Method for high yield of beta-glucan by submerged fermentation of schizophyllum commune

Abstract

This invention discloses a method for high-yield β-glucan production through deep fermentation of *Schizophyllum commune*. Specifically, it includes the step of directly adding an aqueous mycelial suspension of *Schizophyllum commune* cultured on an slant culture medium for segmented controlled fermentation. This method is the first to use an aqueous mycelial suspension for direct fermentation to produce fermentation products, eliminating the need for subculturing and seed culture of the strain, thus significantly improving production efficiency and saving costs. It can be widely applied to the fermentation culture of fungi, molds, and some actinomycetes. Furthermore, by using a modified culture medium with specific types of buffer salts and added specific metabolic regulators, and implementing segmented dissolved oxygen control, the yield of β-glucan is greatly increased. This invention further employs an alternating five-column system for purification, achieving decolorization, desalting, deproteinization, and deionization to obtain a clear and transparent β-glucan solution that can be directly used for subsequent applications.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation culture technology, specifically to a method for high-yield β-glucan production by deep fermentation of Schizophyllum commune, and more particularly to a deep fermentation culture medium for Schizophyllum commune and a method for high-yield β-glucan production. Background Technology

[0002] Schizophyllum commune, belonging to the genus Schizophyllum in the family Schizophyllaceae of the class Basidiomycetes, has fruiting bodies that can be used medicinally and are also edible when young. It is distributed in many provinces and cities in my country, and its resources are abundant. The β-glucan produced by Schizophyllum has immune-boosting, anti-tumor, and moisturizing effects, and can be used in cosmetics, medicine, and other fields.

[0003] Among the existing methods for producing β-glucan using Schizophyllum commune, such as patent document CN111440832A, a fungal culture method for producing high levels of biological polysaccharides is provided. This method changes the growth state and activity of Schizophyllum commune on plates by using modified PDA solid medium for subculturing and adding solid insolubles to the seed medium and / or fermentation medium, thereby increasing the yield of β-glucan in the later fermentation culture (7-100 g / L). However, this method requires cumbersome operations such as subculturing and shake-flask culture before fermentation, making the entire production cycle very long, at least 20 days. Moreover, the method described for polysaccharide testing involves first boiling the fermentation broth (it is uncertain whether it contains Schizophyllum commune) at 80 degrees Celsius for 4 hours, and then sterilizing the culture medium at high temperature before fermentation. Both of these methods can extract polysaccharides from the culture medium (such as corn starch), so the polysaccharide yield measured is inaccurate and is not all of it produced by β-glucan from Schizophyllum commune. In addition, the composition of the culture medium used is not conducive to the observation of bacterial morphology. At the same time, soybean meal coarse powder will produce active peptides after microbial fermentation and hydrolysis, and corn cob coarse powder contains polysaccharides, fats, etc., all of which make subsequent purification of polysaccharides difficult.

[0004] Patent document CN107557407A discloses a method for regulating the molecular weight of schizophyllum polysaccharide, a fermentation product of Schizophyllum commune. This method involves slant inoculation culture, preparation of a fermentation seed culture, fermentation of the seed culture, and subsequent separation and purification to obtain refined schizophyllum polysaccharide. While this method eliminates the need for subculturing, it still requires the preparation of a fermentation seed culture. Furthermore, our previous research has shown that the yield of β-glucan in the fermentation broth obtained using this method is low (not reaching the polysaccharide content of this invention). In addition, its purification steps involve concentration, alcohol precipitation, and reconstitution. Concentration increases energy consumption, and alcohol precipitation and reconstitution use large amounts of ethanol, yet cannot guarantee the complete absence of ethanol residue.

[0005] In summary, existing methods for producing β-glucan via Schizophyllum commune fermentation either result in low β-glucan content in the fermentation broth (ranging from 2 g / L to 8 g / L) or involve numerous steps and long production cycles. No method has been found that balances high yield with a short production cycle. Furthermore, the purification processes used in existing Schizophyllum commune fermentation methods for β-glucan production also have drawbacks for industrial application, such as cumbersome procedures, long processing times, excessive use of organic solvents, and environmental friendliness. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for high-yield β-glucan production through deep fermentation of Schizophyllum commune.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides an application of a mycelial aqueous suspension in the fermentation culture of a bacterial strain, wherein the mycelial aqueous suspension is prepared by the following method:

[0009] The strain is inoculated onto an agar slant culture medium and cultured for 4-10 days. Then, sterile water is added, and the mycelial growth on the agar slant culture medium is scraped into sterile water. The sterile water containing the mycelial growth is collected to obtain the mycelial suspension.

[0010] As a preferred embodiment, the strain includes at least one of fungi, molds, and actinomycetes. For example, when using the aforementioned method to produce polysaccharides (containing β-glucan and / or heteropolysaccharides), the fungi are selected from Schizophyllum commune, Grifola frondosus, Sprassis crispa, Ganoderma lucidum, Cordyceps sinensis, and Morchella esculenta;

[0011] The molds were selected from Rhizonus oligosporus, Trichoderma harzianum, and Rhizopus oryzae.

[0012] The actinomycetes are selected from Streptomyces spp., Actinopolyspora halophila, and Acidophilic actinomycetes.

[0013] As a preferred embodiment, when using the aforementioned method to produce β-glucan, the fungus is selected from Schizophyllum commune, Sprassis crispa, and Polyporus frondosus.

[0014] Secondly, the present invention provides a fermentation culture method based on mycelial aqueous suspension, comprising the following steps:

[0015] A. Preparation of mycelial aqueous suspension: The strain is inoculated onto an agar slant culture medium and cultured for 4-10 days. Then, sterile water is added, and the mycelial growth on the agar slant culture medium is scraped into sterile water. The sterile water containing the mycelial growth is collected to obtain the mycelial aqueous suspension.

