A vitamin D2 derived from mushrooms for calcium absorption and immune system support

Abstract

The present invention falls within the field of vitamin preparation techniques and relates in particular to a process for preparing and applying a fungal-derived vitamin D2 intended to promote calcium absorption, prevent rickets and strengthen immunity, the process comprising the following steps: preparation of the raw material by selecting edible mushrooms rich in ergosterol as starting material; cultivation and optimization by genetic modification of the fermented and cultivated mushrooms; conversion by light exposure by irradiating the culture with ultraviolet rays.The beneficial effects lie in the fact that, through the construction of a gene expression vector, the transformation of fungal cells and the selection and identification of transformants with a high synthesis capacity, the expression of genes related to ergosterol synthesis and conversion to vitamin D2 is increased, making it possible to obtain edible mushroom strains with higher levels of ergosterol and thus improve the yield and efficiency of vitamin D2 production.

Description

1 A vitamin D2 derived from fungi for calcium absorption and immune strengthening Technical field The present invention relates to the field of vitamin preparation techniques, and 5 more particularly a process for preparing and applying a vitamin D2 of fungal origin, intended to promote calcium absorption, prevent rickets and strengthen immunity. Vitamin D2 is available as white to off-white crystals, in flakes or needles, and is unstable in light or air. It is primarily used for the prevention and treatment of vitamin D deficiency; it can also be used in the treatment of chronic hypocalcemia, hypophosphatemia, rickets, osteomalacia associated with chronic renal failure, familial hypophosphatemic rickets, and hypoparathyroidism (post-operative, idiopathic, or pseudohypoparathyroidism). Furthermore, vitamin D2 is effective in cases of acute, chronic, latent, post-operative, or idiopathic tetany.In the current state of the art, vitamin D2 preparation processes are becoming more efficient and environmentally friendly. Researchers are exploring new extraction and purification methods to improve the purity and activity of vitamin D2 while reducing energy consumption and emissions. The use of advanced technologies, such as microwave-assisted extraction or supercritical fluid extraction, allows for more efficient extraction of vitamin D2 from edible mushrooms. However, the optimization of the extraction source—the mushrooms themselves—is often neglected, and there is a lack of solutions aimed at increasing extraction yield by improving the characteristics of the starting biological material.Thus, it is necessary to have a process for preparing and applying a vitamin D2 of fungal origin, intended to promote calcium absorption, prevent rickets and strengthen immunity, to remedy the current deficiency related to the optimization of the raw material and to improve the genetic characteristics of the material at the source to increase the extraction yield of vitamin D2. Content of the invention The objective of the present invention is to provide a method for preparing and applying a vitamin D2 of fungal origin, intended to promote calcium absorption, prevent rickets and strengthen immunity, to solve the problem, mentioned in the prior art 5, of the lack of optimization of the raw material in the extraction of vitamin D2, and to allow the improvement of the genetic characteristics of the starting material in order to increase the yield of vitamin D2 extraction.To achieve this objective, the present invention proposes the following technical solution: a process for preparing a vitamin D2 of fungal origin intended to promote the absorption of calcium, to prevent rickets and to strengthen immunity, said process comprising the following steps: preparation of the raw material, consisting of selecting edible mushrooms rich in engosterol as starting material; culture and optimization, consisting of subjecting the fermented or cultivated mushrooms to a modification by genetic engineering; conversion by light irradiation, consisting of exposing the cultures to ultraviolet radiation; extraction and purification, consisting of purifying vitamin D2 using nanometric technologies.20 Preferably, the raw material preparation step includes the following operations: selecting edible mushrooms rich in energosterol as material intended for the preparation of vitamin D2.Preferably, the specific operations of the culture and optimization stage include: 25 inoculating the fungus into a culture medium containing a carbon source, a nitrogen source as well as minerals and trace elements, controlling the pH of the medium so that it is neutral or slightly acidic, placing the culture vessel in a stirred incubator or fermenter and setting an appropriate temperature of 25 to 30°C; constructing a gene expression vector, transforming the fungal