CN110669681A - Tremella fuciformis strain and application thereof in production of tremella fuciformis polysaccharide - Google Patents
Tremella fuciformis strain and application thereof in production of tremella fuciformis polysaccharide Download PDFInfo
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- CN110669681A CN110669681A CN201911080658.7A CN201911080658A CN110669681A CN 110669681 A CN110669681 A CN 110669681A CN 201911080658 A CN201911080658 A CN 201911080658A CN 110669681 A CN110669681 A CN 110669681A
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Abstract
The invention screens out a Tremella fuciformis strain (Tremella fuciformis) with the strain name of XY, which is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation date of 2019, 4 months and 25 days and the preservation number of CGMCC No. 17675. The invention also provides the application of the tremella strain in the production of tremella polysaccharide, which mainly comprises the step of inoculating seed liquid prepared by CGMCC No.17675 into a solid fermentation culture medium for fermentation to obtain fermentation liquid. The method can prepare high molecular weight polysaccharide with molecular weight of 1000-1500kDa and low molecular weight polysaccharide with molecular weight of 100-300kDa, the total yield can reach 15-25g/L, the protein content is less than 0.3 percent, and the method has obvious cost advantage compared with a liquid fermentation method and has better application prospect in the industrial production of the tremella polysaccharide.
Description
Technical Field
The invention belongs to the field of microorganism and fermentation engineering, and relates to a tremella strain and application thereof in production of tremella polysaccharide.
Background
Tremella, also known as Tremella, belongs to the family Tremellaceae of the subclass of Alternatherae Basidiomycetes, and has high nutritive value and medicinal effect. The white fungus is a special product in China, the planting is distributed in a plurality of provinces such as Zhejiang, Fujian and Hubei, the Fujian province is used as a main production area of the white fungus, and the total production of the white fungus reaches over 90 percent of the total production in China. The tremella contains carbohydrate, protein, vitamins and various amino acids, the most important bioactive substance is tremella polysaccharide which can be obtained by directly extracting tremella sporocarp or preparing tremella polysaccharide by fermentation, and the tremella polysaccharide has multiple effects of regulating immunity, resisting tumors, resisting oxidation and aging, reducing blood sugar and blood fat, resisting blood coagulation and thrombus, resisting ulcer, promoting protein synthesis, resisting viruses, promoting nerve cell growth, improving memory and the like.
The tremella polysaccharide is a polysaccharide with molecular weight ranging from tens of thousands to millions, usually takes alpha- (1 → 3) -D-mannose as a main chain and is linked with side chains such as D-glucuronic acid and the like, so that the tremella polysaccharide also determines the physicochemical property of the tremella polysaccharide, and has good hydrophilicity and certain emulsifying capacity. The tremella polysaccharide with high molecular weight (generally, the molecular weight reaches the million level), has super-strong moisture retention capacity and excellent film forming property, has the defects of low solubility, difficult absorption by skin and the like, and is difficult to be fully applied in the field of daily chemicals.
It is noted that the high molecular weight tremella polysaccharides are basidiomycete polysaccharide immunopotentiators, and have the functions of improving the immune function of the organism and increasing the white blood cells. Experimental research shows that the tremella polysaccharide can obviously improve the phagocytic function of reticuloendothelial cells of mice, and has prevention and treatment effects on rat leucopenia caused by cyclophosphamide. The traditional Chinese medicine composition is clinically used for leucopenia caused by tumor chemotherapy or radiotherapy and leucopenia caused by other reasons, and has obvious effect. In addition, the traditional Chinese medicine composition can also be used for treating chronic bronchitis, and the effective rate is up to more than 80%. Therefore, the high molecular weight tremella polysaccharide has wide application in the field of medicine and is popular with medical workers and patients. The low molecular weight tremella polysaccharide (generally, the molecular weight is below 300 kDa) also has good moisturizing function, relatively low viscosity, no sticky feeling in a cosmetic formula, and the like, and has the advantages of easy absorption by skin, so the low molecular weight tremella polysaccharide is more widely and conveniently applied in the daily chemical field.
At present, the production method of tremella polysaccharide focuses on a fruiting body extraction method, and relatively few research reports about preparation of tremella polysaccharide by a fermentation method are reported. Researches show that the fermentation method has the problems of low polysaccharide yield, high protein impurity content, single produced polysaccharide component quantum, high input-output ratio and the like. Therefore, screening to obtain a high-yield strain for producing tremella polysaccharide by fermentation, solving the problems of high investment cost, breakthrough of single molecular weight of the product and the like becomes urgent.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a tremella strain aiming at the defects of the prior art.
