g., airtight flask with septum or closed bioreactor), maintaining a reproducible working volume / headspace ratio (preferably 40-80% v / v of volume and 20-60% v / v of headspace), is heat sterilized and cooled to <45 °C; in parallel, Part B (thermolabile) comprising the biomimetic carbohydrate block (BBG) is prepared in solution and sterilized by sterile filtration (preferably 0.22 m), then aseptically added to Part A and inoculated with the microorganism, operating without continuous purging of inert gas and with limited agitation (static or minimal mixing for homogeneity) so that the process maintains residual DO >0 mg / L and <5 mg / L during at least part of the culture;DO, pH and ORP are monitored and sterile samples are taken for CFU / mL at least at 0, 12, 24 and 48 h (preferably every 4-6 h for the first 24 h), comparing against an illustrative control with the same Part A but whose Part B lacks BBG, replacing it with a simple carbohydrate and / or a low-complexity non-biomimetic prebiotic.

[0142] Table 7-1. Follow-up under non-zero residual DO with Pareto-type biomimetic block (mean ± standard deviation; n=3).

[0143] Time (h) Temperature (°C) pH DO (mg / L) Conductivity (pS / cm) Turbidity (NTU) CFU / mL 12 37.0 ± 0.1 6.40 ± 0.05 0.50 ± 0.05 1505 ± 12 25.3 ± 2.0 (1.0 ± 0.2)x10 A 9 24 37.1 ± 0.1 6.00 ± 0.03 0.10 ± 0.02 1550 ± 15 80.2 ± 3.5 (4.5 ± 0.15)x10 A 9 36 37.2 ± 0.1 5.80 ± 0.04 0.05 ± 0.02 1610 ± 20 180 ± 5.0 (4.8 ± 0.10)x10 A 9

[0144] 48 37.1 ± 0.2 5.60 ± 0.05 0.05 ± 0.02 1670 ± 25 220 ± 8.0 (3.8 ± 0.15)x10 A 9

[0145] 60 37.0 ± 0.2 5.50 ± 0.05 0.05 ± 0.02 1740 ± 30 250 ± 10.0 (2.5 ± 0.20)x10 A 9

[0146] 72 37.0 ± 0.2 5.40 ± 0.04 0.05 ± 0.02 1800 ± 35 300 ± 12.0 (1.5 ± 0.25)x10 A 9

[0147] Technical interpretation: the process maintains non-zero residual DO (typically 0.05-0.50 mg / L) without sustained ultra-anaerobiosis, with viability on the order of 10 A 9 CFU / mL and behavior consistent with the modular architecture and the biomimetic block.

[0148] 7.1.6 Pareto vs. control comparison (without strict inertization)

[0149] Table 7-2. CFU / mL for Pareto type biomimetic block vs control under conditions without continuous purging of inert gas.

[0150] Time (h) Pareto (CFU / mL) Control (CFU / mL) 0 ~1x10 A 7 ~1 xl0 A 7 12 ~1 xlO A 9 ~2x10 A 8 24 ~(4-6)x10 A 9 ~(2-3)x10 A 8 36 ~(4-6)x10 A 9 ~(4-6)x10 A8 48 ~(3-5)x10 A 9 ~(2-4)x10 A 8 60 ~(2-3)x10 A 9 ~(1-3)x10 A 8 72 ~(1-2)x10 A 9 ~(<3x10 A 8)

[0151] Validation of the technical effect: in the 12-48 h windows, the Pareto-type biomimetic block shows typical increases of ~1 Iog10 or higher compared to the control under non-zero residual OD, consistent with support of growth / viability without requiring ultra-anaerobiosis.

