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

Abstract

The present invention provides a Bacillus subtilis strain and an alcohol-free industrial method for producing low-molecular-weight tremella polysaccharides. Specifically, the present invention provides a Bacillus subtilis strain BS-Y-007, a microbial agent comprising Bacillus subtilis BS-Y-007 and Lactobacillus plantarum CGMCC1.572, application of the Bacillus subtilis BS-Y-007 or the microbial agent in preparing tremella polysaccharides, a method for preparing a fermentation product containing low-molecular-weight tremella polysaccharides, and a method for producing low-molecular-weight tremella polysaccharides. The present invention screens out a Bacillus subtilis strain that is beneficial for preparing tremella polysaccharides. The present invention uses Lactobacillus plantarum and Bacillus subtilis to ferment tremella spore fermentation liquid for the first time, thereby reducing the molecular weight of the polysaccharide and effectively avoiding the interference of organic reagents in the process.

Description

Technical Field

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

[0002] Tremella fuciformis, a specialty of China, boasts both medicinal and edible properties. Tremella polysaccharide, the main active ingredient in Tremella fuciformis, possesses anti-inflammatory, anti-aging, anti-tumor, and moisturizing properties. As a natural active polysaccharide, its development and application are currently a hot topic of research. Research has shown that the activity of Tremella fuciformis polysaccharide is directly related to its molecular weight. In cosmetics, high-molecular-weight Tremella fuciformis polysaccharides offer better moisturizing properties, but their solubility is low and poorly absorbed by the skin. Low-molecular-weight Tremella fuciformis polysaccharides, on the other hand, have a stronger free radical scavenging ability and higher solubility, allowing them to penetrate deeply into the skin's surface and nourish it. In the food industry, low-molecular-weight Tremella fuciformis polysaccharides are also more easily absorbed by the body. In the pharmaceutical industry, low-molecular-weight Tremella fuciformis polysaccharides are effective against inflammation and tumors. Therefore, low-molecular-weight Tremella fuciformis polysaccharides are more suitable for the development of food, cosmetics, and pharmaceuticals.

[0003] Numerous methods for preparing Tremella fuciformis polysaccharides have been reported in the prior art. For example, Chinese patent CN117625713 A (Application Number: 202311590189.X) discloses an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing, and anti-aging properties, as well as its preparation method and application. This method utilizes Candida utilis to ferment Tremella fuciformis fruiting bodies. Chinese patent CN 107459585 A (Application Number: 201710760049.0) discloses a method for producing low-molecular weight Tremella fuciformis polysaccharide. This method utilizes alcohol precipitation and acid hydrolysis to produce the Tremella fuciformis polysaccharide and reduce its molecular weight, making it difficult to ensure product quality. Furthermore, the large-scale use of ethanol poses safety risks. Chinese patent document CN 118286140 A (application number: 202410352697.2) discloses a Tremella fuciformis polysaccharide fermentation product, products containing the same, and preparation methods and applications thereof. Tremella fuciformis polysaccharide is used as a reaction substrate, and the Tremella fuciformis polysaccharide requires corresponding preparation, with a long acquisition cycle and high cost. The quality of each batch of Tremella fuciformis polysaccharide is also uneven, making it difficult to ensure that the fermentation conditions are always suitable and the product is always stable.

[0004] Currently, many methods combine ethanol extraction; there are also reports of producing Tremella polysaccharides using microbial fermentation. The raw material used is primarily Tremella fruiting bodies, and the microbial strains used vary. Different Tremella polysaccharide preparation methods produce Tremella polysaccharides with varying purity and molecular weight. Due to their widespread application, Tremella polysaccharides, especially low-molecular-weight Tremella polysaccharides, are currently in high demand in the market. Research and development of a method for producing Tremella polysaccharides, particularly low-molecular-weight Tremella polysaccharides, has important application value. Limited prior art reports exist on microbial strains suitable for producing low-molecular-weight Tremella polysaccharides, making research and development of such strains of microorganisms of interest. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a Bacillus subtilis spore strain and an alcohol-free industrial method for producing low-molecular-weight Tremella polysaccharide.

[0006] Bacillus subtilis ( Bacillus subtilis )BS-Y-007 is abbreviated as Bacillus subtilis BS-Y-007.

