Preparation method and application of edible fungus and probiotic co-culture fermentation broth

Abstract

The invention belongs to the field of fermentation engineering, and particularly discloses a preparation method and application of edible fungus and probiotic co-culture fermentation liquor. A bidirectionally promoted edible fungus-probiotic symbiotic metabolism system is constructed by adopting a synchronous inoculation technology and combining with an optimized culture medium formula taking grain processing materials such as coarse rice powder as a main substrate. According to the method, in the culture period of 7-14 days, the fermentation liquor with the total triterpene content not lower than 35 mg / g, the total polysaccharide content not lower than 25 mg / g and the viable count of probiotics reaching 1 * 10 < 8 > CFU / mL or above can be obtained. The technology has the advantages of being easy and convenient to operate, low in cost and short in period, the content of the active ingredients of the edible fungi is remarkably increased while the viable count of the probiotics is maintained, and a new technical scheme is provided for developing high-quality functional food or preparations with the probiotic function and the active ingredients of the fungi.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering, specifically relating to a method for preparing a fermentation broth co-cultured with edible fungi and probiotics and its application. Background Technology

[0002] Edible fungi, as important biological resources, have broad application prospects in the development of functional foods and drugs. Representative species such as Ganoderma lucidum, Cordyceps militaris, and Grifola frondosa, rich in polysaccharides, triterpenoids, sterols, and other bioactive substances, have shown significant effects in immune regulation, anti-tumor activity, and blood glucose and lipid regulation, and are increasingly becoming the focus of attention in the health industry.

[0003] Currently, the large-scale production technology of edible fungi mainly follows two technical routes: The first route is fruiting body cultivation technology, which artificially simulates the natural environment to promote the development of mycelium into complete fruiting bodies, obtaining fungal fruiting bodies with structures and compositions close to natural products and high content of active ingredients. However, this traditional method has inherent drawbacks such as excessively long production cycles (usually requiring several months to several years), large land area requirements, high labor costs, high energy consumption, and susceptibility to environmental factors, making it difficult to meet the needs of modern large-scale production. The second route is liquid submerged fermentation technology, which cultivates mycelium in fermenters, enabling the acquisition of a large biomass in a relatively short time (usually several weeks). However, numerous studies have shown that the content of key active ingredients (such as polysaccharides and triterpenes) in mycelium obtained through liquid fermentation is often significantly lower than that in fruiting bodies, which severely limits its application value in high-end functional products.

[0004] To address the aforementioned issues, the industry has begun exploring co-cultivation techniques for edible fungi and probiotics. In the prior art, patent document CN106387652B discloses a stepwise cultivation method. This technique utilizes the differences in growth rates between fungi and probiotics, achieving co-cultivation through sequential inoculation. Specific operations include first cultivating fungal mycelium to a certain biomass before inoculating with probiotics, or using the reverse inoculation order. However, this stepwise cultivation model has significant drawbacks: First, the operation process is complex, requiring multiple inoculations and aseptic techniques, significantly increasing the risk of contamination during production; second, stepwise cultivation leads to a prolonged overall fermentation cycle, generally longer than the cultivation time for a single strain, reducing production efficiency; most importantly, although this technology can ensure that the final product has a certain number of viable probiotics and a suitable flavor, its effect on increasing the content of effective components in edible fungi is unclear, and existing publicly available data fail to demonstrate that its active ingredient content is significantly better than, or even close to, the level of fruiting bodies in single-strain cultivation.