[0016] B. Fermentation culture of mycelial aqueous suspension: Inoculate the mycelial aqueous suspension into the fermentation medium and ferment for 4-10 days to obtain the fermentation product.

[0017] Thirdly, this invention provides a method for high-yield β-glucan production through submerged fermentation of *Schizophyllum commune* based on mycelial aqueous suspension, comprising the following steps:

[0018] S1. Preparation of mycelial aqueous suspension: Inoculate Schizophyllum commune onto slant culture medium, culture on the slant for 5-7 days, add sterile water, scrape the mycelial growth on the slant culture medium into sterile water, collect the sterile water containing the mycelial growth, and obtain the mycelial aqueous suspension.

[0019] S2. Fermentation culture of mycelial aqueous suspension: The mycelial aqueous suspension is inoculated into a fermenter containing the first component of the Schizophyllum commune deep fermentation medium for the first stage of fermentation culture; then the second component of the Schizophyllum commune deep fermentation medium is added to the fermenter for the second stage of fermentation culture, thus obtaining the fermentation broth containing β-glucan.

[0020] As a preferred embodiment, in step S1, the method for preparing the slant culture medium is as follows: peeled potatoes are cut into pieces and boiled in water for 20-40 minutes, then filtered with gauze. Glucose, yeast extract powder, and agar powder are added to the filtrate, heated and stirred to dissolve, and then water is added in a measured amount.

[0021] The temperature for slant culture is 26-30℃;

[0022] The ratio of the volume of sterile water added to the volume of the slant culture medium is 1:0.8-1.2.

[0023] As a preferred embodiment, in step S2, the volume ratio of the mycelial aqueous suspension to the first component of the Schizophyllum commune deep fermentation medium is 0.5-0.7:35.

[0024] The first component of the *Schizophyllum commune* submerged fermentation medium comprises the following components at the following concentrations: glucose 25-30 g / L, yeast extract 2-6 g / L, buffer salt 0.5-1.0 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, and the remainder being water;

[0025] The second component of the Schizophyllum commune deep fermentation medium is a metabolic regulatory factor, and the final concentration of the metabolic regulatory factor is 0.5-5 ppm.

[0026] As a preferred option, the conditions for the first stage of fermentation culture are: fermentation temperature 26-30℃, dissolved oxygen 70-80%, and culture time 3-4 days;

[0027] As a preferred option, the conditions for the second stage of fermentation are: fermentation temperature 26-30℃, dissolved oxygen 15-25%, and culture time 1-3 days.

[0028] As a preferred embodiment, the dissolved oxygen is regulated by controlling the stirring speed and ventilation volume; for example, during the first stage of fermentation, the stirring speed is controlled at 100-200 rpm and the ventilation volume at 1.8-5.5 m³ / h. 3 The dissolved oxygen level is maintained at 70-80% per hour; during the second fermentation culture, the stirring speed is controlled at 50-120 rpm and the aeration rate at 1.0-3.0 m³ / h. 3 / h, to keep dissolved oxygen at 15-25%.

[0029] As a preferred embodiment, the metabolic regulatory factor is selected from one or more of adenine nucleotide, para-aminobenzoic acid, folic acid, pan-aminophen, and N,N-dimethylglycine.

[0030] The buffer salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0031] As a further preferred embodiment, the metabolic regulatory factor is selected from one or more of adenine nucleotides, para-aminobenzoic acid, and folic acid, which can further increase the yield of β-glucan.

[0032] As a further preferred option, the buffer salt is selected from dipotassium hydrogen phosphate, which can further increase the yield of β-glucan. In preliminary experiments of this invention, it was found that the choice of buffer salt had the following effect on β-glucan yield: dipotassium hydrogen phosphate was higher than potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0033] As a preferred embodiment, the method further includes the following steps:

[0034] S3. After inactivating the fermentation broth containing β-glucan, centrifuge the centrifuged liquid and pass it into the "alternating five-column system" for purification. After fine filtration, a colorless and transparent β-glucan liquid is obtained.

[0035] The "alternating five-column system" comprises a granular activated carbon column, an anion exchange resin column, a first macroporous adsorption resin column, a cation exchange resin column, and a second macroporous adsorption resin column arranged in series; the connection order of each column can be arbitrarily adjusted. In preliminary experiments of this invention, it was found that if one or more columns were missing, or if the type of resin column was replaced, the purification effect would be poor, resulting in either a darker color, greater polysaccharide loss, or inadequate protein removal.

[0036] As a further preferred embodiment, the "alternating five-column combined system" includes a granular activated carbon column, an anion exchange resin column, a first macroporous adsorption resin column, a cation exchange resin column, and a second macroporous adsorption resin column arranged in sequence.

[0037] As a preferred embodiment, the diameter-to-height ratio of the granular activated carbon column is 1:4-1:6, more preferably 1:5, and the granular activated carbon used is at least one of 8-30 mesh wood-based activated carbon, fruit shell activated carbon, and coal-based activated carbon.

[0038] The diameter-to-height ratio of the anion exchange resin column is 1:4-1:6, more preferably 1:5. The anion exchange resin used is selected from anion exchange resins containing -N(CH3)2, and its basicity is weaker than that of strong base anion exchange resins; for example, D301.

[0039] The diameter-to-height ratio of the cation exchange resin column is 1:4-1:6, more preferably 1:5, and the cation exchange resin used is selected from cation exchange resins with carboxylic acid groups; for example, D113.

[0040] The diameter-to-height ratio of the first macroporous adsorption resin column and the second macroporous adsorption resin column is 1:2-1:4, more preferably 1:3, and both are selected from the HYA series resins. For example, the first macroporous adsorption resin column is HYA106, and the second macroporous adsorption resin column is HYA605.