[0007] The technical solutions of the present invention are as follows:

[0008] A Bacillus subtilis ( Bacillus subtilis )BS-Y-007, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is 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 comprises the following steps:

[0012] Tremella fuciformis spores are used for liquid fermentation to prepare tremella fuciformis polysaccharide fermentation liquid. Lactobacillus plantarum is first inoculated into the prepared tremella fuciformis 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 fuciformis polysaccharide is obtained.

[0013] According to a preferred embodiment of the present invention, the method for preparing the fermentation product containing low molecular weight Tremella polysaccharide comprises the following steps:

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

[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 fuciformis polysaccharide fermentation liquid prepared in step (2) for fermentation, and then inoculating the Bacillus subtilis seed liquid prepared in step (1) for fermentation. After the fermentation is completed, a fermentation product containing low molecular weight Tremella fuciformis polysaccharide is obtained.

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

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

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

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

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

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

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

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

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

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

[0027] The bacterial solution was inoculated into a seed culture medium at a volume ratio of 1% to 2% for activation. The activation conditions were 25 to 30°C, a rotation speed of 150 to 250 rpm, and activation for 48 to 60 hours. The culture was subcultured 1 to 3 times, with the inoculum volume of each subculture being 1% to 2% by volume. The subculture conditions were the same as those for activation, to obtain a Tremella fuciformis seed solution.

[0028] ② The tremella seed liquid obtained in step ① is inoculated into the 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, the temperature is 25 to 30°C, and aerobic fermentation is carried out 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 15~30 L / min, the dissolved oxygen-related speed is automatically adjusted, the speed is set to 200~900 rpm, the minimum dissolved oxygen value is set to 25%, and 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 comprises: 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 water as the solvent;

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

[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 potassium 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 fuciformis 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 fuciformis 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] Option 1:

[0040] The fermented product containing low molecular weight Tremella fuciformis 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, followed by solid-liquid separation, and the separated liquid is mixed with the retained liquid to extract and prepare low molecular weight Tremella fuciformis polysaccharide;

[0041] Option 2:

[0042] The fermented product containing low molecular weight Tremella fuciformis polysaccharide prepared by the above method 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 fuciformis polysaccharide.

[0043] According to a preferred embodiment of the present invention, the method comprises the following steps:

[0044] adding sodium chloride to a final concentration of 2-4% to the fermentation material containing low-molecular-weight Tremella polysaccharide prepared by the above method, dissolving the material, performing solid-liquid separation, retaining the liquid, adding 1-1.5 times the volume of water to the solid, freezing and thawing the solid, adjusting the pH to 8-9, adding 2-5‰ pancreatin, stirring at 45-55°C for 20-40 min, adding sodium chloride to a final concentration of 2-4% to dissolve the material, then performing solid-liquid separation, mixing the separated liquid with the retained liquid, and subjecting the mixed solution to 200-500 Dal ultrafiltration to remove excess salts, culture medium nutrients, and some pigments, 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] According to a preferred embodiment of the present invention, the second embodiment of the method comprises the following steps:

[0047] The fermentation product containing low molecular weight Tremella fuciformis 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 product is stirred at 45-55°C for 20-40 minutes. Sodium chloride is added to a final concentration of 2-4% to dissolve the product, and then solid-liquid separation is performed. The separated liquid is subjected to ultrafiltration at 200-500 Dal to remove excess salts, culture medium nutrients, and some pigments. The retained polysaccharide solution is then further decolorized and clarified to obtain a low molecular weight Tremella fuciformis polysaccharide solution.

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

[0049] According to a preferred embodiment of the present invention, in the above-mentioned scheme 1 or scheme 2, the solid-liquid separation method 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 10-15 minutes and a rotation speed of 7000-9000 rpm.

[0051] According to a preferred embodiment of the present invention, in the above-mentioned solution 1 or solution 2, the excess salts, culture medium nutrients and part of the pigment are removed by ultrafiltration membrane treatment at 200-500 Dal.

