Phellinus linteus mycelium fermentation method capable of increasing polysaccharide yield and application of phellinus linteus mycelium fermentation method

Abstract

The invention belongs to the technical field of mycelium culture, and provides a phellinus linteus mycelium fermentation method for increasing the polysaccharide yield and application thereof. According to the fermentation method provided by the invention, firstly, the ratio of carbon to nitrogen in the liquid culture medium is reasonably adjusted, and necessary trace elements and growth factors are supplemented to optimize the formula of the culture medium; secondly, key parameters in the fermentation process are further controlled, the key parameters comprise the inoculation amount and the fermentation time, specific test conditions such as monochromatic light red light irradiation are adopted, and the measures jointly promote rapid proliferation of mycelia. Therefore, according to the method, the culture period is greatly shortened, and the thallus yield is remarkably increased. The finally obtained phellinus linteus mycelium fermentation liquor and mycelium are rich in polysaccharide content and show a good blood fat reducing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of mycelium culture, and particularly relates to a Vanilla fusilli mycelium fermentation method for improving polysaccharide yield and application thereof. Background Art

[0002] Phellinus igniarius, also known as mulberry parasite and mulberry god, belongs to the genus Phellinus of the family Polyporaceae. It covers the fruiting bodies of Phellinus baumannii, Phellinus pyrophytes, Phellinus schizophylla, and Phellinus fusca, and has distinct regional characteristics. At present, medicinal fungi have been classified as the fourth category of the traditional Chinese medicine system. Phellinus igniarius contains a variety of compounds, such as polysaccharides, flavonoids, terpenes, etc. Among them, Phellinus igniarius polysaccharides are regarded as the active ingredients with the main pharmacological effects. Its therapeutic and nourishing effects have been widely recognized and applied at home and abroad.

[0003] Vaninii, classified as Populus lindera, is mainly distributed in Northeast China, North America and Russia. However, the existing technology has many disadvantages in culturing Vaninii mycelium, such as long culture cycle, low bacterial yield, significant morphological differences, weak activity, and is also restricted by season and environment. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a Vanilla mulberry mycelium fermentation method for increasing polysaccharide production. By optimizing the liquid culture medium formula, rationally adjusting the carbon-nitrogen ratio, supplementing trace elements and growth factors, the rapid proliferation of mycelium is promoted, the culture cycle is greatly shortened and the mycelium yield is significantly increased. The present invention controls the key parameters (inoculum size, fermentation time) during the fermentation process, uses monochromatic red light irradiation, ensures that the mycelium has uniform morphology and strong activity, and significantly improves the accumulation of metabolites, thereby obtaining high-quality strains, and the obtained mycelium polysaccharides have good lipid-lowering effects.

[0005] The technical solution of the present invention is:

[0006] A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production mainly comprises the following steps:

[0007] (1) Select freshly harvested, intact, healthy and insect-free Vanisan yellow fruiting bodies, dissect and weigh 1-2 g of small pieces of tissue to potato dextrose agar plate medium, adjust the pH to 6.5, and culture at 28°C in the dark for 4-6 days to obtain Vanisan yellow fungus strains;

[0008] (2) Subculture: Place the Vanisanella isolate isolated in (1) in the center of a potato dextrose agar plate and culture at 28°C for 7-8 days;

[0009] (3) Expanding the culture: inoculate the pure colonies of Vaniella fusca cultured in (2) into a liquid culture medium, adjust the pH to 6.5, and culture at 28°C in the dark for 2-4 days, wherein the liquid culture medium is potato dextrose broth;

[0010] (4) Liquid deep fermentation: When the diameter of the Phellinus igniarius colony reaches the diameter of the plate, take a bacterial block with a diameter of 0.8-1.2 cm under sterile conditions and inoculate it into a fermentation tank filled with liquid fermentation medium. The inoculation amount is 6-12% of the mass of the liquid. The culture is carried out for 7-10 days under a tank pressure of 0.03 MPa, an air intake of 3 L / min, a stirring speed of 30-60 rpm, and red light to harvest the Vani Phellinus igniarius mycelium.