[0152] 7.1.7 Description of the drawings

[0153] Figure 1 compares the viability (CFU / mL) over time between a medium with a Pareto-type biomimetic block and a control without said block, under equivalent conditions, according to the method of the invention: culture in a closed container, without continuous purging of inert gas, maintaining non-zero residual dissolved oxygen (0 < OD < 5 mg / L) during at least part of the process. Figure 2 compares the microbial viability (CFU / mL) over time between (i) a reconstituted medium with a Pareto-type biomimetic block (BBG) and (ii) a control without BBG, where Part B is replaced by a simple carbohydrate and / or a non-biomimetic prebiotic, under equivalent conditions, according to the method of the invention: culture in a closed container, without continuous purging of inert gas and with non-zero residual dissolved oxygen (0 < OD < 5 mg / L) during at least part of the process.

[0154] Figure 3 schematically illustrates the operational flow for preparing a culture medium from a heat-stable basal fraction (Part A) and a heat-labile fraction (Part B, comprising BBG), and for carrying out the cultivation or fermentation of at least one oxygen-sensitive microorganism in a closed vessel without continuous purging, with monitoring and control of the operating conditions. In particular, the figure illustrates: (1) the heat sterilization of Part A; (2) subsequent cooling; (3) the aseptic addition of Part B to reconstitute the medium; (4) the inoculation of the microorganism; (5) the operation of the cultivation / fermentation in a closed vessel without continuous purging; (6) the control of the process by adjusting the mixture, headspace, and / or redox conditions, with measurement of variables such as dissolved oxygen, redox potential, pH, and temperature; and (7) obtaining the product, comprising viable biomass and / or cell-free supernatant.

[0155] 7.2 Example 2 - Intestinal embodiment with Akkermansia: A / B kit with fucosylated / sialylated booster / Intestinal embodiment with Akkermansia

[0156] 7.2.1 Objective

[0157] Demonstrate a highly demanding intestinal niche implementation, maintaining the same inventive core: Part A / Part B medullary, biomimetic glucide block and operation with residual OD other than zero.

[0158] 7.2.2 Formulation per 1 L final

[0159] Part A (heat-stable; heat sterilizable): yeast extract 2 g / L; peptone or tryptone 2 g / L; total phosphates 2 g / L; NaCl 0.5 g / L (optional); cysteine ​​HCl 0.3 g / L; and carbon source compatible with the target microorganism (when applicable). Adjust pH to 6.8–7.0.

[0160] Part B (heat-labile; aseptic addition): comprises the biomimetic glucose block (BBG) and includes GIcNAc (1.5 g / L), a branched polysaccharide (e.g., arabinogalactan) (0.5 g / L), and at least one additional glucose component selected from fucosylated and / or sialylated biomimetic oligosaccharides (e.g., 2 g / L total), and optionally heat-labile vitamins or other cofactors. Part B is prepared as an aqueous solution and sterilized by sterile filtration (preferably through a 0.22 µm filter) for aseptic addition to the medium.

[0161] 7.2.3 Procedure

[0162] Dissolve Part A in 800-900 mL of sterile water, adjust pH to 6.8-7.0 and sterilize by heat (e.g., 121 °C for 15-20 min).

[0163] Cool to <40-45 °C.

[0164] Dissolve Part B in 50-100 mL of sterile water at room temperature and filter sterile (0.22 pm).

[0165] Aseptically add Part B to the container with cooled Part A and make up to 1 L with sterile water. Inoculate the microorganism and operate in static culture or with limited agitation, without continuous purging of inert gas, maintaining a non-zero residual OD within a functional window.

[0166] 7.2.4 Representative performance data (residual OD window and viability)

[0167] Table 7-3. Results in culture for 72 h, under residual DO other than zero (typical values; n=3).

[0168] Time (h) Temperature (°C) pH DO (mg / L) CFU / mL 0 37 6.9 0.8-2, 0 ~1 x 10 A 7 24 376, 3-6, 6 0.10-0.40 ~(0.5-2)x10 A 8 48 376, 0-6, 3 0.05-0.20 ~(1-5)x10 A 8 72 37 5, 8-6, 2 0.05-0.15 ~(0.5-3)x10 A 8

[0169] Validation: plate count under anaerobic or microaerophilic conditions suitable for the microorganism, with contamination control (blank plates) and confirmation of morphology / identity by compatible microbiological or molecular method.