[0052] Preferably, according to the present invention, in the above-mentioned solution 1 or 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 fuciformis spore fermentation, wherein the components per liter include: 40-80 g of sucrose, 2-6 g of yeast powder, 0.4-0.6 g of potassium dihydrogen phosphate, 0.5-1 g of dipotassium hydrogen phosphate, 0.5-1 g of magnesium sulfate, 0.2-0.5 g of asparagine, 0.2-0.5 g of sodium glutamate, 0.2-0.5 g of glutamine, 0.2-0.5 g of aspartic acid, 0.2-0.5 g of glycine, and 0.2-0.5 g of inositol. The pH value is natural, and the solvent is water.

[0054] According to the present invention, the culture medium preferably comprises, per liter, the following ingredients: sucrose 60 g, yeast powder 5 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate 1 g, asparagine 0.25 g, sodium glutamate 0.25 g, glutamine 0.25 g, aspartic acid 0.25 g, glycine 0.25 g, inositol 0.25 g, 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 Bacillus subtilis BS-Y-007 that is beneficial for preparing Tremella fuciformis 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 reducing the molecular weight of polysaccharides, and further increasing the polysaccharide yield by freeze-thawing and trypsin hydrolysis. The Tremella fuciformis polysaccharide prepared by the method provided by the present invention reduces the molecular weight of Tremella fuciformis polysaccharide, and the process effectively avoids the interference of organic reagents. While increasing the yield and reducing the molecular weight, it also reduces the production cost and improves the safety of the product and production.

[0059] 3. The polysaccharide prepared by the present invention has a high yield and 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 with reference to the following examples, but the protection scope of the present invention is not limited thereto.

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

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

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

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

[0067] Bacillus subtilis CGMCC 1.821 (purchased from the China General Microbial Culture Collection Center) was subjected to ARTP mutagenesis to screen and obtain the 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 ( Bacillus subtilis )BS-Y-007, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is 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 liquid culture medium

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

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

[0075] The Lactobacillus plantarum seed liquid culture medium was MRS 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 activated at 36°C for 24 h. The culture was continued twice, with the inoculum volume of each subculture being 1% by volume. The subculture conditions were the same as those for activation to obtain Lactobacillus plantarum seed solution.

[0080] Preparation of Bacillus subtilis BS-Y-007 seed solution: Bacillus subtilis was inoculated into LB medium and activated under the conditions of culturing at 36°C for 24 h and 180 rpm. The culture was continued twice, with the inoculum volume of each subculture being 1% by volume. The subculture conditions were the same as those for activation to obtain Bacillus subtilis seed solution.

[0081] Preparation of Tremella fuciformis 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 a volume fraction of 2%, and the activation conditions were 28°C, 180 rpm, and activation for 60 h. 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] For this experiment, a 20-L fermenter was used to maintain a 15-L fermentation medium volume after sterilization. One liter of Tremella fuciformis seed solution (6.6% inoculum by volume) was prepared for each fermenter. The pH of the fermentation solution was adjusted to 5. Once the pH stabilized, the Tremella fuciformis seed solution was inoculated into the fermenter. The fermenter was maintained at 28°C with an aeration rate of 15 L / min. Because Tremella fuciformis is an aerobic bacterium and low dissolved oxygen levels inhibit its growth, an automatic speed-dependent adjustment of dissolved oxygen was configured. Experimental results indicate that the minimum dissolved oxygen value was set at 25% and the maximum at 100%. The speed was set between 200 and 900 rpm, with stirring automatically initiated when the dissolved oxygen value fell below 25%.

[0084] According to the Tremella fuciformis growth curve obtained in 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 decline period, and the bacteria grew slowly. Therefore, the above-mentioned Lactobacillus plantarum seed liquid was inoculated at a volume fraction of 1% at the 120th hour of fermentation. At this time, the pH was adjusted to 6.5 and the temperature was 28°C. After continuing to ferment for 36 hours, Lactobacillus plantarum completed the growth cycle. At this time, the above-mentioned Bacillus subtilis seed liquid was inoculated at a volume fraction of 1%. After inoculation, the culture was continued, and the fermentation temperature and pH remained unchanged. The fermentation was terminated 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] The above Tremella polysaccharide fermentation liquid was taken, sodium chloride was added at a rate of 4% (w / v, g / mL) and stirred to dissolve, centrifuged for 10 min at a speed of 9000 rpm, and the supernatant and bacteria were collected respectively; the bacteria and water were mixed at a volume ratio of 1:1.5, and the mixture was placed in a -20 ° C freezer overnight, taken out and thawed, and the pH was adjusted to 8~9 with sodium hydroxide solution, 2‰ (w / v, g / mL) of pancreatic enzyme was added, and stirred at 55 ° C for 30 min, and then sodium chloride was added at a rate of 4% and stirred to dissolve, and the bacteria were centrifuged for 10 min at a speed of 9000 rpm, 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 to remove excess salts, culture medium nutrients and some pigments; the retained polysaccharide solution is treated three times with an activated carbon fiber membrane (purchased from Beijing Rixin Yuanwang Technology Development Co., Ltd.) to remove fat-soluble pigments to achieve a clear state, and obtain polysaccharide powder by spray drying. The spray drying conditions are as follows: an inlet air temperature of 160°C, an inlet air volume of 95%, and a feed flow rate of 15 mL / min.

