Talaromyces sp. Separated from bran and application of talaromyces sp.

Abstract

The talaromyces sp. Is an endophytic fungus separated from wheat bran, is named as Talaromyces sp. ZY1104, is preserved in the China Center for Type Culture Collection with the number of CCTCC NO: M2025051, the center of a bacterial colony is turquoise, the bacterial colony gradually becomes light white when growing towards the edge, and the optimum pH value is 7.0 + / -0.2. The talaromyces sp. ZY1104 is a strain of endophytic fungus separated from wheat bran, and the talaromyces sp. ZY1104 is preserved in the China Center for Type Culture Collection with the number of CCTCC NO: M2025051. According to the invention, Talaromyces sp.ZY1104 is used for fermentation production research of exopolysaccharides, and the method specifically comprises the following steps: S1, activating a strain to obtain a strain seed solution; S2, carrying out a fermentation process to obtain a fermentation solution; and S3, separating and extracting the exopolysaccharides. The Talaromyces sp.ZY1104 disclosed by the invention can be used for producing the extracellular polysaccharide through fermentation by utilizing a general fermentation culture medium and a bran culture medium, the fermentation temperature range is 28-48 DEG C, the fermentation period is 48-168 hours, 3.1 g or more of the extracellular polysaccharide can be obtained in each liter of fermentation liquor, and the extracellular polysaccharide produced by the Talaromyces sp.ZY1104 does not contain cytotoxic substances and has various biological characteristics of oxidation resistance, anti-inflammatory activity and the like.

Description

Technical Field

[0001] The invention belongs to the fields of microbial technology, polysaccharide technology and natural product extraction technology, and relates to a basket fungus separated from bran and application thereof. Background Art

[0002] The fermentation production technology of extracellular polysaccharides is a process that uses separation and extraction technology to obtain extracellular polysaccharides synthesized and secreted by microorganisms under specific fermentation conditions.

[0003] In existing technologies, yeasts, such as Cryptococcus laurentii, are typically used for production. However, the cost of producing exopolysaccharides using Cryptococcus laurentii is high. This is due, in part, to the high cost of carbon and nitrogen sources in traditional liquid fermentation media. Furthermore, this strain is prone to mutation and decline during the fermentation process. Furthermore, compared to model yeasts, this strain has relatively limited gene editing tools and research foundations. Consequently, the technical foundation for strain improvement through genetic recombination is weak, and the research costs are high.

[0004] The Talaromyces isolated and obtained by the present invention is a fungus belonging to the genus Talaromyces of the family Aspergillaceae. At present, reports on the ability of fungi to secrete and produce extracellular polysaccharides are limited to some marine organisms. For example, the Chinese invention patent application with application number 2006100350123 discloses "A method for preparing extracellular polysaccharides of mangrove fungi and its use". Microdermabranchus was screened and isolated from mangrove fungi. This species belongs to the family Microdermabranchus and the genus Microdermabranchus. The fermentation culture medium of this species is relatively special and requires artificial seawater culture medium. The fermentation cycle is 6 to 7 days and the fermentation temperature is 25°C to 30°C. The culture medium requirements and fermentation conditions are relatively strict. The document does not report the extracellular polysaccharide production of this species. Chinese invention patent application number 2023110198713, "Fungus medium Aspergillus, its exopolysaccharide, and Application of Exopolysaccharides," reports on the fermentation production of exopolysaccharides using Aspergillus medium isolated from marine coral fish intestinal samples. This strain belongs to the Aspergillus family and genus Aspergillus. The fermentation conditions used are more stringent than those reported in the 2006100350123 publication, requiring a temperature of 25°C to 27°C and a fermentation cycle of 6 to 7 days. Furthermore, this strain has higher requirements for culture medium. Although the use of specific culture medium can increase polysaccharide yield, the highest polysaccharide yield reported in the publication is only 1.4322 g / L.

[0005] Existing research reveals that strains of endophytic fungi isolated from marine organisms that secrete exopolysaccharides share a common drawback: strict fermentation temperature requirements and a poor adaptability to higher temperatures. It is generally believed that temperatures above 35°C inhibit exopolysaccharide synthesis, while low-temperature fermentation inevitably impacts the fermentation cycle, resulting in prolonged fermentation times. Due to the limitations of their growth environment, these endophytic fungi are particularly particular about the composition and ratio of their culture media, and can only be fermented in specific culture media with compositions close to seawater. More importantly, even under these stringent fermentation conditions, polysaccharide yields are low.

[0006] It can be seen that neither the existing yeast nor the fungi derived from marine organisms are suitable for the industrial production of exopolysaccharides by microbial fermentation. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a basket fungus derived from bran and its application.

[0008] The technical solution adopted by the present invention is a talaromyces isolated from wheat bran. The key point is that the talaromyces is an endophytic fungus isolated from wheat bran, named Talaromyces sp. ZY1104, preserved in the China Center for Type Culture Collection, number CCTCC NO: M2025051, the collection center address: Wuhan University, Wuhan, China, the collection date: January 6, 2025, the colony center of the talaromyces is turquoise, and gradually turns pale white as it grows towards the edge, and the optimal pH = 7.0±0.2.

[0009] Furthermore, the above-mentioned Bacillaceae can produce extracellular polysaccharides, and the weight average molecular weight of the above-mentioned extracellular polysaccharides is 230kDa~240kDa, and the number average molecular weight is 120kDa~130kDa.

[0010] Specifically, the extracellular polysaccharide is composed of arabinose, galactose, glucose, mannose and glucuronic acid, with a mass ratio of 1:38-42:360-365:28-22:1.5-2.0.