[0041] As a preferred embodiment, the fine filtration is performed using a 0.45μm polypropylene membrane.

[0042] The "alternating five-column combined system" used in this invention first removes some pigments and impurities from the centrifuged liquid through a granular activated carbon column, then deions it through an anion exchange resin column, then removes some pigments and proteins through a first macroporous adsorption resin column, then deions it through a cation exchange resin column, then deodorizes it through a second macroporous adsorption resin column, and finally filters it through a 0.45μm polypropylene membrane. This achieves the purposes of deionization, protein removal, color removal, odor removal, and clarification. The resulting β-glucan solution is clear and transparent with a β-glucan content of more than 1.5%, and the solution can be used directly without drying, pulverizing, or reconstitution.

[0043] Secondly, the present invention provides a deep fermentation culture medium for Schizophyllum commune, comprising a first component and a second component, wherein the first component comprises the following components at the following concentrations: glucose 25-30 g / L, yeast extract 2-6 g / L, buffer salt 0.5-1.0 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, and water to make up a quantitative amount.

[0044] The second component is a metabolic regulatory factor, and the final concentration of the metabolic regulatory factor is 0.5-5 ppm;

[0045] The metabolic regulatory factor is selected from one or more of adenine nucleotide, para-aminobenzoic acid, folic acid, pan-pan-amino acid, and N,N-dimethylglycine.

[0046] The buffer salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0047] As a preferred embodiment, the metabolic regulatory factor is selected from one or more of adenine nucleotides, para-aminobenzoic acid, and folic acid. More preferably, the metabolic regulatory factor is folic acid; and the buffer salt is dipotassium hydrogen phosphate.

[0048] Compared with the prior art, the present invention has the following significant effects:

[0049] 1. This invention is the first to directly ferment and produce fermentation products by suspending activated slant culture strains in water to form a mycelial suspension. This method eliminates the need for subculturing and seed culture of the strains, greatly improving production efficiency and saving costs. It can be widely applied to the fermentation culture of fungi, molds, and some actinomycetes. Furthermore, using mycelia to directly prepare mycelial suspensions allows the strains to quickly adapt to liquid culture media, shortening the lag phase in liquid media, eliminating the need for shake-flask culture, and avoiding the risk of contamination caused by excessive operational steps.

[0050] 2. In this invention, the method for producing β-glucan through submerged fermentation of *Schizophyllum commune* based on mycelial aqueous suspension significantly increases β-glucan yield by modifying the fermentation medium formula, using specific types of buffer salts, and adding specific metabolic regulatory factors. The use of dipotassium hydrogen phosphate serves both as a phosphorus and potassium mineral element and as a suitable buffer for the pH value during the logarithmic phase. This technique allows the strain to maintain a more suitable pH growth environment during the logarithmic phase under the action of the buffer, enabling rapid proliferation. During the stationary phase, the pH value is maintained at a relatively weakly acidic level to inhibit the continued excessive proliferation of *Schizophyllum commune*, while simultaneously stimulating the secretion of extracellular polysaccharides. The use of metabolic regulatory factors promotes the efficient synthesis of extracellular polysaccharides, ultimately achieving high yields.

[0051] 3. In the method for producing β-glucan based on the submerged fermentation culture of *Schizophyllum commune* using mycelial aqueous suspension, this invention further employs segmented fermentation control technology. During the logarithmic growth phase of *Schizophyllum commune*, adjusting the fermentation speed and aeration rate ensures a high dissolved oxygen content (80-70%) in the fermentation broth, promoting rapid cell growth. During the stationary growth phase, adjusting the fermentation speed and aeration rate ensures a lower dissolved oxygen content (25-15%) in the fermentation broth, inhibiting the continued rampant proliferation of *Schizophyllum commune* and stimulating the cell secretion of extracellular polysaccharides. This further increases the yield of β-glucan.

[0052] 4. This invention further addresses the purification of fermentation broth by providing a method for adsorption using an alternating five-column system after centrifugation of the inactivated fermentation broth. This adsorption process achieves decolorization, desalting, deproteinization, and deionization, yielding a clear and transparent β-glucan solution. This purification process is simple to operate, highly efficient, energy-saving, and does not require organic solvents. Furthermore, it can be automated from fermentation culture to purification preparation, or manual operation is also possible. In addition, compared to existing methods that use solid-liquid separation of the soaking liquid, followed by activated carbon adsorption and subsequent solid-liquid separation, alcohol precipitation and drying of the supernatant, and require the resulting β-glucan to be pulverized and reconstituted before application, the β-glucan solution obtained using the purification method of this invention can be directly used for subsequent applications without the need for drying, pulverizing, and reconstitution. Attached Figure Description

[0053] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0054] Figure 1 This is a schematic diagram of the "alternating five-column combined system" used in Example 1;

[0055] Figure 2 The images show the centrifuged liquid before purification and the purified β-glucan liquid after purification using the "alternating five-column system" in Example 1. Detailed Implementation

[0056] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0057] Prior to this invention, existing reports on the deep fermentation of *Schizophyllum commune* to produce β-glucan and its purification processes mostly involved modifying the culture medium components, particularly the activated and subcultured solid medium, to alter the growth state and activity of the strain. Furthermore, the bacterial suspension cultured in the fermenter was generally prepared using the traditional shake-flask method. The purification processes mostly involved boiling the fermentation broth or mycelium, solid-liquid separation, activated carbon decolorization, further solid-liquid separation, concentration followed by either direct alcohol precipitation, and finally drying or further reconstitution. Essentially, these processes did not deviate from the traditional design principles of fermentation and purification processes.