[0170] 7.3 Example 3 - Monoculture and co-culture: robustness under variable OD with the same inventive core

[0171] 7.3.1 Objective

[0172] Demonstrate that the method allows operation in monoculture and / or co-culture, and that it tolerates moderate / reduced / variable DO while maintaining a non-zero residual DO, preserving the inventive unity.

[0173] 7.3.2 Design

[0174] A reconstituted medium of Part A / Part B is used according to the examples above. Two conditions are run: (i) monoculture of an oxygen-sensitive microorganism; (ii) co-culture of a mucinolytic microorganism and a compatible fermentative microorganism (e.g., lactic acid bacteria or bifidobacteria), under the same OD window.

[0175] 7.3.3 Procedure and regime DO

[0176] The reconstituted medium is prepared by combining heat-sterilized and cooled (<45 °C) Part A with aseptically added, heat-labile Part B, where Part B comprises the biomimetic carbohydrate block (BBG). The culture is carried out in a closed vessel, without continuous purging of inert gas, with a reproducible working volume / headspace ratio and with limited mixing (static or minimal agitation for homogeneity), so that oxygen availability comes primarily from the headspace and controlled gas-liquid transfer. The process can be initiated at a moderate OD to favor the establishment of the microorganism(s), and subsequently, the transition to a reduced OD can be induced primarily by biological oxygen consumption in the closed system and / or by controlled reduction of gas-liquid transfer (e.g.,, decreasing effective mixing and / or adjusting headspace), allowing for a reduced or variable flow rate (fluctuating between moderate and reduced) depending on the crop kinetics.

[0177] In co-culture, combined consumption can temporarily lower the DO to very low values; this condition can be compatible with performance without requiring continuous purging or externally imposed strict anaerobiosis. During the process, DO, pH, and Eh / ORP are monitored at predefined points, and operational adjustments (effective mixing, headspace, and / or compatible redox conditioning) are made to maintain reproducible conditions appropriate for the microorganism and the process objective. Aseptic samples are taken to quantify CFU / mL and, when applicable, metabolites in cell-free supernatant, compared to an illustrative control prepared under the same conditions but without BBG in Part B.

[0178] 7.3.4 Performance data in co-cultivation

[0179] Table 7-4. Example of co-cultivation under variable DO (typical values; n=3).

[0180] Hour (h) OD (mg / L) pH CFU / mL total (consortium) 0 0.8-2.0 6, 8-7, 0 ~1x10 A 7

[0181] 12 0.30-0.806, 3-6.6 ~(2-8)x10 A 8

[0182] 24 0.05-0.20 5.9-6.2 ~(1-6)x10 A 9

[0183] 48 0.05-0.20 5.6-6.0 ~(0.5-4)x10 A 9

[0184] Validation: quantification by CFU / mL total and, when disaggregation by species is required, plating in differential / selective media or quantification by equivalent compatible method.

[0185] 7.4 Example 4 - Composition, microbiology and stabilization (external validation; formulations with staged performance)

[0186] 7.4.1 Objective

[0187] Demonstrate that the method produces a obtained or obtainable microbiological composition comprising (i) viable biomass of the oxygen-sensitive microorganism and / or (ii) a cell-free fermentation supernatant (postbiotic), and that these fractions can be recovered and stabilized for industrial use, including their presentation in stabilized and / or reconstitutable forms. An independent external validation (University of Costa Rica - Anaerobic Bacteriology Research Laboratory) evaluates scalable formulations of increasing complexity and quantifies performance in biomass (CFU / mL) and total metabolic product (mg / L), without limiting the scope of the invention.

[0188] 7.4.2 Representative procedure for recovery, stabilization and external validation

[0189] At the end of the cultivation (e.g., close to maximum viability), the biomass is separated by physical operations (e.g., centrifugation or filtration) and supernatant is obtained.