[0005] There is an urgent need in this field to develop a process for co-culturing edible fungi and probiotics. This process should be able to simplify the operation process and shorten the production cycle, while breaking through the existing technical bottlenecks and significantly increasing the content of the main active ingredients (polysaccharides, triterpenes, etc.) of edible fungi while maintaining a high number of live probiotics, thereby providing reliable technical support for the development of high-quality functional products. Summary of the Invention

[0006] This invention aims to overcome existing technological bottlenecks and provide a method for preparing a fermentation broth co-cultured with edible fungi and probiotics, as well as its applications. This preparation method establishes a symbiotic metabolic system of edible fungi and probiotics through the synergistic optimization of simultaneous inoculation technology and a specific culture medium formulation. The resulting fermentation broth not only has a high number of viable probiotics but also significantly increases the content of active ingredients such as total triterpenes and total polysaccharides. This invention further provides application directions for the prepared fermentation broth.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] A method for preparing a co-culture fermentation broth of edible fungi and probiotics includes the following steps: (1) preparing a culture medium containing grain processed matter: adding 1-4 wt% of grain processed matter to water, then adding carbon source, nitrogen source and inorganic salt in sequence, mixing evenly and then sterilizing and adjusting pH value; (2) preparing seed liquid: inoculating activated edible fungi strains and probiotic strains into seed culture medium, and obtaining edible fungi seed liquid and probiotic seed liquid after cultivation; (3) synchronous inoculation: synchronously inoculating 5-10 vol% of edible fungi and probiotic seed liquid into the culture medium prepared in step (1); (4) mixed culture: culturing at 26-29.5℃ for 7-14 days to obtain co-culture fermentation broth.

[0009] Furthermore, the processed grain product is at least one of brown rice flour, corn flour, or potato extract.

[0010] Furthermore, the processed grain product is brown rice flour.

[0011] Furthermore, the edible fungus is Ganoderma lucidum, Cordyceps militaris, or Grifola frondosa.

[0012] Furthermore, the probiotics are Lactobacillus plantarum, Lactobacillus paracasei, or Bacillus subtilis.

[0013] Furthermore, the culture medium contains 1-3 wt% sucrose, 0.5-1 wt% soybean peptone, 0.1-0.2 wt% dipotassium hydrogen phosphate and 0.05-0.1 wt% anhydrous magnesium sulfate.

[0014] Furthermore, the pH of the culture medium is adjusted to 4-7.

[0015] Furthermore, the number of live probiotics in the fermentation broth is not less than 1×10⁻⁶. 8 The CFU / mL content is not less than 35 mg / g for total triterpenes and not less than 25 mg / g for total polysaccharides.

[0016] Furthermore, the above-mentioned fermentation broth can be used to prepare functional foods or preparations.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] Significantly enhanced active ingredients: Through symbiotic metabolic mechanisms, the total triterpenoid content increased by 26.41-65.52%, and the total polysaccharide content increased by 31.67-71.42%, both higher than those of monoculture and stepwise culture;

[0019] Symbiotic metabolic advantages: Probiotics produce acid through metabolism, which promotes fungal enzyme activity. Fungi degrade cellulose and release reducing sugars, which in turn feed the probiotics, forming a positive symbiotic metabolic cycle that promotes both fungi and probiotics.

[0020] Simplified process: Simultaneous inoculation avoids the complex operations and contamination risks of stepwise culture, and shortens the culture cycle to 7-14 days, which is 17.65-58.23% shorter than the stepwise culture cycle;

[0021] Significant cost advantage: Using inexpensive processed grains as a base reduces raw material costs by more than 30%;

[0022] The product has high overall value: it is rich in active ingredients of edible fungi and live probiotics, and no further compounding is required. Attached Figure Description

[0023] Figure 1 The effects of different culture medium substrates on total triterpenoid content and mycelial content Detailed Implementation

[0024] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, the experimental methods used are conventional methods, and the reagents or instruments used are all commercially available conventional products. Unless otherwise specified, all reagents used in the present invention are analytical grade reagents.