[0058] In this invention, the bacterial suspension cultured in the upper fermenter was innovatively replaced with an agar suspension of mycelium from slant culture, avoiding the cumbersome operation of multiple shake flasks and large-volume bacterial suspension transfers, as well as the long shake flask culture time. Furthermore, by improving the fermentation medium formula, specific buffer salts (such as dipotassium hydrogen phosphate) and metabolic regulators were used to achieve rapid growth of *Schizophyllum commune* and promote efficient synthesis of extracellular polysaccharides, thereby increasing β-glucan production. Additionally, by segmentally controlling the dissolved oxygen content during fermentation, the β-glucan production was further increased. The fermentation broth obtained after deep fermentation of this invention showed a β-glucan yield of over 10 g / L, a better yield of over 18 g / L, and an optimal yield of 23 g / L.

[0059] This invention also improves the method for purifying fermentation broth, abandoning traditional purification steps. Instead, it utilizes a specific "alternating five-column system" for sequential exchange and adsorption, followed by membrane filtration. This achieves decolorization, deodorization, protein removal, and deionization, ultimately yielding a colorless, odorless, and clear β-glucan solution. This method effectively avoids the use of organic solvents and the problem of ultrafine particle residues in excipients, enabling continuous production, reducing labor intensity, and improving production efficiency. Furthermore, the purified β-glucan solution can be directly used for subsequent applications.

[0060] In the following examples, the slant culture medium is prepared using the existing PDA culture medium preparation method.

[0061] In the following examples, the specific methods used to determine the β-glucan content are as follows:

[0062] 1. Determination of total sugar content using the phenol-sulfuric acid method

[0063] Accurately weigh 10 mg of anhydrous glucose standard, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the mark to obtain the reference solution.

[0064] Take 7 sets of test tubes. Except for the blank (1 tube), each of the other 6 sets consists of 2 parallel test tubes. Accurately measure 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml, 1.2 ml, and 1.4 ml of the reference solution into stoppered colorimetric tubes, and add water to each tube to a final volume of 2.0 ml. Add 2.0 ml of water to the blank tube. Then, accurately add 1 ml of 5% phenol solution to each tube and mix well. Quickly and accurately add 5 ml of sulfuric acid, cap the tubes, mix well, and place in a boiling water bath for 15 minutes. After cooling to room temperature, measure the absorbance at 490 nm and plot a standard curve.

[0065] Take 0.2 ml of the test sample (fermentation broth prepared in each example and comparative example, and purified β-glucan solution) and place them in two stoppered test tubes, adding water to each to a final volume of 2.0 ml. Then, precisely add 1 ml of 5% phenol solution to each tube and shake well. Quickly and precisely add 5 ml of sulfuric acid, cap the tubes, shake well, and place them in a boiling water bath for 15 minutes. After cooling to room temperature, measure the absorbance at a wavelength of 490 nm. Calculate the total sugar content in the test sample based on the standard curve.

[0066] 2. Determination of free glucose content using a biosensor method

[0067] The content of free glucose in the test sample was directly determined using an SBA-40E biosensor analyzer (Shandong Provincial Institute of Biology, Scientific Research Institute) following the steps in the instrument's instruction manual.

[0068] 3. Calculation of β-glucan content

[0069] The β-glucan content is calculated using the following formula, and the content is expressed as a percentage.

[0070] β-glucan content = total sugar content - free glucose content

[0071] In the following examples, the specific methods used for determining the solid content are as follows:

[0072] Weigh approximately 1g of the fermentation broth (accurate to 0.001g) and place it in a pre-weighed evaporating dish. Place the dish in an oven at 105±2℃ and dry for 4 hours. Remove and place in a desiccator to cool to room temperature, then weigh. Repeat the drying process, each time for 1 hour, until the weight difference between two consecutive drying cycles is less than 5mg. Calculate the solid content using the following formula:

[0073]

[0074] Where: X—solid content, %;

[0075] m1—Weight of empty evaporating dish, g;

[0076] m2 — Weight of the evaporating dish and solids after drying to constant weight, in g;

[0077] m—sample volume of fermentation broth, in grams.

[0078] In the following embodiments, the specific methods used for measuring conductivity are as follows:

[0079] The conductivity was measured directly using a Leici DDS-307 conductivity meter.

[0080] In the following examples, the specific methods used for determining protein content are as follows:

[0081] Chinese Pharmacopoeia 2020 Edition, Volume 4, General Chapter 0731, Determination of Protein Content (Method II, Folin-Ciocalteu Method).

[0082] In the following examples, the folic acid, para-aminobenzoic acid, adenine nucleotide, pan-aminophen, and N,N-dimethylglycine used were all commercially available raw materials, obtained through purchase. In specific use, the purchased solid raw materials were first prepared into solutions of a certain concentration, and then each solution was added to the fermentation tank.

[0083] Example 1

[0084] This embodiment provides a method for producing β-glucan by deep fermentation culture of Schizophyllum commune, the specific steps of which are as follows:

[0085] 1. Preparation of a water suspension of Schizophyllum commune mycelium

[0086] (1) Preparation of slant culture medium: Weigh 120g of peeled potatoes, cut them into small pieces, add 500ml of pure water and boil for 30min, then filter through two layers of gauze. Add 12g of glucose and 3g of yeast extract to the filtrate, preheat and stir to dissolve, then add 11g of agar powder, heat and stir to dissolve, and add water to 600ml. While still hot, dispense the culture medium into sterilized test tubes, 40ml / test tube, and finally stopper the test tubes with silicone foam stoppers, dispensing 15 tubes in total. After sterilization at 121℃ for 30min, place the slants to 2 / 3 of the test tube to set.

[0087] (2) Slant culture: Take a small piece of Schizophyllum commune from the activated slant culture using an inoculation hook (the strain used in each example is "Schizophyllum commune with strain number: CICC 2591, source: China Industrial Microbial Culture Collection Center") and inoculate it into the bottom of the slant. Seal the test tube with silicone foam plug. Repeat this process for 15 slant cultures. Place the tube in a biochemical incubator and incubate at 28°C for 6 days.