[0190] • Viable biomass: it is conditioned with a compatible protectant and stabilized by an industrial drying process (e.g., freeze-drying or spray-drying) preserving viability.

[0191] • Cell-free supernatant: clarified and sterile filtered to obtain a cell-free filtrate, optionally concentrated.

[0192] External validation replicates representative runs and quantifies (i) CFU / mL and (ii) total metabolic product (mg / L), comparing F1-F4 formulations.

[0193] 7.4.3 Data (performance by formulation and retention after stabilization) Table 7-5A. Comparative performance by formulation (representative values; external validation).

[0194] • F1 (basic): ~10 A9 CFU / mL; -1-3 mg / L

[0195] • F2 (intermediate): ~(2-4)x10 A 9 CFU / mL; ~3-6 mg / L

[0196] • F3 (advanced): ~(3-6)x10 A 9 CFU / mL; ~5-9 mg / L

[0197] • F4 (specialized): ~(4-7)x10 A 9 CFU / mL; -7-12 mg / L

[0198] Table 7-5. Retention of viability after stabilization (typical values; n=3).

[0199] • Before stabilization: ~(1-5)X 10 A 9

[0200] • After stabilization: ~(3X10 A 8-3X 10 A 9)

[0201] • Relative retention: ~30%-80%

[0202] Validation: plate count after reconstitution, compared against pre-stabilization control; the metabolic product is reported as total concentration (mg / L) by appropriate analytical method.

[0203] 7.5 Additional realizations per niche library (same inventive core)

[0204] Without limitation, the Part A / Part B architecture can be adapted as a library of kits / media for different niches by adjusting the signature of the biomimetic carbohydrate block and cofactors without altering the general inventive principle. In embodiments, the complexity of the block (low / intermediate / high) is increased to enhance tolerance to residual and variable DO, while maintaining the A / B medullary structure and the aseptic addition of the thermolabile fraction.

[0205] 7.6 Practical aspects of scaling

[0206] At larger scales, thermostable / thermolabile separation reduces degradation of heat-sensitive components and facilitates batch traceability. The redox regime is maintained within the functional window through a combination of reactor geometry, gas-liquid transfer, agitation, headspace volume, selection of the biomimetic block complexity level, and, where applicable, mild redox conditioning. In fed-batch or continuous operation, core integrity is preserved through Part A feeding and Part B aseptic pulses.

[0207] 7.7 Functional equivalents (acceptance criteria)

[0208] For the purposes of the invention, a “functional equivalent” of a component of the biomimetic carbohydrate block is one that, in combination with the Part A / Part B modular architecture and under a non-zero residual OD, verifiably contributes to sustaining growth and / or viability within a functional operating window. A representative validation criterion comprises achieving at least 10 A 7 CFU / mL and / or demonstrate a viability increase of at least 1 log10 compared to a control lacking the glucose biomimetic block, under comparable process conditions. 8. INDUSTRIAL APPLICABILITY

[0209] 8.1 Industrial nature and scalability

[0210] The invention is industrially applicable as it provides a kit, a reconstituted culture medium, and a reproducible method for cultivating or fermenting oxygen-sensitive microorganisms under non-zero residual dissolved oxygen, without requiring ultra-anaerobic conditions. The modular Part A / Part B architecture is compatible with standard manufacturing operations, thermal sterilization, quality control, and scale-up, including batch, fed-batch, and, where applicable, semi-continuous processes. The separation of a heat-sterilizable, heat-stable fraction (Part A) and a heat-labile fraction that can be aseptically added (Part B) facilitates: (i) stability of heat-sensitive components, (ii) reduced degradation during sterilization and storage, (iii) batch traceability and standardization, and (iv) niche configuration flexibility without altering the overall inventive concept.

[0211] 8.2 Main industrial sectors

[0212] The invention is applicable, without limitation, in the following sectors:

[0213] (i) Biotechnology and industrial fermentations: production of viable microbial biomass and / or cell-free fermentation supernatants under realistic operating windows of DO and redox, with robustness against micro-oxygen inputs and process disturbances.