[0011] Preferably, the preparation method of the potato dextrose agar plate medium in (1) is:

[0012] Take 4-8g potato extract powder, 1-3g peptone, 1-3g yeast extract powder, 0.3-0.7g MgSO4·7H2O, 0.4-0.8g KH2PO4, 0.8-1.2g K2HPO4·3H2O, 15-22g glucose, and 15-22g agar, dissolve in 1L distilled water, adjust the pH to 6.5, fill 5mL in a 5cm dish, and sterilize at 121℃ for 30min.

[0013] Preferably, in (3), the preparation method of the medium PDB used for the expanded culture is:

[0014] Cut 180-220 g of fresh potato into pieces and put them into 1 L of water and boil for 30 min. Filter and add 1.5-2.5 g of glucose, 0.8-1.2 g of peptone and 0.3-0.7 g of KH2PO4 to the filtrate. Add distilled water to 100 mL and sterilize at 121°C for 30 min.

[0015] Preferably, the formula of the liquid fermentation medium in (4) is:

[0016] Glucose 35-45g / L, peptone 3-8g / L, MgSO4·7H2O 0.8-1.2g / L, KH2PO4 1-3g / L;

[0017] The preparation method of the fermentation tank containing the liquid fermentation medium is as follows:

[0018] Weigh glucose, peptone, MgSO4·7H2O, and KH2PO4 and dissolve them in water. After fully stirring and dissolving, pour them into a 5L fermenter with a liquid volume of 55-65% of the fermenter capacity. Sterilize the fermenter at 121°C for 20 minutes, then cool it to 25-26°C to obtain a fermenter filled with liquid fermentation medium.

[0019] The polysaccharides in the fermentation liquid of Vanis tinctorius mycelium obtained by the above-mentioned fermentation method and the application of Vanis tinctorius mycelium polysaccharides in the preparation of products with blood sugar lowering effect are also the key protected contents of this patent.

[0020] Compared with the prior art, the present invention has the following advantages and effects:

[0021] (1) Since the present invention adopts liquid deep fermentation technology, the culture period of the strain is shortened (from 15 days to 7-10 days), and the obtained strains show strong vitality and high yield, and are not restricted by seasonal environment. Therefore, in large-scale production applications, the fermentation technology of the present invention can more effectively improve production efficiency and strain quality;

[0022] (2) The dry weight of mycelium obtained by the fermentation method of Vani Sanguisorba officinalis mycelium in the present invention is 856.81 mg, and the polysaccharide mass is 56.77 mg, both of which are significantly improved compared with the relevant indicators obtained by the prior art;

[0023] (3) The Vani mycelium polysaccharide obtained in the present invention can reduce the triglyceride content in zebrafish, especially the triglyceride content (optical density) in the high-dose group is significantly reduced with statistical difference, and can be used as a blood sugar-lowering efficacy ingredient to develop related health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of fermentation time on mycelium dry weight, crude polysaccharide quality and polysaccharide content in fermentation broth;

[0025] Figure 2 The effect of fermentation time on the polysaccharide content of the fermentation liquid;

[0026] Figure 3 The effect of inoculum size on the quality of crude polysaccharides and dry weight of mycelium;

[0027] Figure 4 The effect of inoculum size on polysaccharide content in fermentation broth;

[0028] Figure 5 The effect of different monochromatic light on the quality of crude polysaccharide and dry weight of mycelium;

[0029] Figure 6 The effect of different monochromatic light on the polysaccharide content of fermentation broth;

[0030] Figure 7 The interaction effects on the dry weight and crude polysaccharide quality of Phellinus linterus mycelium;

[0031] Figure 8 This is the cholesterol content in zebrafish blood vessels (the red area pointed by the arrow is cholesterol). DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0033] Example 1

[0034] 1.1 Preparation of culture medium

[0035] (1) Preparation of potato dextrose agar (PDA) plate culture medium:

[0036] Raw materials and dosage: 6g potato extract powder, 2g peptone, 2g yeast extract powder, 0.5g MgSO4·7H2O, 0.6 g KH2PO4, 1g K2HPO4·3H2O, 20g glucose, 20g agar, and 1L distilled water.