The invention also provides application of the strain in production of tremella polysaccharide.
In order to solve the technical problems, the invention discloses a Tremella strain which is classified and named as Tremella (Tremella fuciformis) with the strain name XY and is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, the preservation date is 2019, 4 and 25 days, the preservation number is CGMCC No.17675, and the preservation address is the institute of microbiology of China academy of sciences No. 3, North Cheng Lu No.1, northwest of the Yangxi district, Beijing city.
The obtaining process of the tremella strain comprises the following steps: selecting fresh tremella fuciformis fruiting bodies from different producing areas, carrying out sterile treatment on the surfaces of the tremella fuciformis fruiting bodies, cutting the ear pieces to the size of a nail cover by using a sterile blade, attaching the cut to the surface of a culture medium in a conical flask, culturing for 24 hours at 25-30 ℃, selecting viscous and milky colony in the tremella fuciformis fruiting bodies, and carrying out flat-plate streaking and constant-temperature amplification culture for 3 days at 25-30 ℃ to obtain tremella fuciformis strain monospores. The device of the step consists of a 250mL conical flask, a bottle sealing membrane, a rubber band and the like, wherein the used culture medium is a PDA solid culture medium: potato 20%, glucose 2%, agar 2%, natural pH.
The application of the tremella strain in the production of tremella polysaccharide is also within the protection scope of the invention.
The application specifically comprises the following steps: inoculating the seed liquid prepared by CGMCC No.17675 into a solid fermentation culture medium for fermentation to obtain fermentation liquid.
The preparation method of the seed liquid comprises the following steps:
(1) as shown in figure 1(a), a tremella strain CGMCC No.17675 is smeared in a PDA solid culture medium and cultured to obtain a single colony;
(2) and (2) inoculating the single colony obtained in the step (1) into a seed culture medium for culture to obtain a seed solution.
In the step (1), the PDA solid culture medium is any one of a glucose agar culture medium, a yeast extract peptone glucose agar culture medium (YPD) and a Chua's culture medium, and the pH value is natural; the culture is carried out at the constant temperature of 25-30 ℃ for 5-6 days.
Wherein the culture is preferably carried out at 26 ℃ for 5-6 days; the single colony is yeast-shaped, the diameter of the single colony can reach 4-5 mm, the color of the single colony is milky white to grey white, and the single colony is occasionally light yellow, and the surface of the single colony is transparent, moist and smooth. The streaked portion of the plate showed linear lawn, which was viscous and prominent in the ridge and did not grow into the medium. Its single spore is oval or oval, like a yeast cell (as shown in FIG. 1 (b)).
In the step (2), the seed culture medium is 5-30 g/L of glucose, 1-10 g/L of soybean peptone and MgSO 240.1～1g/L，KH2PO40.1～1g/L，K2HPO40.5 to 5g/L, pH 5.0 to 7.0; the culture is constant temperature shaking culture at the temperature of 25-30 ℃ and the rpm of 150-200 for 60-72 hours.
Wherein, the seed culture medium is preferably 25g/L of glucose, 2g/L of soybean peptone and MgSO40.5g/L，KH2PO40.46g/L，K2HPO41g/L，pH 6.0。
Preferably, the seed liquid is stored in a glycerin tube and frozen in a refrigerator at-20 ℃ for later use.
Wherein the solid state fermentation culture medium is prepared from glucose, (NH)4)2SO4、MgSO4、KH2PO4、K2HPO4Bran, soybean meal and water; the mass ratio of the soybean meal to the bran is 1: 1-1: 3; adding water to make relative humidity 80 + -5%; glucose, MgSO4、KH2PO4、K2HPO4The concentration of the water-soluble organic compound is 10-20 g/L, 0.1-1 g/L, 0.1-3 g/L, and pH is 5.0-7.0.
Wherein the solid state fermentation medium further comprises corn steep liquor and (NH)4)2SO4Any one or a combination of two of;
wherein the concentration of the corn steep liquor is 2-10 g/L; (NH)4)2SO4The concentration of (b) is 1-5 g/L.