[0214] (i) Food and beverage industry: preparation of starter cultures, ferments, functional ingredients and / or biological additives for food and beverage products, including fermented products, by enabling processes without ultra-anaerobic infrastructure and with improved reproducibility.

[0215] (iii) Nutraceuticals and food supplements (non-therapeutic use): formulation of compositions based on viable biomass and / or derived postbiotics, optionally stabilized (e.g., freeze-dried, microencapsulated, granulated or concentrated), for incorporation into supplement matrices or functional ingredients.

[0216] (iv) Biotechnology laboratories and services: development and maintenance of fastidious microbial consortia and microorganisms in environments where absolute oxygen exclusion is impractical, and where core formulation and operational control are required.

[0217] 8.3 Obtainable products and value chains

[0218] The invention enables the production of: (i) viable biomass at industrial concentrations, (ii) cell-free supernatants (postbiotics) with fermentation-derived metabolites, and (iii) stabilized forms suitable for logistics and formulation. These products can be integrated as intermediate or final components in fermentation value chains, functional foods, and supplements, maintaining the same special technical core (carbohydrate biomimetic block + A / B medulla + residual OD).

[0219] 8.4 Technical advantages with industrial impact

[0220] In industrial implementations, the invention provides, without limitation: • Less dependence on ultra-anaerobiosis: enables operation under DO regime below saturation with residual DO other than zero, reducing the need for continuous purging of inert gas and specialized equipment.

[0221] • Greater operational robustness: increases tolerance to process disturbances that introduce transient oxygen (e.g., sampling, inoculation, transfers, and mixing).

[0222] • Improved consistency and quality: The modular A / B architecture protects thermolabile components and promotes batch-to-batch standardization through controlled formulation of the biomimetic block.

[0223] • Cost-performance flexibility: allows supplying Part B at levels of complexity (low / intermediate / high) to optimize performance according to microorganism, niche and scale, without altering the inventive unit.

[0224] • Compatibility with stabilization: facilitates obtaining viable biomass and / or cell-free supernatant (postbiotic) in stabilized forms for distribution and industrial use.

[0225] 8.5 Representative Implementations

[0226] The invention can be implemented in simple closed vessels (flasks or tanks with limited / controlled agitation) as well as in laboratory, pilot, and industrial bioreactors, maintaining an operation aimed at minimizing oxygen exposure and reasonably controlling the physicochemical conditions of the culture. In plant settings, it is integrated into standard practices through thermal sterilization of the heat-stable fraction and subsequent aseptic addition of heat-labile components via sterile filtration, according to the modular A / B architecture, to reduce thermal degradation, improve batch-to-batch reproducibility, and facilitate formulation logistics. The operation may include monitoring and control of key variables (pH, DO, and redox potential) by adjusting gas-liquid transfer, agitation, and redox conditioning to maintain a stable and repeatable window.In all modalities the central technical concept is preserved: A / B modularity + biomimetic support + operating regime compatible with oxygen-sensitive microorganisms.

[0227] 8.6 Conclusion of industrial applicability

[0228] The invention is industrially applicable by providing a modular, biomimetic platform that enables the cultivation or fermentation of oxygen-sensitive microorganisms under realistic operating conditions, including operation with non-zero residual dissolved oxygen, reducing dependence on ultra-anaerobiosis and increasing tolerance to micro-oxygen inputs associated with process operations, while maintaining a functional operating window.

[0229] Reproducibility and scalability are based on the A / B architecture (heat-sterilizable thermostable fraction and aseptically added thermolabile fraction) and on biomimetic blocks that preserve the performance of the target microorganism, being compatible with standard instrumentation and consolidated industrial practices, which facilitates batch-to-batch standardization, technology transfer and the obtaining of viable biomass and / or cell-free fractions for non-therapeutic commercial applications, preserving technical coherence and inventive unity.