[0087] 4. Index determination:

[0088] The total sugar and protein determinations of the polysaccharides prepared above 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, with reference 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 medium components remain unchanged, that is, the same as in Example 1, and the Tremella fuciformis spore fermentation medium is optimized. After optimizing the experimental conditions, it is finally confirmed that the Tremella fuciformis spore fermentation medium: sucrose 60 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 1 g / L, asparagine 0.25 g / L, sodium glutamate 0.25 g / L, glutamine 0.25 g / L, aspartic acid 0.25 g / L, glycine 0.25 g / L, inositol 0.25 g / 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 replaced, 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 no longer undergoes 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. This time, the enzyme was added at 5 g / L to avoid the difference caused by insufficient enzyme dosage. Yield was used as an indicator. Experiment 2 did not perform enzymatic hydrolysis treatment, and other treatments were the same.

[0102] Experimental Group 1: Comparison of screening results of different enzyme treatments is shown 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 enzymatic hydrolysis of bacterial fluid on Tremella polysaccharide production

[0107]

[0108] Comparative Example 3

[0109] Compared with Example 2, this group of experiments only used Tremella fuciformis for fermentation, without adding Lactobacillus plantarum and Bacillus subtilis for co-fermentation, and all other conditions were 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 polysaccharide

[0112]

[0113] 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 that of Comparative Example 2, both freezing and enzymatic hydrolysis can 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.

[0114] Comparative Example 4

[0115] Compared with Example 2, Experiment 1 screened Tremella fuciformis, using the mass of polysaccharide produced per liter of fermentation broth as an indicator; Experiment 2 compared Bacillus bacteria, using the mass of polysaccharide produced per liter of fermentation broth as an indicator.

[0116] Experimental Group 1: Screening of Tremella fuciformis

[0117] Tremella fuciformis strains were purchased from the Shanghai Microbial Culture Collection Center and the China General Microbial Culture Collection Center. The experimental results are shown in Table 6.

[0118] Table 6

[0119]

[0120] Experiment 2: Screening of Bacillus subtilis

[0121] Bacillus subtilis strains CGMCC 1.821, CGMCC 1.15792, and CGMCC 1.7740 were purchased from the China Center for General Microbiological Culture Collection. The Bacillus subtilis strain BS-Y-007 used in this experiment was obtained by laboratory mutagenesis and is currently deposited in the China Center for Type Culture Collection under the strain number CCTCC M 20242282. The experimental results are shown in Table 7.

[0122] Table 7

[0123]

[0124] Comparative Example 5

[0125] 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.

[0126] By comparing the situation of adding two strains at the same time and adding a single strain, when two bacteria act on the fermentation system, the effect is far better than the effect of a single strain.

[0127] Table 8

[0128]

[0129] Effect Examples

[0130] 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:

[0131] 1. Prepare a 0.1 mmol / L DPPH standard stock solution in anhydrous ethanol (keep at low temperature and away from light); prepare a 7 mmol / L ABTS diammonium salt stock solution in water.

[0132] 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.

[0133] 3. DPPH free radical determination method:

[0134] Take 3 mL of Tremella fuciformis polysaccharide solution of varying concentrations, add 3 mL of DPPH solution, mix quickly, and let stand in the dark for half an hour. Measure the absorbance at 517 nm using a spectrophotometer. Repeat the measurement three times. Use anhydrous ethanol as the blank control and VC as the positive control.

