Directional enrichment process of tremella aurantialba functional fermentation metabolite

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a tremella aurantialba functional fermentation metabolite directional enrichment process which comprises the following steps: S1, tremella aurantialba strain activation, S2, seed solution preparation, S3, controllable fermentation culture, S4, metabolite enrichment regulation and S5, separation and purification. According to the method, the fermentation temperature, the pH value, the dissolved oxygen content and the carbon nitrogen ratio are synergistically regulated and controlled, a metabolite synthesis inducer is added in a specific stage, a synthesis channel of polysaccharide and ergosterol in tremella aurantialba mycelia can be accurately activated, the polysaccharide content in the finally obtained mycelia is larger than 15% (on the basis of dry weight), and the polysaccharide content is larger than 15% (on the basis of dry weight). The content of ergosterol is more than 0.5% (on the basis of dry weight) and is increased by more than three to fifty percent compared with a traditional fermentation process, natural limitation of metabolite synthesis is effectively broken through, an automatic fermentation tank control system is adopted, parameters such as fermentation temperature, pH value and dissolved oxygen are regulated in real time, and the method is remarkably superior to a traditional manual regulation process. And the product quality consistency is ensured.

Description

Technical Field

[0001] This invention relates to a process for the targeted enrichment of functional metabolites from *Auricularia auricula-judae* fermentation, and specifically to a process for activating the mycelial synthesis pathway of *Auricularia auricula-judae* under controlled fermentation conditions to achieve the targeted enrichment of specific functional metabolites such as ergosterol and polysaccharides. Background Technology

[0002] *Tremella aurantiaca*, belonging to the phylum Basidiomycota, class Tremella, and family Auriculariaceae, is a rare and precious edible and medicinal fungus endemic to my country. Its natural fruiting body is rich in various active ingredients, including polysaccharides, ergosterol, amino acids, vitamins, and minerals, with polysaccharides and ergosterol being the core functional substances. Modern pharmacological studies have shown that *Tremella aurantiaca* polysaccharides enhance immunity, regulate intestinal flora, and have antioxidant effects, making them suitable for the development of functional foods, health products, and pharmaceutical intermediates. Ergosterol, as a precursor to vitamin D2, promotes calcium absorption and regulates cholesterol metabolism, showing broad application prospects in food additives and cosmetics. Currently, the acquisition of active ingredients from golden ear fungus mainly relies on the collection of natural fruiting bodies and artificial cultivation. However, the growth cycle is long (the natural fruiting body needs 3-6 months to grow, while the artificial cultivation cycle needs 2-3 months), and it is greatly affected by environmental factors (temperature, humidity, and light). In addition, the natural content of polysaccharides and ergosterol in the fruiting body is low (the polysaccharide content is usually 5-8%, and the ergosterol content is usually 0.2-0.4%), which makes it difficult to meet the demand for high-activity raw materials in large-scale industrial production.

[0003] With the development of microbial fermentation technology, the production of metabolites by liquid fermentation of Auricularia auricula-judae mycelium has become an important way to replace natural fruiting bodies, which can effectively shorten the production cycle (the traditional liquid fermentation cycle is about 8-10 days) and reduce environmental dependence.

[0004] The following problems still exist with existing technologies:

[0005] 1. Traditional fermentation processes do not adequately regulate the metabolic pathways of Auricularia auricula-judae mycelium. Simply optimizing the basal culture medium formulation (such as adjusting the type of carbon or nitrogen source) is insufficient to activate the synthesis pathways of specific metabolites. As a result, the content of polysaccharides and ergosterol in fermentation products is still limited by the basal metabolic level of the mycelium, and cannot achieve targeted enrichment.

[0006] 2. Traditional fermentation processes suffer from poor parameter stability. Key conditions such as fermentation temperature, pH, and dissolved oxygen are often controlled manually and intermittently, which can lead to large fluctuations in the internal environment of the fermentation system. This not only affects the uniformity of mycelial growth but also causes significant differences in the content of specific metabolites in different batches of fermentation products, reducing product quality stability and increasing the difficulty and cost of subsequent separation and purification.

[0007] Therefore, developing a process that can precisely control fermentation conditions and achieve targeted enrichment of specific metabolites of Auricularia auricula-judae with high stability is key to promoting the industrial application of functional components of Auricularia auricula-judae. Summary of the Invention

[0008] The purpose of this invention is to provide a process for the targeted enrichment of functional fermentation metabolites of *Auricularia auricula-judae*, in order to solve the problems mentioned in the background art.