cells, then 30 2025 / 5755 BE2025 / 5755 3 selecting and identifying the transformants exhibiting a high capacity for synthesis, so as to increase the expression of genes related to ergosterol synthesis and conversion to vitamin D2. Preferably, the specific operations of the light irradiation conversion step include: 5 exposing the cultured mushroom to ultraviolet radiation, with a light intensity of 10W / m² and an irradiation duration of between six and fourteen hours.Preferably, the specific operations of the extraction and purification step include: destroying the fungal cell wall by grinding to release vitamin D2 into the culture medium, then performing the extraction using ethanol; using an anodilator membrane or a nanometric adsorbent to filter and adsorb the extraction solution, removing impurities and increasing the purity of the vitamin D2; subjecting the vitamin D2 solution obtained after extraction and purification to a crystallization step, separating the crystals from the mother liquor by centrifugation, and then performing drying to remove moisture. Preferably, the process further includes: grinding the vitamin D2 after drying, and then packaging the vitamin D2 in airtight packaging to ensure hygiene and product safety. An application of vitamin D2 obtained by the process of preparing a vitamin D220 of fungal origin intended to promote calcium absorption, to prevent rickets and to strengthen immunity, the prepared vitamin D2 being used to promote calcium absorption.An application of vitamin D2 obtained by the process of preparing vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets and strengthen immunity, the prepared vitamin D2 being used to prevent rickets.25 An application of vitamin D2 obtained by the process of preparing vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets and strengthen immunity, the prepared vitamin D2 being used to strengthen immunity. An application of vitamin D2 obtained by the process of preparing vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets and strengthen immunity, the prepared vitamin D2 being used simultaneously to promote calcium absorption, prevent rickets and strengthen immunity.Compared to the prior art, the beneficial effects of the present invention are as follows: the process for preparing and applying a fungal vitamin D2 intended to promote calcium absorption, prevent rickets, and strengthen immunity allows, through the construction of a gene expression vector, the transformation of fungal cells, and the selection and identification of transformants exhibiting a high synthesis capacity, for increased expression of genes related to ergosterol synthesis and conversion to vitamin D2; this makes it possible to obtain edible mushroom strains with a higher level of ergosterol, thereby improving the yield and efficiency of vitamin D2 production. The present invention also solves the problem of the lack optimization of raw materials in existing extraction methods and allows for improvement of the genetic characteristics of the starting material in order to increase the extraction yield of vitamin D2.15 Specific Embodiments In order to describe clearly and completely the objective, the technical solution, and the advantages of the present invention, it should be understood that the specific embodiments described herein constitute only a part of the embodiments of the invention and not their entirety. They serve only to illustrate the embodiments of the invention and should not be considered as limitations. All other embodiments that a person skilled in the art can obtain without inventive effort also fall within the scope of protection of the present invention. Example of implementation 1 The present invention proposes a technical solution consisting of a process for preparing 25 a vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets and strengthen immunity, said process comprising the following steps: 1. Preparation of the raw material, it is appropriate to select edible mushrooms rich in energosterol as starting material for the preparation of vitamin D2.Ergosterol being the precursor of vitamin D2, it can be converted into the latter by a specific process. Among the different fungal species, shiitake is often chosen because of its high ergosterol content, availability, and ease of processing. The selected mushrooms must be carefully washed to remove impurities and dirt from their surface; this step is essential to guarantee the hygiene of the process and the quality of the final product. Washing can be carried out under running water or with a suitable food-grade cleaning agent. After washing, the mushrooms must be dried to remove excess moisture, either by natural drying or with a dehydrating device. food. Once dried, they are easier to store and transport, and lend themselves better to subsequent grinding and extraction operations. The dried mushrooms must then be ground into powder, manually or with the help of a mechanical grinder, in order to improve the extraction yield.The powder obtained must be stored appropriately in a dry, cool, ventilated environment, protected from direct light, to avoid moisture, contamination, or alteration, and must be regularly monitored to maintain its good condition. 