Claims

CLAIMS It is claimed:

1. A modular kit for preparing a culture medium for the cultivation or fermentation of at least one oxygen-sensitive microorganism under a non-zero residual dissolved oxygen regime, comprising: (i) a physically separated, heat-sterilizable, thermosetting Part A comprising at least one nitrogen and / or peptide source and at least one carbon source, optionally with one or more buffers and / or salts; and (i) a physically separate, heat-labile Part B suitable for aseptic addition after sterilizing and cooling Part A, comprising a biomimetic glucidic block including (a) at least one N-acetylhexosamine motif selected from N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine, and (b) at least one additional glucidic component selected from fucosylated oligosaccharides, sialylated oligosaccharides, branched oligosaccharides and / or sulfated glycans, or combinations thereof; wherein the kit is for use in a method according to claim 3.

2. A reconstituted culture medium composition for the cultivation or fermentation of at least one oxygen-sensitive microorganism under a non-zero residual dissolved oxygen regime, comprising: (i) a basal fraction that includes at least one source of nitrogen and / or peptides and at least one source of carbon, optionally with buffer and / or salts; and (i) a biomimetic glucidic block comprising (a) at least one N-acetylhexosamine motif selected from N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine, and (b) at least one additional glucidic component selected from fucosylated oligosaccharides, sialylated oligosaccharides, branched oligosaccharides and / or sulfated glycans, or combinations thereof; wherein said composition is obtainable by mixing Part A and Part B of the kit of claim 1, and is for use in a method according to claim 3.

3. A method for cultivating or fermenting at least one oxygen-sensitive microorganism in the presence of non-zero residual dissolved oxygen, comprising: (i) provide a thermostable basal fraction that includes at least one source of nitrogen and / or peptides and at least one source of carbon; (i) sterilize said basal fraction by heat and then cool it; (iii) aseptically adding, after cooling, a thermolabile fraction comprising a biomimetic carbohydrate block including (a) at least one N-acetylhexosamine motif selected from N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine, and (b) at least one additional carbohydrate component selected from fucosylated oligosaccharides, sialylated oligosaccharides, branched oligosaccharides and / or sulfated glycans, or combinations thereof; (iv) inoculate at least one microorganism; and (v) cultivate or ferment at least one microorganism in monoculture and / or co-culture in a closed container, without continuous purging of inert gas, maintaining a dissolved oxygen (DO) greater than 0 mg / L and less than or equal to 5 mg / L during at least part of the process.

4. The kit according to claim 1, wherein Part B is formulated to provide, in the reconstituted medium, a total concentration of the glucide biomimetic block of 0.05 g / L to 20 g / L, wherein the N-acetylhexosamine motif represents between 5% and 95% by mass of the glucide biomimetic block.

5. The kit according to claim 4, wherein the N-acetylhexosamine motif comprises N-acetylglucosamine, and wherein the biomimetic glucose block further comprises at least one fucosylated component and at least one sialylated component.

6. The kit according to any of claims 1, 4 or 5, wherein Part B further comprises a branched oligosaccharide and / or polysaccharide selected from arabinogalactans, fructans, pectins, and derivatives and / or fragments thereof.

7. The kit according to any of claims 1 to 6, wherein Part A further comprises: (i) a buffer selected from phosphates, citrates, carbonates or combinations thereof; and (ii) at least one salt selected from chlorides, sulfates or phosphates of sodium, potassium, calcium or magnesium, Part A being adjusted to a pre-sterilization pH of 5.5 to 7.

5.

8. The method according to claim 3, wherein the aseptic addition of the heat-labile fraction of step (iii) comprises sterilizing said fraction by filtration through a filter having a pore size of 0.45 m or less, preferably 0.22 m, and incorporating it when the cooled basal fraction is at a temperature of 45 °C or less.

9. The method according to claim 3 or 8, wherein the cultivation or fermentation is carried out in a closed container without continuous purging of inert gas, maintaining the dissolved oxygen (DO) in a range of 0.05 to 0.50 mg / L for at least 12 hours of the process.