[0135] The scavenging rate of DPPH free radicals by VC and Tremella polysaccharide solution was calculated according to the following formula:

[0136]

[0137] Where: A blank is the absorbance of DPPH working solution; A sample is the absorbance of sample solution

[0138] 4. ABTS Free Radical Assay Method: ABTS working solution: Accurately measure 25 mL each of ABTS diammonium salt stock solution and potassium disulfate solution and mix thoroughly. Incubate in the dark at 4°C for 12 hours to obtain the ABTS stock solution. Dilute with anhydrous ethanol solution before use.

[0139] Take 0.5 mL of different concentrations of Tremella polysaccharide sample solution and mix with 5 mL of ABTS diluent, keep in the dark for 10 minutes, and measure in parallel three times. The experimental group uses anhydrous ethanol, and the positive control group uses VC.

[0140] The scavenging rates of VC and Tremella polysaccharide on ABTS free radicals were calculated according to the following formula:

[0141]

[0142] Where: A blank is the absorbance of ABTS cation working solution; A sample is the absorbance of sample solution

[0143] The DPPH and ABTS free radical scavenging rates of the VC control solution were calculated as follows, see Table 9:

[0144] Table 9

[0145]

[0146] The calculation of the DPPH free radical scavenging rate of the sample solution is as follows, see Table 10:

[0147] Table 10

[0148]

[0149] The calculation of the ABTS free radical scavenging rate of the sample solution is as follows, see Table 11:

[0150] Table 11

[0151]

[0152] Changes in uronic acid content of Tremella polysaccharides prepared in Example 2 and Comparative Example 3

[0153] The carbazole-sulfuric acid method was used to determine uronic acid.

[0154] 1. Borax-sulfuric acid solution: Weigh 9.54 g of sodium tetraborate and dissolve it in 1000 mL of concentrated sulfuric acid.

[0155] 2. Carbazole solution: Accurately weigh 0.125 g, dissolve in 100 mL of anhydrous ethanol, and store in a brown bottle.

[0156] 3. Prepare a 1 mg / mL Tremella polysaccharide solution, weigh 5 g of the solution and dilute it to a 50 mL volumetric flask to obtain the sample solution.

[0157] 4. The specific adding method is as follows:

[0158] Mix 1 mL of the test solution, cool in an ice bath, and slowly add 5 mL of borax-sulfuric acid solution, which has been cooled in an ice bath. Seal the tube tightly and mix thoroughly. Heat in a boiling water bath for 10 minutes, then cool to room temperature in an ice bath. Accurately add 2 mL of carbazole, mix thoroughly, heat in a boiling water bath for 15 minutes, and cool to room temperature in an ice bath. Measure absorbance at 530 nm. Calculate the glucuronic acid concentration based on the calibration curve (see Table 12).

[0159]

[0160] Where: X G : glucuronic acid content, unit is %; glucuronic acid C1: glucuronic acid concentration in the test solution calculated by regression equation, unit is μg / mL; W1: weighed mass of the test sample, unit is g;

[0161] W2: The amount of the diluted test solution in the second step, in g; h: Loss on drying, %

[0162] Table 12

[0163]

[0164] The above experimental results show that, compared with Comparative Example 3, the uronic acid content in Example 2 is increased after co-cultivation, reaching 10 mg / mL, and the ABTS and DPPH free radical scavenging rates after co-cultivation 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 polysaccharide produced by co-cultivation.

[0165] The present invention screened a strain of Bacillus subtilis BS-Y-007 through strain mutagenesis that is beneficial for producing low-molecular-weight Tremella fuciformis polysaccharides. This invention, for the first time, uses Lactobacillus plantarum and Bacillus subtilis to co-ferment Tremella fuciformis spore fermentation broth, achieving a reduction in polysaccharide molecular weight. Freeze-thaw and pancreatic enzymatic hydrolysis further increase polysaccharide yield. The present invention provides a method for preparing Tremella fuciformis polysaccharides, achieving a reduction in molecular weight. The process effectively avoids interference from organic reagents, increasing yield and reducing molecular weight while reducing production costs and improving product and production safety. The polysaccharide prepared by the present invention has a high yield and purity exceeding 94%, a molecular weight of 4 to 6 kDa, and an effective free radical scavenging activity of over 85% against DPPH and ABTS free radicals at 10 mg / mL.