[0011] Furthermore, the isolation method of the above-mentioned basket fungus is: place the bran in a triangular flask, seal the bottle mouth, heat it at 80℃~90℃ for 20min~30min, spread the heated bran on a solid seed culture medium, and culture it at a constant temperature of 28℃ until the strain grows. It is observed that the grown strain has a single color, and the strain is picked up and cultured in a new solid seed culture medium to obtain the above-mentioned basket fungus.

[0012] Specifically, the components of the above-mentioned solid seed culture medium include: 15g / L~25g / L sucrose, 4g / L~6g / L peptone, 2g / L~4g / L beef extract, 0.8g / L~1.2g / L yeast extract powder, 15g / L~25g / L agar, pH value 7.0±0.2; the thickness of the flattened bran is 1mm~1.5mm.

[0013] The invention discloses an application of basket fungi separated from bran, the key of which is to use the above-mentioned basket fungi for the fermentation production of extracellular polysaccharides, specifically including S1 activating the strain to obtain strain seed liquid, S2 obtaining fermentation liquid through the fermentation process and S3 separating and extracting the extracellular polysaccharides.

[0014] Furthermore, the above-mentioned S1 strain activated to obtain the strain seed liquid is:

[0015] Pick colonies from the solid seed culture medium and inoculate them into the liquid seed culture medium with the liquid volume of the liquid seed culture medium being 1 / 2 to 2 / 3. Activate the culture at 32°C for 12 to 36 hours with a stirring speed of 120 to 160 rpm. After the culture has grown to the logarithmic phase, activation is completed to obtain the culture seed solution for later use.

[0016] The components of the liquid seed culture medium include: sucrose 15g / L-25g / L, peptone 4g / L-6g / L, beef extract 2g / L-4g / L, yeast extract powder 0.8g / L-1.2g / L, and pH value 7.0±0.2.

[0017] On the one hand, the fermentation liquid obtained by the fermentation process of S2 is:

[0018] The bacterial seed liquid is inoculated into a fermentation liquid medium, the fermentation liquid volume is 1 / 2 to 2 / 3, the volume ratio of the inoculated bacterial seed liquid to the fermentation liquid medium is 5% to 15%, the stirring speed is 100 rpm to 140 rpm, the ventilation volume is 0.6 vvm to 1.0 vvm, and the temperature is 28° C. to 40° C. for 48 hours to 120 hours to obtain a fermentation liquid;

[0019] The specific components of the above-mentioned fermentation liquid culture medium include: glucose 35g / L~45g / L, peptone 0.8g / L~1.2g / L, yeast extract 0.8g / L~1.2g / L, MgSO4 0.8g / L~1.2g / L, KH2PO4 0.8g / L~1.2g / L and K2HPO4 0.8g / L~1.2g / L.

[0020] On the other hand, the fermentation liquid obtained by the fermentation process of the above-mentioned S2 is:

[0021] The bacterial seed liquid is inoculated into a bran culture medium in an inoculum amount of 50 mL to 150 mL of the bacterial seed liquid per kilogram of the bran culture medium, the bacterial seed liquid is mixed with the bran culture medium, and stirred, and then fermented at a temperature of 32° C. to 48° C. for 96 hours to 168 hours, with stirring every 18 hours to 28 hours. After the fermentation is completed, the fermentation liquid is washed with water and filtered to obtain a fermentation liquid;

[0022] The bran culture medium is obtained by crushing bran into 20-80 meshes, adding 2-4 kg of water per kilogram of bran, and sterilizing.

[0023] Furthermore, the above-mentioned S3 separation and extraction of exopolysaccharides is:

[0024] After the fermentation is completed, the fermentation broth is filtered to remove the mycelia, the filtrate is centrifuged to remove fine insoluble matter, the filtrate is collected and concentrated to 1 / 5 to 1 / 4 of the original volume by rotary evaporation under the conditions of 50°C to 70°C and 0.08MPa to 0.12MPa, 2 to 4 times the volume of the concentrate volume of Sevag reagent is added, and after vigorous shaking, the supernatant is collected by centrifugation;

[0025] Add 3 to 5 times the volume of the collected supernatant and allow to stand overnight at 4°C for precipitation;

[0026] The obtained precipitate was redissolved in water and concentrated to 1 / 5 to 1 / 4 of the original volume by rotary evaporation under conditions of 50°C to 70°C and 0.08 MPa to 0.12 MPa. The precipitate was dialyzed, purified and desalted using a dialysis bag with a molecular weight cutoff of 10.0 kDa to obtain an extracellular polysaccharide product.

[0027] The beneficial effects of the present invention are:

[0028] While researching other projects, the present inventors unexpectedly spread wheat bran, after high-temperature treatment, onto a solid culture medium for culturing. They unexpectedly obtained a fungus capable of secreting exopolysaccharides. This fungus, named Talaromyces sp. ZY1104, is an endophytic fungus of wheat bran and belongs to the Aspergillus family and the genus Talaromyces. This fungus can be fermented using both conventional fermentation media and wheat bran culture medium to produce exopolysaccharides, with a fermentation temperature range of 28°C to 48°C and a fermentation cycle of 48 hours to 168 hours. Compared to other existing fungi derived from marine organisms that can secrete exopolysaccharides, this fungus not only has a stronger adaptability to fermentation temperatures and a shorter fermentation cycle, but can also produce exopolysaccharides from wheat bran. Wheat bran is a byproduct of wheat processing, rich in cellulose, hemicellulose, protein, and minerals, and is an inexpensive and efficient culture medium raw material. The present invention can utilize wheat bran culture medium for the fermentation production of exopolysaccharides, significantly reducing the cost of culture medium raw materials. Although the fermentation cycle using bran medium is relatively longer than that using conventional liquid fermentation media, the yield of exopolysaccharides is comparable to that of liquid fermentation media, with over 3.1g of exopolysaccharides produced per liter of fermentation broth. The advantages of Talaromyces sp. ZY1104 in fermentation production are closely related to its source. The natural growth environment of Talaromyces sp. ZY1104 is subject to high summer temperatures, which makes it more tolerant to high temperatures.

