Bacillus subtilis and alcohol-free industrial method for producing low-molecular-weight tremella polysaccharide

Abstract

The invention provides a strain of bacillus subtilis and an alcohol-free industrial method for producing low-molecular-weight tremella polysaccharide, and particularly provides a strain of bacillus subtilis BS-Y-007, and a microbial agent comprises the bacillus subtilis BS-Y-007 and lactobacillus plantarum CGMCC (China General Microbiological Culture Collection Center) 1.572. The invention further discloses application of the bacillus subtilis BS-Y-007 or the microbial agent in preparation of tremella polysaccharide. The invention relates to a preparation method of a low-molecular-weight tremella polysaccharide-containing fermentation product. The invention discloses a method for producing low-molecular-weight tremella polysaccharide. According to the invention, a strain of bacillus subtilis beneficial to preparation of tremella polysaccharide is screened out; according to the invention, lactobacillus plantarum and bacillus subtilis are mixed to ferment the tremella spore fermentation liquor for the first time, so that the molecular weight of polysaccharide is reduced, and the interference of organic reagents is effectively avoided in the technological process.

Description

Technical Field

[0001] The invention belongs to the field of preparation and application of low molecular weight polysaccharides, and specifically relates to a strain of Bacillus subtilis and an alcohol-free industrial method for producing low molecular weight Tremella polysaccharides. Background Art

[0002] Tremella, as a specialty of China, has the effect of medicine and food. Tremella polysaccharide is the main active substance of Tremella, which has anti-inflammatory, anti-aging, anti-tumor, moisturizing and other effects. As a natural active polysaccharide, its development and application has also become a hot topic in current research. Studies have shown that the activity of Tremella polysaccharide is directly related to its molecular weight. In the field of cosmetics, high molecular weight Tremella polysaccharide has better moisturizing performance, but low solubility and is not easily absorbed by the skin; while low molecular weight Tremella polysaccharide has a stronger ability to scavenge free radicals and higher solubility, can penetrate into the surface of the skin and nourish the skin; in the field of food, low molecular weight Tremella polysaccharide is also easier to be absorbed by the human body; in the field of medicine, low molecular weight Tremella polysaccharide can effectively target inflammation and tumors. Therefore, in the development of food, cosmetics and medicine, low molecular weight Tremella polysaccharide is also more suitable.

[0003] There are many reports on the preparation methods of Tremella polysaccharides in the prior art. For example, the Chinese patent document CN117625713 A (application number: 202311590189.X) discloses an ultra-low molecular weight Tremella polysaccharide extract with moisturizing, soothing and anti-aging effects, and its preparation method and application, which is to ferment Tremella fruiting bodies with Candida utilis. Chinese patent document CN 107459585A (application number: 201710760049.0) discloses a production method of low molecular weight Tremella polysaccharides. This method uses alcohol precipitation and acid hydrolysis to achieve the preparation of Tremella polysaccharides and the reduction of molecular weight, which is not easy to ensure the quality of the product, and the large-scale use of ethanol poses a safety hazard. Chinese patent document CN 118286140 A (application number: 202410352697.2) discloses a Tremella polysaccharide fermentation product, a product containing the same, and a preparation method and application thereof. Tremella polysaccharide is used as a reaction substrate, and the Tremella polysaccharide requires corresponding preparation, has a long acquisition cycle, and a high cost price. The quality of each batch of Tremella polysaccharide produced is also uneven, and it is difficult to ensure that the fermentation conditions are always suitable and the product is always stable.

[0004] At present, many methods are combined with ethanol extraction; there are also reports on the preparation of Tremella polysaccharides by microbial fermentation. The raw materials used are mainly Tremella fruiting bodies, and the microbial strains used are also different. Different Tremella polysaccharide preparation methods have different purity and molecular weight of the prepared Tremella polysaccharides. Because Tremella polysaccharides are widely used, the demand for Tremella polysaccharides, especially low-molecular-weight Tremella polysaccharides, is relatively large in the market. Research and development of a production method for Tremella polysaccharides, especially a production method for low-molecular-weight Tremella polysaccharides, has important application value. There are currently few reports on microbial strains suitable for the preparation of low-molecular-weight Tremella polysaccharides in the prior art, and research and development of microbial strains suitable for the preparation of low-molecular-weight Tremella polysaccharides is of great significance. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a strain of Bacillus subtilis and an alcohol-free industrial method for producing low-molecular-weight Tremella polysaccharide.

[0006] Bacillus subtilis BS-Y-007 is referred to as Bacillus subtilis BS-Y-007.

[0007] The technical solution of the present invention is as follows:

[0008] A Bacillus subtilis BS-Y-007 was deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242282.

[0009] A microbial agent comprises the above-mentioned Bacillus subtilis BS-Y-007 and Lactobacillus plantarum CGMCC1.572.

[0010] The application of the above-mentioned Bacillus subtilis BS-Y-007 or microbial agent in the preparation of Tremella polysaccharide.

[0011] A method for preparing a fermented product containing low molecular weight Tremella polysaccharide, comprising the following steps:

[0012] Tremella fuciformis spores are used for liquid fermentation to prepare tremella polysaccharide fermentation liquid, Lactobacillus plantarum is firstly inoculated into the prepared tremella polysaccharide fermentation liquid for fermentation, and then Bacillus subtilis is inoculated for fermentation, and after the fermentation is completed, a fermentation product containing low molecular weight tremella polysaccharide is obtained.