Therein is added (NH)4)2SO4And corn steep liquor as part of nitrogen source, adjusting the type of nitrogen source, and adding (NH)4)2SO4Is more beneficial to the growth of thalli, and further improves the yield of the high molecular weight tremella polysaccharide. The tremella polysaccharide with high molecular weight has wide application in the field of medicine and can be used in clinical medicine.
Wherein, the seed liquid is inoculated into a solid fermentation culture medium according to the inoculation amount of 5-15 v/v%; the fermentation is carried out for 6-8 days at a constant temperature of 25-30 ℃; wherein v/v is the volume ratio of the seed liquid to the liquid part of the solid fermentation medium.
The application further comprises the following steps: adding water into the fermentation liquor, leaching, centrifuging to obtain supernatant, further concentrating to obtain concentrated solution, and performing alcohol precipitation and drying on the concentrated solution to obtain a tremella polysaccharide product; wherein the addition amount of water is 90-120 mL; the leaching is carried out by hot water at 50-80 ℃ for 0.8-1.2 h; the centrifugation is carried out at 6000-8000 r/min for 5-30 min; wherein the concentration is rotary evaporation concentration at 45-60 ℃; the alcohol precipitation is to precipitate the concentrated solution and ethanol according to the volume ratio of 1: 2-1: 4; the drying is freeze drying; preferably, the concentration is rotary evaporation concentration at 55 ℃, and the concentration multiple is 5-10 times; the alcohol precipitation is to precipitate the concentrated solution and ethanol overnight according to the volume ratio of 1: 3; fishing out the flocculent polysaccharide, and freeze-drying for 2-3 days to obtain the tremella polysaccharide.
And through multiple rounds of screening, detection and verification, selecting spore seeds with the highest yield and the large and small molecular weight tremella polysaccharides to preserve the glycerin pipe, and detecting the stability of the yield after continuous activation, culture and fermentation. Finally, the optimal XY yield and productivity of the Tremella fuciformis are determined.
Preferably, after alcohol precipitation, 20mL of water is added for redissolution, an ultrafiltration centrifugal tube is used, centrifugation is carried out at 8000rpm for 15min, the obtained product is divided into an upper part and a lower part, the upper part and the lower part are respectively tremella polysaccharide with high molecular weight and low molecular weight, and then drying is carried out, so that tremella polysaccharide products with different molecular weights are obtained; wherein the molecular weight cut-off of the ultrafiltration tube in the ultrafiltration centrifugation is 300 kDa; wherein the tremella polysaccharide with high molecular weight is 1000-1500kDa and accounts for 50-80% of the total sugar content, and the tremella polysaccharide with low molecular weight is 100-300kDa and accounts for 20-50% of the total sugar content.
The tremella polysaccharide product prepared by the method can be subjected to infrared spectrum detection, and the detection result in figure 4 shows that the tremella polysaccharide product is polysaccharide.
The tremella polysaccharide product prepared by the method can be used for detecting the sugar content of the tremella polysaccharide product, and the detection method comprises the following steps: the content of total sugar and tremella polysaccharide with large and small molecular weight is detected by anthrone-concentrated sulfuric acid colorimetric method and high performance liquid chromatography.
Wherein, the high performance liquid chromatography is specifically characterized in that after trifluoroacetic acid hydrolysis and PMP derivatization, the high performance liquid chromatography can be used for qualitative detection, and the monosaccharide composition is determined as follows: mannose, glucose, galactose, xylose, rhamnose, fucose, and glucuronic acid. Preferably, the obtained tremella polysaccharide product is prepared into a concentration of 5g/L, trifluoroacetic acid is added for acid hydrolysis, methanol solution is added for overnight drying, and 100 mu L of ultrapure water is added for preparing derivatization reaction. And adding NaOH into the acid hydrolysis sample, adding PMP methanol solution for derivatization reaction for 100min, adding HCl for neutralizing the reaction solution, drying overnight, adding 1mL of ultrapure water and 1mL of chloroform for extraction and layering for 3-5 times, and performing HPLC detection. Through detection, the total sugar content of the tremella polysaccharide in the method can reach 15-25 g/L.
The formula and the method for measuring the total sugar of anthrone-concentrated sulfuric acid are as follows: 0.1g of anthrone is dissolved in 50ml of concentrated sulfuric acid, and the glucose standard solution and the sample are diluted in a gradient manner to obtain a sample: anthrone: mixing concentrated sulfuric acid at a ratio of 1:4, keeping the temperature in boiling water for 10min, cooling the mixture to room temperature with flowing water, measuring the absorbance at 620nm, and calculating the total sugar content of the sample according to the standard curve of the glucose standard solution (as shown in FIG. 2 (a)).