[0037] pH 6.5, preparation method: filling volume 5mL / plate (diameter 5cm), high pressure sterilization at 121℃ for 30min, then pour into a plate and cool for use.

[0038] (2) Preparation of PDB medium

[0039] Peel and cut fresh potatoes into pieces. Take 200 g of the cut potatoes and put them into 1 L of water and boil for 30 min. Filter with three layers of gauze. Add 2 g of food-grade glucose, 1 g of peptone, and 0.5 g of K2HPO4. Add distilled water to 100 mL. Sterilize in an autoclave at 121°C for 30 min and set aside.

[0040] (3) Liquid fermentation medium in the fermenter

[0041] Weigh appropriate amounts of glucose, peptone, MgSO4·7H2O, and KH2PO4 and dissolve them in water to make the concentrations of each component: glucose 40 g / L, peptone 5 g / L, MgSO4·7H2O 1 g / L, KH2PO4 2 g / L. After fully stirring to dissolve, pour into a 5 L fermentation tank with the liquid volume being 60% of the fermentation tank capacity. Sterilize the fermentation tank at 121 ° C for 20 min, then cool to 25 ° C to obtain liquid fermentation medium.

[0042] 1.2 A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production, mainly comprising the following steps:

[0043] (1) Select freshly harvested, intact, healthy and insect-free Vanisan yellow fruiting bodies, dissect and weigh a 1 g piece of tissue to the center of a potato dextrose agar (PDA) plate medium, adjust the pH to 6.5, and culture at 28°C in the dark for 5 days to obtain Vanisan yellow fungus strains;

[0044] (2) Subculture: Subculture the Vani Sanguisorba officinalis strain isolated in step (1), pick the tip hyphae with vigorous growth in the center of the culture medium, and culture it in a constant temperature incubator at 28° C. for 8 days;

[0045] (3) Expansion culture: inoculate the pure colonies of Vaniella fusca cultured in (2) into liquid culture medium, adjust the pH to 6.5, and culture at 28°C in the dark for 3 days, wherein the liquid culture medium is potato dextrose broth;

[0046] (4) Liquid deep fermentation: When the diameter of the Phellinus igniarius colony reaches the diameter of the plate, take a 1 cm diameter bacterial block under sterile conditions and inoculate it into a fermentation tank filled with liquid fermentation medium. The inoculation amount is 10% of the mass of the liquid. The culture is carried out for 8 days under a tank pressure of 0.03 MPa, an air intake of 3 L / min, a stirring speed of 45 rpm, and red light to harvest the Vani Phellinus igniarius mycelium.

[0047] Example 2

[0048] 2.1 Preparation of culture medium

[0049] (1) Preparation of potato dextrose agar (PDA) plate culture medium:

[0050] Raw materials and dosage: potato extract powder 4g, peptone 1g, yeast extract powder 1g, MgSO4·7H2O0.3g, KH2PO40.4 g, K2HPO4·3H2O 0.8g, glucose 15g, agar 15g, distilled water 1L.

[0051] pH 6.5, preparation method: filling volume 5mL / plate (diameter 5cm), high pressure sterilization at 121℃ for 30min, then pour into a plate and cool for use.

[0052] (2) Preparation of PDB medium

[0053] Peel and cut fresh potatoes into pieces. Take 180 g of the cut potatoes and put them into 1 L of water and boil for 30 min. Filter with three layers of gauze. Add 1.5 g of food-grade glucose, 0.8 g of peptone, and 0.3 g of K2HPO4. Add distilled water to 100 mL. Sterilize in an autoclave at 121°C for 30 min and set aside.

[0054] (3) Liquid fermentation medium in the fermenter

[0055] Weigh appropriate amounts of glucose, peptone, MgSO4·7H2O, and KH2PO4 and dissolve them in water to make the concentrations of each component: glucose 35 g / L, peptone 3 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 1 g / L. After fully stirring to dissolve, pour into a 5L fermentation tank with the liquid volume accounting for 55% of the fermentation tank capacity. Sterilize the fermentation tank at 121°C for 20 min, then cool to 26°C to obtain liquid fermentation medium.