[0029] The extracellular polysaccharide produced by Talaromyces sp. ZY1104 is composed of arabinose, galactose, glucose, mannose and glucuronic acid in a mass ratio of 1:38-42:360-365:28-22:1.5-2.0, with a weight average molecular weight range of 230kDa-240kDa and a number average molecular weight range of 120kDa-130kDa, and has a typical and characteristic broad spectrum of polysaccharides.

[0030] In addition, studies have confirmed that the scavenging effect of the exopolysaccharide produced by Talaromyces sp. ZY1104 on ABTS, DPPH, hydroxyl and superoxide anion free radicals increases with increasing concentration and is positively correlated; within the test concentration range of 0.3125 mg / mL to 5.0 mg / mL, the cell viability is close to 100%; the exopolysaccharide of the present invention can effectively reduce the levels of NO, TNF-α, IL-6 and IL-1β in LPS-induced RAW 264.7 macrophages, and is concentration-dependent. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Colony photo of Talaromyces sp. CCTCC NO: M2025051.

[0032] Figure 2 Phylogenetic tree of Talaromyces sp. CCTCC NO: M2025051.

[0033] Figure 3 Fourier transform infrared spectroscopy (FT-IR) analysis results of TSP sample 1 prepared in the present invention.

[0034] Figure 4 1H NMR spectrum results of TSP sample 1 prepared in the present invention.

[0035] Figure 5 13C NMR spectrum results of TSP sample 1 prepared in the present invention.

[0036] Figure 6 The results of the study on the scavenging rate of ABTS free radicals by TSP sample 1 prepared in the present invention.

[0037] Figure 7 The results of the study on the scavenging rate of TSP sample 1 prepared by the present invention on DPPH free radicals.

[0038] Figure 8 The results of the study on the scavenging rate of hydroxyl radicals by TSP sample 1 prepared by the present invention.

[0039] Figure 9 The results of the study on the scavenging rate of superoxide anion free radicals by TSP sample 1 prepared by the present invention.

[0040] Figure 10 Cytotoxicity test results of TSP sample 1 prepared in the present invention.

[0041] Figure 11 The results of TNF-α concentration detection in the anti-inflammatory test of TSP sample 1 prepared by the present invention on LPS-induced RAW 264.7 cells.

[0042] Figure 12 The results of IL-1β concentration detection in the anti-inflammatory test of LPS-induced RAW 264.7 cells by TSP sample 1 prepared in the present invention.

[0043] Figure 13 The IL-6 concentration test results of the TSP sample 1 prepared in the present invention in the anti-inflammatory test on LPS-induced RAW 264.7 cells.

[0044] Figure 14 The NO concentration detection results of the TSP sample 1 prepared in the present invention in the anti-inflammatory test on LPS-induced RAW 264.7 cells.

[0045] Figure 15The SOD concentration detection results of the TSP sample 1 prepared in the present invention in the anti-inflammatory test on LPS-induced RAW 264.7 cells.

[0046] Figure 16 CAT concentration detection results of TSP sample 1 prepared in the present invention in the anti-inflammatory test on LPS-induced RAW 264.7 cells.

[0047] Figure 17 The GSH-Px concentration detection results of the TSP sample 1 prepared in the present invention in the anti-inflammatory test on LPS-induced RAW 264.7 cells.

[0048] Figure 18 The MDA concentration test results of the TSP sample 1 prepared in the present invention in the anti-inflammatory test on LPS-induced RAW 264.7 cells. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described in detail below with reference to specific embodiments.

[0050] Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the raw materials involved are all commercially available conventional raw materials; and the experimental methods involved are all conventional methods.

[0051] Exopolysaccharides were detected by Shanghai Sanshu Biotechnology Co., Ltd. (Shanghai, China).

[0052] All data in the present invention are expressed as mean ± standard deviation of three repeated experiments, and one-way analysis of variance (ANOVA) was performed using Origin software (Origin Pro 8.5).

[0053] Example 1: Method for separating Talaromyces sp. ZY1104 from bran

[0054] Place 30g of bran in a 500mL conical flask, seal the flask with four layers of newspaper, heat it at 80℃ for 30min, spread the heated bran on a solid seed culture medium with a thickness of 1mm to 1.5mm, and transfer it to a 28℃ constant temperature incubator for culture. It was found that the strain grew, and the growing strain was observed to be of a single color. The strain was picked up in a clean bench and cultured on a new solid seed culture medium to obtain basket fungi.

[0055] The components of the solid seed culture medium are 20 g / L sucrose, 5 g / L peptone, 3 g / L beef extract, 1 g / L yeast extract powder, 20 g / L agar, and purified water. The pH value is adjusted to 7.0 using hydrochloric acid or sodium hydroxide. After preparation, the medium is sterilized and ready for use.

[0056] The isolated Talaromyces sp. ZY1104 was named and deposited in China Center for Type Culture Collection with CCTCC NO: M2025051. The address of the collection is Wuhan University, Wuhan, China. The date of deposit is January 6, 2025. The center of the Talaromyces colony is turquoise and gradually turns pale white towards the edge. The colony morphology is shown in Figure 1 , optimum pH = 7.0±0.2.

[0057] The phylogenetic tree of Talaromyces sp. ZY1104 can be found in Figure 2 .

[0058] Example 2: Method for Isolating Talaromyces sp. ZY1104 from Wheat Bran

[0059] The separation method of this embodiment is the same as that of Example 1, except that:

[0060] 1. Change the temperature and time of bran heating: place 30g bran in a 500mL Erlenmeyer flask, seal the flask with four layers of newspaper, and heat at 90℃ for 20min.