In the method for detecting molecular weight by high performance liquid chromatography, a TSK gel G4000 gel chromatographic column is adopted to analyze a glucan standard substance and a sample, an RID differential detector is adopted to detect the glucan standard substance and the sample, and a glucan standard curve is shown in figure 2 (b).
The tremella polysaccharide product prepared by the method contains trace protein, the total protein content is less than 0.5 percent by detecting the protein concentration of the polysaccharide by a Coomassie brilliant blue method, and the tedious steps of removing protein and the like are omitted; the protein concentration detection method comprises the following steps: coomassie brilliant blue G2500.1g is dissolved in 50mL of ethanol, 100mL of phosphoric acid is added, water is added for dilution to 1000mL, and 1G/L bovine serum albumin gradient dilution and Coomassie brilliant blue G250 are mixed according to the proportion of 1:50, and the absorbance is measured at 595 nm. The protein content of the sample was calculated from the standard curve (as shown in FIG. 2 (c)).
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the total sugar content of the tremella polysaccharide can reach 15-25g/L, wherein the tremella polysaccharide with large molecular weight is 1000-1500kDa and accounts for 60-80% of the total sugar content, the tremella polysaccharide with small molecular weight is 100-300kDa and accounts for 20-40% of the total sugar content, and the tremella polysaccharide overcomes the defects of low strain fermentation yield, single product molecular weight and the like in the prior art.
(2) The strain provided by the invention has mild fermentation conditions, and the solid-state fermentation hot water extraction treatment is easy for separation and purification of the polysaccharide at the later stage.
(3) Compared with liquid deep fermentation, the method has the advantages that through solid state fermentation, the protein content in the obtained feed liquid is extremely low, the complex steps of removing protein and the like in actual production are omitted, and the method is suitable for large-scale production; meanwhile, the obtained extracellular polysaccharide has a structure more similar to that of tremella polysaccharide extracted from sporocarp, the substrate cost is reduced greatly, and the tremella polysaccharide has the advantages of easiness in condition control and short growth period, so that the tremella polysaccharide prepared by solid fermentation is a faster and more effective method, and has a certain application in the preparation of plant polysaccharide.
(4) The tremella polysaccharide with different molecular weights can be obtained by adjusting the type and proportion of the nitrogen source in the culture medium, and the tremella polysaccharide is easy to implement and high in input-output ratio in actual production.
Drawings
FIG. 1 is a colony morphology, spore microscopic image, of Tremella fuciformis XY (Tremella fuciformis-XY); wherein, the picture (a) is a colony morphology picture; (b) the spore microscope image is obtained.
FIG. 2 is a standard graph; wherein, the graph (a) is a standard working curve chart of the glucose concentration in the total glucose of anthrone-concentrated sulfuric acid determination; (b) a standard working curve chart for detecting the glucan with different molecular weights by high performance liquid chromatography. FIG. c is a standard working curve diagram of the Bradford method for determining protein concentration in tremella polysaccharide samples.
FIG. 3 is a diagram of a tremella polysaccharide product prepared by solid-state fermentation; wherein, the graph (a) is a tremella polysaccharide product with the molecular weight of 800-1500 kDa; (b) is 100-and 300kDa molecular weight tremella polysaccharide product.
FIG. 4 is a structural identification diagram of Tremella polysaccharides; wherein, the figure (a) is a monosaccharide composition HPLC chromatogram of tremella polysaccharide; (b) the infrared detection spectrum of the tremella polysaccharide; in FIG. (a), 1-mannose, 2-glucuronic acid, 3-glucose, 4-galactose, 5-xylose and 6-fucose are shown.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
The specific techniques or conditions are not indicated in the examples of the present invention, and the techniques or conditions described in the literature in the art are performed in accordance with the description. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.