[0009] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0010] This invention relates to a process for the targeted enrichment of functional fermentation metabolites from *Auricularia auricula-judae*, comprising the following steps:

[0011] S1. Activation of Auricularia auricula-judae strain: The preserved Auricularia auricula-judae mycelium strain is inoculated into an activation medium and revived under specific temperature and humidity conditions to obtain a stable and active Auricularia auricula-judae mother strain.

[0012] S2. Seed liquid preparation: The mother culture of Auricularia auricula-judae obtained in step S1 is inoculated into the seed liquid culture medium. Under the condition of shaking culture, the rotation speed and aeration are adjusted and cultured until the mycelial concentration reaches the preset threshold to obtain Auricularia auricula-judae seed liquid.

[0013] S3. Controlled fermentation culture: The seed liquid of Auricularia auricula-judae obtained in step S2 is inoculated into the fermentation medium at a preset inoculation amount. In the fermenter, the fermentation temperature, pH value, dissolved oxygen and carbon-nitrogen ratio are synergistically controlled to induce Auricularia auricula-judae mycelium to synthesize specific metabolites.

[0014] S4. Metabolite enrichment regulation: During the fermentation process in step S3, a metabolite synthesis inducer is added at a specific fermentation stage to maintain the regulation parameters stable until the fermentation endpoint, thereby obtaining a fermentation broth containing a high concentration of specific metabolites.

[0015] S5. Separation and purification: The fermentation broth obtained in step S4 is subjected to solid-liquid separation, extraction and purification to obtain high-purity auricularia auricula-judae specific metabolites.

[0016] Furthermore, the activation medium mentioned in step S1 is a PDA-modified medium, the components of which, by mass parts, include: 200-250 parts of potato, 15-20 parts of glucose, 5-8 parts of peptone, 18-22 parts of agar, 1000 parts of water, and the pH value is adjusted to 6.0-6.5.

[0017] The resuscitation and culture conditions are: temperature 22-26℃, relative humidity 65-75%, and culture in the dark for 5-7 days.

[0018] Further, the seed culture medium in step S2 comprises, by mass parts: 20-25 parts glucose, 3-5 parts yeast extract, 1.5-2.0 parts potassium dihydrogen phosphate, 0.8-1.2 parts magnesium sulfate heptahydrate, 0.01-0.02 parts vitamin B1, and 1000 parts water, with the pH adjusted to 6.2-6.8.

[0019] The shaking culture conditions are: rotation speed 180-220 r / min, aeration rate 0.5-1.0 vvm, temperature 24-28℃, and culture until the mycelial concentration reaches 8-12 g / L.

[0020] Further, the fermentation culture medium in step S3 comprises, by mass parts: 15-30 parts carbon source, 2-5 parts nitrogen source, 2.0-2.5 parts potassium dihydrogen phosphate, 1.0-1.5 parts magnesium sulfate heptahydrate, 0.3-0.5 parts calcium chloride, and 1000 parts water.

[0021] The carbon source is selected from at least one of glucose, sucrose, and maltose, and the nitrogen source is selected from at least one of peptone, yeast powder, and soybean powder.

[0022] The synergistic control parameters are: fermentation temperature 25-30℃, pH value 6.0-7.2, dissolved oxygen 10-25%, and carbon-nitrogen ratio 10:1-25:1.

[0023] Further, the specific fermentation stage mentioned in step S4 is day 3-5 of fermentation culture, and the metabolite synthesis inducer is selected from at least one of methyl jasmonate, salicylic acid, and chitosan, with an addition concentration of 50-200 μmol / L.

[0024] The criteria for determining the fermentation endpoint are: the dry weight of mycelium in the fermentation broth no longer increases, and the content of specific metabolites reaches its peak.

[0025] Furthermore, the solid-liquid separation in step S5 is carried out by centrifugation or plate and frame filtration. The centrifugation conditions are: rotation speed 8000-10000 r / min and time 15-20 min.

[0026] The extraction process for polysaccharide metabolites uses hot water extraction at a temperature of 80-90℃ for 2-3 hours.

[0027] Ergosterol metabolites were extracted using an ethanol reflux extraction method with an ethanol concentration of 90-95%, a reflux temperature of 75-85℃, and a reflux time of 3-4 hours.

[0028] Further, the purification process described in step S5 includes: after extraction of polysaccharide metabolites, adding 3-5 times the volume of 95% ethanol for alcohol precipitation, letting stand for 12-16 hours, centrifuging to collect the precipitate, and then deproteinizing using the Sevag method and decolorizing by adsorption with macroporous resin to obtain polysaccharides with a purity ≥90%.

[0029] After extraction, ergosterol metabolites were separated by silica gel column chromatography. The target eluent was collected and concentrated under reduced pressure using a mixture of petroleum ether and ethyl acetate (volume ratio 8:2-9:1) as the eluent to obtain ergosterol with a purity ≥85%.