[0025] 1. Methods for detecting active ingredients

[0026] 1.1 Determination of fungal mycelium content

[0027] The fermentation broth was centrifuged at 2500 g for 25 minutes to collect the fungal mycelial precipitate. The precipitate was washed with distilled water and centrifuged again, repeated three times to remove any remaining culture medium. The washed mycelium was pre-frozen at -80°C and then dried in a freeze dryer to constant weight. The weight of the dried mycelium was accurately weighed, and the mycelial content in the fermentation broth (g / L fermentation broth) was calculated according to formula (1):

[0028] (1)

[0029] 1.2 Determination of viable probiotic count

[0030] The viable cell count was determined using the plate count method. The fermentation broth was serially diluted with sterile physiological saline to an appropriate concentration. 100 μL of the diluted solution was spread onto an MRS agar plate and incubated upside down at 37°C for 48 hours. Plates with colony counts between 30 and 300 were selected for counting, and the viable cell count (CFU / mL) was calculated using formula (2):

[0031] (2)

[0032] Each dilution was performed in triplicate, and results are expressed as mean ± standard deviation.

[0033] 1.3 Determination of total triterpenoid content

[0034] The vanillin-perchloric acid method was used for determination. The fermentation broth was freeze-dried to remove all moisture, obtaining a powdered solid. 1.0 g of the fermentation broth powder was accurately weighed and added to 50 mL of 95% ethanol. After disruption using a cell disruptor, the mixture was ultrasonically extracted at 75℃ and 400W for 1 hour. This extraction was repeated once. The extracts were combined and diluted to 100 mL. 0.1 mL of the extract was placed in a test tube, and the solvent was evaporated at 60℃. Then, 0.2 mL of 5% (m / v) vanillin-glacial acetic acid solution and 0.5 mL of perchloric acid were added sequentially. The mixture was reacted in a 60℃ water bath for 20 minutes. After cooling with ice water, 5 mL of glacial acetic acid was added, and the absorbance was measured at 550 nm. A standard curve was plotted using oleanolic acid as a standard (linear equation: Y = 0.0011X + 0.8728, R0). 2 =0.9978), calculate the total triterpenoid content (mg / g) according to formula (3):

[0035] (3)

[0036] 1.4 Determination of total polysaccharide content

[0037] The phenol-sulfuric acid method was used for determination. 1.0 g of the freeze-dried fermentation broth powder obtained in step 1.3 was accurately weighed, added to 20 mL of distilled water, and disrupted using a cell disruptor. The mixture was then ultrasonically extracted at 65℃ and 800 W for 1 hour, and the extraction was repeated once. The extracts were combined, and 4 volumes of 95% ethanol were added. The mixture was precipitated at 4℃ for 24 hours, and the precipitate was collected by centrifugation at 3000 r / min for 20 minutes. The precipitate was washed twice with 95% ethanol, then extracted in 1 M NaOH solution at 60℃ for 1 hour. The supernatant was collected by centrifugation and brought to a final volume of 100 mL. 1 mL of the sample solution was taken, and the volume was increased to 2 mL with deionized water. 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid were added, and the mixture was reacted in a boiling water bath for 15 minutes. After cooling, the absorbance was measured at 490 nm. A standard curve was plotted using glucose as the standard (the linear regression equation for the total sugar standard curve was: Y = 18.452X - 0.0007, R0). 2 =0.998); reducing sugar content was determined by the DNS method (standard curve: Y=30.801X+0.9528, R0). 2 =0.9982), calculate the total polysaccharide content (mg / g) according to formula (4):

[0038] (4)

[0039] Example 1: Preparation of fermentation broth from co-culture of Ganoderma lucidum and Lactobacillus plantarum

[0040] (1) Materials

[0041] Ganoderma lucidum was purchased from the China General Microbiological Culture Collection Center, strain number CGMCC 5.616;

[0042] Lactiplantibacillus plantarum was isolated in the laboratory and the strain is deposited in the China General Microbiological Culture Collection Center, with the number CGMCC No. 34512.

[0043] Seed culture medium formula: potato extract 1 wt%, glucose 2 wt%, peptone 1.8 wt%, potassium dihydrogen phosphate 0.3 wt%, magnesium sulfate 0.15 wt%, vitamin B1 0.005 wt%, pH 5.5.