2. Cultivation and optimization: Genetically modifying fermented and cultivated edible mushrooms; to support the growth and metabolism of the mushroom, a culture medium rich in carbon sources, nitrogen sources, minerals, and trace elements is prepared. Carbon sources, such as glucose or sucrose, provide the energy necessary for the mushroom; nitrogen sources, such as yeast extract or lapeptone, support cell growth; Minerals and trace elements guarantee the normal course of biological activities. In parallel, it is necessary to control the pH of the culture medium, which must be neutral or slightly acidic, to reproduce the natural environment of the fungus.The selected fungus is inoculated into the prepared medium, taking care to operate aseptically to avoid any contamination. The inoculated container is then placed in a stirred incubator or fermenter, with the temperature set between 25 and 30°C to ensure optimal growth and reproduction.25 Furthermore, depending on the fungus's growth characteristics, the stirring speed or aeration rate of the fermenter must be adjusted to meet its oxygen requirements. To further improve the fungus's vitamin D2 synthesis capacity, genetic engineering techniques are implemented. Initially, a gene expression vector is constructed by cloning the genes encoding the key enzymes for synthesis.30 2025 / 5755 BE2025 / 5755 6 of ergosterol and of conversion into vitamin D2. These vectors are then introduced into fungal cells by transformation, thus allowing the expression of genes of interest. The transformed cells must be selected and identified, and the strains exhibiting a higher capacity for synthesis are retained.To further increase vitamin D2 yield, culture conditions must be optimized, including medium composition, pH, temperature, humidity, and lighting parameters, to allow the fungus to grow and metabolize under optimal conditions. The fungal strains obtained after culture and optimization must be stored appropriately to ensure the durability and longevity of their genetic characteristics; methods such as freezing, dehydration, or storage in liquid nitrogen can be used. When necessary, the strains can be subcultured and regenerated to ensure their continued availability in vitamin D2 production. 3. Conversion by light exposure, consisting of irradiating the culture with ultraviolet rays; an appropriate UV source and corresponding irradiation equipment should be prepared.The choice of ultraviolet radiation source is essential, as the emitted wavelength must allow the conversion of ergosterol present in fungal cells into vitamin D2. The irradiation equipment must also ensure stable emission and adjustable light intensity to meet the needs of different fungal strains or growth stages. Before light conversion, the fungi must be cultured to an appropriate growth stage, generally in exponential or stationary phase, to guarantee sufficient engergosterol content as a precursor. The concentration and homogeneity of the culture must also be controlled to ensure uniformity of the reaction during irradiation. Key conversion parameters include the light intensity and the duration of irradiation. In the present example, the intensity is fixed at 10 W per square meter, an optimized parameter allowing to maintain an efficient conversion while limiting cellular damage.The irradiation time is adjusted according to the strain characteristics and experimental requirements, generally between six and fourteen hours. Excessive irradiation can lead to cell damage or product degradation, while insufficient irradiation can result in incomplete conversion. The prepared fungal culture is placed in the irradiation equipment so that it receives uniform exposure to UV rays, while controlling the temperature and humidity of the light environment to reproduce the natural growth conditions of the fungus. At the end of irradiation, the culture must be processed appropriately, including interrupting the exposure, collecting the culture, and performing extraction and Purification of vitamin D2. During the extraction and purification stages, it is necessary to avoid any additional exposure to light in order to prevent degradation of the product and to guarantee the quality and purity of the vitamin D2 obtained. 