[0013] Preferably, according to the present invention, the method for preparing the fermented product containing low molecular weight Tremella polysaccharide comprises the following steps:

[0014] (1) activating and culturing Lactobacillus plantarum and Bacillus subtilis respectively to obtain Lactobacillus plantarum seed liquid and Bacillus subtilis seed liquid;

[0015] (2) activating and culturing the Tremella fuciformis spores and then fermenting them to obtain Tremella fuciformis polysaccharide fermentation liquid;

[0016] (3) inoculating the Lactobacillus plantarum seed liquid prepared in step (1) into the Tremella polysaccharide fermentation liquid prepared in step (2) for fermentation, and then inoculating the Bacillus subtilis seed liquid prepared in step (1) for fermentation, and after the fermentation is completed, obtaining a fermentation product containing low molecular weight Tremella polysaccharide.

[0017] Preferably, according to the present invention, in step (1), activating and culturing Lactobacillus plantarum comprises the following steps:

[0018] The plant lactobacillus is inoculated into a lactic acid bacteria culture medium, and the activation condition is culturing at 35-37° C. for 18-24 hours. After activation, it is directly used or continued to be subcultured for 1-3 times, and the inoculation amount of each subculture is 1%-3% by volume fraction. The subculture conditions are the same as the activation conditions to obtain the plant lactobacillus seed liquid.

[0019] Further preferably, the lactic acid bacteria culture medium is MRS culture medium.

[0020] Preferably, according to the present invention, in step (1), activating and culturing Bacillus subtilis comprises the following steps:

[0021] The Bacillus subtilis is inoculated into a bacterial culture medium, and the activation conditions are culturing at 35-37° C. for 18-24 hours and 170-230 rpm. After activation, it is directly used or continuously subcultured for 1-3 times, and the inoculation amount of each subculture is 1%-3% by volume fraction. The subculture conditions are the same as the activation conditions to obtain a Bacillus subtilis seed solution.

[0022] Further preferably, the bacterial culture medium is LB culture medium;

[0023] LB medium consists of 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the solvent is water.

[0024] Preferably, according to the present invention, in step (2), the fermentation after activation culture of Tremella fuciformis spores comprises the following steps:

[0025] ①Inoculate Tremella fuciformis spores into the seed culture medium and culture at 25-30℃ and 150-250rpm until OD 560 13 to 16, to obtain a culture, which is used as a bacterial liquid;

[0026] Alternatively, the obtained culture is mixed with 50% glycerol and directly activated as a bacterial liquid for use as a preserved bacterial strain;

[0027] The bacterial liquid is inoculated into a seed culture medium at a volume ratio of 1% to 2% for activation, the activation conditions are 25 to 30° C., a rotation speed of 150 to 250 rpm, and an activation time of 48 to 60 hours, and the culture is subcultured 1 to 3 times, and the inoculation amount of each subculture is 1% to 2% by volume, and the subculture conditions are the same as the activation conditions, to obtain a Tremella seed liquid;

[0028] ② The tremella seed liquid obtained in step ① is inoculated into a fermentation medium at a volume ratio of 2% to 7%. Before inoculation, the pH value of the fermentation medium is adjusted to 4 to 5, 25 to 30° C., and aerobic fermentation is performed for 120 to 150 hours to obtain a tremella polysaccharide fermentation liquid.

[0029] Further preferably, in step ②, the aerobic fermentation conditions are a ventilation volume of 15 to 30 L / min, the dissolved oxygen-related speed is automatically adjusted, the speed is set to 200 to 900 rpm, the minimum dissolved oxygen value is set to 25%, the maximum is 100%, and stirring is automatically started when the dissolved oxygen value is lower than 25% dissolved oxygen.

[0030] Further preferably, in step ①, the seed culture medium components include: 20 g / L glucose, 2 g / L yeast powder, 2 g / L peptone, 0.46 g / L potassium dihydrogen phosphate, 1 g / L potassium hydrogen phosphate, 1 g / L magnesium sulfate, natural pH, and the solvent is water;

[0031] In step ②, the fermentation medium comprises, per liter, 40-80 g sucrose, 2-6 g yeast powder, 0.4-0.6 g potassium dihydrogen phosphate, 0.5-1 g dipotassium hydrogen phosphate, 0.5-1 g magnesium sulfate, 0.2-0.5 g asparagine, 0.2-0.5 g sodium glutamate, 0.2-0.5 g glutamine, 0.2-0.5 g aspartic acid, 0.2-0.5 g glycine, and 0.2-0.5 g inositol, with a natural pH and water as the solvent;

[0032] Or the fermentation medium comprises, per liter, 20 g of glucose, 2 g of yeast powder, 2 g of peptone, 0.46 g of potassium dihydrogen phosphate, 1 g of dipotassium hydrogen phosphate, 1 g of magnesium sulfate, natural pH, and water as the solvent.

[0033] More preferably, in step ②, the fermentation medium comprises per liter: 60 g sucrose, 5 g yeast powder, 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, 0.25 g asparagine, 0.25 g sodium glutamate, 0.25 g glutamine, 0.25 g aspartic acid, 0.25 g glycine, 0.25 g inositol, the pH is natural, and the solvent is water.

[0034] Preferably, according to the present invention, in step (3), the Lactobacillus plantarum seed solution prepared in step (1) is inoculated in an inoculum amount of 0.5% to 1% by volume into the Tremella polysaccharide fermentation liquid prepared in step (2), the pH is adjusted to 6.0 to 7.0, the temperature is 25 to 35° C., and the culture is continued for 24 to 48 hours to obtain a culture, and then the Bacillus subtilis seed solution prepared in step (1) is inoculated in the culture in an inoculum amount of 0.5% to 1% by volume, the temperature is 25 to 35° C., and the culture is continued until the dissolved oxygen value rises and stabilizes, and the fermentation is terminated to obtain a fermentation product containing low molecular weight Tremella polysaccharide.

[0035] Preferably according to the present invention, in the preparation method, the Lactobacillus plantarum is Lactobacillus plantarum CGMCC1.572, and the Bacillus subtilis is the above-mentioned Bacillus subtilis BS-Y-007.