[0044] Preparation of seed solution:

[0045] The activated Ganoderma lucidum strain was inoculated into the seed culture medium and cultured statically for 7 days to obtain Ganoderma lucidum seed liquid;

[0046] The activated *Lactobacillus plantarum* strain was inoculated into a seed culture medium with the same formulation and cultured with shaking for 16 hours to obtain *Lactobacillus plantarum* seed liquid.

[0047] (2) Fermentation process

[0048] Weigh 40 g of brown rice flour, add 1 L of deionized water, and then add 30 g of sucrose, 10 g of soybean peptone, 2 g of dipotassium hydrogen phosphate, and 1 g of anhydrous magnesium sulfate in sequence. Adjust the pH to 7.0 with 1 mol / L sodium hydroxide solution and autoclave at 121 ℃. After the culture medium cools to room temperature, simultaneously inoculate 50 mL of Ganoderma lucidum seed culture and 50 mL of Lactobacillus plantarum seed culture, and incubate in a constant temperature incubator at 29.5 ℃ for 14 days.

[0049] (3) Test results

[0050] The test results showed that the total polysaccharide content of the co-culture fermentation products was 38.10±1.32 mg / g, the total triterpenoid content reached 48.18±2.65 mg / g, the Ganoderma lucidum mycelium content was 16.91±0.59 g / L, and the viable count of Lactobacillus plantarum was 2.0×10⁻⁶. 8 CFU / mL.

[0051] Comparative Example 1: Preparation of Ganoderma lucidum monoculture fermentation broth

[0052] Following the seed culture medium formulation, culture medium formulation, and culture conditions of Example 1, only Ganoderma lucidum seed liquid was inoculated. The results of detecting the active ingredients in the single-culture product of Ganoderma lucidum showed that the total polysaccharide content was 10.89±0.34 mg / g, the total triterpenoid content was 19.44±0.23 mg / g, and the mycelium content was 3.76±0.09 g / L.

[0053] Referring to the seed culture medium formulation, culture medium formulation, and culture conditions of Example 1, only *Lactobacillus plantarum* seed culture was inoculated, and the viable count of *Lactobacillus plantarum* monoculture in the fermentation broth was 7.2 × 10⁻⁶. 7 CFU / mL.

[0054] Compared with single-culture of Ganoderma lucidum, co-culture fermentation products increased the total polysaccharide content by 71.42%, the total triterpenoid content by 59.65%, the mycelial content of Ganoderma lucidum and the viable count of Lactobacillus plantarum by 77.76% and 64.00%, respectively.

[0055] Comparative Example 2: Comparison of active ingredients in Ganoderma lucidum fruiting bodies, spore powder, and co-culture products.

[0056] The fruiting bodies and spores of *Ganoderma lucidum* were purchased from Dalian Yichuntang Pharmacy, originating from Shangluo, Shaanxi Province. The total polysaccharide and total triterpenoid contents were tested. These data were compared with the co-culture product prepared in Example 1 and the single-culture product in Comparative Example 1 for active ingredients. The results are detailed in Table 1. Analysis showed that the total triterpenoid content in the co-culture fermentation broth was significantly higher than that in the fruiting bodies and approached the level of the spores; while the total polysaccharide content was significantly better than that in both the fruiting bodies and spores, demonstrating the advantages of the co-culture process used in this invention in enhancing active ingredients.

[0057] Table 1

[0058] ,

[0059] Note: Lowercase letters in the table indicate statistically significant differences, p < 0.05.

[0060] Examples 2-6: Co-culture experiments under different conditions

[0061] Referring to the seed culture medium formulation and culture method in Example 1, multiple co-culture experiments were conducted with relevant parameters adjusted. Specific parameters for adjustment are detailed in Table 2.