[0056] 2.2 A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production, mainly comprising the following steps:

[0057] (1) Select freshly harvested, intact, healthy and insect-free Vanisan yellow fruiting bodies, dissect and weigh 2 g of small pieces of tissue to the center of a potato dextrose agar (PDA) plate medium, adjust the pH to 6.5, and culture at 28°C in the dark for 4 days to obtain Vanisan yellow fungus strains;

[0058] (2) Subculture: Subculture the Vani Sanguisorba officinalis strain isolated in step (1), pick the tip hyphae with vigorous growth in the center of the culture medium, and culture it in a constant temperature incubator at 28° C. for 7 days;

[0059] (3) Expansion culture: inoculate the pure colonies of Vaniella fusca cultured in (2) into liquid culture medium, adjust the pH to 6.5, and culture at 28°C in the dark for 2 days, wherein the liquid culture medium is potato dextrose broth;

[0060] (4) Liquid deep fermentation: When the diameter of the Phellinus igniarius colony reaches the diameter of the plate, take a 0.8 cm diameter bacterial block under sterile conditions and inoculate it into a fermentation tank filled with liquid fermentation medium. The inoculation amount is 6% of the mass of the liquid. The culture is carried out for 7 days under a tank pressure of 0.03 MPa, an air intake of 3 L / min, a stirring speed of 30 rpm, and red light to harvest the Vanilla Phellinus igniarius mycelium.

[0061] Example 3

[0062] 3.1 Preparation of culture medium

[0063] (1) Preparation of potato dextrose agar (PDA) plate culture medium:

[0064] Raw materials and dosage: potato extract powder 8g, peptone 3g, yeast extract powder 3g, MgSO4·7H2O0.7g, KH2PO40.8 g, K2HPO4·3H2O 1.2g, glucose 22g, agar 22g, distilled water 1L.

[0065] pH 6.5, preparation method: filling volume 5mL / plate (diameter 5cm), high pressure sterilization at 121℃ for 30min, then pour into a plate and cool for use.

[0066] (2) Preparation of PDB medium

[0067] Peel and cut fresh potatoes into pieces. Take 220 g of the cut potatoes and put them into 1 L of water and boil for 30 min. Filter with three layers of gauze. Add 2.5 g of food-grade glucose, 1.2 g of peptone, and 0.7 g of K2HPO4. Add distilled water to 100 mL. Sterilize in an autoclave at 121°C for 30 min and set aside.

[0068] (3) Liquid fermentation medium in the fermenter

[0069] Weigh appropriate amounts of glucose, peptone, MgSO4·7H2O, and KH2PO4 and dissolve them in water to make the concentrations of each component: glucose 45 g / L, peptone 8 g / L, MgSO4·7H2O 1.2 g / L, KH2PO4 3 g / L. After fully stirring to dissolve, pour into a 5 L fermentation tank with the liquid volume being 65% of the fermentation tank capacity. Sterilize the fermentation tank at 121 °C for 20 min, then cool to 26 °C to obtain liquid fermentation medium.

[0070] 3.2 A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production, mainly comprising the following steps:

[0071] (1) Select freshly harvested, intact, healthy and insect-free Vanisan yellow fruiting bodies, dissect and take a 1 g piece of tissue to the center of a potato dextrose agar (PDA) plate medium, adjust the pH to 6.5, and culture at 28°C in the dark for 6 days to obtain Vanisan yellow fungus strains;

[0072] (2) Subculture: Subculture the Vani Sanguisorba officinalis strain isolated in step (1), pick the tip hyphae with vigorous growth in the center of the culture medium, and culture it in a constant temperature incubator at 28° C. for 8 days;

[0073] (3) Expansion culture: inoculate the pure colonies of Vaniella fusca cultured in (2) into liquid culture medium, adjust the pH to 6.5, and culture at 28°C in the dark for 4 days, wherein the liquid culture medium is potato dextrose broth;

[0074] (4) Liquid deep fermentation: When the diameter of the Phellinus igniarius colony reaches the diameter of the plate, take a 1.2 cm diameter bacterial block under sterile conditions and inoculate it into a fermentation tank filled with liquid fermentation medium. The inoculation amount is 12% of the mass of the liquid. The culture is carried out for 10 days under a tank pressure of 0.03 MPa, an air intake of 3 L / min, a stirring speed of 60 rpm, and red light to harvest the Vani Phellinus igniarius mycelium.