[0061] 2. Adjust the components of the solid seed culture medium to 25 g / L sucrose, 4 g / L peptone, 4 g / L beef extract, 0.8 g / L yeast extract powder, 25 g / L agar, and purified water. Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.2. After preparation, sterilize and set aside.

[0062] The strain isolated in this example and the Talaromyces sp. ZY1104 isolated in Example 1 were identified and found to be the same strain.

[0063] Example 3: Method for Isolating Talaromyces sp. ZY1104 from Wheat Bran

[0064] The separation method of this embodiment is the same as that of Example 1, except that:

[0065] 1. Change the temperature and time of bran heating: place 30 g bran in a 500 mL Erlenmeyer flask, seal the flask with four layers of newspaper, and heat at 85°C for 25 min.

[0066] 2. Adjust the components of the solid seed culture medium to 15 g / L sucrose, 6 g / L peptone, 2 g / L beef extract, 1.2 g / L yeast extract powder, 15 g / L agar, and purified water. Use hydrochloric acid or sodium hydroxide to adjust the pH to 6.8. After preparation, sterilize and set aside.

[0067] The strain isolated in this example and the Talaromyces sp. ZY1104 isolated in Example 1 were identified and found to be the same strain.

[0068] Example 4: Production of exopolysaccharides using liquid fermentation medium and Talaromyces sp. ZY1104

[0069] S1 strain activated to obtain strain seed liquid:

[0070] Select a Talaromyces sp. ZY1104 colony from the solid seed culture medium and inoculate it into a liquid seed culture medium with the liquid volume of the liquid seed culture medium at 1 / 2. Activate the culture at 32°C for 36 hours with a stirring speed of 120 rpm. After the culture grows to the logarithmic phase, the culture is activated to obtain a seed solution for later use.

[0071] The components of the liquid seed culture medium of this embodiment are 20 g / L sucrose, 5 g / L peptone, 3 g / L beef extract, 1.0 g / L yeast extract powder and purified water. The pH value is adjusted to 7.0 using hydrochloric acid or sodium hydroxide. After preparation, it is sterilized and used for later use.

[0072] S2 is fermented to obtain fermentation liquid:

[0073] The bacterial seed liquid was inoculated into the fermentation medium, the fermentation liquid volume was 2 / 3, the volume ratio of the inoculated bacterial seed liquid to the fermentation liquid medium was 5%, the stirring speed was 140 rpm, the ventilation volume was 1.0 vvm, the fermentation temperature was 28° C., and the fermentation culture time was 120 h to obtain a fermentation liquid;

[0074] The components of the fermentation medium are glucose 45 g / L, peptone 0.8 g / L, yeast extract 1.2 g / L, MgSO4 0.8 g / L, KH2PO4 1.2 g / L, K2HPO4 0.8 g / L and purified water. After preparation, sterilize and set aside.

[0075] S3 separation and extraction of exopolysaccharides:

[0076] After fermentation, the fermentation broth was filtered to remove mycelia, and the filtrate was centrifuged to remove fine insoluble matter. The filtrate was collected and concentrated to 1 / 4 of the original volume by rotary evaporation at 70°C and 0.08 MPa. Sevag reagent (2 times the volume of the concentrate) was added, and after vigorous shaking, the supernatant was collected by centrifugation at 10,000 × g for 10 min.

[0077] Add 5 times the volume of the collected supernatant to anhydrous ethanol and let it stand at 4°C overnight to precipitate;

[0078] The obtained precipitate was redissolved in water and concentrated to 1 / 5 of the original volume by rotary evaporation at 50°C and 0.12 MPa. The product was purified and desalted by dialysis using a dialysis bag with a molecular weight cutoff of 10.0 kDa to obtain an extracellular polysaccharide product, which was recorded as TSP sample 1.

[0079] Example 5: Production of exopolysaccharides using liquid fermentation medium and Talaromyces sp. ZY1104

[0080] S1 strain activated to obtain strain seed liquid:

[0081] Select a Talaromyces sp. ZY1104 colony from the solid seed culture medium and inoculate it into a liquid seed culture medium with the liquid volume of the culture medium at 2 / 3. Activate the culture medium at 32°C for 24 hours with a stirring speed of 140 rpm. After the culture has grown to the logarithmic phase, activation is completed to obtain a seed solution for later use.

[0082] The components of the liquid seed culture medium of this embodiment are 25 g / L sucrose, 4 g / L peptone, 2 g / L beef extract, 0.8 g / L yeast extract powder and purified water. The pH value is adjusted to 7.2 using hydrochloric acid or sodium hydroxide. After preparation, it is sterilized and used for later use.

[0083] S2 is fermented to obtain fermentation liquid:

[0084] The bacterial seed liquid was inoculated into the fermentation medium, the fermentation liquid volume was 1 / 2, the volume ratio of the inoculated bacterial seed liquid to the fermentation liquid medium was 10%, the stirring speed was 120 rpm, the ventilation volume was 0.8 vvm, the fermentation temperature was 32° C., and the fermentation culture time was 72 h to obtain a fermentation liquid;

[0085] The components of the fermentation medium are glucose 40 g / L, peptone 1.0 g / L, yeast extract 1.0 g / L, MgSO4 1.0 g / L, KH2PO4 1.0 g / L, K2HPO4 1.0 g / L and purified water. After preparation, sterilize and set aside.

[0086] S3 separation and extraction of exopolysaccharides:

[0087] After fermentation, the fermentation broth was filtered to remove mycelia, and the filtrate was centrifuged to remove fine insoluble matter. The filtrate was collected and concentrated to 1 / 5 of the original volume by rotary evaporation at 60°C and 0.10 MPa. Sevag reagent (3 times the volume of the concentrate) was added, and after vigorous shaking, the supernatant was collected by centrifugation at 10,000 × g for 10 min.