[0062] Table 2

[0063] ,

[0064] The content of active ingredients in the co-culture products obtained from each group of experiments was detected separately, and the specific detection results are shown in Table 3.

[0065] Table 3

[0066] ,

[0067] Under different co-culture conditions, the experimental results showed that the yields of total triterpenes and total polysaccharides were both increased, and the viable count of probiotics remained at a high level. Even in a culture environment with low substrate concentration, the total triterpenes content reached 39.28±0.34 mg / g, the total polysaccharide content was 30.22±0.78 mg / g, and the viable count remained at 1.2×10⁻⁶. 8 The CFU / mL concentration indicates that the present invention still possesses good fermentation efficiency under simplified culture medium conditions. Furthermore, a total of 12 inoculation operations were performed in Examples 1-6, and no contamination occurred.

[0068] The reason for this success lies in the establishment of a highly efficient symbiotic metabolic mechanism between fungi and probiotics in this co-culture system. Organic acids such as lactic acid produced during probiotic metabolism naturally regulate the system's pH, creating a suitable acidic environment. This environment not only promotes the rapid growth of edible fungal mycelia but also activates their intracellular enzyme systems, accelerating the decomposition of structural polysaccharides such as cellulose and hemicellulose in the culture medium, releasing a large amount of reducing sugars. These reducing sugars further serve as a high-quality carbon source, promoting the growth and reproduction of probiotics, thus constructing a bidirectional metabolic cycle system of "fungi-probiotics".

[0069] Example 7: Comparison of different grain substrates

[0070] Following the cultivation method of Example 1, comparative experiments were conducted using different processed grains (brown rice flour, corn flour, and potato extract) as substrates. The composition of the substrates was as follows: processed grains 1 wt%, sucrose 1 wt%, soybean peptone 0.5 wt%, dipotassium hydrogen phosphate 0.1 wt%, anhydrous magnesium sulfate 0.05 wt%, pH 7, *Ganoderma lucidum* inoculum 5 vol%, and *Lactobacillus plantarum* inoculum 5 vol%. The mixture was cultured at 26 °C for 7 days to prepare a co-culture fermentation broth of *Ganoderma lucidum* and *Lactobacillus plantarum*. Following the cultivation method of Comparative Example 1, single-culture fermentation broths of *Ganoderma lucidum* and *Lactobacillus plantarum* were prepared separately. The total triterpenoid content and mycelial content of the prepared fermentation broths were analyzed, and the specific results are as follows: Figure 1 As shown.

[0071] Experimental data showed that different processed grains, when used as substrates, yielded higher total triterpenoid and mycelial contents compared to monoculture. Among them, brown rice flour showed the highest total triterpenoid content (48.91±1.32 mg / g) and mycelial content (16.80±0.32 g / L). This indicates that the vitamins and niacin antioxidants abundant in brown rice flour are more conducive to mycelial growth and metabolite synthesis, and that the nutrients in brown rice are more beneficial to human health. Compared to refined rice, brown rice is lower in cost and more suitable for microbial culture and industrial production.

[0072] Example 8: Co-culture of Cordyceps militaris and Bacillus subtilis

[0073] Cordyceps militaris was purchased from the China Agricultural Microbial Culture Collection Center, accession number ACCC52353;

[0074] Bacillus subtilis was purchased from the China General Microbiological Culture Collection Center, with the strain number CGMCC 1.102.

[0075] The seed culture medium used the same formula as in Example 1.

[0076] 5 vol% *Cordyceps militaris* and 5 vol% *Bacillus subtilis* seed culture were simultaneously inoculated into a culture medium (1 wt% brown rice flour, 1 wt% sucrose, 0.5 wt% soybean peptone, 0.1 wt% dipotassium hydrogen phosphate, 0.05 wt% anhydrous magnesium sulfate, pH=4) and cultured at 26℃ for 7 days to prepare a co-culture fermentation broth of *Cordyceps militaris* and *Bacillus subtilis*. Following the culture method of Comparative Example 1, single-culture fermentation broths of *Cordyceps militaris* and *Bacillus subtilis* were prepared separately.