[0075] Example 4 Fermentation conditions

[0076] In the fermentation method proposed in the present invention, the fermentation time, inoculation amount and monochromatic illumination of the liquid deep fermentation step were explored, and the remaining experimental steps were the same as in Example 1.

[0077] Polysaccharides in fermentation broth: Fermentation broth polysaccharides are polysaccharides produced by microorganisms (such as fungi, bacteria, etc.) during fermentation growth in liquid culture medium. These polysaccharides usually dissolve in the fermentation broth or form soluble polysaccharides (such as monosaccharides, low molecular weight polysaccharides, etc.).

[0078] Mycelial crude polysaccharide: Mycelial crude polysaccharide refers to the polysaccharide accumulated in the mycelium (i.e., the vegetative growth stage of the microorganism) when the microorganism grows in a solid or liquid culture medium. Most of these polysaccharides exist in the mycelial cell wall or extracellular space.

[0079] By studying the polysaccharide content of fermentation broth and mycelium, we can help optimize fermentation parameters such as culture medium composition, temperature, pH, dissolved oxygen conditions, and thus improve the yield and quality of polysaccharides. Studying the changes in the content of the two polysaccharides under different culture conditions can provide a theoretical basis and practical guidance for improving production processes and increasing production efficiency.

[0080] 4.1 Fermentation time

[0081] Table 1 Effect of fermentation time on the quality of crude polysaccharides and dry weight of mycelium

[0082]

[0083] Effects of fermentation time on the quality of crude polysaccharides and dry weight of mycelium Figure 1 As shown in the figure, with the continuous increase of fermentation days, the quality of crude polysaccharides shows a trend of first increasing and then decreasing. From the difference analysis in the figure, it can be seen that there is no obvious difference between the 5th and 7th day of fermentation time, and there is no significant difference between the 3rd, 9th and 11th day of fermentation time. It can be seen that the 5th to 7th day of fermentation is significantly different from other fermentation times and is better than other fermentation times. Therefore, it is judged that the 5th to 7th day of fermentation is the best fermentation time, and the polysaccharide content fermented is as high as 21.3 mg / 100 g (see Table 1). On the 9th day, the mycelium showed autolysis, the quality of crude polysaccharides began to decline rapidly, and the mycelium entered the growth decline period. By the end of the 11th day of fermentation, the quality dropped to 11.9 mg / 100 g.

[0084] Depend on Figure 1 Combined with the data in Table 1, it can be seen that the dry weight of mycelium shows an increasing trend with the extension of fermentation time, and the increase is the largest when the fermentation time is 9 days, increasing from 622.0 mg / 100 g on the 7th day to 827.6 mg / 100 g, an increase of 33.05%.

[0085] Table 2 Effect of fermentation time on polysaccharide content in fermentation broth

[0086]

[0087] Depend on Figure 2 Combined with the data in Table 2, it can be seen that the polysaccharide content in the fermentation broth shows a trend of first increasing and then decreasing with the increase in the number of culture days (first increasing from 4.9 mg / 100g to 8.2 mg / 100g and then decreasing to 5.8 mg / 100g). It can be seen from the significant differences in the analysis diagram that there is no significant difference in the polysaccharide content in the fermentation broth within 5-9 days of fermentation time, and there is no significant difference in the polysaccharide content in the fermentation broth on the 3rd day of fermentation and the 11th day of fermentation. There is a significant difference in the polysaccharide content in the fermentation broth on the 7th day of fermentation and the 3rd day of fermentation and the 11th day of fermentation. The fermentation time has a large difference from the 9th day to the 11th day, indicating that in the subsequent culture, the polysaccharide content in the fermentation broth decreases, the polysaccharide concentration difference decreases, the diffusion rate slows down, and the polysaccharide has been basically dissolved and reached a state of equilibrium.