[0030] Furthermore, the fermenter mentioned in step S3 is a mechanically stirred fermenter with a stirring speed of 100-150 r / min. During the fermentation process, the carbon source concentration is maintained at 5-8 g / L by an automatic feeding system, the pH value is adjusted in real time by adding hydrochloric acid or sodium hydroxide solution, and the dissolved oxygen is adjusted by an aeration flow controller.

[0031] Furthermore, the specific metabolites include Auricularia auricula polysaccharide and ergosterol. After treatment in step S5, the content of the Auricularia auricula polysaccharide product is ≥15% (based on the dry weight of mycelium), and the content of the ergosterol product is ≥0.8% (based on the dry weight of mycelium).

[0032] Furthermore, the auricularia auricularia mycelium strain mentioned in step S1 is the auricularia auricularia strain with the preservation number Cgmccno.41097. This strain is activated after being passaged on slant culture 3-4 times to ensure that the mycelium is free from contamination by other microorganisms and has stable activity.

[0033] The present invention has the following beneficial effects:

[0034] (1) This invention can precisely activate the synthesis pathways of polysaccharides and ergosterol in Auricularia auricula-judae mycelium by synergistic regulation of fermentation temperature, pH value, dissolved oxygen and carbon-nitrogen ratio, combined with the addition of metabolite synthesis inducers at specific stages. The resulting mycelium has a polysaccharide content of more than 15% (based on dry weight) and an ergosterol content of more than 0.8% (based on dry weight), which is 30% to 50% higher than traditional fermentation processes, effectively breaking through the natural limitations of metabolite synthesis.

[0035] (2) The content of specific metabolites obtained by the present invention is much higher than that of natural golden ear fruiting bodies (natural fruiting bodies have a polysaccharide content of 5-8% and an ergosterol content of 0.2-0.4%). The polysaccharide content is one to three times that of natural fruiting bodies, and the ergosterol content is two to four times that of natural fruiting bodies. It does not rely on natural resources for collection and can stably provide highly active raw materials.

[0036] (3) The present invention adopts an automated fermenter control system to adjust the fermentation temperature, pH value, dissolved oxygen and other parameters in real time, ensuring that the temperature fluctuation range in the fermentation system is less than ±0.5 degrees, the pH value is ±0.2, the dissolved oxygen is ±2%, and the content of specific metabolites in different batches of fermentation products is less than 5%, which is significantly better than the traditional manual control process and ensures the consistency of product quality.

[0037] (4) The optimized fermentation cycle of this invention is only 5-7 days, which is more than 30% shorter than the traditional liquid fermentation process (8-10 days). Furthermore, the seed liquid preparation and fermentation culture can be carried out continuously, and the metabolite yield per unit time is increased by more than 40%, thus reducing production costs.

[0038] (5) Based on the physicochemical properties of polysaccharides and ergosterol, the present invention designs special extraction and purification processes for each. The polysaccharides are purified to a purity of more than 90% after hot water extraction, alcohol precipitation and deproteinization treatment, and the ergosterol is purified to a purity of more than 80% after ethanol reflux and silica gel column chromatography. Compared with the traditional mixed extraction process, the purification efficiency is improved by 15% to 20%, reducing the impact of impurities on subsequent applications.

[0039] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is a flowchart of the process flow of the present invention. Detailed Implementation

[0042] like Figure 1 As shown, this invention provides a process for the targeted enrichment of functional fermentation metabolites from *Auricularia auricula-judae*, comprising the following steps:

[0043] S1. Activation of Auricularia auricula-judae strain: The preserved Auricularia auricula-judae mycelium strain is inoculated into an activation medium and revived under specific temperature and humidity conditions to obtain a stable and active Auricularia auricula-judae mother strain.

[0044] In this step, the activation medium is a PDA-modified medium, whose components by mass parts include: 200-250 parts potato, 15-20 parts glucose, 5-8 parts peptone, 18-22 parts agar, and 1000 parts water, with the pH value adjusted to 6.0-6.5.

[0045] The resuscitation and culture conditions are: temperature 22-26℃, relative humidity 65-75%, and culture in the dark for 5-7 days.

[0046] S2. Seed liquid preparation: The mother culture of Auricularia auricula-judae obtained in step S1 is inoculated into the seed liquid culture medium. Under the condition of shaking culture, the rotation speed and aeration are adjusted and cultured until the mycelial concentration reaches the preset threshold to obtain Auricularia auricula-judae seed liquid.

