Preparation method of fermentation liquor containing inonotus obliquus intracellular polysaccharide

Abstract

The invention relates to microbial fermentation, in particular to a preparation method of fermentation liquor containing inonotus obliquus intracellular polysaccharide. By optimizing the variety and dosage of a carbon source, a nitrogen source and inorganic salt in the culture medium and combining the factors, a series of culture conditions such as inoculum size, pH, temperature, loading capacity, stirring speed and the like are further optimized, so that the crude polysaccharide content and biomass in the inonotus obliquus intracellular polysaccharide after fermentation are obviously improved compared with those before optimization; and the content of crude polysaccharide can be increased by 16.56% at most.

Description

Technical Field

[0001] This invention relates to microbial fermentation, and in particular to a method for preparing a fermentation broth containing intracellular polysaccharides from Inonotus obliquus. Background Technology

[0002] Inonotus obliquus (Fr.) Pilat is a medicinal fungus belonging to the genus *Poria hypobrunnea* of the family Polyporacea. It is mainly distributed in northern Russia, Northern Europe, and regions north of 40-50 degrees latitude, including Heilongjiang and Changbai Mountains in Jilin, China, growing under the bark of birch trees. The applicant disclosed a method for preparing Inonotus obliquus fermentation broth in invention patent 201510736450.1, using Inonotus obliquus (accession number CGMCC No. 1535) as the production strain. The method primarily aims to reduce production costs and effectively increase the extracellular polysaccharide content in the fermentation broth by optimizing the culture medium and cultivation conditions.

[0003] Existing research indicates that Inonotus obliquus polysaccharides are divided into extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS). EPS is mainly composed of glucose, mannose, and small amounts of rhamnose, arabinose, xylose, and galactose. The two types differ significantly in structure, function, and yield. Structurally, IPS is mainly composed of glucose, mannose, and small amounts of rhamnose, arabinose, xylose, and galactose, with glucose accounting for over 59%. EPS has a wider molecular weight distribution, while IPS has a relatively concentrated molecular weight, indicating a more complex EPS structure. Functionally, EPS exhibits strong scavenging activity against DPPH, ABTS, and hydroxyl radicals. 200 mg / kg - 1 Se-EPS reduced fasting blood glucose by 42%, increased insulin levels, and improved oxidative indicators. At 43% relative humidity, the EOEPS-1 component maintained a moisturizing rate of over 85% after 48 hours, superior to hyaluronic acid. Skin patch tests showed no irritation, indicating its potential as a natural moisturizer. Further studies have shown its protective effect against oxidative damage, and it also possesses emulsifying and anti-cancer functions. IPS also exhibits antioxidant, immune-enhancing, and tumor-inhibiting effects. In terms of hypoglycemic activity, EPS primarily works by repairing pancreatic β-cell damage (e.g., reducing hydrogen peroxide-induced apoptosis) and improving insulin sensitivity. IPS, on the other hand, focuses on regulating the expression of key enzymes in glucose metabolism (such as glucokinase and pyruvate kinase), accelerating glycolysis and inhibiting gluconeogenesis. In STZ-induced diabetic mice, IPS reduced blood glucose levels by 30%–40%, while increasing liver and muscle glycogen content and inhibiting α-glucosidase activity (IC50). 50It is 1 / 280th the yield of acarbose. In terms of immune activity regulation, IPS has a more pronounced regulatory effect on Th2 cytokines (such as IL-4). In terms of yield, EPS produced in the same fermentation tank is typically about three times that of IPS.

[0004] The applicant has not found any patent literature reports that are the same as or similar to this application. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing fermentation broth containing intracellular polysaccharides from Inonotus obliquus. By optimizing the selection of components, dosage control, and culture conditions of the Inonotus obliquus fermentation culture medium, the method aims to effectively increase the content of intracellular polysaccharides in the fermentation broth.

[0006] The main technical concept of this invention is:

[0007] A method for preparing fermentation broth containing intracellular polysaccharides from Inonotus obliquus involves inoculating Inonotus obliquus (accession number CGMCC No. 1535) into a sterile culture medium and culturing it. The method is characterized by using shake flask culture, with a shaker speed of 150-180 rpm and a culture temperature of 25-31 degrees Celsius. The culture medium consists of the following ingredients: 25-35 g / L glucose, 25-35 g / L soybean meal, 0.3-1 g / L magnesium sulfate, 1.5-3 g / L potassium dihydrogen phosphate, with the remainder being water, and an initial pH of 5-6.

[0008] The specific process steps and process conditions in this invention also include:

[0009] The preferred technical implementation method is that the culture medium content is 32% to 48%.