[0036] Preferably according to the present invention, in the preparation method, the Tremella fuciformis strain includes Tremella fuciformis CGMCC 5.163, Tremella fuciformis CGMCC5.466, Tremella fuciformis SHMCC D83250, Tremella fuciformis SHMCC D82536, Tremella fuciformis SHMCC D24212, SHMCC D24214 or SHMCCD67684.

[0037] Further preferably, in the preparation method, the Tremella fuciformis strain includes Tremella fuciformis SHMCC D82536 or Tremella fuciformis SHMCCD24212.

[0038] A method for producing low molecular weight Tremella polysaccharide, comprising any of the following schemes:

[0039] Solution 1:

[0040] The fermented product containing low molecular weight Tremella polysaccharide prepared by the above method is subjected to solid-liquid separation, the liquid is retained, the solid is frozen and thawed, and then treated with pancreatic enzyme, and then the solid-liquid separation is performed, and the separated liquid is mixed with the retained liquid to extract and prepare low molecular weight Tremella polysaccharide;

[0041] Option 2:

[0042] The fermented product containing low molecular weight Tremella polysaccharide prepared by the above method is frozen and thawed, treated with pancreatic enzyme, and then solid-liquid separation is performed. The separated liquid is used to extract and prepare low molecular weight Tremella polysaccharide.

[0043] Preferably, according to the present invention, the method in scheme 1 comprises the following steps:

[0044] Adding sodium chloride to a final concentration of 2-4% to the fermented product containing low molecular weight Tremella polysaccharide prepared by the above method, dissolving it, performing solid-liquid separation, retaining the liquid, adding 1-1.5 times the volume of water to the solid for freezing and thawing, adjusting the pH to 8-9, adding 2-5‰ pancreatin, stirring at 45-55°C for 20-40 minutes, adding sodium chloride with a final concentration of 2-4% to dissolve, and then performing solid-liquid separation, mixing the separated liquid with the retained liquid, subjecting the mixed solution to ultrafiltration treatment at 200-500Dal to remove excess salt, culture medium nutrients and part of the pigment, and then further decolorizing and clarifying the retained polysaccharide solution to obtain a low molecular weight Tremella polysaccharide solution;

[0045] Alternatively, the low molecular weight Tremella polysaccharide solution is spray-dried to obtain the low molecular weight Tremella polysaccharide powder.

[0046] Preferably, according to the present invention, the method in scheme 2 comprises the following steps:

[0047] The fermented product containing low molecular weight Tremella polysaccharide prepared by the above method is frozen and thawed, and then the pH is adjusted to 8-9, 2-5‰ pancreatin is added, and the mixture is stirred at 45-55°C for 20-40 minutes, and sodium chloride with a final concentration of 2-4% is added to dissolve, and then solid-liquid separation is performed, and the separated liquid is subjected to 200-500Dal ultrafiltration treatment to remove excess salt, culture medium nutrients and part of the pigment, and then the retained polysaccharide solution is further decolorized and clarified to obtain a low molecular weight Tremella polysaccharide solution;

[0048] Alternatively, the low molecular weight Tremella polysaccharide solution is spray-dried to obtain the low molecular weight Tremella polysaccharide powder.

[0049] Preferably according to the present invention, in the above scheme 1 or scheme 2, the method of solid-liquid separation includes one or both of centrifugation and filtration.

[0050] More preferably, the solid-liquid separation method is centrifugation, and the centrifugation conditions are centrifugation for 10 to 15 minutes and a rotation speed of 7000 to 9000 rpm.

[0051] Preferably according to the present invention, in the above scheme 1 or scheme 2, the excess salt, culture medium nutrients and part of the pigment are removed by ultrafiltration membrane treatment at 200 to 500 Dal.

[0052] Preferably according to the present invention, in the above scheme 1 or scheme 2, the retained polysaccharide solution is decolorized and clarified by passing through an activated carbon fiber membrane.

[0053] The invention discloses a culture medium for tremella spore fermentation, wherein the components per liter include: 40-80g of sucrose, 2-6g of yeast powder, 0.4-0.6g of potassium dihydrogen phosphate, 0.5-1g of dipotassium hydrogen phosphate, 0.5-1g of magnesium sulfate, 0.2-0.5g of asparagine, 0.2-0.5g of sodium glutamate, 0.2-0.5g of glutamine, 0.2-0.5g of aspartic acid, 0.2-0.5g of glycine and 0.2-0.5g of inositol, the pH value is natural and the solvent is water.

[0054] Preferably according to the present invention, the culture medium comprises per liter the following ingredients: 60 g sucrose, 5 g yeast powder, 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, 0.25 g asparagine, 0.25 g sodium glutamate, 0.25 g glutamine, 0.25 g aspartic acid, 0.25 g glycine, 0.25 g inositol, natural pH, and water as the solvent.

[0055] Preferably according to the present invention, the pH value of the culture medium is adjusted to 4-5 before inoculation.

[0056] The beneficial effects of the present invention include at least the following:

[0057] 1. The present invention screened out a strain of Bacillus subtilis BS-Y-007 that is beneficial for preparing Tremella polysaccharide through strain mutagenesis.

[0058] 2. The present invention uses Lactobacillus plantarum and Bacillus subtilis to mix and ferment Tremella fuciformis spore fermentation liquid for the first time, thereby achieving a reduction in the molecular weight of polysaccharides, and further increases the polysaccharide yield by freeze-thawing and pancreatic enzymolysis. The Tremella fuciformis polysaccharide prepared by the method provided by the present invention achieves a reduction in the molecular weight of the Tremella fuciformis polysaccharide, and the process effectively avoids the interference of organic reagents. While increasing the yield and reducing the molecular weight, the production cost is reduced, and the safety of the product and the safety of production are improved.