[0047] In this step, the seed culture medium includes, by weight, 20-25 parts glucose, 3-5 parts yeast extract, 1.5-2.0 parts potassium dihydrogen phosphate, 0.8-1.2 parts magnesium sulfate heptahydrate, 0.01-0.02 parts vitamin B1, and 1000 parts water, with the pH adjusted to 6.2-6.8.

[0048] The shaking culture conditions are: rotation speed 180-220 r / min, aeration rate 0.5-1.0 vvm, temperature 24-28℃, and culture until the mycelial concentration reaches 8-12 g / L.

[0049] S3. Controlled fermentation culture: The seed liquid of Auricularia auricula-judae obtained in step S2 is inoculated into the fermentation medium at a preset inoculation amount. In the fermenter, the fermentation temperature, pH value, dissolved oxygen and carbon-nitrogen ratio are synergistically controlled to induce Auricularia auricula-judae mycelium to synthesize specific metabolites.

[0050] In this step, the fermentation medium includes, by mass, 15-30 parts carbon source, 2-5 parts nitrogen source, 2.0-2.5 parts potassium dihydrogen phosphate, 1.0-1.5 parts magnesium sulfate heptahydrate, 0.3-0.5 parts calcium chloride, and 1000 parts water.

[0051] The carbon source is selected from at least one of glucose, sucrose, and maltose, and the nitrogen source is selected from at least one of peptone, yeast powder, and soybean powder.

[0052] The synergistic control parameters are: fermentation temperature 25-30℃, pH value 6.0-7.2, dissolved oxygen 10-25%, and carbon-nitrogen ratio 10:1-25:1.

[0053] In this step, it should be noted that the fermenter is a mechanically stirred fermenter with a stirring speed of 100-150 r / min. During the fermentation process, the carbon source concentration is maintained at 5-8 g / L by an automatic feeding system, the pH value is adjusted in real time by adding hydrochloric acid or sodium hydroxide solution, and the dissolved oxygen is adjusted by an aeration flow controller.

[0054] S4. Metabolite enrichment regulation: During the fermentation process in step S3, a metabolite synthesis inducer is added at a specific fermentation stage to maintain the regulation parameters stable until the fermentation endpoint, thereby obtaining a fermentation broth containing a high concentration of specific metabolites.

[0055] In this step, the specific fermentation stage is day 3-5 of fermentation culture, and the metabolite synthesis inducer is selected from at least one of methyl jasmonate, salicylic acid, and chitosan, with an addition concentration of 50-200 μmol / L.

[0056] The criteria for determining the fermentation endpoint are: the dry weight of mycelium in the fermentation broth no longer increases, and the content of specific metabolites reaches its peak.

[0057] S5. Separation and purification: The fermentation broth obtained in step S4 is subjected to solid-liquid separation, extraction and purification to obtain high-purity auricularia auricula-judae specific metabolites.

[0058] In this step, solid-liquid separation is performed by centrifugation or plate and frame filtration. The centrifugation conditions are: rotation speed 8000-10000 r / min and time 15-20 min.

[0059] For the extraction of polysaccharide metabolites, hot water extraction was used at an extraction temperature of 80-90℃ for 2-3 hours.

[0060] Ergosterol metabolites were extracted using an ethanol reflux extraction method with an ethanol concentration of 90-95%, a reflux temperature of 75-85℃, and a reflux time of 3-4 hours.

[0061] In this step, it should be noted that the purification process includes: after extraction of polysaccharide metabolites, 3-5 times the volume of 95% ethanol is added for alcohol precipitation, the precipitate is collected by centrifugation after standing for 12-16 hours, and then deproteinized by Sevag method and decolorized by macroporous resin adsorption to obtain polysaccharides of Auricularia auricula-judae with a purity of ≥90%.

[0062] After extraction, ergosterol metabolites were separated by silica gel column chromatography. The target eluent was collected and concentrated under reduced pressure using a mixture of petroleum ether and ethyl acetate (volume ratio 8:2-9:1) as the eluent to obtain ergosterol with a purity ≥85%.

[0063] In steps S1-S5 above, it should be noted that the specific metabolites include Tremella aurantium polysaccharide and ergosterol. After treatment in step S5, the content of the Tremella aurantium polysaccharide product is ≥15% (based on the dry weight of mycelium), and the content of the ergosterol product is ≥0.8% (based on the dry weight of mycelium). In addition, the selected Tremella aurantium mycelium strain is the Tremella aurantium strain with the preservation number Cgmccno.41097. This strain is used for activation after being subcultured on slant agar 3-4 times to ensure that the mycelium is free from contamination by other microorganisms and has stable activity.