[0010] Furthermore, the inoculation amount is 2 to 5 bacterial blocks with a diameter of 5 mm to 1 cm per 100 ml of culture medium.

[0011] Furthermore, the cultivation period is 11 to 18 days.

[0012] To verify the effectiveness of this invention, the applicant, referring to the method described in agricultural industry standard NY / T1676-2008 (with slight modifications), determined the crude polysaccharide content in the intracellular polysaccharide after fermentation, as follows:

[0013] Weigh 0.2 g to 0.5 g (accurate to 0.000 l g) of the dry sample into an Erlenmeyer flask, add 20 mL of distilled water, shake to mix thoroughly, refrigerate overnight at 4°C, then mix again using a vortex mixer, and extract in an autoclave at 120°C for 30 minutes. After cooling the solution to room temperature, slowly add 250 mL of anhydrous ethanol while gently stirring for 30 seconds. Cover the Erlenmeyer flask with a film and place it in a refrigerator at 4°C for 12 hours. Transfer the contents of the Erlenmeyer flask to a centrifuge tube and centrifuge at 9000 rpm for 15 minutes. Discard the supernatant, transfer the precipitate to the Erlenmeyer flask, wash the centrifuge tube, and add the washings to the Erlenmeyer flask. Heat the Erlenmeyer flask on a hot plate and stir until the precipitate is evenly dispersed. Transfer to a 250 mL volumetric flask, cool, add water to make up to volume, shake well, and filter quickly through filter paper. Discard the initial 10 mL to 15 mL of filtrate and collect 20 mL to 30 mL.

[0014] Standard curve:

[0015] Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose standard solution (5.5) into 10 mL centrifuge tubes, respectively. Add water to a final volume of 1.0 mL, then add 1 mL of 5% phenol solution and mix well. Accurately and quickly add 5 mL of sulfuric acid using a pipette (add perpendicularly to the liquid surface, avoiding contact with the centrifuge tube wall to ensure thorough mixing with the reaction solution). Let stand for 10 minutes, then shake well. Place in a 30°C water bath for 20 minutes, and measure the absorbance at a wavelength of 490 nm. Plot a standard curve with absorbance on the ordinate and glucose mass (µg) on ​​the abscissa.

[0016] Measurement:

[0017] Pipette 0.2 mL to 1.0 mL of the test solution and measure it according to the standard curve. At the same time, prepare a reagent blank (without weighing the sample, but other steps are the same) and calculate the crude polysaccharide content in the intracellular polysaccharide based on the absorbance of the sample extract (subtract the absorbance of the reagent blank).

[0018] Result calculation:

[0019]

[0020] The crude polysaccharide content in the sample was calculated using the following formula:

[0021] In the formula:

[0022] X -- The value of crude polysaccharide content in the intracellular polysaccharide of the sample, in grams per 100 grams (g / 100g);

[0023] m1 -- The standard curve corresponds to the sugar content in the sample solution, expressed in micrograms (μg).

[0024] V1 -- The numerical value of the sample volume after finalization, in milliliters (ml);

[0025] V2 -- The volume of sample solution transferred during colorimetric determination, expressed in milliliters (ml);

[0026] m2 -- The numerical value of sample mass, in grams (g);

[0027] 0.9 -- Correction factor for converting glucose to dextran;

[0028] 106 -- Conversion factor.

[0029] The calculation results are expressed as the arithmetic mean of two independent measurements obtained under repeatability conditions, and the results are retained to three significant figures.

[0030] The essential features and significant technical advancements of this invention are as follows:

[0031] This invention optimizes the types and amounts of carbon sources, nitrogen sources, and inorganic salts in the culture medium. It also further optimizes a series of culture conditions such as inoculum size, pH, temperature, loading volume, and stirring speed. As a result, the crude polysaccharide content and biomass in the intracellular polysaccharide of Inonotus obliquus after fermentation are significantly improved compared with those before optimization. The crude polysaccharide content is increased by at least 8.5% and up to 16.56%. Attached Figure Description

[0032] Figure 1 This is a schematic diagram showing the effect of the type of nitrogen source in the culture medium of this invention on polysaccharides.

[0033] Figure 2 This is a schematic diagram showing the effect of nitrogen source concentration on polysaccharides in the culture medium of this invention.

[0034] Figure 3 This is a schematic diagram showing the effect of pH on polysaccharides in the culture medium of this invention.

[0035] Figure 4 This is a schematic diagram showing the effect of the type of carbon source in the culture medium of this invention on polysaccharides.

[0036] Figure 5 This is a schematic diagram showing the effect of carbon source concentration on polysaccharides in the culture medium of this invention.