4.Extraction and purification, using nanometric technologies to purify vitamin D2; in order to release the vitamin D2 contained in fungal cells, it is necessary to destroy the cell wall of the fungus, an operation that can be carried out by physical or chemical methods such as high-pressure homogenization, ultrasonic disintegration, or enzymatic hydrolysis, the choice of method being adapted to the characteristics of the fungus in order to avoid any alteration of the vitamin D2. After cell rupture, vitamin D2, as well as other intracellular components, are released into the culture medium, which facilitates extraction. A suitable solvent, generally ethanol due to its good solubility for vitamin D2 and its ease of use, is then used to extract vitamin D2, controlling the temperature and extraction time to ensure sufficient dissolution and limit the extraction of undesirable impurities.The extraction solution containing vitamin D2 may contain various impurities; to eliminate them and improve the purity of the vitamin D2, anofiltration membranes or nanometric adsorbents are used to filter and selectively adsorb these impurities, which makes it possible to obtain a purified solution suitable for crystallization. The vitamin D2 is then crystallized by adjusting the temperature, concentration and precipitation conditions; the crystals formed are separated from the mother liquid by centrifugation, then dried, in particular by video drying or spray drying, in order to eliminate residual moisture and obtain a final product of dry vitamin D2. Example implementation 2 Based on example implementation 1, the following method is proposed: Preparation of vitamin D2 1. Preparation of the raw material 30 2025 / 5755 BE2025 / 5755 8 Fresh shiitake mushrooms are selected as the raw material for the preparation of vitamin D2. The shiitake mushrooms must be washed, dried and ground to obtain a homogeneous fungal powder. 2.Fungal Culture: Medium Preparation: Glucose, powdered yeast, inorganic salts, and trace elements are mixed in a specific ratio to prepare a liquid medium. The pH of the medium is adjusted to 6.5. Inoculation and Culture: Streptomyces is inoculated into the medium and then placed in a shaker at a temperature of 28°C and a speed of 150 rpm. The culture duration is 48 hours to ensure sufficient fungal growth and metabolism. 10 3. Genetic Modification: Vector Construction: The genes encoding the key enzymes for ergosterol synthesis and conversion to vitamin D2 are cloned into an expression vector. Transformation and selection: The constructed expression vector is transformed in Streptomyces, and positive transformants are obtained by selection on a medium containing a resistance agent. Verification of expression: The positive transformants are cultured and the production of ergosterol and vitamin D2 is analyzed in order to select strains exhibiting a high capacity for synthesis. 4.Conversion by light exposure 20 The cultured fungal cell suspension is uniformly spread on a Petri dish and then placed under a UV lamp for light conversion. The light intensity is set at 5 W / m² and the irradiation time at 8 hours. UV exposure stimulates the conversion of intracellular ergosterol to vitamin D2. 5. Extraction and purification 25 Extraction: The fungal cells after light conversion are lysed, and then extraction is carried out using ethanol as the solvent. The extraction temperature is set at 40°C, the duration at 2 hours, and the solvent / biomass ratio at 3:1. Filtration and concentration: The extraction solution is filtered to remove impurities, then concentrated using a rotary evaporator to obtain a crude vitamin D2 extract. 30 2025 / 5755 BE2025 / 5755 9 Purification by nanotechnology: A nanofiltration membrane is used to filter the crude extract, further remove impurities and increase the purity of vitamin D2. 6. Crystallization and drying: The purified vitamin D2 solution undergoes a crystallization step.By controlling the crystallization conditions, vitamin D2 precipitates in the form of crystals. The five crystals obtained are then dried using a vacuum dryer to remove moisture and obtain the final vitamin D2 product. 7. Packaging and Storage The dried vitamin D2 product is packaged using airtight packaging materials to guarantee its hygiene and safety. The packaged product is then stored in a cool, dry environment, protected from light, to avoid humidity and light exposure. 8. Quality Control The final vitamin D2 product is subject to quality control, including tests for purity, content and activity. By comparing it to reference standards, it is possible to ensure that the quality of the product meets the applicable standards and requirements.Example implementation 3: An application of vitamin D2 obtained according to the process for preparing vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets, and strengthen immunity; the prepared vitamin D2 is used to promote calcium absorption. Example implementation 4: An application of vitamin D2 obtained according to the process for preparing vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets, and strengthen immunity; the prepared vitamin D2 is used to prevent rickets. Example implementation 5: An application of vitamin D2 obtained according to the process for preparing vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets, and strengthen immunity; the prepared vitamin D2 is used to strengthen immunity.Example of implementation 6 An application of vitamin D2 obtained according to the process for preparing a vitamin D2 of fungal origin intended to promote calcium absorption, prevent rickets and strengthen immunity, the prepared vitamin D2 being used simultaneously to promote calcium absorption, prevent rickets and strengthen immunity. Comparative example 1 An enzymatic process for preparing vitamin D2, comprising the following steps: 5 1. Preparation of the raw material An energy-rich yeast is selected as the raw material. The yeast is cultured, collected and then dried in preparation for the subsequent steps. 