[0059] 3. The polysaccharide prepared by the present invention has a high yield, a purity of more than 94%, a molecular weight of 4 to 6 kDa, and a scavenging rate of DPPH free radicals and ABTS free radicals of more than 85% at 10 mg / mL, achieving a good free radical scavenging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a process flow chart for the preparation of low molecular weight Tremella polysaccharide.

[0061] Figure 2 This is the standard curve of mannose for polysaccharide detection. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0063] The contents not described in detail in the embodiments are based on the prior art in the art.

[0064] The Tremella fuciformis strains used in the following examples were purchased from Shanghai Collection of Microorganisms Center with a strain number of SHMCC (SHBCC) D82536, and the implanted Lactobacillus was purchased from China General Microorganism Culture Collection Administration Center with a strain number of CGMCC1.572.

[0065] Pancreatin: purchased from MacLean, USP grade.

[0066] The screening method of Bacillus subtilis BS-Y-007 is as follows:

[0067] Bacillus subtilis CGMCC 1.821 (purchased from China General Microbiological Culture Collection Center) was subjected to ARTP mutagenesis to screen and obtain a mutant strain BS-Y-007; compared with the initial strain, the mutant strain BS-Y-007 can promote the rupture of Tremella fuciformis, release intracellular polysaccharides, and increase the yield of Tremella fuciformis polysaccharides.

[0068] The mutant strain BS-Y-007 was deposited as a strain, and the specific deposit information is as follows:

[0069] A Bacillus subtilis BS-Y-007 was deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242282.

[0070] Figure 1 This is a process flow chart for the preparation of low molecular weight Tremella polysaccharide.

[0071] Example 1

[0072] 1. Preparation of seed culture medium

[0073] Tremella fuciformis seed culture medium and fermentation medium: glucose 20g / L, yeast powder 2g / L, peptone 2g / L, potassium dihydrogen phosphate 0.46g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 1g / L, natural pH, solvent is water;

[0074] The Bacillus subtilis seed liquid culture medium is LB medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, and the solvent is water;

[0075] The Lactobacillus plantarum seed liquid culture medium was MRS culture medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.).

[0076] Take 4.83 g of MRS and add 100 mL of distilled water. Autoclave at 121°C to obtain MRS medium.

[0077] The culture medium can be prepared accordingly according to actual requirements.

[0078] 2. Fermentation process

[0079] Preparation of Lactobacillus plantarum seed solution: Lactobacillus plantarum CGMCC1.572 was inoculated into MRS medium, and the activation condition was culturing at 36°C for 24 hours. The culture was continued for 2 times, and the inoculation amount for each subculture was 1% by volume. The subculture conditions were the same as the activation conditions to obtain Lactobacillus plantarum seed solution.

[0080] Preparation of Bacillus subtilis BS-Y-007 seed solution: Bacillus subtilis was inoculated into LB medium, and the activation conditions were 36°C for 24 hours and 180 rpm. The culture was continued for 2 times, and the inoculation amount for each subculture was 1% by volume. The subculture conditions were the same as the activation conditions to obtain Bacillus subtilis seed solution.

[0081] Preparation of Tremella seed solution: Tremella fuciformis spores D82536 were inoculated into Tremella fuciformis seed culture medium and cultured at 28°C and 180 rpm until OD 560 The culture was mixed with 50% glycerol for seed preservation and used as bacterial liquid.

[0082] The above bacterial liquid was inoculated into Tremella fuciformis seed culture medium at 2% by volume, and the activation conditions were 28°C, 180rpm, and activation for 60h. The culture was continued for 2 times, and the inoculation amount for each subculture was 1% by volume. The subculture conditions were the same as the activation conditions to obtain Tremella fuciformis seed liquid.

[0083] The experiment uses a 20L fermentation tank for fermentation, ensuring that the volume of the fermentation medium after sterilization is 15L. Prepare 1L of Tremella fuciformis seed liquid (inoculation volume 6.6% by volume fraction) for each fermentation tank. First adjust the pH of the fermentation liquid to 5. After the pH of the fermentation tank is stable, inoculate the Tremella fuciformis seed liquid into the fermentation tank, 28°C, ventilation 15L / min, because Tremella fuciformis is an aerobic bacteria, low dissolved oxygen will inhibit bacterial growth, set the dissolved oxygen associated speed automatic adjustment, after experimental verification, set the minimum dissolved oxygen value to 25%, the maximum to 100%, the speed is set to 200-900 rpm, and automatically start stirring when the dissolved oxygen value is lower than 25% dissolved oxygen.

[0084] According to the Tremella fuciformis growth curve obtained from the experiment, at the 120th hour of fermentation, the Tremella fuciformis fermentation reached the late stage of the stable period and the early stage of the decay period, and the bacterial body grew slowly. Therefore, the above-mentioned Lactobacillus plantarum seed solution was inoculated at the 120th hour of fermentation, and the inoculation amount was 1% by volume. At this time, the pH was adjusted to 6.5 and 28°C. When the fermentation was continued for 36 hours, the Lactobacillus plantarum completed the growth cycle. At this time, the above-mentioned Bacillus subtilis seed solution was accessed, and the inoculation amount was 1% by volume. After access, the culture was continued, and the fermentation temperature and pH remained unchanged. The fermentation end point was taken after the dissolved oxygen value recovered and stabilized (the total fermentation cycle was 190 hours), and the Tremella fuciformis polysaccharide fermentation liquid was obtained.