[0088] (3) Preparation of mycelial aqueous suspension: Take 15 sterilized test tubes, add 40ml of pure water to each tube, and stopper with silicone foam. Sterilize at 121℃ for 30min to obtain sterile water. After cooling, pour 40ml of sterile water into each tube slant, scrape the mycelial growth into the sterile water with an inoculation scraper, and finally collect them into a 1000ml sterile Erlenmeyer flask (600ml of mycelial aqueous suspension), stopper with silicone foam, and refrigerate for later use.

[0089] 2. Suspension culture of mycelium in fermentation tank

[0090] (1) Culture medium preparation: Weigh 1.0 kg of glucose, 105 g of yeast extract, 26.3 g of dipotassium hydrogen phosphate and 13.1 g of magnesium sulfate heptahydrate, dissolve them in pure water, transfer them to a 50 L fermenter, and make up to 35 L with pure water. After sterilization at 115 °C for 20 min, cool down to 28 °C and inoculate with 600 ml of mycelial suspension.

[0091] (2) 50L tank fermentation: 0-80h, temperature controlled at 28℃, rotation speed at 170rpm, and ventilation rate at 2.0m³ / h. 3 Cultured at 80h (dissolved oxygen 80%-70%). At 80h, add a single folic acid solution (final concentration 3ppm) as a feed. Maintain a temperature of 28℃, rotation speed of 110rpm, and aeration rate of 1.2m³ / h from 80-120h. 3 Cultured at 120h (dissolved oxygen 25%-15%). Fermentation stopped after 120h. At this point, the β-glucan yield in the fermentation broth reached 2.3%, or 23g / L, with a characteristic odor, brownish-yellow color, electrical conductivity of 3900μS / cm, and protein content of 0.62%.

[0092] 3. β-glucan purification

[0093] The fermentation broth was heated to 90°C and kept at that temperature for 30 minutes to inactivate the fermentation agent. Then, it was centrifuged in a disc centrifuge via tubing. The centrifuged liquid ( Figure 2The centrifuged liquid before purification (yellow in color) enters the storage tank and is then pumped into the "alternating five-column system," which consists of a granular activated carbon column (diameter-to-height ratio 1:5, 8-30 mesh wood-based activated carbon, size 100*500mm), an anion exchange resin column (diameter-to-height ratio 1:5, model D301, size 100*500mm), and a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA106, size 100*300mm). The product was processed using a cation exchange resin column (diameter-to-height ratio 1:5, model D113, size 100*500mm) and a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA605, size 100*300mm) (flow rate controlled at 3-4 BV / h), followed by fine filtration through a 0.45μm membrane to obtain a β-glucan liquid product with a solid content of 2.1% and a β-glucan content of 1.9%. The product was colorless, odorless, and transparent. Figure 2 The purified β-glucan liquid shown is colorless, has a conductivity of 37 μS / cm, and a protein content of 0.12%, and can be applied directly.

[0094] Example 2

[0095] This embodiment provides a method for producing β-glucan by deep fermentation culture of Schizophyllum commune, the specific steps of which are as follows:

[0096] 1. Preparation of a water suspension of Schizophyllum commune mycelium

[0097] (1) Preparation of slant culture medium: Weigh 120g of peeled potatoes, cut them into small pieces, add 500ml of pure water and boil for 30min, then filter through two layers of gauze. Add 12g of glucose and 3g of yeast extract to the filtrate, preheat and stir to dissolve, then add 11g of agar powder, heat and stir to dissolve, and add water to 600ml. While still hot, dispense the culture medium into sterilized test tubes, 40ml / test tube, and finally stopper the test tubes with silicone foam stoppers, dispensing 15 tubes in total. After sterilization at 121℃ for 30min, place the slants to 2 / 3 of the test tube to set.

[0098] (2) Slant culture: Take a small piece of bacterial growth from the activated slant culture with an inoculation hook and inoculate it into the bottom of the slant. Seal the test tube with a silicone foam stopper. Repeat this process for 15 slant cultures. Place the tube in a biochemical incubator and incubate at 29°C for 5 days.

[0099] (3) Preparation of mycelial aqueous suspension: Take 15 sterilized test tubes, add 40ml of pure water to each tube, and stopper with silicone foam. Sterilize at 121℃ for 30min to obtain sterile water. After cooling, pour 40ml of sterile water into each tube slant, scrape the mycelial growth into the sterile water with an inoculation scraper, and finally collect them into a 1000ml sterile Erlenmeyer flask (600ml of mycelial aqueous suspension), stopper with silicone foam, and refrigerate for later use.

[0100] 2. Suspension culture of mycelium in fermentation tank

[0101] (1) Culture medium preparation: Weigh 0.875 kg of glucose, 70 g of yeast extract, 17.5 g of dipotassium hydrogen phosphate and 7 g of magnesium sulfate heptahydrate, dissolve them in pure water, transfer them to a 50 L fermenter, and make up to 35 L with pure water. After sterilization at 115 °C for 20 min, cool down to 29 °C and inoculate with 600 ml of mycelial suspension.

[0102] (2) Fermentation in a 50L tank for 0-80 hours, with controlled temperature at 29℃, rotation speed at 200rpm, and ventilation rate at 2.2m³ / h. 3 Cultured at 80h (dissolved oxygen 80%-70%). At 80h, add a single para-aminobenzoic acid solution (final concentration 0.5ppm) in a fed-batch manner. Maintain a temperature of 29℃, rotation speed of 120rpm, and aeration rate of 1.5m³ / h from 80-120h. 3 Cultured at 120h (dissolved oxygen 25%-15%). Fermentation stopped after 120h. At this point, the β-glucan yield in the fermentation broth was 1.8%, or 18g / L, with a characteristic odor, yellow color, electrical conductivity of 3870μS / cm, and protein content of 0.60%.