[0088] 4.2 Inoculum size

[0089] Table 3 Effect of inoculation amount on crude polysaccharide content and mycelium dry weight

[0090]

[0091] Effect of inoculation amount on the quality of crude polysaccharides and dry weight of mycelium Figure 3 As shown. Figure 3 It can be seen that with the increase of inoculation amount, the quality of crude polysaccharides in mycelium shows an increasing trend. Through the difference analysis in the figure, it can be seen that there is a significant difference between the inoculation amount of 10% and other inoculation amounts. Therefore, the single factor experiment can conclude that the optimal inoculation amount is above 10%.

[0092] The dry weight of mycelium increased with the increase of inoculation amount, showing an upward trend. There was no significant difference between the dry weight of mycelium at an inoculation amount of 8% and 10%, which was 862.4mg / 100g and 919.7mg / 100g respectively from the data in Table 3; there was no significant difference between the dry weight of mycelium at an inoculation amount of 2% and 4%, which was 556.2mg / 100g and 630.9mg / 100g respectively; there were significant differences between the dry weight of mycelium at an inoculation amount of 4%, 6% and 8%, which increased from 630.9mg / 100g to 862.4mg / 100g, a total increase of 231.5mg / 100g.

[0093] Table 4 Effect of inoculation amount on polysaccharide content in fermentation broth

[0094]

[0095] Effect of inoculation amount on polysaccharide content in fermentation broth Figure 4 As shown. Figure 4It can be seen that the polysaccharide content of the fermentation broth decreases with the increase of the inoculation amount. There is no significant difference in the polysaccharide content of the fermentation broth when the inoculation amount is 2%, 4%, 6%, 8% and 10%. From the data in Table 4, it can be seen that when the inoculation amount reaches 6%, the polysaccharide content in the fermentation broth gradually decreases to 5.6 mg / mL, and at 10%, the polysaccharide content decreases to a minimum of 4.0 mg / mL.

[0096] 4.3 Different monochromatic lights

[0097] Table 5 Effects of different monochromatic light on crude polysaccharide content and mycelium dry weight

[0098]

[0099]

[0100] Effects of different monochromatic light on the quality of crude polysaccharides and dry weight of mycelium Figure 5 As shown. Figure 5 It can be seen that after the samples were treated with different light sources, the mass of crude polysaccharides in mycelium increased the most under the condition of red light irradiation, reaching 25.7 mg (Table 5). Through the difference analysis in the figure, it can be seen that there is little difference between red light and white light, but there is a significant difference between red light, white light and other lighting conditions.

[0101] Different monochromatic lights have different effects on the dry weight of mycelium. The difference analysis in the figure shows that under the irradiation of red light and green light, the dry weight of mycelium is significantly different from that under dark treatment conditions. Under the irradiation of red, green and white light, the dry weight of mycelium shows an increasing trend. Under the irradiation of blue light, there is no significant difference in the dry weight of mycelium from that under dark treatment conditions, and the dry weight of mycelium shows a decreasing trend.

[0102] Table 6 Effects of different monochromatic light on polysaccharide content in fermentation broth

[0103]

[0104] Effects of different monochromatic light on polysaccharide content in fermentation broth Figure 6 As shown. Figure 6 It can be seen that different monochromatic lights have different effects on the polysaccharide content of the fermentation broth. When the light source is blue or white light, the polysaccharide content in the fermentation broth shows a decreasing trend (see the data in Table 6, decreasing from 4.7 mg / 100 g to 4.0 mg / 100 g). When the light source is red light, there is no significant difference in the polysaccharide content of the fermentation broth compared with the dark treatment.

[0105] 4.4 Summary

[0106] According to the results of the single factor experiment, this experiment took mycelium dry weight Y1 and polysaccharide content Y2 as response values, analyzed the effects of A fermentation time, B inoculation amount, and C different monochromatic light on the response value, and adopted a 3-factor 3-level Box-Behnken response surface model. The experimental design and results are shown in Table 7.