2. Enzyme Selection and Preparation: An enzyme with a high capacity to catalyze the conversion of ergosterol to vitamin D2, such as a vitamin D2 synthesis enzyme, is selected. This enzyme can be expressed in a microorganism using genetic engineering techniques, then purified and concentrated. 3. Enzymatic Conversion: The prepared yeast is mixed with an appropriate amount of enzyme solution.The temperature, pH, and duration of the reactions are controlled to allow the enzyme to catalyze the conversion of ergosterol to vitamin D2. The specific reaction conditions can be optimized according to the characteristics of the enzyme and the properties of the raw material. 4. Extraction and Purification After enzymatic conversion, vitamin D2 is extracted from the reaction system using a suitable extraction solvent and appropriate conditions. Purification methods such as chromatography or crystallization are then used to remove impurities and improve the purity of the vitamin D2. 5. Drying and Packaging: The purified vitamin D2 is dried to remove moisture. It is then packaged using appropriate packaging material to ensure product hygiene and safety. The product is stored in a cool, dry environment, away from light, to prevent moisture and light exposure. 6.Quality Control The final vitamin D2 product is subject to quality control including the analysis of its purity, atherosclerosis, and activity. By comparing it to reference standards, it is possible to guarantee that the product meets the relevant standards and requirements. Comparative Example 2 A process for preparing vitamin D2 by photo-induced biosynthesis is proposed, comprising the following steps: 1. Preparation of the raw material Plant leaves or engosterol-rich microorganisms are selected as raw material. The raw materials are washed, ground, and pre-treated to facilitate the subsequent photo-induced biosynthesis reaction. 2. Optimization of Irradiation Conditions 10 The conditions of photo-induced biosynthesis are optimized by adjusting parameters such as light intensity, wavelength, and irradiation duration. An appropriate light source and suitable irradiation equipment are chosen, ensuring the elasticity and uniformity of light emission. 3.Photo-induced biosynthesis reaction 15 The pre-treated raw material is placed under irradiation conditions so that ergosterol is converted to vitamin D2 under the effect of light. By controlling the irradiation conditions and the reaction time, efficient photo-induced biosynthesis is obtained. 4. Extraction and purification After photo-induced biosynthesis, vitamin D2 is extracted from the raw material using 20 an appropriate solvent and extraction conditions. Then, purification techniques such as chromatography or crystallization are used to remove impurities and increase the purity of vitamin D2. 5. Drying and Packaging: The purified vitamin D2 is dried to remove moisture, then packaged in airtight packaging material to ensure product hygiene and safety. The packaged product is stored in a cool, dry environment, away from light, to avoid moisture and light exposure. 6.Quality control The final vitamin D2 product is subject to quality analyses focusing in particular on 30 2025 / 5755 BE2025 / 5755 12 purity, latency and activity. By comparing it to reference standards, it is possible to verify that the product meets the corresponding standards and requirements.By comparison, implementation example 2 presents the best results: thanks to the construction of a gene expression vector, the transformation of fungal cells, as well as the selection and identification of transformers exhibiting a high capacity for synthesis, 5 the expression of genes linked to the synthesis of ergosterol and to the conversion into vitamin D2 is increased, making it possible to obtain strains of edible mushrooms exhibiting higher levels of ergosterol and to improve the yield and efficiency of vitamin D2 production; this also makes it possible to solve the problem of the lack of optimization of the raw material in existing extraction methods and to improve the genetic characteristics 10 of the starting material in order to increase the yield of vitamin D2 extraction.Although embodiments of the present invention have been described and illustrated, it is understandable to those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention. The scope of the present invention is defined by the attached claims and their equivalents. 2025 / 5755 BE2025 / 5755.