[0088] Add 4 times the volume of the collected supernatant to anhydrous ethanol and let it stand at 4°C overnight to precipitate;

[0089] The obtained precipitate was redissolved in water and concentrated to 1 / 5 of the original volume by rotary evaporation at 60°C and 0.10 MPa. The product was purified and desalted by dialysis using a dialysis bag with a molecular weight cutoff of 10.0 kDa to obtain an extracellular polysaccharide product, which was recorded as TSP sample 2.

[0090] Example 6: Production of exopolysaccharides using liquid fermentation medium and Talaromyces sp. ZY1104

[0091] S1 strain activated to obtain strain seed liquid:

[0092] Select a Talaromyces sp. ZY1104 colony from the solid seed culture medium and inoculate it into a liquid seed culture medium with the liquid volume of the liquid seed culture medium at 2 / 3. Activate the culture at 32°C for 12 hours with a stirring speed of 160 rpm. After the culture grows to the logarithmic phase, the culture is activated to obtain a seed solution for later use.

[0093] The components of the liquid seed culture medium of this embodiment are 15 g / L sucrose, 6 g / L peptone, 4 g / L beef extract, 1.2 g / L yeast extract powder and purified water. The pH value is adjusted to 6.8 using hydrochloric acid or sodium hydroxide. After preparation, it is sterilized and used for later use.

[0094] S2 is fermented to obtain fermentation liquid:

[0095] The bacterial seed liquid was inoculated into the fermentation medium, the fermentation liquid volume was 2 / 3, the volume ratio of the inoculated bacterial seed liquid to the fermentation liquid medium was 15%, the stirring speed was 160 rpm, the ventilation volume was 0.6 vvm, the fermentation temperature was 40° C., and the fermentation culture time was 48 h to obtain a fermentation liquid;

[0096] The components of the fermentation medium are glucose 35 g / L, peptone 1.2 g / L, yeast extract 0.8 g / L, MgSO4 1.2 g / L, KH2PO4 0.8 g / L, K2HPO4 1.2 g / L and purified water. After preparation, sterilize and set aside.

[0097] S3 separation and extraction of exopolysaccharides:

[0098] After fermentation, the fermentation broth was filtered to remove mycelia, and the filtrate was centrifuged to remove fine insoluble matter. The filtrate was collected and concentrated to 1 / 5 of the original volume by rotary evaporation at 50°C and 0.12 MPa. Four times the volume of the concentrate was added with Sevag reagent, and after vigorous shaking, the supernatant was collected by centrifugation at 10,000 × g for 10 min.

[0099] Add 3 times the volume of the collected supernatant to anhydrous ethanol and let it stand overnight at 4°C to precipitate;

[0100] The obtained precipitate was redissolved in water and concentrated to 1 / 4 of the original volume by rotary evaporation at 70°C and 0.08 MPa. The product was purified and desalted by dialysis using a dialysis bag with a molecular weight cutoff of 10.0 kDa to obtain an extracellular polysaccharide product, which was recorded as TSP sample 3.

[0101] Example 7: Production of exopolysaccharides using semi-solid bran culture medium and Talaromyces sp. ZY1104

[0102] S1: Activate the bacterial strain to obtain bacterial seed liquid: This step is the same as in Example 5;

[0103] S2 is fermented to obtain fermentation liquid:

[0104] The bacterial seed liquid is inoculated into the bran medium at an inoculum amount of 100 mL of the bacterial seed liquid per kilogram of the bran medium, the bacterial seed liquid is mixed with the bran medium, and stirred, and then fermented at a temperature of 38° C. for 120 hours, stirring once every 24 hours, and after the fermentation is completed, the fermentation liquid is washed with water and filtered to obtain a fermentation liquid;

[0105] The bran culture medium is prepared by crushing bran into 20-80 mesh sizes, adding 3 kg of water per kilogram of bran, and sterilizing the bran culture medium used in this embodiment. The particle size distribution (mass percentage) of the crushed bran is measured by screening: 20 mesh 25.6%, 40 mesh 33.8%, 60 mesh 28.3%, and 80 mesh 12.3%.

[0106] S3 separation and extraction of extracellular polysaccharides: This step is the same as Example 5, and the obtained extracellular polysaccharide product is recorded as TSP sample 4.

[0107] Example 8: Production of exopolysaccharides using semi-solid bran culture medium and Talaromyces sp. ZY1104

[0108] S1: Activate the bacterial strain to obtain bacterial seed liquid: This step is the same as in Example 5;

[0109] S2 is fermented to obtain fermentation liquid:

[0110] The bacterial seed liquid is inoculated into the bran medium at an inoculum size of 150 mL of the bacterial seed liquid per kilogram of the bran medium. The bacterial seed liquid and the bran medium are mixed and stirred, and fermented at a temperature of 32° C. for 168 hours, with stirring every 28 hours. After the fermentation is completed, the fermentation liquid is washed with water and filtered to obtain a fermentation liquid.

[0111] The bran culture medium is prepared by crushing bran into 20-80 mesh sizes, adding 2 kg of water per kilogram of bran, and sterilizing the bran culture medium used in this embodiment. The particle size distribution (mass percentage) of the crushed bran after screening is as follows: 36.7% for 20 mesh, 46.8% for 40 mesh, 10.3% for 60 mesh, and 6.2% for 80 mesh.

[0112] S3 separation and extraction of extracellular polysaccharides: This step is the same as Example 5, and the obtained extracellular polysaccharide product is recorded as TSP sample 5.