[0037] Figure 6 This is a schematic diagram showing the effect of temperature on polysaccharides in the culture medium of this invention.

[0038] Figure 7 This is a schematic diagram showing the effect of the types of inorganic salts in the culture medium of this invention on polysaccharides.

[0039] Figure 8This is a schematic diagram showing the effect of the type of nitrogen source on biomass in the culture medium of this invention.

[0040] Figure 9 This is a schematic diagram showing the effect of nitrogen source concentration on biomass in the culture medium of this invention.

[0041] Figure 10 This is a schematic diagram showing the effect of pH on biomass in the culture medium of this invention.

[0042] Figure 11 This is a schematic diagram showing the effect of the type of carbon source on biomass in the culture medium of this invention.

[0043] Figure 12 This is a schematic diagram showing the effect of carbon source concentration on biomass in the culture medium of this invention.

[0044] Figure 13 This is a schematic diagram showing the effect of temperature on biomass in the culture medium of this invention.

[0045] Figure 14 This is a schematic diagram showing the effect of the types of inorganic salts in the culture medium of this invention on biomass.

[0046] Figure 15 This is a schematic diagram of the 2D and 3D response surface curves showing the effect of the interaction of various factors on the crude polysaccharide production of the strain in this invention. Detailed Implementation

[0047] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention. The scope of protection of the present invention shall be determined by the contents of the claims. Any equivalent means substitution made in accordance with the description of the present invention shall not depart from the scope of protection of the present invention.

[0048] Example 1

[0049] Single-factor experiment

[0050] 1. Preparation of microbial culture

[0051] (1) Solid culture: The slant of Inonotus obliquus, which has the preservation number CGMCC No.1535 and Latin name, was inoculated onto PDA plate medium with an inoculation stick and placed in a biochemical incubator and cultured at 25 degrees Celsius in the dark for 10 days.

[0052] (2) Liquid culture: Three 5 mm diameter holes were punched in the well-grown Inonotus obliquus agar plates using a sterile puncher. These holes were then inoculated into Erlenmeyer flasks (250 mL capacity) containing 100 mL of liquid culture medium. The flasks were cultured under single-factor conditions and in sequence. Nitrogen source screening was performed first, and the best result was used for the next screening step. The basal culture medium consisted of the following ingredients: 20 g / L glucose, 25 g / L yeast extract, and natural pH. The culture conditions were: 25°C, 150 rpm, and a culture period of 13 days. Each experiment was repeated three times.

[0053] 2. Using the mycelial biomass and crude polysaccharide content of Inonotus obliquus (accession number CGMCC No. 1535) as indicators, seven variables were set up for the study, including carbon source, nitrogen source, inorganic salt, temperature, and pH value.

[0054] (1) Types of nitrogen sources: Soybean meal powder, yeast powder, peptone, corn steep liquor powder, ammonium chloride, ammonium sulfate (all 25 g / L), and soybean meal powder + yeast powder (20 g + 5 g) were selected as the sole nitrogen source to replace the yeast extract in the basal culture medium. The experimental results are as follows: Figure 1 , Figure 8 .

[0055] (2) Carbon source types: sucrose, corn flour, soluble starch, glucose, maltose, fructose (all 20 g / L), glucose + corn flour (10 g + 10 g), used as the sole carbon source to replace glucose in the basal medium. The experimental results are as follows: Figure 4 , Figure 11 .

[0056] (3) Types of inorganic salts: magnesium sulfate, potassium dihydrogen phosphate, sodium chloride, manganese sulfate, calcium chloride (all 4 g / L), and magnesium sulfate + potassium dihydrogen phosphate (1 g + 3 g) were used to replace potassium dihydrogen phosphate in the basal culture medium. The experimental results are as follows: Figure 7 , Figure 14 .

[0057] (4) Set concentration gradients using the selected optimal carbon source: 15 g / L, 20 g / L, 25 g / L, 30 g / L, and 35 g / L. The experimental results are as follows: Figure 5 , Figure 12 .

[0058] (5) Set up concentration gradients using the selected optimal nitrogen source: 15 g / L, 20 g / L, 25 g / L, 30 g / L, and 35 g / L. The experimental results are as follows: Figure 2 , Figure 9 .

[0059] (6) Initial pH: 4, 5, 6, 7, 8, experimental results are as follows Figure 3 , Figure 10 .

[0060] (7) Temperature: 22, 25, 28, 31, 34 degrees Celsius, test results are as follows Figure 6 , Figure 13 .