[0107] Table 7 Response surface experimental design and results

[0108]

[0109] The three-factor three-level Box-Behnken response surface model was adopted, and the experimental design and results are shown in Table 7. Design-expert software was used to perform polynomial regression fitting on the experimental data, and the factors affecting the stability coefficient were further studied and optimized to make a response surface diagram. The quadratic equation model of the stability coefficient and each factor was obtained by multiple regression fitting:

[0110] Y1=798.09+26.33A+69.23B+32.92C-44.94AB+17.82AC-1.25BC-31.97A2-13.50B2-38.57C2

[0111] Y2=48.88+1.26A+6.69B+0.4C+0.79AB+2.41AC+0.15BC-3.49A2-1.16B2-1.17C2

[0112] The established regression model reached a significant level for mycelium dry weight and polysaccharide quality (P<0.01), indicating that the regression equation model is extremely significant, and the difference in the lack of fit terms is not obvious (P>0.05), indicating that the regression equation has a small error in fitting the test, which well shows the relationship between the influencing factors and the response value and can better predict the test value. Among them, fermentation time (A), inoculation amount (B), monochromatic light (C), BC, A 2 , C 2 The dry weight of mycelium was significantly affected (P<0.05), and the other factors were not significant. The order of influence of the three factors on juice yield was inoculation amount > fermentation time > monochromatic light. 2 It had a significant effect on the quality of mycelium polysaccharides (P<0.05), and the other effects were not significant. The order of influence of the three factors on the quality of polysaccharides was inoculation amount>fermentation time>monochromatic light.

[0113] Table 8 Analysis of variance of mycelium dry weight in regression model

[0114]

[0115]

[0116] Note: *Significant difference (P<0.05), **Very significant difference (P<0.01).

[0117] Table 9 Variance analysis of polysaccharide quality based on regression model

[0118]

[0119] Note: *Significant difference (P<0.05), **Very significant difference (P<0.01).

[0120] Depend on Figure 7 It can be seen that the slope of the response surface of inoculation amount and fermentation time on the dry weight of Phellinus mycelium, inoculation amount and fermentation time on the quality of mycelium polysaccharides is steep, and the interaction between the two is significant. The optimal conditions for mycelium fermentation were obtained by Design-Expert V8 software analysis: fermentation time 7.936d, inoculation amount 10%, red light, predicted theoretical mycelium dry weight 847.71mg, polysaccharide quality 55.14mg.

[0121] For ease of operation, the above process was optimized to fermentation time of 8 days, inoculation size of 10%, and red light. The actual mycelium dry weight was measured to be 856.81 mg, and the polysaccharide mass was 56.77 mg, which were close to the model prediction values, indicating that the process parameters optimized by response surface methodology are reliable.

[0122] Example 5 Evaluation of lipid-lowering activity of polysaccharides from the mycelium of Vaniella tinctorius

[0123] Feeding zebrafish fry with high-fat food (egg yolk powder) rapidly increased the fat content in the zebrafish intestine and plasma, and staining with Oil Red O to establish a fat absorption model. Based on the fat absorption model, drug treatment was performed to detect the effect of polysaccharides on zebrafish blood lipid levels.

[0124] Through experiments, the maximum detection concentration was explored: 400μg / mL. A normal group (zebrafish did not receive any treatment), a model group (no drug), a positive group (lovastatin 1μg / mL), and a test group (low, medium, and high) were set up. After drug treatment, the fluorescence method was used to detect changes in cholesterol content in zebrafish to determine the effect of Vanilla mycelium polysaccharide on zebrafish blood lipids.

[0125] Table 10 Statistics of total triglyceride optical density in each test group

[0126]

[0127] It can be seen from the data in Table 10 that compared with the model control group, the triglyceride content of the model group (hyperlipidemia group) after medication was reduced, especially the triglyceride content (optical density) of the high concentration group was significantly reduced, with statistical significance, indicating that the Vanilla mycelium polysaccharide produced after liquid fermentation has a good lipid-lowering effect and can be used as an effective ingredient to develop health foods.