Claims

1. A method for preparing a fermented product containing low molecular weight Tremella polysaccharide, characterized in that: The steps include: The method comprises the following steps: using Tremella fuciformis spores to carry out liquid fermentation to prepare Tremella fuciformis polysaccharide fermentation liquid, first inoculating Lactobacillus plantarum into the prepared Tremella fuciformis polysaccharide fermentation liquid for fermentation, continuing to culture for 24 to 48 hours, and then inoculating Bacillus subtilis for fermentation, and obtaining a fermentation product containing low molecular weight Tremella fuciformis polysaccharide after the fermentation is completed; The plant lactobacillus is plant lactobacillus CGMCC1.

572.

2. The preparation method according to claim 1, wherein The steps include: (1) activating and culturing Lactobacillus plantarum and Bacillus subtilis to obtain Lactobacillus plantarum seed liquid and Bacillus subtilis seed liquid respectively; (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 fuciformis polysaccharide fermentation liquid prepared in step (2) for fermentation, and then inoculating the Bacillus subtilis seed liquid prepared in step (1) for fermentation. After the fermentation is completed, a fermentation product containing low molecular weight Tremella fuciformis polysaccharide is obtained.

3. The preparation method according to claim 2, wherein In step (1), activating and culturing Lactobacillus plantarum comprises the following steps: Lactobacillus plantarum is inoculated into a lactic acid bacteria culture medium and cultured at 35-37°C for 18-24 hours for activation. After activation, it can be used directly or subcultured 1-3 times. The inoculation amount for each subculture is 1%-3% by volume. The subculture conditions are the same as the activation conditions to obtain a Lactobacillus plantarum seed solution.

4. The preparation method according to claim 3, wherein The lactic acid bacteria culture medium is MRS culture medium.

5. The preparation method according to claim 2, wherein In step (1), activating and culturing Bacillus subtilis includes the following steps: Bacillus subtilis was inoculated into bacterial culture medium and activated under the conditions of culturing at 35-37°C for 18-24 h and 170-230 rpm. After activation, it was used directly or continued to be subcultured 1-3 times. The inoculation amount for each subculture was 1%-3% by volume. The subculture conditions were the same as the activation conditions to obtain Bacillus subtilis seed solution.

6. The preparation method according to claim 5, wherein The bacterial culture medium is LB culture medium; LB medium contains 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and water as the solvent.

7. The preparation method according to claim 2, wherein In step (2), the Tremella fuciformis spores are activated and then fermented, which includes the following steps: ①Inoculate Tremella fuciformis spores into the seed culture medium and culture at 25-30℃ and 150-250 rpm until OD 560 The temperature is 13-16, and the culture is obtained and used as bacterial liquid; Alternatively, the obtained culture can be mixed with 50% glycerol and used as a preserved strain, and then directly activated into a bacterial liquid for use; 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 activation for 48 to 60 hours. The culture is subcultured 1 to 3 times, and the inoculation amount for each subculture is 1% to 2% by volume. The subculture conditions are the same as the activation conditions to obtain a Tremella fuciformis seed liquid. ② The tremella seed liquid obtained in step ① is inoculated into the 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, the temperature is 25 to 30°C, and aerobic fermentation is carried out for 120 to 150 hours to obtain a tremella polysaccharide fermentation liquid.

8. The preparation method according to claim 7, wherein In step ②, the aerobic fermentation condition is a ventilation volume of 15~30L / min, the dissolved oxygen-related speed is set to automatically adjust, the speed is set to 200~900 rpm, the minimum dissolved oxygen value is set to 25%, and the maximum is 100%. When the dissolved oxygen value is lower than 25%, stirring is automatically started.

9. The preparation method according to claim 7, wherein In step ①, the seed culture medium comprises: 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 water as the solvent; In step ②, the fermentation medium comprises, per liter, 40-80 g of sucrose, 2-6 g of yeast powder, 0.4-0.6 g of potassium dihydrogen phosphate, 0.5-1 g of dipotassium hydrogen phosphate, 0.5-1 g of magnesium sulfate, 0.2-0.5 g of asparagine, 0.2-0.5 g of monosodium glutamate, 0.2-0.5 g of glutamine, 0.2-0.5 g of aspartic acid, 0.2-0.5 g of glycine, and 0.2-0.5 g of inositol; the pH is natural, and the solvent is water; 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 potassium hydrogen phosphate, 1 g of magnesium sulfate, natural pH, and water as the solvent.