[0113] Example 9: Production of exopolysaccharides using semi-solid bran culture medium and Talaromyces sp. ZY1104

[0114] S1: Activate the bacterial strain to obtain bacterial seed liquid: This step is the same as in Example 5;

[0115] S2 is fermented to obtain fermentation liquid:

[0116] The bacterial seed liquid is inoculated into the bran medium at an inoculum size of 50 mL of the bacterial seed liquid per kilogram of the bran medium. The bacterial seed liquid and the bran medium are mixed and stirred, and fermented at a temperature of 48° C. for 96 hours, stirring once every 18 hours. After the fermentation is completed, the fermentation liquid is washed with water and filtered to obtain a fermentation liquid.

[0117] The bran culture medium is prepared by crushing bran into 20-80 mesh sizes, adding 4 kg of water per kilogram of bran, and sterilizing the bran culture medium used in this embodiment. The particle size distribution (mass percentage) of the crushed bran after screening is as follows: 12.3% for 20 mesh, 25.7% for 40 mesh, 35.3% for 60 mesh, and 26.7% for 80 mesh.

[0118] S3 separation and extraction of extracellular polysaccharides: This step is the same as Example 5, and the obtained extracellular polysaccharide product is recorded as TSP sample 6.

[0119] Example 10: Analysis of basic components of the obtained samples

[0120] The carbohydrate content in TSP samples 1 to 6 was determined by the anthrone-sulfuric acid colorimetric method (using glucose as a standard), and the protein content was determined by the Coomassie brilliant blue method (using bovine serum albumin as a standard). The test results are shown in Table 1.

[0121] Table 1: Basic component analysis results of samples

[0122] Name of sample to be tested Polysaccharide yield (g / L) Mass ratio of carbohydrates in the sample (%) Mass ratio of protein in the sample (%) TSP Sample 1 3.34 93.1 2.82 TSP Sample 2 3.89 96.0 2.89 TSP Sample 3 3.11 90.2 2.75 TSP Sample 4 3.93 94.6 2.92 TSP Sample 5 3.56 95.2 2.86 TSP Sample 6 3.17 95.3 2.72

[0123] The polysaccharide yields in Table 1 are the mass of extracellular polysaccharides extracted per liter of fermentation broth.

[0124] As can be seen from the results in Table 1, the polysaccharide yield can reach 3.11 g / L when the Basilisk fungus of the present invention is used to ferment and produce extracellular polysaccharides, and the highest is even close to 4 g / L; the main components of the obtained samples are carbohydrates and proteins, among which the carbohydrate content is above 90%, and the highest reaches 96%, indicating that the obtained extracellular polysaccharides have few impurities and high purity.

[0125] Example 11: Determination of Monosaccharide Composition and Molecular Weight of Extracellular Polysaccharides

[0126] The monosaccharide composition, ratio, and molecular weight of TSP samples 1 to 6 were determined by Shanghai Sanshu Biotechnology Co., Ltd. (Shanghai, China) using high-performance anion exchange chromatography (HPAEC) and high-performance gel filtration chromatography (HPGFC), respectively. The experimental results are shown in Tables 2 and 3.

[0127] In the present invention, 13 common monosaccharides and uronic acids were selected as standard substances, including: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid.

[0128] Sample processing:

[0129] TSP samples 1 to TSP samples 6 were added to TFA acid solution respectively, heated at 121°C for 2 hours; nitrogen was passed through and blown dry; methanol was added for washing, and the mixture was blown dry again. The methanol washing was repeated 2 to 3 times, and sterile water was added for dissolution to prepare the test samples 1 to 6 corresponding to each sample.

[0130] Table 2: Monosaccharide composition and mass percentage of samples

[0131]

[0132]

[0133] Table 3: Molecular weight determination results of samples

[0134] Name of sample to be tested Corresponding original sample name Weight average molecular weight (kDa) Number average molecular weight (kDa) Sample 1 to be tested TSP Sample 1 235.187 124.712 Sample 2 to be tested TSP Sample 2 239.657 129.568 Sample 3 TSP Sample 3 230.124 120.432 Sample 4 TSP Sample 4 237.216 122.568 Sample 5 TSP Sample 5 234.561 126.782 Sample 6 TSP Sample 6 236.267 127.021

[0135] As can be seen from the results in Tables 2 and 3, the extracellular polysaccharide prepared by the present invention contains five monosaccharides or uronic acids, specifically arabinose, galactose, glucose, mannose and glucuronic acid, and the mass ratio of these five monosaccharides or uronic acids ranges from 1:38 to 42:360 to 365:18 to 22:1.5 to 2.0; the weight average molecular weight of the extracellular polysaccharide prepared by the present invention is in the range of 230 kDa to 240 kDa, and the number average molecular weight is in the range of 120 kDa to 130 kDa.

[0136] In addition to the above five monosaccharides or uronic acids, fucose, rhamnose, xylose, fructose, ribose, galacturonic acid, mannuronic acid and guluronic acid were not detected in the extracellular polysaccharide prepared by the present invention.

[0137] Example 12: Structural confirmation of exopolysaccharide (I) Fourier transform infrared spectroscopy (FT-IR) analysis

[0138] 1 mg of TSP sample 1 and 100 mg of potassium bromide were accurately weighed and thoroughly ground in an agate mortar and pressed into pellets. The samples were scanned and detected using a Nexus 470 Fourier transform infrared spectrometer (Nicolet, USA) with a scanning wavenumber range of 500. -1 ~4000cm -1 , the results are shown in Figure 3 .

[0139] Depend on Figure 3 The results show that 3400cm -1 to 3200cm -1 The absorption peaks between 3000 cm and 3000 cm may be related to the intermolecular hydrogen bonding and OH stretching vibration of OH groups. -1 to 2900cm -1 The absorption peaks between 1800 cm and 1800 cm may be attributed to the antisymmetric stretching vibration of CH2, which are characteristic absorption peaks of polysaccharides. -1 to 1700cm -1 The absorption peak between 1600 cm and 1600 cm may be related to the C=O stretching vibration of COOH group or acetyl group. -1 to 1400cm -1 The absorption peaks between 1400cm and 1400cm may be related to the symmetrical ring stretching vibration of CH2 or the CH2 scissor vibration. -1 Up to 1100cm -1 The absorption peaks between 900 cm and 900 cm may be related to the in-plane deformation of OH, the antisymmetric stretching vibration of COC, and the stretching vibration of CO. -1 Up to 500cm -1 The absorption peaks between them may be related to the stretching vibration of the C-terminal group and the stretching vibration of the pyran ring.