The detection method of the invention is as follows:
determination of total sugar content/anthrone-concentrated sulfuric acid colorimetry:
(1) preparing a color developing agent and a standard solution: accurately weighing 0.1g of anthrone to be dissolved in 50ml of concentrated sulfuric acid (used as prepared), accurately weighing 100mg of anhydrous glucose, and metering the volume to 1L with RO water to obtain a standard glucose mother liquor;
(2) determination of glucose standard curve: the glucose stock solution was gradient diluted to 0. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 30. mu.g/ml, 40. mu.g/ml, 60. mu.g/ml, 80. mu.g/ml, respectively, as samples: after the developer is mixed uniformly in a ratio of 1:4, the mixture is quickly immersed into a boiling water bath for heat preservation for 10min, the flowing water is cooled to the room temperature, the light absorption value is measured at 620nm, and the calculation standard curve is shown in figure 2 (a).
(3) Fermentation broth treatment for total sugar content determination: centrifuging the upper and lower filtrates at 8000rpm for 15min, diluting the supernatant, measuring light absorption value according to flow path in (2), and substituting into the standard curve in FIG. 2(a) to calculate total sugar content.
And (3) determining the molecular weight of the tremella polysaccharide:
(1) determination of dextran standard curve: dextran with molecular weight of 5w, 11w, 28w, 50w, 75w and 100w was prepared at the same concentration and passed through a 0.22 μm aqueous filter for use. HPLC detection shows that the peak-off time correlation curves of different molecular weights are obtained, as shown in FIG. 2 (b).
(2) Fermentation treatment for molecular weight determination: centrifuging the upper and lower filtrates at 8000rpm for 15min, and filtering the supernatant with 0.22 μm water-based filter membrane. The molecular weight was calculated by substituting the time to peak into the dextran standard curve in FIG. 2(b) by HPLC detection.
Identification of tremella polysaccharide purity/detection of protein content:
(1) determination of protein concentration standard curve: bovine serum albumin mother liquor is respectively diluted to 0.1G/L, 0.2G/L, 0.4G/L, 0.6G/L, 0.8G/L and 1G/L, 0.1mL of each gradient diluent and sample solution is taken, 5mL of Coomassie brilliant blue G250 solution is added and mixed evenly, the absorbance is measured at 595nm, and the calculated standard is shown in figure 2 (c).
(2) Fermentation broth treatment for protein content determination: preparing the tremella polysaccharide freeze-dried sample to be 1g/L concentration, mixing Coomassie brilliant blue according to the proportion of 1:50, measuring the light absorption value at 595nm, substituting the light absorption value into the protein concentration standard curve in the figure 2(c) to calculate the protein content, and obtaining the purity percentage of the tremella polysaccharide freeze-dried sample.
And (3) monosaccharide composition determination: preparing a tremella polysaccharide sample into a concentration of 5g/L, adding 4mol/M trifluoroacetic acid, hydrolyzing at 120 ℃ for 2h, cooling, adding a methanol solution, drying overnight, and adding 100 mu L of ultrapure water for derivatization reaction. Adding 0.6M NaOH into an acid hydrolysis sample, adding 0.5M PMP methanol solution, uniformly mixing, heating to 70 ℃, reacting for 100min, cooling to room temperature, adding 0.3M HCl to neutralize the reaction solution, drying overnight, adding 1mL of ultrapure water and 1mL of chloroform, extracting and layering for 3 times, passing through a 0.22 mu M water system filter head, and detecting the sample by using a C18 chromatographic column.
Example 1
As shown in figure 1(a), a tremella strain CGMCC No.17675 is smeared in a PDA solid culture medium (glucose agar culture medium), and is cultured at a constant temperature of 26 ℃ for 5 days to obtain a single colony (as shown in figure 1 (b)) and a 18s sequence of the single colony is sequenced, and the tremella strain is determined after Blast comparison. The sequence of 18s is shown in a sequence table SEQ ID No. 1.
Example 2: preparation of fermentation broth
Single colonies were prepared as in example 1.
Solid-state fermentation culture of high-yield strains: the obtained strain was picked up in seed medium (glucose 25g/L, soyapeptone 2g/L, MgSO)40.5g/L，KH2PO40.46g/L，K2HPO41g/L, pH 6.0), 26 ℃, and constant temperature culture at 170rpm for 3 days. 3.6mL (10 v/v%) of the biomass was inoculated into a solid medium (15 g/L glucose, MgSO 2)40.5g/L，KH2PO40.5g/L，K2HPO40.5g/L, 8g of bran, 12g of soybean meal, 80 +/-5% of relative humidity, and culturing at the constant temperature of 26 ℃ for 6-8 days. The yield of the tremella polysaccharide is 12.9g/L through detection of an anthrone sulfate method, wherein the large molecular weight is 1000-1500kDa and accounts for 54% of the total amount of the polysaccharide, and the small molecular weight is 100-300kDa and accounts for 46% of the total amount of the polysaccharide.