[0085] 3. Post-processing process

[0086] Take the above Tremella polysaccharide fermentation liquid, add sodium chloride in an amount of 4% (w / v, g / mL) and stir to dissolve, centrifuge for 10 minutes, rotate at 9000rpm, collect the supernatant and bacteria respectively; mix the bacteria and water in a volume ratio of 1:1.5, freeze the mixture at -20°C overnight, take it out and thaw it, adjust the pH to 8-9 with sodium hydroxide solution, add 2‰ (w / v, g / mL) of pancreatin, stir at 55°C for 30 minutes, then add sodium chloride in an amount of 4% and stir to dissolve, centrifuge the bacteria for 10 minutes , speed 9000rpm, collect the supernatant and mix it with the original supernatant to obtain a mixed solution; pass the mixed solution through an ultrafiltration separation device, select a 500Dal ultrafiltration membrane, and remove excess salt, culture medium nutrients and some pigments; treat the retained polysaccharide solution through an activated carbon fiber membrane (purchased from Beijing Rixin Yuanwang Technology Development Co., Ltd.) for 3 times to remove fat-soluble pigments to achieve a clear state, and obtain a polysaccharide powder by spray drying. The spray drying conditions are an inlet air temperature of 160°C, an inlet air volume of 95%, and a feed flow rate of 15mL / min.

[0087] 4. Index determination:

[0088] The total sugar and protein determination of the above-prepared polysaccharides refer to the enterprise standard Q / FCG 1001S-2024. The standard curve of mannose in the total sugar determination is shown in Figure 2 The molecular weight of polysaccharides was determined by the Ubbelohde viscometer method, referring to the enterprise standard Q / FCG1002S-2024. The mass of polysaccharides per liter of fermentation broth was directly weighed according to the polysaccharides obtained after processing per liter of fermentation broth.

[0089] Example 2

[0090] 1. Various sub-liquid culture conditions and culture medium components remain unchanged, i.e. the same as in Example 1, the Tremella fuciformis spore fermentation medium is optimized, and after optimal experimental conditions, it is finally confirmed that the Tremella fuciformis spore fermentation medium: sucrose 60g / L, yeast powder 5g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 1g / L, asparagine 0.25g / L, sodium glutamate 0.25g / L, glutamine 0.25g / L, aspartic acid 0.25g / L, glycine 0.25g / L, inositol 0.25g / L, pH is natural, and the solvent is water.

[0091] 2. The fermentation process and post-treatment process refer to the 2nd and 3rd items of Example 1, except that the Tremella fuciformis fermentation medium is changed, and the other items remain unchanged. The index determination refers to the method of 4th item of Example 1.

[0092] The difference in Tremella polysaccharide produced before and after changing the culture medium is shown in Table 1:

[0093] Table 1 Effects of culture medium composition changes on Tremella polysaccharide production

[0094]

[0095] Comparative Example 1

[0096] Compared with Example 2, the difference is that this group is no longer subjected to freezing treatment.

[0097] The comparison of the results before and after freezing is shown in Table 2:

[0098] Table 2 Effects of freezing treatment on Tremella polysaccharide production

[0099]

[0100] Comparative Example 2

[0101] Compared with Example 2, Experiment 1 screened different enzymes. The enzyme was added at 5 g / L to avoid the difference caused by insufficient enzyme dosage. The yield was used as an indicator. Experiment 2 did not perform enzymatic hydrolysis, and other treatments were the same.

[0102] Experimental group 1: The screening results of different enzyme treatments are compared in Table 3:

[0103] Table 3 Comparison of screening results of different enzymes

[0104]

[0105] Experimental Group 2: Comparison of results before and after enzymatic hydrolysis is shown in Table 4:

[0106] Table 4 Effects of bacterial fluid enzymatic hydrolysis on Tremella polysaccharide production

[0107]

[0108] Comparative Example 3

[0109] Compared with Example 2, this group of experiments only used Tremella fuciformis for fermentation, and did not add Lactobacillus plantarum and Bacillus subtilis for co-fermentation, and everything else was the same.

[0110] The comparison of the results before and after co-culture is shown in Table 5:

[0111] Table 5 Effects of co-culture on Tremella polysaccharides

[0112]

[0113]

[0114] The above experimental results confirm that, by comparing Example 1 with Example 2, the change in the formula of the culture medium is more conducive to the fermentation process and increases the yield of polysaccharides; by comparing the treatment methods of Example 2 with those of Comparative Example 2, freezing and enzymatic hydrolysis can both contribute to the outflow of intracellular polysaccharides and the acquisition of cell wall polysaccharides; by comparing Example 2 with Comparative Example 3, it is confirmed that the co-cultivation process can naturally reduce the molecular weight of polysaccharides.

[0115] Comparative Example 4

[0116] Compared with Example 2, Experiment 1 screened Tremella fuciformis, taking the mass of polysaccharides obtained per liter of fermentation broth as an indicator; Experiment 2 compared Bacillus bacteria, taking the mass of polysaccharides obtained per liter of fermentation broth as an indicator.

[0117] Experimental Group 1: Screening of Tremella fuciformis

[0118] Tremella fuciformis strains were purchased from Shanghai Collection of Microorganisms Center and China General Microorganism Culture Collection Administration Center. The experimental results are shown in Table 6.

[0119] Table 6

[0120] Strain No. Polysaccharide mass (g / L) CGMCC 5.466 10.53 CGMCC 5.163 12.25 SHMCC(SHBCC)D83250 11.36 SHMCC(SHBCC)D82536 15.67 SHMCC(SHBCC)D24212 14.23 SHMCC(SHBCC)D24214 7.70 SHMCC(SHBCC)D67684 9.09

[0121] Experimental Group 2: Screening of Bacillus subtilis

[0122] Bacillus subtilis CGMCC 1.821, CGMCC 1.15792, and CGMCC 1.7740 were purchased from the China General Microbiological Culture Collection Administration Center; the Bacillus subtilis BS-Y-007 in this experiment was obtained by laboratory mutagenesis and is currently preserved in the China Center for Type Culture Collection with a strain number of CCTCC M 20242282. The experimental results are shown in Table 7.

[0123] Table 7

[0124]

[0125]

[0126] Comparative Example 5

[0127] Compared with Example 2, the difference from Example 2 is that in this comparative example, only one of Lactobacillus plantarum and Bacillus subtilis is added to the Tremella polysaccharide fermentation liquid for fermentation, and the difference in the synergistic effect of the two strains and co-culture is compared. The experimental results are shown in Table 8.