[0103] 3. β-glucan purification

[0104] Heat the fermentation broth to 90℃ and keep it warm for 30 minutes to inactivate it. The solution is then fed into a disc centrifuge via pipeline for centrifugation. The centrifuged liquid enters a storage tank and is then pumped into an alternating five-column system, which consists of a granular activated carbon column (diameter-to-height ratio 1:5, 8-30 mesh wood-based activated carbon, size 100*500mm), an anion exchange resin column (diameter-to-height ratio 1:5, model D301, size 100*500mm), a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA106, size 100*300mm), a cation exchange resin column (diameter-to-height ratio 1:5, model D113, size 100*500mm), and a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA605, size 100*300mm) connected in sequence for treatment (flow rate controlled at 3-4 BV / h). Finally, it is finely filtered through a 0.45μm membrane to obtain the β-glucan liquid product with a solid content of 1.7%. The resulting product contains 1.5% β-glucan, is colorless, odorless, and transparent, has an electrical conductivity of 32 μS / cm, and a protein content of 0.13%, and can be used directly.

[0105] Example 3

[0106] This embodiment provides a method for producing β-glucan by deep fermentation culture of Schizophyllum commune, the specific steps of which are as follows:

[0107] 1. Preparation of a water suspension of Schizophyllum commune mycelium

[0108] (1) Preparation of slant culture medium: Weigh 120g of peeled potatoes, cut them into small pieces, add 500ml of pure water and boil for 30min, then filter through two layers of gauze. Add 12g of glucose and 3g of yeast extract to the filtrate, preheat and stir to dissolve, then add 11g of agar powder, heat and stir to dissolve, and add water to 600ml. While still hot, dispense the culture medium into sterilized test tubes, 40ml / test tube, and finally stopper the test tubes with silicone foam stoppers, dispensing 15 tubes in total. After sterilization at 121℃ for 30min, place the slants to 2 / 3 of the test tube to set.

[0109] (2) Slant culture: Take a small piece of bacterial growth from the activated slant culture with an inoculation hook and inoculate it into the bottom of the slant. Seal the test tube with a silicone foam plug. Repeat this process for 15 slant cultures. Place the tube in a biochemical incubator and incubate at 27°C for 7 days.

[0110] (3) Preparation of mycelial aqueous suspension: Take 15 sterilized test tubes, add 40ml of pure water to each tube, and stopper with silicone foam. Sterilize at 121℃ for 30min to obtain sterile water. After cooling, pour 40ml of sterile water into each tube slant, scrape the mycelial growth into the sterile water with an inoculation scraper, and finally collect them into a 1000ml sterile Erlenmeyer flask (600ml of mycelial aqueous suspension), stopper with silicone foam, and refrigerate for later use.

[0111] 2. Suspension culture of mycelium in fermentation tank

[0112] (1) Culture medium preparation: Weigh 1.05 kg of glucose, 210 g of yeast extract, 35 g of dipotassium hydrogen phosphate and 17.5 g of magnesium sulfate heptahydrate, dissolve them in pure water, transfer them to a 50 L fermenter, and make up to 35 L with pure water. After sterilization at 115 °C for 20 min, cool down to 27 °C and inoculate with 600 ml of mycelial suspension.

[0113] (2) 50L tank fermentation: 0-66h, temperature 27℃, rotation speed 150rpm, ventilation 1.8m 3 / h culture (dissolved oxygen at 80%-70%).

[0114] (3) Preparation of culture medium in a 500L tank: Weigh 10.5kg of glucose, 2.1kg of yeast extract, 350g of dipotassium hydrogen phosphate, and 175g of magnesium sulfate heptahydrate, dissolve them in pure water, and transfer them to a 500L fermenter. Make up the volume to 350L with pure water. After sterilization at 115℃ for 20min, cool to 27℃, and then transfer the 66h-hour-old inoculum from the 50L fermenter into the 500L fermenter. The 500L fermenter maintains a temperature of 27℃, a rotation speed of 100rpm, and a ventilation rate of 5.5m³ / h for 0-80h. 3Cultured at 80h (dissolved oxygen 80%-70%). At 80h, a single adenine nucleotide solution (final concentration 5ppm) is added as a feed. From 80-120h, maintain a temperature of 27℃, a rotation speed of 50rpm, and an aeration rate of 3.0m³ / h. 3 Cultured at 120h (dissolved oxygen 25%-15%). Fermentation stopped after 120h. At this point, the β-glucan yield in the fermentation broth was 2.1%, or 21g / L, with a characteristic odor, yellow color, electrical conductivity of 3930μS / cm, and protein content of 0.57%.

[0115] 3. β-glucan purification

[0116] The fermentation broth was heated to 90°C and kept at that temperature for 30 minutes to inactivate the fermentation agent. Then, it was piped into a disc centrifuge for centrifugation. The centrifuged liquid was transferred to a storage tank and then pumped into an alternating five-column system (e.g., [system name missing]). Figure 1 As shown), that is Figure 1 The following columns, connected sequentially from left to right, are used for treatment (flow rate controlled at 3-4 BV / h): granular activated carbon column (diameter-to-height ratio 1:5, 8-30 mesh wood-based activated carbon, size 300*1500mm), anion exchange resin column (diameter-to-height ratio 1:5, model D301, size 300*1500mm), macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA106, size 300*900mm), cation exchange resin column (diameter-to-height ratio 1:5, model D113, size 300*1500mm), and macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA605, size 300*900mm). Finally, the product is finely filtered through a 0.45μm membrane to obtain a β-glucan liquid product with a solid content of 1.9%. The resulting product contains 1.7% β-glucan, is colorless, odorless, and transparent, has an electrical conductivity of 41 μS / cm, and a protein content of 0.11%, and can be used directly.