Example 3: preparation of fermentation broth
Single colonies were prepared as in example 1.
Solid-state fermentation culture of high-yield strains: the obtained strain was picked up in seed medium (glucose 25g/L, soyapeptone 2g/L, MgSO)40.5g/L，KH2PO40.46g/L，K2HPO41g/L, pH 6.0), 26 ℃, and constant temperature culture at 170rpm for 3 days. 3.6mL (10 v/v%) of the biomass was inoculated into a solid medium (glucose 15g/L, (NH)4)2SO43g/L，MgSO40.5g/L，KH2PO40.5g/L，K2HPO40.5g/L, 8g of bran, 12g of soybean meal, 80 +/-5% of relative humidity, and culturing at the constant temperature of 26 ℃ for 6-8 days. The yield of the tremella polysaccharide is 22.5g/L through detection of an anthrone sulfate method, wherein the large molecular weight is 1000-1500kDa and accounts for 75% of the total amount of the polysaccharide, and the small molecular weight is 100-300kDa and accounts for 25% of the total amount of the polysaccharide.
Example 4: preparation of fermentation broth
Single colonies were prepared as in example 1.
Solid-state fermentation culture of high-yield strains: the obtained strain was picked up in liquid seed medium (glucose 25g/L, soytone 2g/L, MgSO)40.5g/L，KH2PO40.46g/L，K2HPO41g/L, pH 6.0), 26 ℃, and constant temperature culture at 170rpm for 3 days. 3.6mL (10 v/v%) of the biomass was inoculated into a solid medium (glucose 40g/L, corn steep liquor 5g/L, MgSO40.5g/L，KH2PO40.5g/L，K2HPO40.5g/L, 8g of bran, 12g of soybean meal, 80 +/-5 percent of relative humidity, and constant-temperature culture at 26 ℃ for 6-8 days. The yield of the tremella polysaccharide is 19.4g/L through detection of an anthrone sulfate method, wherein the large molecular weight is 1000-1500kDa and accounts for 65% of the total amount of the polysaccharide, and the small molecular weight is 100-300kDa and accounts for 35% of the total amount of the polysaccharide.
Example 5: preparation of fermentation broth
Single colonies were prepared as in example 1.
Solid-state fermentation culture of high-yield strains: the obtained strain is picked up in a liquid seed culture medium (Glucose 25g/L, Soy peptone 2g/L, MgSO40.5g/L，KH2PO40.46g/L，K2HPO41g/L, pH 6.0), 26 ℃, and constant temperature culture at 170rpm for 3 days. 3.6mL (10 v/v%) of the biomass was inoculated into a solid medium (glucose 40g/L, (NH)4)2SO43g/L, 5g/L corn steep liquor and MgSO40.5g/L，KH2PO40.5g/L，K2HPO40.5g/L, 8g of bran, 12g of soybean meal, 80 +/-5 percent of relative humidity, and constant-temperature culture at 26 ℃ for 6-8 days. The yield of the tremella polysaccharide is 21.0g/L through detection of an anthrone sulfate method, wherein the large molecular weight is 1000-1500kDa and accounts for 72% of the total amount of the polysaccharide, and the small molecular weight is 100-300kDa and accounts for 28% of the total amount of the polysaccharide.
Example 5: preparation of the supernatant
Treating and detecting fermentation yield: selecting the fermentation culture medium in the example 2 with higher yield for post-treatment, adding 100mL of hydrothermal water to extract polysaccharide in the culture medium, and extracting for 1h at 70 ℃; adding absolute ethanol at a ratio of 1:3, precipitating with ethanol, taking out flocculent polysaccharide, adding 20mL water, redissolving, centrifuging at 8000rpm for 15min with an ultrafiltration centrifugal tube with 300kDa molecular weight cut-off. The total sugar content of the two parts of liquid is respectively detected to be 20.42g/L, wherein the proportion of high molecular weight tremella polysaccharide is about 73 percent, the proportion of low molecular weight tremella polysaccharide is 27 percent, and the total protein content is less than 0.5 percent (figure 3). And then, detecting, wherein the monosaccharide composition is as follows: mannose, glucuronic acid, glucose, galactose, xylose and fucose; the infrared spectrum contains characteristic peaks such as mannose and uronic acid (FIG. 4).