[0128] By comparing the situation of adding two strains at the same time and adding only one strain, when two bacteria act on the fermentation system, the effect is much better than the single effect.

[0129] Table 8

[0130]

[0131] Effect example

[0132] The Tremella polysaccharides prepared in Example 2 and Comparative Example 3 were subjected to DPPH and ABTS free radical scavenging experiments. The experimental methods and experimental results are as follows:

[0133] 1. Use anhydrous ethanol to prepare 0.1mmol / L DPPH standard stock solution (keep at low temperature and away from light); use water to prepare 7mmol / L ABTS diammonium salt stock solution.

[0134] 2. Prepare Tremella polysaccharide solutions at concentrations of 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg / mL respectively, with VC as the positive control and anhydrous ethanol as the blank control group.

[0135] 3.DPPH free radical determination method:

[0136] Take 3 mL of Tremella polysaccharide experimental solution of different concentrations, add 3 mL of DPPH solution, mix quickly and keep it away from light for half an hour, measure the absorbance at 517 nm with a spectrophotometer, and measure three times in parallel. The blank group chooses anhydrous ethanol, and the positive control group chooses VC.

[0137] The scavenging rate of DPPH free radicals by VC and Tremella polysaccharide solution was calculated according to the following formula:

[0138]

[0139] Where: A blank is the absorbance of DPPH working solution; A sample is the absorbance of sample solution

[0140] 4. ABTS free radical determination method: ABTS working solution: Accurately measure 25 mL of ABTS diammonium salt stock solution and potassium disulfate solution, mix thoroughly. Place in a dark place at 4°C for 12 hours to obtain ABTS stock solution. Dilute with anhydrous ethanol solution when using.

[0141] Take 0.5mL of different concentrations of Tremella polysaccharide sample experimental solution and mix with 5mL ABTS diluent, keep it in dark for 10 minutes, and measure it three times in parallel. The experimental group uses anhydrous ethanol, and the positive control group uses VC.

[0142] The scavenging rate of ABTS free radicals by VC and Tremella polysaccharide was calculated according to the following formula:

[0143]

[0144] Where: A blank is the absorbance of ABTS cation working solution; A sample is the reference solution with the absorbance VC of the sample solution. The scavenging rate of DPPH and ABTS free radicals is calculated as follows, see Table 9:

[0145] Table 9

[0146] Concentration (mg / mL) DPPH free radical scavenging rate (%) ABTS free radical scavenging rate (%) 6 98.21 97.89 4 97.85 97.74 2 97.36 97.56 1 95.54 97.84 0.5 93.57 92.14 0.1 80.21 80.13

[0147] The calculation of the scavenging rate of the sample solution for DPPH free radicals is as follows, see Table 10:

[0148] Table 10

[0149]

[0150]

[0151] The calculation of the scavenging rate of the sample solution for ABTS free radicals is as follows, see Table 11:

[0152] Table 11

[0153] No co-cultivation (Comparative Example 3) Co-cultivation (Example 2) Concentration (mg / mL) ABTS free radical scavenging rate (%) ABTS free radical scavenging rate (%) 10 73.85 86.33 8 63.66 76.84 6 49.76 71.22 4 46.25 66.58 2 34.74 38.85 1 14.32 19.14

[0154] Changes in uronic acid content of Tremella polysaccharides prepared in Example 2 and Comparative Example 3

[0155] The carbazole-sulfuric acid method was used to determine uronic acid.

[0156] 1. Borax sulfuric acid solution: Weigh 9.54 g of sodium tetraborate and dissolve it in 1000 mL of concentrated sulfuric acid.

[0157] 2. Carbazole solution: Accurately weigh 0.125 g, dissolve in 100 mL of anhydrous ethanol, and store in a brown bottle.

[0158] 3. Prepare a 1 mg / mL Tremella polysaccharide solution, weigh 5 g of the solution and dilute it into a 50 mL volumetric flask to obtain a sample solution.

[0159] 4. The specific adding method is as follows:

[0160] 1 mL of test solution, mix well, cool in an ice bath, slowly add 5 mL of borax sulfuric acid solution that has been cooled in an ice bath, seal, and mix well. Heat in a boiling water bath for 10 minutes, cool to room temperature in an ice bath. Accurately add 2 mL of carbazole, mix well, heat in a boiling water bath for 15 minutes, and cool to room temperature in an ice bath. Measure the absorbance at a wavelength of 530 nm. Calculate the glucuronic acid concentration based on the standard curve, see Table 12.

[0161] X G =(C1*50*100*100*10 -6 ) / W1*(100-h)*W2

[0162] Where: X G : glucuronic acid content, in %; glucuronic acid C1: glucuronic acid concentration in the test solution calculated by regression equation, in μg / mL; W1: the weight of the test sample weighed, in g;

[0163] W2: the amount of the diluted test solution in the second step, in g; h: loss on drying, %

[0164] Table 12

[0165]

[0166]

[0167] The above experimental results show that, compared with Comparative Example 3, the uronic acid content in Example 2 is increased after co-culture, reaching 10 mg / mL, and the ABTS and DPPH free radical scavenging rates after co-culture are measured to be more than 85%. The uronic acid content is proportional to the free radical scavenging rate, further confirming the advantages of low molecular weight Tremella polysaccharides produced by co-culture.