10. The kit according to any of claims 1 to 7, wherein Part A and Part B are supplied in physically separate packages or sachets, comprising at least a first package for Part A and a second package for Part B, and optionally including a third package containing one or more additional thermolabile components selected from vitamins, trace elements and / or a redox conditioner, for aseptic addition.

11. The kit according to any of claims 1, 4 to 7 or 10, wherein the biomimetic glucose block of Part B comprises galacto-oligosaccharides (GOS).

12. The kit according to any of claims 1, 4 to 7, 10 or 11, wherein at least one of the glucose components of the glucose biomimetic block comprises an oligosaccharide with a degree of polymerization (DP) of 3 to 5.

13. The kit according to any of claims 1, 4 to 7 or 10 to 12, wherein the additional glucide component of the glucide biomimetic block comprises at least one fucosylated component selected from fucose, fucosyl-lactose, fucosylated oligosaccharides, and combinations thereof.

14. The kit according to any of claims 1, 4 to 7 or 10 to 13, wherein the additional glucide component of the glucide biomimetic block comprises at least one sialylated component selected from N-acetylneuraminic acid, sialyl-lactose, sialylated oligosaccharides, and combinations thereof.

15. The kit according to any of claims 11 to 14, wherein the biomimetic glucose block comprises a Pareto-type biomimetic signature in which, as a mass percentage of the total biomimetic glucose block: (i) galacto-oligosaccharides represent from 60% to 80%; (ii) the N-acetylhexosamine motif represents from 10% to 25%; (iii) the fucosylated component represents from 2% to 10%; (iv) the branched and / or sulfated glycan component represents from 2% to 10%; and (v) the sialylated component represents from 2% to 10%.

16. The kit according to any of claims 1, 4 to 7 or 10 to 15, wherein Part B is provided in a plurality of formulations selected from: (i) a low complexity formulation comprising the N-acetylhexosamine motif and galacto-oligosaccharides; (ii) an intermediate complexity formulation comprising the N-acetylhexosamine motif, galacto-oligosaccharides and at least one fucosylated and / or sialylated component; and (iii) a high complexity formulation comprising the N-acetylhexosamine motif, galacto-oligosaccharides, at least one fucosylated and / or sialylated component, and at least one sulfated glycan and / or a branched polysaccharide.

17. The kit according to any of claims 1, 4 to 7 or 10 to 16, further comprising a redox conditioner supplied (i) within Part A, or (ii) in a separate container for aseptic addition, for use in the method according to claim 3.

18. The kit according to claim 17, wherein the redox conditioner is selected from cysteine, ascorbic acid or ascorbate, thioglycolate, glutathione, and combinations thereof.

19. The method according to any of claims 3, 8 or 9, wherein the heat sterilization of step (i) comprises a heat treatment equivalent to 115 °C to 125 °C for 10 to 30 minutes, followed by cooling prior to the aseptic addition of step (i).

20. The method according to any one of claims 3, 8, 9 or 19, wherein the dissolved oxygen regime comprises at least one of: (a) a moderate regime, wherein the dissolved oxygen is greater than 0.5 mg / L and up to 5 mg / L; (b) a reduced regime, wherein the dissolved oxygen is greater than 0 mg / L and equal to or less than 0.5 mg / L; and (c) a variable regime, wherein the dissolved oxygen fluctuates between values ​​of the moderate regime and the reduced regime during cultivation or fermentation, maintaining non-zero residual dissolved oxygen during at least part of the process.

21. The method according to any of claims 3, 8, 9, 19 or 20, further comprising measuring the redox potential (Eh / ORP) of the medium using an ORP electrode, reported in mV, and optionally adjusting said redox potential by adding a redox conditioner selected from cysteine, ascorbate, thioglycolate, glutathione, and combinations thereof.