[0140] (2) Nuclear magnetic resonance (NMR) spectroscopy analysis

[0141] Accurately weigh 0.1 g of TSP sample 1 and dissolve it in 0.5 mL of deuterated water (D2O). After vigorous shaking, let it stand at room temperature for 12 h to ensure complete dissolution. The solution was then centrifuged at 10,000 × g for 10 min, and the supernatant was transferred to an NMR tube. 1H NMR and 13C NMR spectra were collected using a Bruker AVANCE III 500 MHz NMR spectrometer (Bruker, Germany). The results are shown in Table 1. Figure 4 and Figure 5 .

[0142] Depend on Figure 4 The results show that the large peak at about 4.8 ppm may be related to the D2O used in this study, the peak between 4.62 ppm and 4.63 ppm may be attributed to the ectopic atoms of β-1,3-D-glucose, the peak at about 4.5 ppm may be caused by the β-isomer proton, the peak between 3.7 ppm and 3.8 ppm may be related to the H in -OH, and the peak between 3.4 ppm and 3.6 ppm may be related to the CC signal;

[0143] Depend on Figure 5 The results show that the signal between 96 ppm and 100 ppm may be attributed to C=C; the signal between 65 ppm and 75 ppm may be related to HC≡CH, the signal between 60 ppm and 65 ppm may be attributed to CH, CH2 and CH3, and the signal between 10 ppm and 35 ppm may be related to the methyl groups of rhamnose and galactose.

[0144] Based on the above test results, the extracellular polysaccharide sample prepared by the present invention has a typical and characteristic spectrum of polysaccharide.

[0145] Example 13: Determination of the Antioxidant Activity of Extracellular Polysaccharides in Vitro

[0146] TSP sample 1 was dissolved in deionized water to prepare solutions of different concentrations (0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5.0 mg / mL). After filtration through a 0.22 μm water filter membrane, the scavenging ability of TSP sample 1 on DPPH, ABTS, hydroxyl, and superoxide free radicals was measured (expressed as TSP). The same concentration of ascorbic acid (expressed as Vc) was used for comparison. The results are shown in Table 1. Figures 6 to 9 .

[0147] Depend on Figures 6 to 9 The results show that the scavenging effect of the exocellular polysaccharide of the present invention on ABTS, DPPH, hydroxyl, and superoxide anion free radicals increased with increasing concentration, and showed a positive correlation. At a concentration of 5.0 mg / mL, the exocellular polysaccharide of the present invention had a scavenging rate of 33.4% ± 1.48% for ABTS, 25.16% ± 2.13% for DPPH, 56.54% ± 2.31% for hydroxyl, and 71.05% ± 2.04% for superoxide anion free radicals, respectively.

[0148] This is because the high carbohydrate content may be a factor in the antioxidant activity of TSP. Furthermore, the high molecular weight of the exopolysaccharides of the present invention may enable them to form a network structure in solution, thereby binding free radicals and exerting antioxidant activity. Furthermore, the type of monosaccharide may also affect the antioxidant activity of the exopolysaccharides of the present invention.

[0149] Example 14: Cytotoxicity test of extracellular polysaccharides

[0150] RAW 264.7 macrophages were cultured in DMEM supplemented with 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. TSP sample 1 was prepared into solutions of different concentrations in DMEM, filtered through a 0.22 μm filter, and then added to the inoculated RAW 264.7 cells (2×10 4 cells / mL) in a 96-well plate and continue to culture for 24 hours. Finally, the cell activity was detected using the CCK-8 kit according to the instructions. Figure 10 .

[0151] Depend on Figure 10 The results show that in the cytotoxicity test, within the test concentration range of 0.3125 mg / mL to 5.0 mg / mL, the cell viability is close to 100%, indicating that the extracellular polysaccharide of the present invention is highly safe and can be used for subsequent anti-inflammatory activity test evaluation.

[0152] Example 15: Evaluation of the anti-inflammatory activity of exopolysaccharides

[0153] The anti-inflammatory effect of TSP sample 1 on LPS-induced RAW 264.7 cells was determined.

[0154] RAW 264.7 cells (4×10 4 The inflammatory model was established by adding different concentrations of TSP for 24 hours. The same amount of DMEM was used as a control (i.e., the control group). The cell culture supernatant was collected and the levels of TNF-α, IL-1β, IL-6, and NO were measured according to the kit instructions. The collected cells were lysed and centrifuged, and the supernatant was used to measure the activities of SOD, CAT, MDA, and GSH-Px using ELISA kits. The results are shown in the table. Figures 11 to 18 .

[0155] Depend on Figures 11 to 18The results show that the extracellular polysaccharides of the present invention can effectively reduce the levels of NO, TNF-α, IL-6 and IL-1β in LPS-induced RAW 264.7 macrophages, and are concentration-dependent. This is because the high content of carbohydrates may give the extracellular polysaccharides of the present invention better anti-inflammatory activity, and the galactose, arabinose and glucuronic acid in the extracellular polysaccharides of the present invention and their molecular weight may also give it stronger anti-inflammatory activity. At the same time, regulating oxidative stress in the body can also relieve and alleviate the inflammatory state. The extracellular polysaccharides of the present invention significantly increased the levels of SOD, CAT and GSH-Px in LPS-induced RAW 264.7 macrophages, and reduced the MDA level, and were concentration-dependent. This is because the high in vitro antioxidant activity may enable the extracellular polysaccharides of the present invention to effectively regulate oxidative factors in RAW 264.7 macrophages.