[0064] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and examples of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0065] Implementation Plan

[0066] like Figure 1 As shown, the present invention provides an embodiment, which specifically includes the following steps:

[0067] (1) Material preparation

[0068] The strain was *Tremella aurantialba*, which was passaged three times on slant agar to ensure that the mycelium was free from contamination (verified by the streak plate method, with no contaminant colonies growing), and that the mycelium was white, fluffy, and growing vigorously.

[0069] Culture medium:

[0070] a. Activation medium (PDA modified medium): 220g potato (peeled and diced, boiled for 30min and then filtered to obtain juice), 18g glucose, 6g peptone, 20g agar, 1000mL distilled water, adjust pH to 6.3 with 1mol / L HCl or NaOH, autoclave at 121℃ for 20min, cool and pour into plates (20-25mL per plate).

[0071] b. Seed culture medium: 22g glucose, 4g yeast extract, 1.8g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 0.015g vitamin B1, 1000mL distilled water, adjust pH to 6.5, autoclave at 121℃ for 20min, cool and dispense into 500mL Erlenmeyer flasks (200mL culture medium per flask).

[0072] c. Fermentation medium: 25g glucose (carbon source), 3g peptone + 1g yeast extract (nitrogen source), 2.2g potassium dihydrogen phosphate, 1.2g magnesium sulfate heptahydrate, 0.4g calcium chloride, 1000mL distilled water, adjust the carbon-nitrogen ratio to 20:1, adjust the pH to 6.8, autoclave at 121℃ for 20min, and cool before use.

[0073] Reagents and equipment:

[0074] a. Reagents: Methyl jasmonate (analytical grade, purity ≥98%), salicylic acid (analytical grade), chitosan (degree of deacetylation ≥90%), 95% ethanol (food grade), petroleum ether (analytical grade, boiling range 60-90℃), ethyl acetate (analytical grade), macroporous resin (D101 type), silica gel (100-200 mesh);

[0075] b. Equipment: Clean bench (SW-CJ-2FD type), constant temperature incubator (LRH-250 type), shaker (THZ-300 type), 50L mechanically stirred fermenter (BLBIO-50JS type, equipped with online monitoring and automatic control system for temperature, pH and dissolved oxygen), high-speed centrifuge (TGL-20M type), rotary evaporator (RE-52AA type), high performance liquid chromatograph (HPLC, Agilent 1260 type, used for ergosterol content detection), ultraviolet spectrophotometer (UV-2550 type, used for polysaccharide content detection).

[0076] (2) Process operation steps

[0077] a. Activation of Auricularia auricula-judae strains (step S1):

[0078] In a clean bench, use a sterile inoculation needle to pick up a small amount of CGMCC No. 25689 auricularia auricula slant culture and inoculate it onto an activation medium plate. Place the plate in a constant temperature incubator and incubate for 6 days at 24℃, 70% relative humidity and in the dark, until the plate is covered with white fluffy mycelium (mycelium diameter ≥ 5cm). This is the active and stable auricularia auricula mother culture, which can be used for later use.

[0079] b. Seed liquid preparation (step S2):

[0080] In a clean bench, use a sterile punch (1 cm in diameter) to take 5 mycelial blocks from the mother culture plate obtained in step S1, and inoculate them into an Erlenmeyer flask containing 200 mL of seed culture medium. Place the Erlenmeyer flask on a shaker and set the rotation speed to 200 r / min, the aeration rate to 0.8 vvm, and the temperature to 26 ℃. Shake and culture for 48 h. Take a sample to detect the mycelial concentration. When the mycelial concentration reaches 10 g / L, stop the culture. This is the seed culture of Auricularia auricula-judae.

[0081] c. Controlled fermentation culture (step S3):

[0082] The golden ear seed liquid obtained in step S2 was inoculated into a 50L fermenter containing 30L of fermentation medium at an inoculation rate of 10% (v / v). The fermenter control system was started and the initial parameters were set as follows: fermentation temperature 28℃, pH value 6.8, dissolved oxygen 20%, and stirring speed 120r / min.

[0083] During fermentation, the carbon source concentration in the fermentation broth was maintained at 6-7 g / L by the automatic feeding system of the fermenter (feeding with 200 g / L glucose solution). The pH value was adjusted in real time by adding 1 mol / L HCl or 1 mol / L NaOH solution to stabilize it at 6.8 ± 0.2. The dissolved oxygen content was maintained at 20% ± 2% by adjusting the aeration flow rate (0.5-1.5 vvm). The fermentation was carried out for 4 days.

[0084] d. Regulation of metabolite enrichment (step S4):

[0085] On the third day of fermentation in step S3 (i.e., the middle of fermentation, when the mycelium enters the late logarithmic growth phase), methyl jasmonate (final concentration 100 μmol / L) was added to the fermenter as a metabolite synthesis inducer, and the fermentation parameters of step S3 were maintained until the end of fermentation.