[0168] The present invention screens out a strain of Bacillus subtilis BS-Y-007 that is beneficial for preparing low-molecular-weight Tremella polysaccharides through strain mutagenesis. The present invention uses Lactobacillus plantarum and Bacillus subtilis to mix and ferment Tremella fuciformis spore fermentation liquid for the first time, realizes the reduction of polysaccharide molecular weight, and further increases polysaccharide yield by freeze-thaw and pancreatic enzyme hydrolysis. The Tremella polysaccharide prepared by the method provided by the present invention realizes the reduction of Tremella polysaccharide molecular weight, and the process effectively avoids the interference of organic reagents, while increasing the yield and reducing the molecular weight, reducing the production cost, and improving the safety of the product and the safety of production. The polysaccharide prepared by the present invention has a high yield, a purity of more than 94%, a molecular weight of 4 to 6 kDa, and at 10 mg / mL, it reaches more than 85% for DPPH free radicals and ABTS free radicals, achieving a good free radical scavenging effect.

Claims

1. A Bacillus subtilis BS-Y-007, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242282.

2. A microbial agent, characterized in that: It comprises the Bacillus subtilis BS-Y-007 and Lactobacillus plantarum CGMCC1.572 described in claim 1.

3. Use of the Bacillus subtilis BS-Y-007 described in claim 1 or the microbial agent described in claim 2 in the preparation of Tremella polysaccharide.

4. A method for preparing a fermented product containing low molecular weight Tremella polysaccharide, characterized in that: The steps include: Tremella fuciformis spores are used for liquid fermentation to prepare tremella polysaccharide fermentation liquid. Lactobacillus plantarum is firstly inoculated into the prepared tremella polysaccharide fermentation liquid for fermentation, and then Bacillus subtilis is inoculated for fermentation. After the fermentation is completed, a fermentation product containing low molecular weight tremella polysaccharide is obtained.

5. The preparation method according to claim 4, characterized in that: The steps include: (1) activating and culturing Lactobacillus plantarum and Bacillus subtilis respectively to obtain Lactobacillus plantarum seed liquid and Bacillus subtilis seed liquid; (2) activating and culturing the Tremella fuciformis spores and then fermenting them to obtain Tremella fuciformis polysaccharide fermentation liquid; (3) inoculating the Lactobacillus plantarum seed liquid prepared in step (1) into the Tremella polysaccharide fermentation liquid prepared in step (2) for fermentation, and then inoculating the Bacillus subtilis seed liquid prepared in step (1) for fermentation, and after the fermentation is completed, obtaining a fermentation product containing low molecular weight Tremella polysaccharide.

6. The preparation method according to claim 5, characterized in that: In step (1), activating and culturing plant lactobacillus comprises the following steps: Inoculate Lactobacillus plantarum into a lactic acid bacteria culture medium, and culture it at 35-37° C. for 18-24 hours under the activation condition. After activation, use it directly or continue to subculture it for 1-3 times, and the inoculation amount of each subculture is 1%-3% by volume fraction. The subculture conditions are the same as the activation conditions to obtain a Lactobacillus plantarum seed solution; Preferably, the lactic acid bacteria culture medium is MRS culture medium; Preferably, in step (1), activating and culturing Bacillus subtilis comprises the following steps: Inoculate Bacillus subtilis into bacterial culture medium, and activate it under the conditions of culturing at 35-37°C for 18-24h and 170-230rpm. After activation, use it directly or continue to subculture it for 1-3 times, and the inoculation amount of each subculture is 1%-3% by volume. The subculture conditions are the same as the activation conditions to obtain Bacillus subtilis seed liquid; Preferably, the bacterial culture medium is LB medium; LB medium is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the solvent is water; Preferably, in step (2), the fermentation after activation culture of Tremella fuciformis spores comprises the following steps: ①Inoculate Tremella fuciformis spores into the seed culture medium and culture at 25-30℃ and 150-250rpm until OD 560 13 to 16, to obtain a culture, which is used as a bacterial liquid; Alternatively, the obtained culture is mixed with 50% glycerol and directly activated as a bacterial liquid for use as a preserved strain; The bacterial liquid is inoculated into a seed culture medium at a volume ratio of 1% to 2% for activation, the activation conditions are 25 to 30° C., a rotation speed of 150 to 250 rpm, and an activation time of 48 to 60 hours, and the culture is subcultured 1 to 3 times, and the inoculation amount of each subculture is 1% to 2% by volume, and the subculture culture conditions are the same as the activation conditions, to obtain a Tremella seed liquid; ② Inoculate the Tremella fuciformis seed liquid obtained in step ① into the fermentation medium at a volume ratio of 2% to 7%, adjust the pH value of the fermentation medium to 4 to 5 before inoculation, and ferment aerobically for 120 to 150 hours to obtain Tremella fuciformis polysaccharide fermentation liquid; Preferably, in step ②, the aerobic fermentation condition is a ventilation volume of 15 to 30 L / min, the dissolved oxygen-related speed is automatically adjusted, the speed is set to 200 to 900 rpm, the minimum dissolved oxygen value is set to 25%, the maximum is 100%, and stirring is automatically started when the dissolved oxygen value is lower than 25% dissolved oxygen; Preferably, in step ①, the seed culture medium components include: 20 g / L glucose, 2 g / L yeast powder, 2 g / L peptone, 0.46 g / L potassium dihydrogen phosphate, 1 g / L potassium hydrogen phosphate, 1 g / L magnesium sulfate, natural pH, and the solvent is water; In step ②, the fermentation medium comprises, per liter, 40-80 g sucrose, 2-6 g yeast powder, 0.4-0.6 g potassium dihydrogen phosphate, 0.5-1 g dipotassium hydrogen phosphate, 0.5-1 g magnesium sulfate, 0.2-0.5 g asparagine, 0.2-0.5 g sodium glutamate, 0.2-0.5 g glutamine, 0.2-0.5 g aspartic acid, 0.2-0.5 g glycine, and 0.2-0.5 g inositol, with a natural pH and water as the solvent; Or the fermentation medium comprises, per liter, 20 g glucose, 2 g yeast powder, 2 g peptone, 0.46 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, natural pH, and water as solvent; Preferably, the fermentation medium comprises, per liter, 60 g sucrose, 5 g yeast powder, 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, 0.25 g asparagine, 0.25 g sodium glutamate, 0.25 g glutamine, 0.25 g aspartic acid, 0.25 g glycine, and 0.25 g inositol, with a natural pH and water as the solvent.