Claims

1. A basket fungus isolated from bran, characterized in that The Talaromyces is an endophytic fungus isolated from wheat bran, named Talaromyces sp. ZY1104, and preserved in the China Center for Type Culture Collection with the number CCTCC NO: M2025051. The center of the Talaromyces colony is turquoise and gradually turns pale white as it grows towards the edge. The optimal pH is 7.0±0.

2.

2. The method of claim 1, wherein the fungus is isolated from wheat bran. The basket fungus can produce extracellular polysaccharide, and the weight average molecular weight of the extracellular polysaccharide is 230kDa-240kDa, and the number average molecular weight is 120kDa-130kDa.

3. The Basilisk fungus isolated from bran according to claim 2, characterized in that: The extracellular polysaccharide consists of arabinose, galactose, glucose, mannose and glucuronic acid, with a mass ratio of 1:38-42:360-365:18-22:1.5-2.

0.

4. The method of claim 1, wherein the fungus is isolated from wheat bran. The isolation method of the basket fungus is as follows: placing bran in a triangular flask, sealing the bottle mouth, heating it at 80°C to 90°C for 20min to 30min, spreading the heated bran on a solid seed culture medium, and culturing it at a constant temperature of 28°C until a strain grows. When it is observed that the grown strain has a single color, the strain is picked up and cultured in a new solid seed culture medium to obtain the basket fungus.

5. The method of separating the tricholoma from wheat bran according to claim 4, wherein: The solid seed culture medium comprises the following components: 15g / L-25g / L sucrose, 4g / L-6g / L peptone, 2g / L-4g / L beef extract, 0.8g / L-1.2g / L yeast extract powder, 15g / L-25g / L agar, and a pH value of 7.0±0.

2. The thickness of the flattened bran is 1mm-1.5mm.

6. The use of the Basilisk fungus isolated from bran according to claim 1, characterized in that: The Basilisk fungus is used for the fermentation production of extracellular polysaccharides, specifically comprising S1 activating the strain to obtain strain seed liquid, S2 obtaining fermentation liquid through a fermentation process, and S3 separating and extracting the extracellular polysaccharides.

7. The use of the Basilisk fungus isolated from bran according to claim 6, characterized in that: The S1 strain activated to obtain the strain seed liquid is: Pick colonies from the solid seed culture medium and inoculate them into the liquid seed culture medium with the liquid volume of the liquid seed culture medium being 1 / 2 to 2 / 3. Activate the culture at 32°C for 12 to 36 hours with a stirring speed of 120 to 160 rpm. After the culture has grown to the logarithmic phase, activation is completed to obtain the culture seed solution for later use. The components of the liquid seed culture medium include: 15g / L-25g / L sucrose, 4g / L-6g / L peptone, 2g / L-4g / L beef extract, 0.8g / L-1.2g / L yeast extract powder, and a pH value of 7.0±0.

2.

8. The use of the Basilisk fungus isolated from bran according to claim 6, characterized in that: The fermentation liquid obtained by the fermentation process of S2 is: The bacterial seed liquid is inoculated into a fermentation liquid medium, the fermentation liquid volume is 1 / 2 to 2 / 3, the volume ratio of the inoculated bacterial seed liquid to the fermentation liquid medium is 5% to 15%, the stirring speed is 100 rpm to 140 rpm, the ventilation volume is 0.6 vvm to 1.0 vvm, and the temperature is 28° C. to 40° C. for 48 hours to 120 hours to obtain a fermentation liquid; The specific components of the fermentation liquid culture medium include: 35g / L~45g / L glucose, 0.8g / L~1.2g / L peptone, 0.8g / L~1.2g / L yeast extract, 0.8g / L~1.2g / L MgSO4, 0.8g / L~1.2g / L KH2PO4 and 0.8g / L~1.2g / L K2HPO4.

9. The use of the Basilisk fungus isolated from bran according to claim 6, characterized in that: The fermentation liquid obtained by the fermentation process of S2 is: The bacterial seed liquid is inoculated into a bran culture medium in an inoculum amount of 50 mL to 150 mL of the bacterial seed liquid per kilogram of the bran culture medium, the bacterial seed liquid is mixed with the bran culture medium, and stirred, and then fermented at a temperature of 32° C. to 48° C. for 96 hours to 168 hours, with stirring every 18 hours to 28 hours. After the fermentation is completed, the fermentation liquid is washed with water and filtered to obtain a fermentation liquid; The bran culture medium is obtained by crushing bran into 20-80 meshes, adding 2-4 kg of water per kilogram of bran, and sterilizing.

10. The use of the Basilisk fungus isolated from bran according to claim 6, characterized in that: The S3 separation and extraction of exopolysaccharides is: After the fermentation is completed, the fermentation broth is filtered to remove the mycelia, the filtrate is centrifuged to remove fine insoluble matter, the filtrate is collected and concentrated to 1 / 5 to 1 / 4 of the original volume by rotary evaporation under the conditions of 50°C to 70°C and 0.08MPa to 0.12MPa, 2 to 4 times the volume of the concentrate volume of Sevag reagent is added, and after vigorous shaking, the supernatant is collected by centrifugation; Add 3 to 5 times the volume of the collected supernatant and allow to stand overnight at 4°C for precipitation; The obtained precipitate was redissolved in water and concentrated to 1 / 5 to 1 / 4 of the original volume by rotary evaporation under conditions of 50°C to 70°C and 0.08 MPa to 0.12 MPa. The precipitate was dialyzed, purified and desalted using a dialysis bag with a molecular weight cutoff of 10.0 kDa to obtain an extracellular polysaccharide product.

Images