[0086] Fermentation endpoint determination: Samples were taken every 12 hours to detect the dry weight of mycelium and the content of specific metabolites. When the dry weight of mycelium (after centrifugation and drying at 60℃ to constant weight) no longer increased in two consecutive tests (fluctuation ≤0.5g / L), and the polysaccharide content and ergosterol content reached their peak values, fermentation was stopped, and a fermentation broth containing a high concentration of specific metabolites was obtained.

[0087] e. Separation and purification (step S5):

[0088] e1. Solid-liquid separation: Transfer the fermentation broth obtained in step S4 to a high-speed centrifuge and centrifuge for 18 minutes at a speed of 9000 r / min and a temperature of 25℃. Collect the precipitate (Auricularia auricula mycelium) and the supernatant (containing some soluble polysaccharides).

[0089] e2. Polysaccharide extraction and purification:

[0090] 1) Extraction: Combine the centrifuged precipitate with the supernatant, add distilled water to adjust the solid-liquid ratio to 1:20 (g / mL), and place in an 85℃ constant temperature water bath for hot water extraction for 2.5h, stirring once every 30min during the extraction. After extraction, cool to room temperature and centrifuge (8000r / min, 15min) to obtain the supernatant.

[0091] 2) Alcohol precipitation: Slowly add 3 times the volume of 95% ethanol to the supernatant, stir well, and place in a 4℃ refrigerator for 14 hours. Centrifuge (9000r / min, 20min) to collect the polysaccharide precipitate.

[0092] 3) Deproteinization and decolorization: Dissolve the polysaccharide precipitate in distilled water (concentration 10 mg / mL), add Sevag reagent (chloroform: n-butanol = 5:1) at a volume ratio of 1:4, shake for 30 min, centrifuge (6000 r / min, 10 min), and take the supernatant. Repeat the operation 3 times to remove protein.

[0093] The deproteinized polysaccharide solution was then passed through a D101 macroporous resin column (column diameter:column height = 1:8), eluted with distilled water (elution flow rate 1.5 BV / h), the eluent was collected, concentrated by rotary evaporation to 1 / 5 of the original volume, and then freeze-dried to obtain the final product of Auricularia auricula polysaccharide.

[0094] e3. Ergosterol extraction and purification:

[0095] 1) Extraction: The mycelium obtained by centrifugation in step e1 (after freeze-drying) is crushed to 80 mesh, 5 times the mass of 95% ethanol is added, and the mixture is refluxed in an 80℃ constant temperature water bath for 3.5h. After extraction, the mixture is cooled to room temperature and filtered to obtain the filtrate.

[0096] 2) Column chromatography separation: The filtrate was concentrated by rotary evaporation until no ethanol odor was detected (concentration temperature 60℃, vacuum degree -0.08MPa), dissolved in petroleum ether (concentration 50mg / mL), and loaded onto a silica gel column (column diameter:column height = 1:10, silica gel volume 20 times the sample mass). A mixture of petroleum ether and ethyl acetate (volume ratio 8.5:1.5) was used as the eluent at a flow rate of 1.0 BV / h. The eluted components were detected by HPLC, and the target component containing ergosterol was collected.

[0097] 3) Concentration and drying: The target eluent is concentrated to dryness by rotary evaporation and then vacuum dried (temperature 50℃, vacuum degree -0.09MPa) to obtain the gold ear ergosterol product.

[0098] (3) Test results

[0099] The content and purity of the *Auricularia auricula-judae* polysaccharide and ergosterol products obtained in step e were determined:

[0100] 1) Polysaccharide content detection (phenol-sulfuric acid method): Using glucose as the standard, a standard curve was plotted, and the polysaccharide content of the finished product was found to be 16.2% (based on mycelial dry weight), with a purity of 92.5%.

[0101] 2) Ergosterol content detection (HPLC method): Using ergosterol standard (purity ≥99%) as a reference, the chromatographic conditions were: C18 column (250mm×4.6mm, 5μm), mobile phase: methanol-water (volume ratio 95:5), flow rate: 1.0mL / min, column temperature: 30℃, detection wavelength: 282nm. The content of ergosterol in the finished product was 0.88% (based on mycelial dry weight), and the purity was 87.3%.

[0102] 3) Batch stability verification: The experiment was repeated for 3 batches according to the above implementation plan. The polysaccharide content in the 3 batches was 16.2%, 15.8% and 16.5% respectively, and the ergosterol content was 0.88%, 0.85% and 0.91% respectively. The batch differences were 2.1% and 3.5% respectively, all ≤5%, which met the quality stability requirements.