7. The preparation method according to claim 5, characterized in that: In step (3), the Lactobacillus plantarum seed solution prepared in step (1) is inoculated in an amount of 0.5% to 1% by volume into the Tremella polysaccharide fermentation liquid prepared in step (2), the pH is adjusted to 6.0 to 7.0, the temperature is 25 to 35° C., and the culture is continued for 24 to 48 hours to obtain a culture, and then the Bacillus subtilis seed solution prepared in step (1) is inoculated in the culture in an amount of 0.5% to 1% by volume, the temperature is 25 to 35° C., and the culture is continued until the dissolved oxygen value rises and stabilizes, and the fermentation is terminated to obtain a fermentation product containing low molecular weight Tremella polysaccharide; Preferably, in the preparation method, Lactobacillus plantarum is Lactobacillus plantarum CGMCC1.572, and Bacillus subtilis is Bacillus subtilis BS-Y-007 described in claim 1; Preferably, in the preparation method, the Tremella fuciformis strain includes Tremella fuciformis CGMCC 5.163, Tremella fuciformis CGMCC 5.466, Tremella fuciformis SHMCC D83250, Tremella fuciformis SHMCC D82536, Tremella fuciformis SHMCC D24212, SHMCC D24214 or SHMCC D67684; Preferably, in the preparation method, the Tremella fuciformis strain includes Tremella fuciformis SHMCC D82536 or Tremella fuciformis SHMCC D24212.

8. A method for producing low molecular weight Tremella polysaccharide, characterized in that: This includes any of the following options: Solution 1: The fermented product containing low molecular weight Tremella polysaccharide prepared by the method according to any one of claims 4 to 7 is subjected to solid-liquid separation, the liquid is retained, the solid is frozen and thawed, and then treated with pancreatic enzyme, and then the solid-liquid separation is performed, and the separated liquid is mixed with the retained liquid to extract and prepare low molecular weight Tremella polysaccharide; Option 2: The fermented product containing low molecular weight Tremella polysaccharide prepared by the method according to any one of claims 4 to 7 is frozen and thawed, treated with pancreatic enzyme, and then solid-liquid separated. The separated liquid is used to extract and prepare low molecular weight Tremella polysaccharide.

9. The method according to claim 8, characterized in that The method includes the following steps: Adding sodium chloride to a final concentration of 2-4% to the fermented product containing low molecular weight Tremella polysaccharide prepared by the method according to any one of claims 4 to 7, dissolving, performing solid-liquid separation, retaining the liquid, adding 1-1.5 times the volume of water to the solid for freeze-thawing, adjusting the pH to 8-9, adding 2-5‰ pancreatin, stirring at 45-55°C for 20-40 minutes, adding sodium chloride with a final concentration of 2-4% to dissolve, and then performing solid-liquid separation, mixing the separated liquid with the retained liquid, treating the mixed liquid through a 200-500Dal ultrafiltration membrane to remove excess salt, culture medium components and part of the pigment, and then further decolorizing and clarifying the retained polysaccharide solution to obtain a low molecular weight Tremella polysaccharide solution; Alternatively, the low molecular weight Tremella polysaccharide solution is spray-dried to obtain low molecular weight Tremella polysaccharide powder; Preferably, the method according to scheme 2 comprises the following steps: The fermented product containing low molecular weight Tremella polysaccharide prepared by the method according to any one of claims 4 to 7 is frozen and thawed, and then the pH is adjusted to 8-9, 2-5‰ pancreatin is added, and the mixture is stirred at 45-55°C for 20-40 minutes, and sodium chloride with a final concentration of 2-4% is added to dissolve the mixture, and then solid-liquid separation is performed, and the separated liquid is ultrafiltered at 200-500 Da1 to remove excess salt, culture medium nutrients and part of the pigment, and then the retained polysaccharide solution is further decolorized and clarified to obtain a low molecular weight Tremella polysaccharide solution; Alternatively, the low molecular weight Tremella polysaccharide solution is spray-dried to obtain low molecular weight Tremella polysaccharide powder; Preferably, in the scheme 1 or 2, the solid-liquid separation method includes one or both of centrifugation and filtration; Preferably, the solid-liquid separation method is centrifugation, and the centrifugation conditions are centrifugation for 10 to 15 minutes and a rotation speed of 7000 to 9000 rpm; Preferably, in the scheme 1 or 2, excess salts, culture medium nutrients and part of the pigment are removed by ultrafiltration at 200-500 Dal; Preferably, in the scheme 1 or 2, the retained polysaccharide solution is decolorized by passing through an activated carbon fiber membrane.

10. A culture medium for Tremella fuciformis spore fermentation, characterized in that: The ingredients per liter include: 40-80g sucrose, 2-6g yeast powder, 0.4-0.6g potassium dihydrogen phosphate, 0.5-1g potassium hydrogen phosphate, 0.5-1g magnesium sulfate, 0.2-0.5g asparagine, 0.2-0.5g sodium glutamate, 0.2-0.5g glutamine, 0.2-0.5g aspartic acid, 0.2-0.5g glycine, 0.2-0.5g inositol, natural pH, solvent is water; Preferably, the culture medium comprises, per liter, 60 g sucrose, 5 g yeast powder, 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, 0.25 g asparagine, 0.25 g sodium glutamate, 0.25 g glutamine, 0.25 g aspartic acid, 0.25 g glycine, and 0.25 g inositol, with a natural pH and water as the solvent; Preferably, the pH value of the culture medium is adjusted to 4-5 before inoculation.

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