[0117] Example 4

[0118] This comparative example provides a method for producing β-glucan by deep fermentation of *Schizophyllum commune*. The only difference from Example 1 is that in step (2) of the mycelial fermentation tank suspension culture, a single pan-amyline solution (final concentration 3 ppm) is added as a feedstock at 80 h instead of folic acid. After 120 h of fermentation, the β-glucan yield in the fermentation broth was 1.46%, or 14.6 g / L.

[0119] Example 5

[0120] This comparative example provides a method for producing β-glucan by submerged fermentation of *Schizophyllum commune*. The only difference from Example 1 is that in step (2) of the mycelial fermentation tank suspension culture, a single N,N-dimethylglycine solution (final concentration 3 ppm) is added as a feedstock at 80 h to replace folic acid. After 120 h of fermentation, the β-glucan yield in the fermentation broth was 1.37%, or 13.7 g / L.

[0121] Example 6

[0122] This embodiment provides a method for producing β-glucan through deep fermentation of Schizophyllum commune. Compared with Example 1, the only difference is that in the subsequent β-glucan purification step, the fermentation broth obtained in Example 1 is processed using a series of interconnected granular activated carbon columns (diameter-to-height ratio 1:5, 8-30 mesh wood-based activated carbon, size 100*500mm), anion exchange resin column (diameter-to-height ratio 1:5, model D301, size 100*500mm), cation exchange resin column (diameter-to-height ratio 1:5, model D113, size 100*500mm), and macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA605, size 100*300mm) (with a flow rate controlled at 3-4 BV / h). Finally, the mixture is finely filtered through a 0.45 μm membrane to obtain the β-glucan liquid product. The resulting product contained 1.87% β-glucan, was light yellow, odorless and transparent, had an electrical conductivity of 35 μS / cm, and a protein content of 0.36%. Compared with Example 1, the final product obtained using the four-column combined system of this example showed a significantly higher protein content, comparable deionization effect, and reduced decolorization effect.

[0123] Example 7

[0124] This embodiment provides a method for producing β-glucan through deep fermentation of Schizophyllum commune. Compared with Example 1, the only difference is that in the subsequent β-glucan purification step of the fermentation broth obtained in Example 1, the following columns are used in sequence: a granular activated carbon column (diameter-to-height ratio 1:5, 8-30 mesh wood-based activated carbon, size 100*500mm), a cation exchange resin column (diameter-to-height ratio 1:5, model D113, size 100*500mm), a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA605, size 100*300mm), an anion exchange resin column (diameter-to-height ratio 1:5, model D301, size 100*500mm), and a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA106, size 100*300mm) (flow rate controlled at 3-4 BV / h), and finally fine filtration through a 0.45μm membrane to obtain the β-glucan liquid product. The resulting product contained 1.63% β-glucan, was colorless, odorless, and transparent, had an electrical conductivity of 40 μS / cm, and a protein content of 0.14%. Compared to Example 1, the final product obtained using the five-column combined system of this example showed a decrease in β-glucan content, but achieved comparable deionization, deproteinization, and decolorization effects, and could be used directly.

[0125] Example 8

[0126] This embodiment provides a method for producing β-glucan through deep fermentation of Schizophyllum commune. Compared with Example 1, the only difference is that in the subsequent β-glucan purification step of the fermentation broth obtained in Example 1, the following columns are used in sequence: a granular activated carbon column (diameter-to-height ratio 1:5, 8-30 mesh wood-based activated carbon, size 100*500), a cation exchange resin column (diameter-to-height ratio 1:5, model 724, size 100*500), a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA605, size 100*300), an anion exchange resin column (diameter-to-height ratio 1:5, model D301, size 100*500), and a macroporous adsorption resin column (diameter-to-height ratio 1:3, model HYA106, size 100*300) (flow rate controlled at 3-4 BV / h), and finally fine filtration through a 0.45 μm membrane to obtain the β-glucan liquid product. The obtained product contained 1.82% β-glucan, was colorless, odorless, and transparent, had an electrical conductivity of 900 μS / cm, and a protein content of 0.11%. Compared with Example 1, the final product obtained using the five-column combined system in this example had a higher electrical conductivity, while other characteristics were similar.

[0127] Example 9

[0128] This embodiment provides a method for producing β-glucan by deep fermentation of *Schizophyllum commune*. The only difference from Example 1 is that in step (1) of the mycelial fermentation tank suspension culture, potassium dihydrogen phosphate is used instead of dipotassium hydrogen phosphate in the culture medium preparation. Other culture medium components and fermentation parameters (including pH) are the same as in Example 1. After 120 hours of fermentation, the β-glucan yield in the fermentation broth was 1.1%, or 11 g / L.

[0129] Example 10

[0130] This embodiment provides a method for producing β-glucan by deep fermentation of Schizophyllum commune. Compared with Example 1, the only difference is that in step (2) of mycelial fermentation in a suspension culture tank, the dissolved oxygen content of the fermentation broth is controlled at 60-50% during 0-80 h. After 120 h of fermentation, the yield of β-glucan in the fermentation broth is 1.43%, i.e., 14.3 g / L.

[0131] Comparative Example 1

[0132] This comparative example provides a method for producing β-glucan by deep fermentation of *Schizophyllum commune*. The only difference from Example 1 is that in step (2) of the mycelial fermentation tank suspension culture, the dissolved oxygen content of the fermentation broth is controlled at 25%-15% during the first 0-80 hours and at 80%-70% during the second 80-120 hours. After 120 hours of fermentation, the β-glucan yield in the fermentation broth is 0.56%, or 5.6 g / L.