[0103] Example 1: This embodiment uses Auricularia auricula-judae polysaccharide as the targeted enrichment target, and adjusts the key parameters based on the implementation plan, as follows:

[0104] 1. Adjustment of process parameters

[0105] a. Fermentation medium: The carbon source is 30g of glucose, the nitrogen source is 2g of peptone, the carbon-to-nitrogen ratio is adjusted to 25:1, and the pH value is adjusted to 6.5;

[0106] b. Fermentation parameters: fermentation temperature 26℃, dissolved oxygen 25%, stirring speed 130r / min;

[0107] c. Metabolite inducer: Add salicylic acid (final concentration 150 μmol / L) on day 3 of fermentation.

[0108] 2. Operating Procedures

[0109] The operation was carried out according to steps S1-S5 of the implementation plan, wherein the hot water extraction temperature was increased to 90℃ and the extraction time was extended to 3h during polysaccharide extraction; and the elution flow rate of the macroporous resin was adjusted to 1.2BV / h during purification.

[0110] 3. Test Results

[0111] Testing revealed that the polysaccharide content in the *Auricularia auricula-judae* mycelium obtained in this embodiment was 17.5% (dry weight), and the purity of the finished polysaccharide product was 94.2%. Compared with the polysaccharide content in the previous implementation plan (16.2%), this represents an 8.0% increase, achieving targeted enrichment of polysaccharides. The fermentation cycle was 6 days, and the batch-to-batch variation was 2.8%, meeting the requirements for industrial production.

[0112] Example 2: This embodiment uses ergosterol from the ear as the targeted enrichment agent, and adjusts the key parameters based on the implementation plan, as follows:

[0113] 1. Adjustment of process parameters

[0114] a. Fermentation medium: The carbon source is 20g sucrose, the nitrogen source is 3g yeast powder + 1g soybean powder, the carbon-nitrogen ratio is adjusted to 15:1, and the pH value is adjusted to 7.0;

[0115] b. Fermentation parameters: fermentation temperature 30℃, dissolved oxygen 15%, stirring speed 110r / min;

[0116] c. Metabolite inducer: Add chitosan (final concentration 200 μmol / L, dissolved in 1% acetic acid solution beforehand) on day 4 of fermentation.

[0117] 2. Operating Procedures

[0118] Perform the operation according to steps S1-S5 of the implementation plan, wherein the ethanol reflux temperature is increased to 85℃ and the reflux time is extended to 4h during ergosterol extraction; the eluent for silica gel column chromatography is adjusted to petroleum ether-ethyl acetate (volume ratio 8:2) and the elution flow rate is adjusted to 0.8 BV / h.

[0119] 3. Test Results

[0120] The test results showed that the ergosterol content in the auricularia auricula mycelium obtained in this embodiment was 0.98% (based on dry weight), and the purity of the ergosterol product was 89.5%. Compared with the ergosterol content in the implementation plan (0.88%), the ergosterol content was increased by 11.4%, achieving targeted enrichment of ergosterol. The fermentation cycle was 7 days, and the batch difference was 3.2%, meeting the production requirements of high-activity ergosterol.

[0121] The fermentation equipment used in this invention is a conventional mechanically stirred fermenter, which can achieve scale-up production of 50L-5000L. The culture medium raw materials (glucose, peptone, etc.) are readily available and low in cost. The amount of metabolite inducer added is small (50-200μmol / L), and there are no toxic or harmful reagent residues. It meets food-grade and pharmaceutical-grade production standards and can be widely used in functional foods, health products and pharmaceutical fields.

[0122] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A process for the targeted enrichment of gold ear functional fermentation metabolites, characterized in that, Comprise the following steps: S1. Activation of Chrysosporium sp: inoculate the preserved Chrysosporium mycelium spore into an activation medium, and recover culture under specific temperature and humidity conditions to obtain active and stable Chrysosporium master strain; S2. Seed liquid preparation: inoculate the Chrysosporium master strain obtained in step S1 into a seed liquid medium, and regulate the rotation speed and aeration under the condition of shaking culture until the mycelium concentration reaches a preset threshold to obtain Chrysosporium seed liquid; S3. Controllable fermentation culture: inoculate the Chrysosporium seed liquid obtained in step S2 into a fermentation medium according to a preset inoculation amount, and induce Chrysosporium mycelium to synthesize specific metabolites by synergistically regulating the fermentation temperature, pH value, dissolved oxygen content and carbon-nitrogen ratio in the fermentation tank; S4. Metabolite enrichment regulation: during the fermentation process in step S3, add a metabolite synthesis inducer at a specific fermentation stage, maintain the regulation parameters stable to the end of fermentation, and obtain a fermentation broth containing high concentration of specific metabolites; S5. Separation and purification: solid-liquid separation, extraction and purification treatment are performed on the fermentation broth obtained in step S4 to obtain high-purity Chrysosporium specific metabolite finished product.