[0128] Combination Figure 8 Analyze the information in:

[0129] Red represents fluorescence intensity. The darker the red, the higher the cholesterol content. Compared with the model group, the fluorescence intensity of the model group after medication was significantly weakened, and the higher the polysaccharide concentration, the lighter the red, indicating that the crude polysaccharide from Vanilla mycelium has a good lipid-lowering effect.

[0130] In summary, the fermentation method of Vanis lindera mycelium provided by the present invention not only greatly shortens the culture period of Vanis lindera mycelium, but also significantly increases the mycelium yield. The finally obtained Vanis lindera mycelium fermentation liquid and mycelium are rich in polysaccharides and show good lipid-lowering effects.

[0131] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production, characterized in that: The main steps include: (1) Select freshly harvested, intact, healthy and insect-free Vanisan yellow fruiting bodies, dissect and weigh 1-2 g of small pieces of tissue to potato dextrose agar plate medium, adjust the pH to 6.5, and culture at 28°C in the dark for 4-6 days to obtain Vanisan yellow fungus strains; (2) Subculture: Place the Vanisanella isolate isolated in (1) in the center of a potato dextrose agar plate and culture at 28°C for 7-8 days; (3) Expanding the culture: inoculate the pure colonies of Vaniella fusca cultured in (2) into a liquid culture medium, adjust the pH to 6.5, and culture at 28°C in the dark for 2-4 days, wherein the liquid culture medium is potato dextrose broth; (4) Liquid deep fermentation: When the diameter of the Phellinus igniarius colony reaches the diameter of the plate, take a bacterial block with a diameter of 0.8-1.2 cm under sterile conditions and inoculate it into a fermentation tank filled with liquid fermentation medium. The inoculation amount is 6-12% of the mass of the liquid. The culture is carried out for 7-10 days under a tank pressure of 0.03 MPa, an air intake of 3 L / min, a stirring speed of 30-60 rpm, and red light to harvest the Vani Phellinus igniarius mycelium.

2. A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production as claimed in claim 1, characterized in that: (1) The preparation method of potato dextrose agar plate medium is as follows: Take 4-8g potato extract powder, 1-3g peptone, 1-3g yeast extract powder, 0.3-0.7g MgSO4·7H2O, 0.4-0.8g KH2PO4, 0.8-1.2g K2HPO4·3H2O, 15-22g glucose, and 15-22g agar, dissolve in 1L distilled water, adjust the pH to 6.5, fill 5mL in a 5cm dish, and sterilize at 121℃ for 30min.

3. A method for fermenting Vanilla fusiformis mycelium for increasing polysaccharide yield as claimed in claim 1, characterized in that: (3), the preparation method of the medium PDB used for the expanded culture is: Cut 180-220 g of fresh potato into pieces and put them into 1 L of water and boil for 30 min. Filter and add 1.5-2.5 g of glucose, 0.8-1.2 g of peptone and 0.3-0.7 g of KH2PO4 to the filtrate. Add distilled water to 100 mL and sterilize at 121°C for 30 min.

4. A method for fermenting Vanilla fusilli mycelium to increase polysaccharide production as claimed in claim 1, characterized in that: (4) The formula of the liquid fermentation medium is: Glucose 35-45g / L, peptone 3-8g / L, MgSO4·7H2O 0.8-1.2g / L, KH2PO4 1-3g / L; The preparation method of the fermentation tank containing the liquid fermentation medium is as follows: Weigh glucose, peptone, MgSO4·7H2O, and KH2PO4 and dissolve them in water. After fully stirring and dissolving, pour them into a 5L fermenter with a liquid volume of 55-65% of the fermenter capacity. Sterilize the fermenter at 121°C for 20 minutes, then cool it to 25-26°C to obtain a fermenter filled with liquid fermentation medium.

5. Use of polysaccharides in the fermentation liquid of Vanilla fuciformis mycelium obtained by the fermentation method of claim 1 in the preparation of products with blood sugar lowering effect.

6. Use of the Vaniella fusca mycelium polysaccharide obtained by the fermentation method of claim 1 in the preparation of a product with blood sugar lowering effect.

Images