[0133] Comparative Example 2

[0134] This comparative example provides a method for producing β-glucan by deep fermentation of Schizophyllum commune. The only difference from Example 1 is that in the step (2) of mycelial fermentation tank suspension culture, the dissolved oxygen content in the fermentation broth is not controlled in stages and is always kept at a high dissolved oxygen content (80-70%). After 120 hours of fermentation, the β-glucan yield in the fermentation broth is 0.8%, i.e., 8 g / L.

[0135] Comparative Example 3

[0136] This comparative example provides a method for producing β-glucan by deep fermentation of Schizophyllum commune. The only difference from Example 1 is that in the step (2) of mycelial fermentation tank suspension culture, the dissolved oxygen content in the fermentation broth is not controlled in stages and is always kept at a low dissolved oxygen content (25-15%). After 120 hours of fermentation, the β-glucan yield in the fermentation broth is 0.9%, i.e., 9 g / L.

[0137] Comparative Example 4

[0138] This comparative example provides a method for producing β-glucan by deep fermentation of Schizophyllum commune. The only difference from Example 1 is that in step (2) of mycelial fermentation tank suspension culture, no metabolic regulator (folic acid) is added at 80h. After 120h of fermentation, the yield of β-glucan in the fermentation broth is 0.6%, i.e. 6g / L.

[0139] Comparative Example 5

[0140] This comparative example provides a method for producing β-glucan by deep fermentation of Schizophyllum commune. The only difference from Example 1 is that in step (2) of mycelial fermentation tank suspension culture, sodium dihydrogen phosphate (final concentration of 3 ppm) is added at 80 h to replace the metabolic regulator (folic acid). After fermentation for 120 h, the yield of β-glucan in the fermentation broth is 0.83%, i.e., 8.3 g / L.

[0141] It should be noted that the method of this invention has proven to prepare a mycelial suspension using *Schizophyllum commune* hyphae, and then directly ferment this mycelial suspension to obtain fermentation products. Based on the same principle, this method can also be applied to the preparation of mycelial suspensions of other fungi, molds, and some actinomycetes, and can also be applied to their fermentation culture to obtain fermentation products, thereby greatly improving production efficiency. This invention will not list them all.

[0142] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A method for high yield production of β-glucans by submerged fermentation of Schizophyllum commune based on aqueous suspension of mycelium, characterized by, Includes the following steps: S1. Preparation of mycelial aqueous suspension: Inoculate Schizophyllum commune onto slant culture medium, culture on the slant for 5-7 days, add sterile water, scrape the mycelial growth on the slant culture medium into sterile water, collect the sterile water containing the mycelial growth, and obtain the mycelial aqueous suspension. S2. Fermentation culture of mycelial aqueous suspension: The mycelial aqueous suspension is inoculated into a fermenter containing the first component of the Schizophyllum commune deep fermentation medium for the first stage of fermentation culture; then the second component of the Schizophyllum commune deep fermentation medium is added to the fermenter for the second stage of fermentation culture, thus obtaining the fermentation broth containing β-glucan. In step S2, the first component of the Schizophyllum commune deep fermentation medium comprises the following components at the following concentrations: glucose 25-30 g / L, yeast extract 2-6 g / L, buffer salt 0.5-1.0 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, and the remainder being water; The second component of the Schizophyllum commune deep fermentation medium is a metabolic regulatory factor, and the final concentration of the metabolic regulatory factor is 0.5-5 ppm. The metabolic regulatory factor is selected from pan-amino acid or N,N-dimethylglycine; The conditions for the first stage of fermentation culture are: fermentation temperature 26-30℃, dissolved oxygen 70-80%, and culture time 3-4 days; The conditions for the second stage of fermentation are: fermentation temperature 26-30℃, dissolved oxygen 15-25%, and culture time 1-3 days.

2. The method for high-yield β-glucan production by submerged fermentation of *Schizophyllum commune* based on mycelial aqueous suspension according to claim 1, characterized in that, In step S1, the method for preparing the slant culture medium is as follows: peeled potatoes are cut into pieces and boiled in water for 20-40 minutes. Then, the mixture is filtered through gauze. Glucose, yeast extract, and agar powder are added to the filtrate. After heating and stirring to dissolve, water is added in a measured amount. The temperature for slant culture is 26-30℃; The ratio of the volume of sterile water added to the volume of the slant culture medium is 1:0.8-1.

2.

3. The method for high-yield β-glucan production by submerged fermentation of *Schizophyllum commune* based on mycelial aqueous suspension according to claim 1, characterized in that, In step S2, the volume ratio of the mycelial suspension to the first component of the Schizophyllum commune deep fermentation medium is 0.5-0.7:

35.

4. The method for high-yield β-glucan production by submerged fermentation of *Schizophyllum commune* based on mycelial aqueous suspension according to claim 1, characterized in that, The buffer salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

5. The method for high-yield β-glucan production by submerged fermentation of *Schizophyllum commune* based on mycelial aqueous suspension according to claim 1, characterized in that, The method further includes the following steps: S3. After inactivating the fermentation broth containing β-glucan, centrifuge the broth and pass the resulting centrifuged liquid into an "alternating five-column system" for purification. After fine filtration, a colorless and transparent β-glucan liquid is obtained. The "alternating five-column combined system" includes a granular activated carbon column, an anion exchange resin column, a first macroporous adsorption resin column, a cation exchange resin column, and a second macroporous adsorption resin column arranged in series.

6. A deep fermentation culture medium for Schizophyllum commune, characterized in that, It includes a first component and a second component. The first component includes the following components at the following concentrations: glucose 25-30 g / L, yeast extract 2-6 g / L, buffer salt 0.5-1.0 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, and water to make up to a certain amount. The second component is a metabolic regulatory factor, and the final concentration of the metabolic regulatory factor is 0.5-5 ppm; The metabolic regulatory factor is selected from pan-amino acid or N,N-dimethylglycine; The buffer salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

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