[0061] 3. Mycelial pretreatment

[0062] The fermented bacterial solution was filtered through gauze, the mycelium was washed three times with distilled water, and dried in a 55°C forced-air drying oven. After drying to constant weight, the mycelium biomass (dry weight) was weighed. The weighed mycelium was then ground in a mortar and passed through a 40-mesh sieve to prepare the test sample. The sample was then stored in a sealed container at room temperature for later use.

[0063] 4. The methods for determining biomass and crude polysaccharides are as described above.

[0064] The above experiments show that, regarding the pH of the culture medium, the biomass (dry weight) was highest at pH 6 (15.63 g / L), while the crude polysaccharide yield was highest at pH 5 (6.19%), followed by pH 6. This indicates that the optimal pH for crude polysaccharide production by *Inonotus obliquus* (CGMCC No. 1535) is 5. Therefore, pH 5 was selected as the center point of the response surface methodology. Specific results can be found in […]. Figure 3 , Figure 10 Regarding the types of inorganic salts, the highest biomass (16.20 g / L) was observed with the addition of magnesium sulfate and potassium dihydrogen phosphate; followed by potassium dihydrogen phosphate alone at 15.20 g / L. However, the crude polysaccharide yield was highest with the addition of magnesium sulfate (5.78%), followed by the addition of magnesium sulfate and potassium dihydrogen phosphate at 5.75%. This may be due to the inhibitory effect of trace additives on the growth of *Inonotus obliquus*, leading to increased polysaccharide yield. However, the crude polysaccharide yields of both were not significantly different. Therefore, magnesium sulfate and potassium dihydrogen phosphate, which have more mycelium, were selected as the response surface methodology medium in later stages. Specific results are shown in [link to results]. Figure 7 , Figure 14 Regarding cultivation temperature, the highest biomass and crude polysaccharide yield were both achieved at 28℃, with a crude polysaccharide yield of 6.19%. Furthermore, preliminary experiments revealed that temperature variations significantly impacted biomass; the biomass at 34℃ was only 22.84% of that at 28℃. This aligns with the fact that *Inonotus obliquus* thrives in cooler regions such as Northeast my country and the Russian Far East. Specific results can be found in [link to relevant documentation]. Figure 6 , 13 This study also conducted single-factor screening for the types and concentrations of nitrogen and carbon sources, finding that the optimal conditions for biomass and crude polysaccharide yield were basically similar. The optimal nitrogen source was soybean meal, and the optimal carbon source was glucose. Specific results can be found in [link to study]. Figure 1 , 24, 5, 8, 9, 11, 12. This indicates that the Inonotus obliquus, with accession number CGMCC No. 1535 and Latin name, is more suitable for direct energy utilization for growth and reproduction, which is similar to previous results obtained in the liquid fermentation of other edible fungi.

[0065] Example 2

[0066] Response surface methodology for optimizing culture conditions

[0067] Based on the single-factor experiment, a three-factor, three-level Box-Behnken experimental design was used with Design-Expert V8.0.6 software to optimize the process of intracellular polysaccharide production by Inonotus obliquus. The intracellular polysaccharide content (Y) was used as the response value, and the culture temperature, carbon source concentration and culture pH that affected the intracellular polysaccharide yield were used as experimental factors. The experimental factors and levels are shown in Table 1.

[0068] Table 1 Factors and levels in the response surface methodology for optimizing fermentation conditions

[0069]

[0070] Example 3

[0071] Based on the results of single-factor experiments, a Box-Behnken design was established for the fermentation experiment of *Inonotus obliquus* (CGMCC No. 1535) to determine the optimal center point. Three analytical gradients (-1, 0, 1) were set for the three relevant factors. The response surface methodology and results are shown in Table 2. Figure 15 A quadratic regression analysis was performed on the experimental results in Table 2. The regression model is: Y = 6.10 + 0.010A + 0.28B - 0.10C - 0.22AB - 0.048AC - 0.73A 2 -0.49B 2 -0.69C 2 Simultaneously, 2D and 3D response surface analysis plots were generated. The steeper the slope of the response surface, the greater the impact of the corresponding factor on the crude polysaccharide yield; and the closer the contour lines are to ellipses, the more significant the interaction between the two factors. Figure 15The effects of different factors on the production of crude polysaccharides from *Inonotus obliquus* are clearly demonstrated. Each pair of interacting factors exhibits an elliptical response surface, with the interaction between temperature and pH showing the most pronounced curvature. This clearly indicates that the interaction between temperature and pH has the most significant impact on the yield of crude polysaccharides within the *Inonotus obliquus* intracellular polysaccharides. Response surface methodology optimization yielded the optimal fermentation conditions as follows: a culture temperature of 28.26°C, a carbon source concentration of 29.96 g / L, and an initial pH of 5.19. Under these conditions, the predicted crude polysaccharide yield was 6.138%. Further adjustments to the fermentation conditions, including equipment control factors, were made: a culture temperature of 28°C, a carbon source concentration of 30.00 g / L, an initial pH of 5.19, a culture medium composition including 30.00 g / L soybean meal, 1 g / L magnesium sulfate, and 3 g / L potassium dihydrogen phosphate, a rotation speed of 150 rpm, a culture medium volume of 40%, and inoculation with three 5 mm diameter mycelial blocks. The experiment was repeated three times under these conditions, and the average crude polysaccharide content was measured to be 6.34%, which is close to the theoretical value predicted by the model. This indicates that the model prediction of optimized fermentation conditions is reliable, and it is also 16.56% higher than the polysaccharide content of 5.29% measured under the basic culture conditions.