[0077] The test results showed that the total polysaccharide content in the co-culture product of Cordyceps militaris and Bacillus subtilis was 21.23±1.34 mg / g, the total triterpenoid content was 13.44±0.21 mg / g, the mycelium content was 6.26±0.33 g / L, and the viable Bacillus subtilis count reached 4.5×10⁻⁶. 8 The CFU / mL concentration met the recommended dosage for probiotics. In the single-cultured Cordyceps militaris fermentation broth, the total polysaccharide content was 7.32±0.51 mg / g, the total triterpenoid content was 9.89±0.81 mg / g, the mycelial content was 4.72±0.67 g / L, and the viable Bacillus subtilis count was 3.2×10⁻⁶. 8 CFU / mL. After co-culture, the total polysaccharide content increased by 65.52%, the total triterpenoid content increased by 26.41%, and the mycelium content of Cordyceps militaris and the viable count of Bacillus subtilis increased by 24.60% and 28.89%, respectively.

[0078] Example 9: Co-culture of Grifola frondosa and Lactobacillus paracasei

[0079] Grifola frondosa was purchased from the China Agricultural Microbial Culture Collection Center, accession number ACCC52761;

[0080] Lactobacillus paracasei was purchased from the China General Microbiological Culture Collection Center, CGMCC No. 1.12731.

[0081] The seed culture medium used the same formula as in Example 1.

[0082] Ten vol% *Grifola frondosa* and ten vol% *Lactobacillus paracasei* seed cultures were simultaneously inoculated into a culture medium (4 wt% brown rice flour, 3 wt% sucrose, 1 wt% soybean peptone, 0.2 wt% dipotassium hydrogen phosphate, 0.1 wt% anhydrous magnesium sulfate, pH=7) and cultured at 29.5℃ for 14 days to prepare a co-culture fermentation broth of *Grifola frondosa* and *Lactobacillus paracasei*. Following the culture method of Comparative Example 1, single-culture fermentation broths of *Grifola frondosa* and *Lactobacillus paracasei* were prepared separately.

[0083] The test results showed that the total polysaccharide content of the co-culture product of Grifola frondosa and Lactobacillus paracasei was 25.10±2.31 mg / g, the total triterpenoid content was 18.07±1.30 mg / g, the mycelial content was 11.74±0.98 g / L, and the viable count of Lactobacillus paracasei reached 2.4×10⁻⁶. 8 The CFU / mL concentration met the recommended dosage for probiotics. In the monoculture fermentation broth of *Grifola frondosa*, the total polysaccharide content was 17.15 ± 1.33 mg / g, the total triterpenoid content was 9.34 ± 0.18 mg / g, the mycelial content was 6.24 ± 0.11 g / L, and the viable count of *Bacillus subtilis* was 8.9 × 10⁻⁶. 7 CFU / mL. After co-culture, the total polysaccharide content increased by 31.67%, the total triterpenoid content increased by 48.31%, and the mycelial content of Grifola frondosa and the viable count of Lactobacillus paracasei increased by 46.85% and 62.92%, respectively.

[0084] Comparative Example 3: Stepwise inoculation experiment (fungus first, then probiotics)

[0085] Referring to the seed culture medium, culture medium formula and culture conditions of Example 1, 5 vol% Ganoderma lucidum seed liquid was first inoculated and cultured for 3 days, and then 5 vol% Lactobacillus plantarum was inoculated and cultured for another 14 days, for a total culture period of 17 days.

[0086] The results showed that the total triterpenoid content was only 8.33±0.23 mg / g, the total polysaccharide content was 4.07±0.14 mg / g, and the viable probiotic count was 5.1×10⁻⁶. 7 The concentration was CFU / mL. Three experiments were conducted, and contamination occurred once, resulting in a contamination rate of 33.33%.