22. The method according to any of claims 3, 8, 9, 19, 20 or 21, further comprising monitoring at least two selected parameters of pH, dissolved oxygen, redox potential, turbidity or DO, and temperature, and optionally adjusting at least one selected operating condition of stirring, gas-liquid exchange, headspace volume and / or addition of redox conditioner, to maintain the dissolved oxygen within the regime according to claim 20 and, when measured, the redox potential around a predetermined target value.

23. The method according to any of claims 3, 8, 9, 19, 20, 21 or 22, wherein the at least one oxygen-sensitive microorganism comprises a strict anaerobe, microaerotolerant or facultative anaerobe with growth inhibited by oxygen, selected from intestinal, ruminal, environmental or marine microorganisms, including lactic acid bacteria, bifidobacteria, short-chain fatty acid-producing bacteria and mucinolytic bacteria, or combinations thereof.

24. The method according to claim 23, wherein the at least one microorganism comprises Akkermansia spp. and / or Bifidobacterium spp., optionally in co-culture with at least one lactic acid bacterium.

25. The method according to any of claims 3, 8, 9, 19, 20, 21, 22, 23 or 24, wherein the cultivation or fermentation produces a biomass with a viable concentration of at least 10 7CFU / mL, and / or in which, under comparable conditions and a predetermined culture time, the culture medium including the biomimetic carbohydrate block provides a viability increase of at least 1 Iog10 compared to a control medium lacking said biomimetic carbohydrate block.

26. A microbiological composition obtained or obtainable by the method according to any of claims 3, 8, 9 or 19 to 25, comprising (i) viable biomass of at least one oxygen-sensitive microorganism and / or (ii) a cell-free fermentation supernatant (postbiotic), optionally in combination with a carrier, excipient, cryoprotectant and / or stabilizing agent.

27. The microbiological composition according to claim 26, wherein the viable biomass and / or cell-free supernatant is in a stabilized form selected from lyophilized, spray-dried, microencapsulated, granulated or concentrated, optionally reconstitutable in an aqueous or food-grade vehicle.

28. A food product or beverage comprising the microbiological composition according to claim 26 or 27 and at least one food ingredient, wherein the microbiological composition is incorporated as a culture, ferment, functional ingredient and / or biological additive.

29. A nutraceutical composition or food supplement comprising the microbiological composition according to claim 26 or 27, optionally combined with pharmaceutically and / or food-acceptable prebiotics, fibers, proteins, minerals, vitamins and / or excipients, for non-therapeutic use as a functional ingredient.

30. A packaged kit product comprising the kit according to claim 1 and / or the reconstituted culture medium composition according to claim 2, together with instructions for performing the method according to claim 3, wherein the instructions describe the modular preparation by heat sterilization of Part A and subsequent aseptic addition of Part B, and describe the closed-container operation without continuous purging of inert gas maintaining non-zero residual dissolved oxygen according to a regime according to claim 20, and optionally describe obtaining the microbiological composition according to claim 26 and its incorporation into the product according to claim 28 and / or the composition according to claim 29.

31. The kit according to claim 6, wherein the derivatives and / or fragments of the branched oligosaccharide and / or polysaccharide are functionally equivalent when, when employed in the method according to claim 3 under a dissolved oxygen regime according to claim 20, they meet at least one of the performance criteria of claim 25.

32. The method according to any of claims 3, 8 or 9, wherein the biomimetic carbohydrate block of the thermolabile fraction of step (iii) comprises a Pareto-type biomimetic signature in which, as a mass percentage of the total biomimetic carbohydrate block: (i) galacto-oligosaccharides represent from 60% to 80%; (ii) the N-acetylhexosamine motif represents from 10% to 25%; (iii) the fucosylated component represents from 2% to 10%; (iv) the branched and / or sulfated glycan component represents from 2% to 10%; and (v) the sialylated component represents from 2% to 10%.

33. The microbiological composition according to claim 26 or 27, wherein the cell-free supernatant (postbiotic) contains culture-derived bioactive metabolites.

Images