Example 6: fermentation post-treatment and product preparation
Fermentation post-treatment and product preparation: centrifuging by a 300kDa ultrafiltration centrifugal tube, and freeze-drying the two obtained filtrates by a freeze dryer for 2-3 days to obtain high-molecular-weight and low-molecular-weight tremella polysaccharide products respectively, wherein the molecular weights of the tremella polysaccharide products are 1000-1500kDa and 100-300 kDa.
The invention provides a tremella strain and a thought and a method for applying the tremella strain in the production of tremella polysaccharide, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
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<120> tremella strain and application thereof in production of tremella polysaccharide
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<213> Tremella (Tremella fuciformis)
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ctttcaacaa cggatctctt ggctctcgca tcgatgaaga acgcagcgaa ttgcgaaaag 180
taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacctt gcgccttttg 240
gtattccgaa aggcatgcct gtttgagtgt catgtagact caaccccctg ggtttctgac 300
ccggcggtgt tggatttggg ccctgcctct ctggctggcc ttaaatgcgt tagtggtttc 360
acgcagacgt cgtaagttac gcgtcgactg tgggccgctc acaaccccct ttacttttgc 420
actctggcct caaatcaggt agggctaccc gctgaactt 459
Claims (10)
1. The Tremella fuciformis strain is classified and named as Tremella fuciformis (Tremella fuciformis), is XY, has been preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has the preservation date of 2019, 4 and 25 days, and has the preservation number of CGMCC No. 17675.
2. The use of the tremella strain of claim 1 in the production of tremella polysaccharide.
3. Use according to claim 2, characterized in that it comprises the following steps: inoculating the seed liquid prepared by CGMCC No.17675 into a solid fermentation culture medium for fermentation to obtain fermentation liquid.
4. The use of claim 3, wherein said solid fermentation medium is composed of glucose, MgSO4、KH2PO4、K2HPO4Bran, soybean meal and water;
wherein the mass ratio of the soybean meal to the bran is 1: 1-1: 3; adding water to make relative humidity 80 + -5%; glucose, MgSO4、KH2PO4、K2HPO4The concentration of (b) is 10-20 g/L, 0.1-1 g/L, 0.1-3 g/L, respectively.
5. The use of claim 4, wherein said solid state fermentation medium further comprises corn steep liquor, (NH)4)2SO4Any one or a combination of two of;
wherein the concentration of the corn steep liquor is 2-10 g/L; (NH)4)2SO4The concentration of (b) is 1-5 g/L.
6. The use according to claim 3, wherein the seed solution is inoculated into the solid state fermentation medium in an inoculum size of 5-15 v/v%.
7. The use of claim 3, wherein the fermentation is carried out at a constant temperature of 25-30 ℃ for 6-8 days.
8. Use according to claim 3, characterized in that it further comprises the following steps: adding water into the fermentation liquor, leaching, centrifuging to obtain supernatant, and further concentrating to obtain concentrated solution; precipitating the concentrated solution with ethanol, and drying to obtain Tremella polysaccharide product.
9. The use according to claim 8, wherein the amount of water added is 90-120 mL; the leaching is carried out by hot water at 50-80 ℃ for 0.8-1.2 h; the centrifugation is carried out at 6000-8000 r/min for 5-30 min.
10. The application of the tremella polysaccharide product as claimed in claim 8, wherein the tremella polysaccharide product with different molecular weights is obtained by adding water for redissolution after alcohol precipitation, ultrafiltration and centrifugation, and drying.
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| CN111690543A (en) * | 2020-06-30 | 2020-09-22 | 南京工业大学 | Tremella aurantialba and application thereof in preparation of tremella aurantialba fermented soybean milk |
| CN111920779A (en) * | 2020-07-20 | 2020-11-13 | 源合盛(吉林)药业有限公司 | Tremella fuciformis polysaccharide capsule medicament and preparation method thereof |
| CN111808777A (en) * | 2020-07-28 | 2020-10-23 | 南京工业大学 | Pantoea camelina and application thereof |
| CN111808777B (en) * | 2020-07-28 | 2022-02-11 | 南京工业大学 | Pantoea camelina and application thereof |
| CN113354754A (en) * | 2021-07-05 | 2021-09-07 | 上海应用技术大学 | Method for extracting tremella polysaccharide by using eutectic solvent |
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