[0087] Comparative Example 4: Stepwise inoculation experiment (probiotics first, then fungi)

[0088] First, inoculate with 5 vol% *Lactobacillus plantarum* and culture for 3 days, then inoculate with 5 vol% *Ganoderma lucidum* and continue culturing for 14 days, for a total culture period of 17 days.

[0089] The results showed that the total triterpenoid content was only 4.72±0.41 mg / g, the total polysaccharide content was 2.96±0.09 mg / g, and the viable probiotic count was 7.1×10⁻⁶. 7 The concentration of CFU / mL was high, and the contamination rate reached 20.00%.

[0090] The results of the stepwise inoculation experiment showed that the stepwise inoculation method was inferior to the synchronous inoculation method of the present invention in terms of both effectiveness and stability. Compared with the present invention, the contamination rate of stepwise inoculation was significantly increased. Compared with the culture cycle of stepwise inoculation, the culture time of the present invention is only 7-14 days, which is a reduction of 17.65-58.23%.

[0091] Application Example 1: Development of Functional Foods

[0092] The fermentation broth obtained in Example 1 was freeze-dried into powder, and functional food products were developed using this powder as a raw material.

[0093] Probiotic solid beverage: The formula contains 10% baking powder, 20% fructooligosaccharides, and 70% maltodextrin. After being mixed with warm water, the number of live bacteria is ≥1×10⁻⁶. 10 CFU / serving

[0094] Application Example 2: Development of Functional Foods

[0095] The fermentation broth obtained in Example 1 was homogenized and used as a raw material to prepare functional beverages:

[0096] Probiotic liquid beverage: The formula contains 80% water, 6% white sugar, 2% fructooligosaccharides, 2% skim milk powder, 9% baking powder, 1% citric acid, and a live bacteria count ≥1×10⁻⁶. 10 CFU / 100mL.

Claims

1. A method for preparing a fermentation broth co-cultured with edible fungi and probiotics, characterized in that, Includes the following steps: (1) Preparation of culture medium containing grain processing: Add 1-4 wt% of grain processing to water, then add carbon source, nitrogen source and inorganic salt in sequence, mix evenly and sterilize, and adjust pH value; (2) Preparation of seed liquid: The activated edible fungi strains and probiotic strains were inoculated into the seed culture medium and cultured to obtain edible fungi seed liquid and probiotic seed liquid; (3) Synchronous inoculation: Simultaneously inoculate 5-10 vol% of edible fungi and probiotic seed liquid into the culture medium prepared in step (1); (4) Co-culture: Culture at 26-29.5℃ for 7-14 days to obtain co-culture fermentation broth.

2. The preparation method according to claim 1, characterized in that, The processed grain product is at least one of brown rice flour, corn flour, or potato extract.

3. The preparation method according to claim 2, characterized in that, The processed grain product is brown rice flour.

4. The preparation method according to claim 1, characterized in that, The edible fungi mentioned are Ganoderma lucidum, Cordyceps militaris, or Grifola frondosa.

5. The preparation method according to claim 1, characterized in that, The probiotics are Lactobacillus plantarum, Lactobacillus paracasei, or Bacillus subtilis.

6. The preparation method according to claim 1, characterized in that, The culture medium contains 1-3 wt% sucrose, 0.5-1 wt% soybean peptone, 0.1-0.2 wt% dipotassium hydrogen phosphate and 0.05-0.1 wt% magnesium sulfate.

7. The preparation method according to claim 1, characterized in that, The pH of the culture medium is adjusted to 4-7.

8. The fermentation broth prepared by the method according to any one of claims 1-7, characterized in that, The number of live probiotics in the fermentation broth is not less than 1×10⁻⁶. 8 The CFU / mL content is not less than 35 mg / g for total triterpenes and not less than 25 mg / g for total polysaccharides.

9. The fermentation broth as described in claim 8 is used in the preparation of functional foods or formulations.

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