2. The process for the targeted enrichment of gold ear functional metabolites according to claim 1, characterized in that, The activation medium in step S1 is a PDA modified medium, which comprises, by mass fraction: potato 200-250 parts, glucose 15-20 parts, peptone 5-8 parts, agar 18-22 parts, water 1000 parts, and the pH value is adjusted to 6.0-6.5; The recovery culture conditions are: temperature 22-26℃, relative humidity 65-75%, and light-free culture for 5-7d.

3. The process as claimed in claim 1, wherein the process is characterized by, The seed liquid medium in step S2 comprises, by mass fraction: glucose 20-25 parts, yeast extract 3-5 parts, potassium dihydrogen phosphate 1.5-2.0 parts, magnesium sulfate heptahydrate 0.8-1.2 parts, vitamin B1 0.01-0.02 parts, water 1000 parts, and the pH value is adjusted to 6.2-6.8; The shaking culture conditions are: rotation speed 180-220r / min, aeration 0.5-1.0vvm, temperature 24-28℃, and the mycelium concentration reaches 8-12g / L.

4. The process as claimed in claim 1, wherein the process is characterized by, The fermentation medium in step S3 comprises, by mass fraction: carbon source 15-30 parts, nitrogen source 2-5 parts, potassium dihydrogen phosphate 2.0-2.5 parts, magnesium sulfate heptahydrate 1.0-1.5 parts, calcium chloride 0.3-0.5 parts, and water 1000 parts; The carbon source is selected from at least one of glucose, sucrose and maltose, and the nitrogen source is selected from at least one of peptone, yeast powder and soybean powder; The synergistically regulated parameters are: fermentation temperature 25-30℃, pH value 6.0-7.2, dissolved oxygen content 10-25%, and carbon-nitrogen ratio 10:1-25:

1.

5. The process as claimed in claim 1, wherein the process is characterized by, The specific fermentation stage in step S4 is the 3rd-5th day of fermentation, and the metabolite synthesis inducer is selected from at least one of methyl jasmonate, salicylic acid and chitosan, and the addition concentration is 50-200μmol / L; The fermentation end point determination standard is that the mycelium dry weight in the fermentation broth no longer increases, and the content of specific metabolites reaches the peak value.

6. The process as claimed in claim 1, wherein the process is characterized by, The solid-liquid separation in step S5 is performed by centrifugal separation or plate and frame filtration, and the centrifugal separation is performed at a speed of 8000-10000 r / min for 15-20 min. The extraction treatment is performed by hot water extraction on polysaccharide metabolites at a temperature of 80-90℃ for 2-3 h. The ergosterol metabolites are extracted by ethanol reflux extraction at an ethanol concentration of 90-95%, a reflux temperature of 75-85℃, and a reflux time of 3-4 h.

7. The process of claim 6, wherein the process is characterized by, The purification treatment in step S5 includes: after the polysaccharide metabolites are extracted, 3-5 times of 95% ethanol is added for alcohol precipitation, and the precipitate is collected by centrifugation after standing for 12-16 h, and then deproteinized by the Sevag method and decolorized by macroporous resin adsorption to obtain gold ear polysaccharide with a purity of ≥90%; After the ergosterol metabolites are extracted, they are separated by silica gel column chromatography with petroleum ether-ethyl acetate mixed liquid (volume ratio 8:2-9:1) as the eluent, and the target elution fraction is collected and concentrated under reduced pressure to obtain gold ear ergosterol with a purity of ≥85%.

8. The process as claimed in claim 1, wherein the process is characterized by, The fermenter in step S3 is a mechanical stirring fermenter, and the stirring speed is 100-150 r / min. During the fermentation process, the carbon source concentration is maintained at 5-8 g / L by an automatic feeding system, the pH value is adjusted in real time by adding hydrochloric acid or sodium hydroxide solution, and the dissolved oxygen content is adjusted by an aeration flow controller.

9. The process as claimed in claim 1, wherein the process is characterized by, The specific metabolites include gold ear polysaccharide and ergosterol, and after step S5, the content of the gold ear polysaccharide product is ≥15% (based on the dry weight of mycelium), and the content of the ergosterol product is ≥0.8% (based on the dry weight of mycelium).

10. The process as claimed in claim 1, wherein the process is characterized by, The gold ear mycelium strain in step S1 is a gold ear (Tremella aurantialba) strain with a preservation number of Cgmcc no. 41097, which is activated after 3-4 times of slant subculture to ensure that the mycelium is free of bacterial contamination and has stable activity.

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