[0072] Table 2. Analysis of polysaccharide-related genes in strain JW-4

[0073]

[0074] Example 4

[0075] A method for preparing fermentation broth containing intracellular polysaccharides from Inonotus obliquus is characterized by inoculating Inonotus obliquus (preservation number CGMCC No. 1535) into a sterile culture medium for cultivation; using shake flask cultivation, with a shaker speed of 150 rpm and a cultivation temperature of 25 degrees Celsius; the culture medium consists of the following raw materials: 25 g / L glucose, 25 g / L soybean meal powder, 0.3 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, with the remainder being water, and an initial pH of 5.

[0076] The culture medium volume was 32%, and the inoculation amount was two mycelial blocks with a diameter of 5 mm to 1 cm per 100 ml of medium. The culture period was 18 days. The average crude polysaccharide yield was measured to be 5.74%.

[0077] Example 5

[0078] A method for preparing fermentation broth containing intracellular polysaccharides from Inonotus obliquus is characterized by inoculating Inonotus obliquus (accession number CGMCC No. 1535) into a sterile culture medium and culturing it in a shake flask at a shaking speed of 180 rpm and a temperature of 31 degrees Celsius. The culture medium consists of the following ingredients: 35 g / L glucose, 35 g / L soybean meal, 0.6 g / L magnesium sulfate, 2.3 g / L potassium dihydrogen phosphate, with the remainder being water, and an initial pH of 6.

[0079] The culture medium volume was 48%, and the inoculation amount was 5 1 cm diameter mycelial blocks per 100 ml of medium. The culture period was 11 days. The average crude polysaccharide yield was measured to be 6.07%.

[0080] Example 6

[0081] A method for preparing fermentation broth containing intracellular polysaccharides from Inonotus obliquus is characterized by inoculating Inonotus obliquus (preservation number CGMCC No. 1535) into a sterile culture medium for cultivation; using shake flask cultivation, with a shaker speed of 170 rpm and a cultivation temperature of 28 degrees Celsius; the culture medium consists of the following ingredients: 30 g / L glucose, 30 g / L soybean meal powder, 1 g / L magnesium sulfate, 3 g / L potassium dihydrogen phosphate, with the remainder being water, and an initial pH of 5.19.

[0082] The culture medium was 40% full, and the inoculation amount was 3 mycelial blocks with a diameter of 5 mm per 100 ml of medium. The culture period was 13 days. The average crude polysaccharide yield was measured to be 6.20%.

Claims

1. A method for preparing fermentation broth containing intracellular polysaccharides from Inonotus obliquus, characterized in that... The method involves inoculating Inonotus obliquus (accession number CGMCC No. 1535) into a sterile culture medium and culturing it. The method is characterized by using a shake flask for cultivation, with a shaker speed of 150-180 rpm and a cultivation temperature of 25-31 degrees Celsius. The culture medium consists of the following ingredients: 25-35 g / L glucose, 25-35 g / L soybean meal, 0.3-1 g / L magnesium sulfate, 1.5-3 g / L potassium dihydrogen phosphate, with the remainder being water, and an initial pH of 5-6.

2. The method for preparing fermentation broth containing Inonotus obliquus intracellular polysaccharides according to claim 1, characterized in that... The culture medium volume is 32%–48%.

3. The method for preparing fermentation broth containing Inonotus obliquus intracellular polysaccharides according to claim 1, characterized in that... The inoculation amount is 2 to 5 pieces of bacteria with a diameter of 5 mm to 1 cm per 100 ml of culture medium.

4. The method for preparing fermentation broth containing Inonotus obliquus intracellular polysaccharides according to claim 1, characterized in that... The culture period is 11 to 18 days.

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