10. The preparation method according to claim 9, wherein The fermentation medium comprises the following components per liter: sucrose 60 g, yeast powder 5 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate 1 g, asparagine 0.25 g, sodium glutamate 0.25 g, glutamine 0.25 g, aspartic acid 0.25 g, glycine 0.25 g, inositol 0.25 g, pH is natural, and the solvent is water.

11. The preparation method according to claim 2, wherein In step (3), the Lactobacillus plantarum seed solution prepared in step (1) is inoculated into the Tremella polysaccharide fermentation liquid prepared in step (2) at an inoculation amount of 0.5% to 1% by volume, 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 into the culture at an inoculation 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.

12. The preparation method according to claim 2, wherein In the preparation method, the Bacillus subtilis is Bacillus subtilis BS-Y-007; The specific deposit information of the Bacillus subtilis BS-Y-007 is as follows: A Bacillus subtilis ( 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, with the deposit number being CCTCC NO: M 20242282.

13. The preparation method according to claim 2, wherein 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 SHMCCD24212, SHMCC D24214 or SHMCC D67684.

14. The preparation method according to claim 13, wherein In the preparation method, the Tremella fuciformis strain includes Tremella fuciformis SHMCC D82536 or Tremella fuciformis SHMCC D24212.

15. A method for producing low molecular weight Tremella polysaccharide, characterized in that: This includes any of the following options: Option 1: The fermentation product containing low molecular weight Tremella fuciformis polysaccharide prepared by the method according to any one of claims 1 to 14 is subjected to solid-liquid separation, the liquid is retained, the solid is frozen and thawed, and then treated with pancreatic enzyme, followed by solid-liquid separation, and the separated liquid is mixed with the retained liquid to extract and prepare low molecular weight Tremella fuciformis polysaccharide; Option 2: The fermented product containing low molecular weight Tremella polysaccharide prepared by the method according to any one of claims 1 to 14 is frozen and thawed, treated with pancreatic enzyme, and then solid-liquid separated. The low molecular weight Tremella polysaccharide is extracted and prepared using the separated liquid.

16. The method according to claim 15, wherein The method includes the following steps: adding sodium chloride to a final concentration of 2-4% to the fermentation product containing low-molecular-weight tremella polysaccharide prepared by the method according to any one of claims 1 to 14, dissolving the product, performing solid-liquid separation, retaining the liquid, adding 1-1.5 times the volume of water to the solid and freezing and thawing it, adjusting the pH to 8-9, adding 2-5‰ of pancreatin, stirring at 45-55°C for 20-40 min, adding sodium chloride to a final concentration of 2-4% to dissolve it, then performing solid-liquid separation, mixing the separated liquid with the retained liquid, and passing the mixed solution through a 200-500 Dal ultrafiltration membrane to remove excess salts, 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; The second method comprises the following steps: The fermented product containing low molecular weight Tremella fuciformis polysaccharide prepared by the method according to any one of claims 1 to 14 is frozen and thawed, and the pH is adjusted to 8-9, 2-5‰ pancreatin is added, and the product is stirred at 45-55°C for 20-40 min, and sodium chloride is added to a final concentration of 2-4% to dissolve the product, followed by solid-liquid separation, and the separated liquid is ultrafiltered at 200-500 Dal to remove excess salts, culture medium nutrients and part of the pigment, and the retained polysaccharide solution is further decolorized and clarified to obtain a low molecular weight Tremella fuciformis polysaccharide solution; Alternatively, the low molecular weight Tremella polysaccharide solution is spray-dried to obtain the low molecular weight Tremella polysaccharide powder.

17. The method according to claim 16, wherein In the first or second solution, the solid-liquid separation method includes one or both of centrifugation and filtration.

18. The method according to claim 17, wherein 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.

19. The method according to claim 16, wherein In the first or second solution, the retained polysaccharide solution is decolorized by passing through an activated carbon fiber membrane.

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